research article retrotransposon-based molecular...
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Research ArticleRetrotransposon-Based Molecular Markers for Analysis ofGenetic Diversity within the Genus Linum
Nataliya V Melnikova1 Anna V Kudryavtseva1 Alexander V Zelenin1
Valentina A Lakunina1 Olga Yu Yurkevich1 Anna S Speranskaya12 Alexey A Dmitriev1
Anastasia A Krinitsina2 Maxim S Belenikin13 Leonid A Uroshlev1
Anastasiya V Snezhkina1 Asiya F Sadritdinova1 Nadezda V Koroban1
Alexandra V Amosova1 Tatiana E Samatadze1 Elena V Guzenko4 Valentina A Lemesh4
Anastasya M Savilova5 Olga A Rachinskaia1 Natalya V Kishlyan6 Tatiana A Rozhmina6
Nadezhda L Bolsheva1 and Olga V Muravenko1
1 Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow 119991 Russia2 Department of Higher Plants Lomonosov Moscow State University Moscow 119991 Russia3 Research Institute of Physico-Chemical Medicine Moscow 119435 Russia4 Institute of Genetics and Cytology National Academy of Science of Belarus 220072 Minsk Belarus5 Research Center for Obstetrics Gynecology and Perinatology Moscow 117997 Russia6All-Russian Research Institute for Flax of the Russian Academy of Agricultural Sciences Torzhok 172002 Russia
Correspondence should be addressed to Nadezhda L Bolsheva nlbolshevamailru
Received 25 April 2014 Revised 18 July 2014 Accepted 1 August 2014 Published 27 August 2014
Academic Editor Peter F Stadler
Copyright copy 2014 Nataliya V Melnikova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
SSAP method was used to study the genetic diversity of 22 Linum species from sections Linum Adenolinum DasylinumStellerolinum and 46 flax cultivars All the studied flax varieties were distinguished using SSAP for retrotransposons FL9 and FL11Thus the validity of SSAP method was demonstrated for flax marking identification of accessions in genebank collections andcontrol during propagation of flax varieties Polymorphism of Fl1a Fl1b and Cassandra insertions were very low in flax varietiesbut these retrotransposons were successfully used for the investigation of Linum species Species clusterization based on SSAPmarkers was in concordance with their taxonomic division into sections Dasylinum Stellerolinum Adenolinum and Linum Allspecies of sect Adenolinum clustered apart from species of sect Linum The data confirmed the accuracy of the separation in thesesections Members of section Linum are not as closely related as members of other sections so taxonomic revision of this sectionis desirable L usitatissimum accessions genetically distant frommodern flax cultivars were revealed in our work These accessionsare of utmost interest for flax breeding and introduction of new useful traits into flax cultivars The chromosome localization ofCassandra retrotransposon in Linum species was determined
1 Introduction
The genus Linum comprises about 200 species which are dis-tributed throughout the temperate and subtropical regions ofthe worldThe genus is subdivided by Ockendon andWaltersinto five sectionsLinumDasylinum (Planch) JuzLinastrum(Planchon) Bentham Syllinum Griseb and Cathartolinum
(Reichenb) Griseb [1] Some taxonomists classified themembers of the L perenne group from section Linum toan independent section Adenolinum (Reichenb) Juz [2 3]The species L stelleroides (Planch) distributed in Far Eastand China was classified by Yuzepchuk [2] to a monotypesection Stellerolinum Juz ex Prob The phylogenetic analysesbased on chloroplast (ndhF trnL-F and trnK 31015840 intron) and
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 231589 14 pageshttpdxdoiorg1011552014231589
2 BioMed Research International
nuclear ITS (internal transcribed spacer) DNA sequencesrevealed that genus Linum was not monophyletic It con-tains two major lineages a yellow-flowered clade (sectionsLinopsis Syllinum and Cathartolinum) and a blue-floweredclade (sections LinumDasylinum and Stellerolinum) [4]Thecultivated flax (L usitatissimum L) belongs to sec Linumfrom a blue-flowered clade L usitatissimum is believed tohave originated as a result of domestication of wild speciesL angustifolium Huds approximately 8000 years ago [5ndash8]For a long time flax has been cultivating as a dual-purposecrop grown for its fiber and linseed oil
According to morphological and qualitative traits cul-tivated flax was divided into five main types (1) fiber flax(L usitatissimum subsp usitatissimum) (2) oil flax (L usi-tatissimum L subsp humileCzernom) (3) dual-purpose flax(L usitatissimum L subsp intermedium Czemom) that wasan intermediate form between the first two ones cultivatedfor fiber and oil (4) large seeded flax (L usitatissimum Lsubsp latifolium Snankev) which is characterized by a set ofspecific morphological features and cultivated for oil in theMediterranean region and North Africa (5) winter flax (Lusitatissimum L subsp bienne Mill Snankev) cultivated forfiber and oil in theCaucasus Turkey Balkans and some othersouth regions of Europe [9 10] In addition collections offlax germplasm maintain accessions of primitive flax formswith dehiscent capsules (L usitatissimum convar crepitans[Boenningh] Kulpa et Danert) [11]
The taxonomyof the genus cannot be considered as finallyestablished one because the phylogenetic linkages betweenthe individual taxa have not been sufficiently investigatedThe phylogeny of species of the genus Linum was previouslystudied by the use of molecular and cytogenetic approaches[4 12ndash17] but there are problems that still remain to besolved
Transposon-based molecular markers are successfullyused in phylogenic studies Transposable elements wereshown to influence changing in genomic structure as well astranscriptional regulation occurring during the evolution [1819] The presence of transposons in various species of plantstheir high integration activity conservative sequences and alarge number of copies encouraged the use of transposonsin the studies of genetic diversity and profiling of plantvarieties [20ndash22] Several molecular marker systems basedon the information available for the transposable elementssequences were developed for plants [20 22ndash27] SSAP(sequence-specific amplified polymorphism) method wasshown to have a number of advantages as compared to othermarker systems SSAP method produces many polymorphicfragments and allows differentiation of most samples usingonly a single combination of specific primers [23 28ndash30]Different plants were successfully studied by SSAP analysisbut the method has not been applied for the investigation ofspecies of the genus Linum yet Only recently flax sequenceshave appeared in databases [31ndash34] and development of amarker system based on flax transposable elements for theinvestigation of cultivated and wild species of the genusLinum has become possible
In this study the SSAP method was used for assessmentof genetic diversity Besides the possibilities of application
of marker-based profiling for identification of L usitatissi-mum varieties were analyzed We studied 46 varieties of Lusitatissimummainly bred inRussia and a number of varietieswhich were grown in geographically close or distant regionsWe also analyzed different types of cultivated flax (fiberoilseed large seeded winter and dehiscent flax) togetherwith 21 wild species and subspecies from sections LinumAdenolinum Dasylinum and Stellerolinum to estimate thepossibility of using the SSAP method for the investigation offlax domestication history and phylogenic linkages betweendifferent taxa of the genus Linum
2 Materials and Methods
21 Plant Materials For the investigation of genetic diversityof cultivated flax 46 L usitatissimum varieties mainly ofRussian origin were obtained from the All-Russian ResearchInstitute for Flax (VNIIL) (Table 1) For analyzing the repro-ducibility of the SSAP analysis flax variety ldquoStormont cirrusrdquo(IPK genbank Gatersleben accession number LIN 261) wasused
For the investigation of genetic diversity 47 flax acces-sions belonging to 22 species from sections Linum Ade-nolinum Dasylinum and Stellerolinum were used (Table 2)Most of these accessions were obtained from genebank ofLeibniz Institute of Plant Genetics and Crop Plant Research(IPK) (Gatersleben Germany) seed collections of All-Russian Flax Institute (VNIIL) (Torzok Russian Federation)NI Vavilov Research Institute of Plant Industry (VIR) (StPetersburg Russian Federation) and Institute of Geneticsand Cytology of NAS Belarus (IGC) (Minsk Belarus) Theaccessions of L amurense and L stelleroides were kindlyprovided by Dr L N Mironova Botanic Garden Institute ofthe Far-Eastern Branch of the Russian Academy of Sciences(BGI) (Vladivostok Russian Federation) Some accessionswere collected in the wild by Dr A A Svetlova KomarovBotanical Institute RAS (St Petersburg Russian Federation)by Dr N L Bolsheva Engelhardt Institute of MolecularBiology RAS (Moscow Russian Federation) and by Dr MPavelka Euroseeds (Novy Jicin Chech Rep)
22 Confirmation of Species Determination Species determi-nation of some accessions of wild Linum species was doneduring the course of our earlier cytogenetic investigations[14 17 37] To confirm the species determination the restof the accessions were planted in the ground and addition-ally chromosome analysis (determination of chromosomenumber) using acetocarmine staining according to previouslydeveloped approach was performed [14] Chromosome num-bers of all the studied accessions of wild flax are representedin Table 2
23 SSAP Analysis Genomic polymorphism of differentLinum species was studied using SSAP method [23] withmodifications described earlier through genomic studies ofwheat [38] and strawberry [39] Total genomic DNA wasextracted from young flax leaves according to Edwards etal [40] with minor modifications 30 ng of genomic DNA
BioMed Research International 3
Table 1 Studied flax varieties
Number Variety Type Originator Variety pedigree1 Alfa Fiber VNIIL L-1120 Tomskij 4 T-10 Torzhokskij-4 G-4887
2 Gorizont Fiber VNIIL L-1120 T-5 T-10 I-2 I-17 Tvertsa Zarya Marit Smolenskij Severyanin SilvaNatasha Viera
3 Lavina Fiber Smolenskaja GOSHOS Sadko times S-108 [X-5 (selected from L-1120) times 1-7]4 Vasilyok Fiber Belarus I-7 L-1120 X-5 (selected from L-1120) 221-84-45 Soyuz Fiber Smolenskaja GOSHOS6 Lada Fiber VNIIL Viking Fibra7 Slavnyj 82 Fiber VNIIL from Shokinskij variety by single plant selection8 Krom Fiber Pskovskij NIISH9 Tverskoj Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-4 I-2 G-278210 Rosinka Fiber VNIIL L-1120 T-5 I-17 Fibra 12881211 Lenok Fiber VNIIL L-1120 M-34 (L-1120 times T-4) T-6 Lazurnyj12 A-93 Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-413 Tost 3 Fiber Sibirskij NIISH14 Mogilevskij-2 Fiber Belarus Stahanovets L-1120 T-5 T-915 Belochka Fiber Vjatskaja GSHA L-1120 Tvertsa16 Torzhokskij-4 Fiber VNIIL M-34 (L-1120 times T-4) times T-10 (G-360 times G-354)17 Tvertsa Fiber VNIIL T-5 times L-112018 Smolich Fiber Smolenskaja GOSHOS Zarya (X-5 times T-5) mutant A-710 8063 severyanin19 A-29 Fiber VNIIL L-1120 T-4 T-5 T-1020 Antey Fiber Pskovskij NIISH
21 Rusich Fiber Pskovskij NIISH Pskovskij 83 (Pryadilshhik L-1120 T-5 Pobeditel) times Rodnik(T-9 Progress I-9 L-1120 VNIIL11 Spartak)
22 Veralin Fiber Netherlands Torzhokskij-4 times Lidiya23 Merilin Fiber Netherlands24 Smolenskij Fiber Smolenskaja GOSHOS Tvertsa (T-5 times L-1120) times Zarya (X-5mdashselected from L-1120 times T-5)25 Tost-5 Fiber Sibirskij NIISH26 S-108 Fiber Smolenskaja GOSHOS X-5 (selected from L-1120) times I-727 Impuls Fiber Smolenskaja GOSHOS S-108 X-5 I-7 Zarya28 Praleska Fiber Belarus29 Lider Fiber Smolenskaja GOSHOS Tayga (France selection) mutagen treatment selection
30 Voronezhskij1308138 Oil VNIIMK
31 Svetoch Fiber VNIIL Selected from Cherskij kryazh32 Ki-5 Oil Ukraine33 LM-98 Oil VNIIL34 Novotorzhskij Fiber VNIIL L-1120 I-2 I-17 selected from Bogotolskij kryazh G-2307 G-4523-6-1335 TMP 1919 china 1 Fiber china36 k-1147 local form Oil Ethiopia37 v-29 Oil China38 Aleksim Fiber VNIIL L-1120 T-4 T-5 T-10 Tekstilshhik M-34 Pryadilshhik Tvertsa39 Ocean k-4497 Oil France40 Orshanskij-2 Fiber Belarus I-16 times L-112041 Κ-6 Fiber Pskovskij NIISH L 1120 times T-542 Vih oil v-2 Oil France43 Donskoj-95 Oil Donskaja opst44 Diplomat Fiber VNIIL Viking times Fibra (with subsequent selection)45 Ford Fiber Belarus46 Tomskij-16 Fiber Sibirskij NIISH and T T-9 times G-1077
4 BioMed Research InternationalTa
ble2Th
eaccessio
nsof
studied
specieso
fgenus
Linu
m
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)SectA
denolin
um1
Lperenn
eL
IPK
LIN1807
RussianFederatio
n2n
=18
[14]
2Lperenn
eLsub
spextraaxillare(Kit)
Nym
anIPK
LIN1651
Poland
2n=36
[17]
3Laltaicu
mLedebex
Juz
IPK
LIN1632
Unk
nown
2n=18
[17]
4LkomaroviiJuz
IPK
LIN1716
Unk
nown
2n=18
[17]
5Lperenn
eLsub
spperenne
IPK
LIN1521
Slovakia
2n=18
[17]
6Lperenn
eLsub
spalpinum
(Jacq)
Stojand
Stef
IPK
LIN1905
Austr
ia2n
=18
[17]
7Lperenn
eLsub
spanglicum
(Mill)
Ockendo
nIPK
LIN1524
GB
2n=36
[17]
8Lperenn
eL
VNIIL
K5500
Unk
nown
2n=36
[17]
9Lleo
niiFW
Schultz
IPK
LIN1672
Germany
2n=18
[14]
10LpallescensB
unge
IPK
LIN1645
Tajik
istan
2n=18
[17]
11LpallescensB
unge
Wild
popu
latio
ncollected
byNLB
olsheva
RussianFederatio
nAltai
2n=18
(present
study)
12Lmesostylum
Juz
IPK
LIN1774
Tajik
istan
2n=18
[17]
13Lmesostylum
Juz
IPK
LIN1662
Tajik
istan
2n=18
[17]
14Llew
isiiP
ursh
IPK
LIN1648
USA
2n=18
[17]
15Llew
isiiP
ursh
IPK
LIN1550
USA
2n=18
[17]
16Lau
striacum
LIPK
LIN1608
Germany
2n=18
[17]
17Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaRu
ssianFederatio
nRo
stov
2n=18
(present
study)
18Lau
striacum
Lsubspeuxinu
m(Ju
z)Ockendo
nIPK
LIN1546
Ukraine
2n=18
[17]
19Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaCr
imea
2n=18
(present
study)
20Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaUkraine
2n=18
(present
study)
21Lam
urenseAlef
BGI
outdoo
rscultivatedcollection
RussianFederatio
nFar
East
near
Pokrovka
settlem
ent
2n=18
[17]
SectD
asylinu
m22
Lhirsutum
Lsubsphirsutum
LIPK
LIN1676
Hun
gary
2n=16
[35]
23Lhirsutum
subsphirsutum
LIPK
LIN1649
Romania
2n=16
[36]
24Lhirsutum
Lsubsppseudoanatolicu
mPH
Davis
Wild
popu
latio
ncollected
byMPavelka
TurkeyK
aram
an2n
=32
(present
study)
25Lhirsutum
Lsubspan
atolicu
m(Boiss)Hayek
Wild
popu
latio
ncollected
byMPavelka
TurkeyA
ksaray
2n=32
(present
study)
SectLinum
26Lmargina
leACun
nex
Planch
IPK
LIN1920
Austr
alia
2n=84
(present
study)
27LnarbonenseL
IPK
LIN2002
Unk
nown
2n=28
[14]
28LnarbonenseL
IPK
LIN1653
France
2n=28
[14]
29Ldecumbens
Desf
IPK
LIN1754
Italy
2n=16
[14]
30Ldecumbens
Desf
IPK
LIN1913
Italy
2n=16
[14]
BioMed Research International 5
Table2Con
tinued
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)31
Lgrandiflorum
Desf
IPK
LIN2000
Unk
nown
2n=16
[14]
32Lgrandiflorum
Desf
IPK
LIN974
Unk
nown
2n=16
[14]
33Langustifoliu
mHud
sIPK
LIN1692
France
2n=30
(present
study)
34Langustifoliu
mHud
sVNIIL
K5695
Unk
nown
2n=30
(present
study)
35Langustifoliu
mHud
sVNIIL
K3108
Belgium
2n=30
(present
study)
36Langustifoliu
mHud
sIG
C15
Belarus
2n=30
[14]
37Langustifoliu
mHud
sVNIIL
K4731
EastGermany
2n=30
(present
study)
38Lbienne
Mill
(syn
Langustifolium
Hud
s)IG
C14
Hun
gary
2n=30
[14]
39Lusita
tissim
umLsubspbienne
Mill
Snankev
VIR
u-303794
Unk
nown
winterfl
ax2n
=30
(present
study)
40Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IGC
260
Belarus
dehiscentfl
ax2n
=30
(present
study)
41Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IPK
LIN119
Portugal
dehiscentfl
ax2n
=30
(present
study)
42Lusita
tissim
umLvarie
tyGiza
IPK
LIN277
Egypt
larges
eededflax
2n=30
(present
study)
43Lusita
tissim
umLG12Ru
zokvety
IPK
LIN633
Czecho
slovakia
dual-purpo
seflax
2n=30
(present
study)
44Lusita
tissim
umLvarietyColchidsky1
VIR
u099845
Abkh
azia
winterfl
ax2n
=30
(present
study)
45Lusita
tissim
umL
VNIIL
k7131
Morocco
larges
eededflax
2n=30
(present
study)
SectStellerolinum
46Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
TelyakovskyInlet
2n=20
(present
study)
47Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
near
Kraskino
settlem
ent
2n=20
(present
study)
6 BioMed Research International
was treated with 10U of TaqI restriction enzyme (ThermoScientific USA) for 3 h at 65∘b Ligation was performedby adding 25U of T4 DNA-ligase (Thermo ScientificUSA) 5mM ATP and 50 pmol of double-strand adapter[51015840-ACTCGATTCTCAACCCGAAAGTATAGATCCCA 51015840-PO4-CGTGGGATCTATACTT-(C6linker)-NH
2] and incu-
bation for 7 h at 37∘b The product was diluted twice withdeionized water The DNA sequence between the LTR (longterminal repeat) region of retrotransposons and the 119879119886119902120572119868restriction site was amplified using the adapter primer (51015840-GTTTACTCGATTCTCAACCCGAAAG 31015840) and primers tothe LTR regions of FL1a FL1b FL4 Cassandra FL10 FL8FL7 FL12 and FL9 retrotransposons [32]
1826 51015840-ACCCCTTGAGCTAACTTTTGGGGTAAG-31015840 (FL1a FL1b)1833 51015840-CTTGCTGGAAAGTGTGTGAGAGG-31015840(FL4)1838 51015840-TGTTAATCGCGCTCGGGTGGGAGCA-31015840(FL1a FL1b Cassandra)1845 51015840-AGCCTGAAAGTGTTGGGTTGTCG-31015840(FL11)1846 51015840-CTGGCATTTCCATTGTCGTCGATGC-31015840(FL10)1854 51015840-GCATCAGCCTGGACCAGTCCTCGTCC-31015840 (FL8)1868 51015840-CACTTCAAATTTTGGCAGCAGCGGATC-31015840 (FL1a FL1b)1881 51015840-TCGAGGTACACCTCGACTCAGG-31015840 (FL7)1886 51015840-ATTCTCGTCCGCTGCGCCCCTACA-31015840(FL12)1899 51015840-TGAGTTGCAGGTCCAGGCATCA-31015840(FL9)
Amplification was performed in two stages At the firststage only the primer to LTRof the retrotransposonwas usedAmplificationwas carried out in 25120583L of PCRmix containing5 120583L of ligation mix 1 U of TrueStart Hot Start Taq DNApolymerase (Thermo Scientific USA) TrueStart Taq DNApolymerase buffer 05mM MgCl
2 20120583M dNTP (Thermo
Scientific) and 5 pmol of the LTR primer The program foramplification for the first stage was 95∘C for 15min 30 cycles(95∘C for 30 s 62∘C for 1min 72∘C for 2min) and 72∘Cfor 10min At the second stage of amplification the adapterprimer 51015840-GTTTACTCGATTCTCAACCCGA-31015840 and one ofthe primers to the LTR region of retrotransposons wereused Amplification was carried out in 25 120583L of PCR mixcontaining 12 120583L of the first-stage PCR product 1 U ofTaq DNA polymerase (Thermo Scientific USA) Taq DNApolymerase buffer 15mM MgCl
2 200120583M dNTP (Thermo
Scientific USA) 25 pmol of LTR primer and 25 pmol of theadapter primerThe program for amplification for the secondstage was 95∘C for 15min 35 cycles (95∘C for 30 s 62∘C for1min 72∘C for 2min) and 72∘C for 10minThePCRproductswere separated in 25 agarose gel using fj buffer andthen stained with ethidium bromide Ten PCR products were
excised from agarose gel and characterized by sequencingon Applied Biosystems 3730 DNA Analyzer to confirm thespecificity of SSAP reaction The Bio-Rad Gel Doc systemwas used for gel documentation and photography as well asfor visual detection of presence or absence of polymorphicfragments in the samples from different accessions Thesedata were recorded in the form of a binary matrix in whichthe presence of a fragment was coded as 1 and its absence as0
The genetic distances between varieties were calculatedbased on the binary matrix of amplified fragments usingDicersquos formula [41]The dendrogramswere constructed usingSplitsTree 410 software [42] Cluster analysis was performedusing neighbor-joining method [43] and bootstrap valueswere determined based on 5000 permutations
24 Preparation of Cassandra Retrotransposon DNA ProbePCR primers were designed for amplification of Cassandraretrotransposon We amplified an internal domain (primersIntDom-F AGTGGTATCCGAGCCTCT and IntDom-R CCCATAGGACTCAACGTC) and the LTR with theexception of the 5S rDNA region of this retrotransposon(primers LTR-1-86-F TGTAATGTAACACGTTAGGCA andLTR-1-86-R TTAGTTAGGGACGGATTGTT LTR-206-279-F AAATAAATCTGTGAGGGATTAGT and LTR-206-279-RACTTGTAACACCCCGTACT) The amplification wascarried out in 20 120583L of PCR mixture that contained 1Uof TaqF DNA polymerase (Amplisens Russia) 1x TaqFbuffer 25 pmol of the forward and reverse primer 200120583MdNTP (Amplisens Russia) and 10 ng of genomic DNA Theprogram for amplification was 95∘C for 15min 40 cycles(95∘C for 10 s 62∘C for 20 s 72∘C for 30 s) and 72∘C for10min The amplicons were analyzed in 2 agarose gel andthen used as a template for biotin PCR labeling to obtainbiotin-labeled probes for FISH PCR labeling was carriedout using Biotin PCR Labeling Core Kit (Jena BioscienceGermany) according to the manufacturersquos protocol LabeledPCR products were precipitated with ethanol
25 FISH with Cassandra Retrotransposon DNA ProbeChromosome preparation was carried out according to thetechnique developed earlier for plants having small-sizedchromosomes [14] The hybridization mixture contained 2xSSC 50 formamide 10 dextran sulphate and 2 ng120583L ofa biotinylated DNA probe of Cassandra retrotransposonTheprobe was hybridized overnight at 31∘C After hybridizationthe slides were washed twice with 01x SSC at 38∘C for10min followed by two washes with 2x SSC at 44∘C for5min and a final 5min wash in 2x SSC at room temperatureThe biotin-labeled DNA probe was detected using a highlysensitive Alexa Fluor 488 Tyramide Signal Amplificationsystem (Invitrogen) according tomanufacturerrsquos instructions
3 Results
31 Analysis of SSAP Fingerprints For analyzing of thereproducibility of the SSAP method DNA of flax variety
BioMed Research International 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder
ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod
All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons
32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties
33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)
All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed
34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
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[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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2 BioMed Research International
nuclear ITS (internal transcribed spacer) DNA sequencesrevealed that genus Linum was not monophyletic It con-tains two major lineages a yellow-flowered clade (sectionsLinopsis Syllinum and Cathartolinum) and a blue-floweredclade (sections LinumDasylinum and Stellerolinum) [4]Thecultivated flax (L usitatissimum L) belongs to sec Linumfrom a blue-flowered clade L usitatissimum is believed tohave originated as a result of domestication of wild speciesL angustifolium Huds approximately 8000 years ago [5ndash8]For a long time flax has been cultivating as a dual-purposecrop grown for its fiber and linseed oil
According to morphological and qualitative traits cul-tivated flax was divided into five main types (1) fiber flax(L usitatissimum subsp usitatissimum) (2) oil flax (L usi-tatissimum L subsp humileCzernom) (3) dual-purpose flax(L usitatissimum L subsp intermedium Czemom) that wasan intermediate form between the first two ones cultivatedfor fiber and oil (4) large seeded flax (L usitatissimum Lsubsp latifolium Snankev) which is characterized by a set ofspecific morphological features and cultivated for oil in theMediterranean region and North Africa (5) winter flax (Lusitatissimum L subsp bienne Mill Snankev) cultivated forfiber and oil in theCaucasus Turkey Balkans and some othersouth regions of Europe [9 10] In addition collections offlax germplasm maintain accessions of primitive flax formswith dehiscent capsules (L usitatissimum convar crepitans[Boenningh] Kulpa et Danert) [11]
The taxonomyof the genus cannot be considered as finallyestablished one because the phylogenetic linkages betweenthe individual taxa have not been sufficiently investigatedThe phylogeny of species of the genus Linum was previouslystudied by the use of molecular and cytogenetic approaches[4 12ndash17] but there are problems that still remain to besolved
Transposon-based molecular markers are successfullyused in phylogenic studies Transposable elements wereshown to influence changing in genomic structure as well astranscriptional regulation occurring during the evolution [1819] The presence of transposons in various species of plantstheir high integration activity conservative sequences and alarge number of copies encouraged the use of transposonsin the studies of genetic diversity and profiling of plantvarieties [20ndash22] Several molecular marker systems basedon the information available for the transposable elementssequences were developed for plants [20 22ndash27] SSAP(sequence-specific amplified polymorphism) method wasshown to have a number of advantages as compared to othermarker systems SSAP method produces many polymorphicfragments and allows differentiation of most samples usingonly a single combination of specific primers [23 28ndash30]Different plants were successfully studied by SSAP analysisbut the method has not been applied for the investigation ofspecies of the genus Linum yet Only recently flax sequenceshave appeared in databases [31ndash34] and development of amarker system based on flax transposable elements for theinvestigation of cultivated and wild species of the genusLinum has become possible
In this study the SSAP method was used for assessmentof genetic diversity Besides the possibilities of application
of marker-based profiling for identification of L usitatissi-mum varieties were analyzed We studied 46 varieties of Lusitatissimummainly bred inRussia and a number of varietieswhich were grown in geographically close or distant regionsWe also analyzed different types of cultivated flax (fiberoilseed large seeded winter and dehiscent flax) togetherwith 21 wild species and subspecies from sections LinumAdenolinum Dasylinum and Stellerolinum to estimate thepossibility of using the SSAP method for the investigation offlax domestication history and phylogenic linkages betweendifferent taxa of the genus Linum
2 Materials and Methods
21 Plant Materials For the investigation of genetic diversityof cultivated flax 46 L usitatissimum varieties mainly ofRussian origin were obtained from the All-Russian ResearchInstitute for Flax (VNIIL) (Table 1) For analyzing the repro-ducibility of the SSAP analysis flax variety ldquoStormont cirrusrdquo(IPK genbank Gatersleben accession number LIN 261) wasused
For the investigation of genetic diversity 47 flax acces-sions belonging to 22 species from sections Linum Ade-nolinum Dasylinum and Stellerolinum were used (Table 2)Most of these accessions were obtained from genebank ofLeibniz Institute of Plant Genetics and Crop Plant Research(IPK) (Gatersleben Germany) seed collections of All-Russian Flax Institute (VNIIL) (Torzok Russian Federation)NI Vavilov Research Institute of Plant Industry (VIR) (StPetersburg Russian Federation) and Institute of Geneticsand Cytology of NAS Belarus (IGC) (Minsk Belarus) Theaccessions of L amurense and L stelleroides were kindlyprovided by Dr L N Mironova Botanic Garden Institute ofthe Far-Eastern Branch of the Russian Academy of Sciences(BGI) (Vladivostok Russian Federation) Some accessionswere collected in the wild by Dr A A Svetlova KomarovBotanical Institute RAS (St Petersburg Russian Federation)by Dr N L Bolsheva Engelhardt Institute of MolecularBiology RAS (Moscow Russian Federation) and by Dr MPavelka Euroseeds (Novy Jicin Chech Rep)
22 Confirmation of Species Determination Species determi-nation of some accessions of wild Linum species was doneduring the course of our earlier cytogenetic investigations[14 17 37] To confirm the species determination the restof the accessions were planted in the ground and addition-ally chromosome analysis (determination of chromosomenumber) using acetocarmine staining according to previouslydeveloped approach was performed [14] Chromosome num-bers of all the studied accessions of wild flax are representedin Table 2
23 SSAP Analysis Genomic polymorphism of differentLinum species was studied using SSAP method [23] withmodifications described earlier through genomic studies ofwheat [38] and strawberry [39] Total genomic DNA wasextracted from young flax leaves according to Edwards etal [40] with minor modifications 30 ng of genomic DNA
BioMed Research International 3
Table 1 Studied flax varieties
Number Variety Type Originator Variety pedigree1 Alfa Fiber VNIIL L-1120 Tomskij 4 T-10 Torzhokskij-4 G-4887
2 Gorizont Fiber VNIIL L-1120 T-5 T-10 I-2 I-17 Tvertsa Zarya Marit Smolenskij Severyanin SilvaNatasha Viera
3 Lavina Fiber Smolenskaja GOSHOS Sadko times S-108 [X-5 (selected from L-1120) times 1-7]4 Vasilyok Fiber Belarus I-7 L-1120 X-5 (selected from L-1120) 221-84-45 Soyuz Fiber Smolenskaja GOSHOS6 Lada Fiber VNIIL Viking Fibra7 Slavnyj 82 Fiber VNIIL from Shokinskij variety by single plant selection8 Krom Fiber Pskovskij NIISH9 Tverskoj Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-4 I-2 G-278210 Rosinka Fiber VNIIL L-1120 T-5 I-17 Fibra 12881211 Lenok Fiber VNIIL L-1120 M-34 (L-1120 times T-4) T-6 Lazurnyj12 A-93 Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-413 Tost 3 Fiber Sibirskij NIISH14 Mogilevskij-2 Fiber Belarus Stahanovets L-1120 T-5 T-915 Belochka Fiber Vjatskaja GSHA L-1120 Tvertsa16 Torzhokskij-4 Fiber VNIIL M-34 (L-1120 times T-4) times T-10 (G-360 times G-354)17 Tvertsa Fiber VNIIL T-5 times L-112018 Smolich Fiber Smolenskaja GOSHOS Zarya (X-5 times T-5) mutant A-710 8063 severyanin19 A-29 Fiber VNIIL L-1120 T-4 T-5 T-1020 Antey Fiber Pskovskij NIISH
21 Rusich Fiber Pskovskij NIISH Pskovskij 83 (Pryadilshhik L-1120 T-5 Pobeditel) times Rodnik(T-9 Progress I-9 L-1120 VNIIL11 Spartak)
22 Veralin Fiber Netherlands Torzhokskij-4 times Lidiya23 Merilin Fiber Netherlands24 Smolenskij Fiber Smolenskaja GOSHOS Tvertsa (T-5 times L-1120) times Zarya (X-5mdashselected from L-1120 times T-5)25 Tost-5 Fiber Sibirskij NIISH26 S-108 Fiber Smolenskaja GOSHOS X-5 (selected from L-1120) times I-727 Impuls Fiber Smolenskaja GOSHOS S-108 X-5 I-7 Zarya28 Praleska Fiber Belarus29 Lider Fiber Smolenskaja GOSHOS Tayga (France selection) mutagen treatment selection
30 Voronezhskij1308138 Oil VNIIMK
31 Svetoch Fiber VNIIL Selected from Cherskij kryazh32 Ki-5 Oil Ukraine33 LM-98 Oil VNIIL34 Novotorzhskij Fiber VNIIL L-1120 I-2 I-17 selected from Bogotolskij kryazh G-2307 G-4523-6-1335 TMP 1919 china 1 Fiber china36 k-1147 local form Oil Ethiopia37 v-29 Oil China38 Aleksim Fiber VNIIL L-1120 T-4 T-5 T-10 Tekstilshhik M-34 Pryadilshhik Tvertsa39 Ocean k-4497 Oil France40 Orshanskij-2 Fiber Belarus I-16 times L-112041 Κ-6 Fiber Pskovskij NIISH L 1120 times T-542 Vih oil v-2 Oil France43 Donskoj-95 Oil Donskaja opst44 Diplomat Fiber VNIIL Viking times Fibra (with subsequent selection)45 Ford Fiber Belarus46 Tomskij-16 Fiber Sibirskij NIISH and T T-9 times G-1077
4 BioMed Research InternationalTa
ble2Th
eaccessio
nsof
studied
specieso
fgenus
Linu
m
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)SectA
denolin
um1
Lperenn
eL
IPK
LIN1807
RussianFederatio
n2n
=18
[14]
2Lperenn
eLsub
spextraaxillare(Kit)
Nym
anIPK
LIN1651
Poland
2n=36
[17]
3Laltaicu
mLedebex
Juz
IPK
LIN1632
Unk
nown
2n=18
[17]
4LkomaroviiJuz
IPK
LIN1716
Unk
nown
2n=18
[17]
5Lperenn
eLsub
spperenne
IPK
LIN1521
Slovakia
2n=18
[17]
6Lperenn
eLsub
spalpinum
(Jacq)
Stojand
Stef
IPK
LIN1905
Austr
ia2n
=18
[17]
7Lperenn
eLsub
spanglicum
(Mill)
Ockendo
nIPK
LIN1524
GB
2n=36
[17]
8Lperenn
eL
VNIIL
K5500
Unk
nown
2n=36
[17]
9Lleo
niiFW
Schultz
IPK
LIN1672
Germany
2n=18
[14]
10LpallescensB
unge
IPK
LIN1645
Tajik
istan
2n=18
[17]
11LpallescensB
unge
Wild
popu
latio
ncollected
byNLB
olsheva
RussianFederatio
nAltai
2n=18
(present
study)
12Lmesostylum
Juz
IPK
LIN1774
Tajik
istan
2n=18
[17]
13Lmesostylum
Juz
IPK
LIN1662
Tajik
istan
2n=18
[17]
14Llew
isiiP
ursh
IPK
LIN1648
USA
2n=18
[17]
15Llew
isiiP
ursh
IPK
LIN1550
USA
2n=18
[17]
16Lau
striacum
LIPK
LIN1608
Germany
2n=18
[17]
17Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaRu
ssianFederatio
nRo
stov
2n=18
(present
study)
18Lau
striacum
Lsubspeuxinu
m(Ju
z)Ockendo
nIPK
LIN1546
Ukraine
2n=18
[17]
19Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaCr
imea
2n=18
(present
study)
20Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaUkraine
2n=18
(present
study)
21Lam
urenseAlef
BGI
outdoo
rscultivatedcollection
RussianFederatio
nFar
East
near
Pokrovka
settlem
ent
2n=18
[17]
SectD
asylinu
m22
Lhirsutum
Lsubsphirsutum
LIPK
LIN1676
Hun
gary
2n=16
[35]
23Lhirsutum
subsphirsutum
LIPK
LIN1649
Romania
2n=16
[36]
24Lhirsutum
Lsubsppseudoanatolicu
mPH
Davis
Wild
popu
latio
ncollected
byMPavelka
TurkeyK
aram
an2n
=32
(present
study)
25Lhirsutum
Lsubspan
atolicu
m(Boiss)Hayek
Wild
popu
latio
ncollected
byMPavelka
TurkeyA
ksaray
2n=32
(present
study)
SectLinum
26Lmargina
leACun
nex
Planch
IPK
LIN1920
Austr
alia
2n=84
(present
study)
27LnarbonenseL
IPK
LIN2002
Unk
nown
2n=28
[14]
28LnarbonenseL
IPK
LIN1653
France
2n=28
[14]
29Ldecumbens
Desf
IPK
LIN1754
Italy
2n=16
[14]
30Ldecumbens
Desf
IPK
LIN1913
Italy
2n=16
[14]
BioMed Research International 5
Table2Con
tinued
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)31
Lgrandiflorum
Desf
IPK
LIN2000
Unk
nown
2n=16
[14]
32Lgrandiflorum
Desf
IPK
LIN974
Unk
nown
2n=16
[14]
33Langustifoliu
mHud
sIPK
LIN1692
France
2n=30
(present
study)
34Langustifoliu
mHud
sVNIIL
K5695
Unk
nown
2n=30
(present
study)
35Langustifoliu
mHud
sVNIIL
K3108
Belgium
2n=30
(present
study)
36Langustifoliu
mHud
sIG
C15
Belarus
2n=30
[14]
37Langustifoliu
mHud
sVNIIL
K4731
EastGermany
2n=30
(present
study)
38Lbienne
Mill
(syn
Langustifolium
Hud
s)IG
C14
Hun
gary
2n=30
[14]
39Lusita
tissim
umLsubspbienne
Mill
Snankev
VIR
u-303794
Unk
nown
winterfl
ax2n
=30
(present
study)
40Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IGC
260
Belarus
dehiscentfl
ax2n
=30
(present
study)
41Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IPK
LIN119
Portugal
dehiscentfl
ax2n
=30
(present
study)
42Lusita
tissim
umLvarie
tyGiza
IPK
LIN277
Egypt
larges
eededflax
2n=30
(present
study)
43Lusita
tissim
umLG12Ru
zokvety
IPK
LIN633
Czecho
slovakia
dual-purpo
seflax
2n=30
(present
study)
44Lusita
tissim
umLvarietyColchidsky1
VIR
u099845
Abkh
azia
winterfl
ax2n
=30
(present
study)
45Lusita
tissim
umL
VNIIL
k7131
Morocco
larges
eededflax
2n=30
(present
study)
SectStellerolinum
46Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
TelyakovskyInlet
2n=20
(present
study)
47Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
near
Kraskino
settlem
ent
2n=20
(present
study)
6 BioMed Research International
was treated with 10U of TaqI restriction enzyme (ThermoScientific USA) for 3 h at 65∘b Ligation was performedby adding 25U of T4 DNA-ligase (Thermo ScientificUSA) 5mM ATP and 50 pmol of double-strand adapter[51015840-ACTCGATTCTCAACCCGAAAGTATAGATCCCA 51015840-PO4-CGTGGGATCTATACTT-(C6linker)-NH
2] and incu-
bation for 7 h at 37∘b The product was diluted twice withdeionized water The DNA sequence between the LTR (longterminal repeat) region of retrotransposons and the 119879119886119902120572119868restriction site was amplified using the adapter primer (51015840-GTTTACTCGATTCTCAACCCGAAAG 31015840) and primers tothe LTR regions of FL1a FL1b FL4 Cassandra FL10 FL8FL7 FL12 and FL9 retrotransposons [32]
1826 51015840-ACCCCTTGAGCTAACTTTTGGGGTAAG-31015840 (FL1a FL1b)1833 51015840-CTTGCTGGAAAGTGTGTGAGAGG-31015840(FL4)1838 51015840-TGTTAATCGCGCTCGGGTGGGAGCA-31015840(FL1a FL1b Cassandra)1845 51015840-AGCCTGAAAGTGTTGGGTTGTCG-31015840(FL11)1846 51015840-CTGGCATTTCCATTGTCGTCGATGC-31015840(FL10)1854 51015840-GCATCAGCCTGGACCAGTCCTCGTCC-31015840 (FL8)1868 51015840-CACTTCAAATTTTGGCAGCAGCGGATC-31015840 (FL1a FL1b)1881 51015840-TCGAGGTACACCTCGACTCAGG-31015840 (FL7)1886 51015840-ATTCTCGTCCGCTGCGCCCCTACA-31015840(FL12)1899 51015840-TGAGTTGCAGGTCCAGGCATCA-31015840(FL9)
Amplification was performed in two stages At the firststage only the primer to LTRof the retrotransposonwas usedAmplificationwas carried out in 25120583L of PCRmix containing5 120583L of ligation mix 1 U of TrueStart Hot Start Taq DNApolymerase (Thermo Scientific USA) TrueStart Taq DNApolymerase buffer 05mM MgCl
2 20120583M dNTP (Thermo
Scientific) and 5 pmol of the LTR primer The program foramplification for the first stage was 95∘C for 15min 30 cycles(95∘C for 30 s 62∘C for 1min 72∘C for 2min) and 72∘Cfor 10min At the second stage of amplification the adapterprimer 51015840-GTTTACTCGATTCTCAACCCGA-31015840 and one ofthe primers to the LTR region of retrotransposons wereused Amplification was carried out in 25 120583L of PCR mixcontaining 12 120583L of the first-stage PCR product 1 U ofTaq DNA polymerase (Thermo Scientific USA) Taq DNApolymerase buffer 15mM MgCl
2 200120583M dNTP (Thermo
Scientific USA) 25 pmol of LTR primer and 25 pmol of theadapter primerThe program for amplification for the secondstage was 95∘C for 15min 35 cycles (95∘C for 30 s 62∘C for1min 72∘C for 2min) and 72∘C for 10minThePCRproductswere separated in 25 agarose gel using fj buffer andthen stained with ethidium bromide Ten PCR products were
excised from agarose gel and characterized by sequencingon Applied Biosystems 3730 DNA Analyzer to confirm thespecificity of SSAP reaction The Bio-Rad Gel Doc systemwas used for gel documentation and photography as well asfor visual detection of presence or absence of polymorphicfragments in the samples from different accessions Thesedata were recorded in the form of a binary matrix in whichthe presence of a fragment was coded as 1 and its absence as0
The genetic distances between varieties were calculatedbased on the binary matrix of amplified fragments usingDicersquos formula [41]The dendrogramswere constructed usingSplitsTree 410 software [42] Cluster analysis was performedusing neighbor-joining method [43] and bootstrap valueswere determined based on 5000 permutations
24 Preparation of Cassandra Retrotransposon DNA ProbePCR primers were designed for amplification of Cassandraretrotransposon We amplified an internal domain (primersIntDom-F AGTGGTATCCGAGCCTCT and IntDom-R CCCATAGGACTCAACGTC) and the LTR with theexception of the 5S rDNA region of this retrotransposon(primers LTR-1-86-F TGTAATGTAACACGTTAGGCA andLTR-1-86-R TTAGTTAGGGACGGATTGTT LTR-206-279-F AAATAAATCTGTGAGGGATTAGT and LTR-206-279-RACTTGTAACACCCCGTACT) The amplification wascarried out in 20 120583L of PCR mixture that contained 1Uof TaqF DNA polymerase (Amplisens Russia) 1x TaqFbuffer 25 pmol of the forward and reverse primer 200120583MdNTP (Amplisens Russia) and 10 ng of genomic DNA Theprogram for amplification was 95∘C for 15min 40 cycles(95∘C for 10 s 62∘C for 20 s 72∘C for 30 s) and 72∘C for10min The amplicons were analyzed in 2 agarose gel andthen used as a template for biotin PCR labeling to obtainbiotin-labeled probes for FISH PCR labeling was carriedout using Biotin PCR Labeling Core Kit (Jena BioscienceGermany) according to the manufacturersquos protocol LabeledPCR products were precipitated with ethanol
25 FISH with Cassandra Retrotransposon DNA ProbeChromosome preparation was carried out according to thetechnique developed earlier for plants having small-sizedchromosomes [14] The hybridization mixture contained 2xSSC 50 formamide 10 dextran sulphate and 2 ng120583L ofa biotinylated DNA probe of Cassandra retrotransposonTheprobe was hybridized overnight at 31∘C After hybridizationthe slides were washed twice with 01x SSC at 38∘C for10min followed by two washes with 2x SSC at 44∘C for5min and a final 5min wash in 2x SSC at room temperatureThe biotin-labeled DNA probe was detected using a highlysensitive Alexa Fluor 488 Tyramide Signal Amplificationsystem (Invitrogen) according tomanufacturerrsquos instructions
3 Results
31 Analysis of SSAP Fingerprints For analyzing of thereproducibility of the SSAP method DNA of flax variety
BioMed Research International 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder
ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod
All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons
32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties
33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)
All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed
34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
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[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Nucleic AcidsJournal of
Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 3
Table 1 Studied flax varieties
Number Variety Type Originator Variety pedigree1 Alfa Fiber VNIIL L-1120 Tomskij 4 T-10 Torzhokskij-4 G-4887
2 Gorizont Fiber VNIIL L-1120 T-5 T-10 I-2 I-17 Tvertsa Zarya Marit Smolenskij Severyanin SilvaNatasha Viera
3 Lavina Fiber Smolenskaja GOSHOS Sadko times S-108 [X-5 (selected from L-1120) times 1-7]4 Vasilyok Fiber Belarus I-7 L-1120 X-5 (selected from L-1120) 221-84-45 Soyuz Fiber Smolenskaja GOSHOS6 Lada Fiber VNIIL Viking Fibra7 Slavnyj 82 Fiber VNIIL from Shokinskij variety by single plant selection8 Krom Fiber Pskovskij NIISH9 Tverskoj Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-4 I-2 G-278210 Rosinka Fiber VNIIL L-1120 T-5 I-17 Fibra 12881211 Lenok Fiber VNIIL L-1120 M-34 (L-1120 times T-4) T-6 Lazurnyj12 A-93 Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-413 Tost 3 Fiber Sibirskij NIISH14 Mogilevskij-2 Fiber Belarus Stahanovets L-1120 T-5 T-915 Belochka Fiber Vjatskaja GSHA L-1120 Tvertsa16 Torzhokskij-4 Fiber VNIIL M-34 (L-1120 times T-4) times T-10 (G-360 times G-354)17 Tvertsa Fiber VNIIL T-5 times L-112018 Smolich Fiber Smolenskaja GOSHOS Zarya (X-5 times T-5) mutant A-710 8063 severyanin19 A-29 Fiber VNIIL L-1120 T-4 T-5 T-1020 Antey Fiber Pskovskij NIISH
21 Rusich Fiber Pskovskij NIISH Pskovskij 83 (Pryadilshhik L-1120 T-5 Pobeditel) times Rodnik(T-9 Progress I-9 L-1120 VNIIL11 Spartak)
22 Veralin Fiber Netherlands Torzhokskij-4 times Lidiya23 Merilin Fiber Netherlands24 Smolenskij Fiber Smolenskaja GOSHOS Tvertsa (T-5 times L-1120) times Zarya (X-5mdashselected from L-1120 times T-5)25 Tost-5 Fiber Sibirskij NIISH26 S-108 Fiber Smolenskaja GOSHOS X-5 (selected from L-1120) times I-727 Impuls Fiber Smolenskaja GOSHOS S-108 X-5 I-7 Zarya28 Praleska Fiber Belarus29 Lider Fiber Smolenskaja GOSHOS Tayga (France selection) mutagen treatment selection
30 Voronezhskij1308138 Oil VNIIMK
31 Svetoch Fiber VNIIL Selected from Cherskij kryazh32 Ki-5 Oil Ukraine33 LM-98 Oil VNIIL34 Novotorzhskij Fiber VNIIL L-1120 I-2 I-17 selected from Bogotolskij kryazh G-2307 G-4523-6-1335 TMP 1919 china 1 Fiber china36 k-1147 local form Oil Ethiopia37 v-29 Oil China38 Aleksim Fiber VNIIL L-1120 T-4 T-5 T-10 Tekstilshhik M-34 Pryadilshhik Tvertsa39 Ocean k-4497 Oil France40 Orshanskij-2 Fiber Belarus I-16 times L-112041 Κ-6 Fiber Pskovskij NIISH L 1120 times T-542 Vih oil v-2 Oil France43 Donskoj-95 Oil Donskaja opst44 Diplomat Fiber VNIIL Viking times Fibra (with subsequent selection)45 Ford Fiber Belarus46 Tomskij-16 Fiber Sibirskij NIISH and T T-9 times G-1077
4 BioMed Research InternationalTa
ble2Th
eaccessio
nsof
studied
specieso
fgenus
Linu
m
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)SectA
denolin
um1
Lperenn
eL
IPK
LIN1807
RussianFederatio
n2n
=18
[14]
2Lperenn
eLsub
spextraaxillare(Kit)
Nym
anIPK
LIN1651
Poland
2n=36
[17]
3Laltaicu
mLedebex
Juz
IPK
LIN1632
Unk
nown
2n=18
[17]
4LkomaroviiJuz
IPK
LIN1716
Unk
nown
2n=18
[17]
5Lperenn
eLsub
spperenne
IPK
LIN1521
Slovakia
2n=18
[17]
6Lperenn
eLsub
spalpinum
(Jacq)
Stojand
Stef
IPK
LIN1905
Austr
ia2n
=18
[17]
7Lperenn
eLsub
spanglicum
(Mill)
Ockendo
nIPK
LIN1524
GB
2n=36
[17]
8Lperenn
eL
VNIIL
K5500
Unk
nown
2n=36
[17]
9Lleo
niiFW
Schultz
IPK
LIN1672
Germany
2n=18
[14]
10LpallescensB
unge
IPK
LIN1645
Tajik
istan
2n=18
[17]
11LpallescensB
unge
Wild
popu
latio
ncollected
byNLB
olsheva
RussianFederatio
nAltai
2n=18
(present
study)
12Lmesostylum
Juz
IPK
LIN1774
Tajik
istan
2n=18
[17]
13Lmesostylum
Juz
IPK
LIN1662
Tajik
istan
2n=18
[17]
14Llew
isiiP
ursh
IPK
LIN1648
USA
2n=18
[17]
15Llew
isiiP
ursh
IPK
LIN1550
USA
2n=18
[17]
16Lau
striacum
LIPK
LIN1608
Germany
2n=18
[17]
17Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaRu
ssianFederatio
nRo
stov
2n=18
(present
study)
18Lau
striacum
Lsubspeuxinu
m(Ju
z)Ockendo
nIPK
LIN1546
Ukraine
2n=18
[17]
19Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaCr
imea
2n=18
(present
study)
20Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaUkraine
2n=18
(present
study)
21Lam
urenseAlef
BGI
outdoo
rscultivatedcollection
RussianFederatio
nFar
East
near
Pokrovka
settlem
ent
2n=18
[17]
SectD
asylinu
m22
Lhirsutum
Lsubsphirsutum
LIPK
LIN1676
Hun
gary
2n=16
[35]
23Lhirsutum
subsphirsutum
LIPK
LIN1649
Romania
2n=16
[36]
24Lhirsutum
Lsubsppseudoanatolicu
mPH
Davis
Wild
popu
latio
ncollected
byMPavelka
TurkeyK
aram
an2n
=32
(present
study)
25Lhirsutum
Lsubspan
atolicu
m(Boiss)Hayek
Wild
popu
latio
ncollected
byMPavelka
TurkeyA
ksaray
2n=32
(present
study)
SectLinum
26Lmargina
leACun
nex
Planch
IPK
LIN1920
Austr
alia
2n=84
(present
study)
27LnarbonenseL
IPK
LIN2002
Unk
nown
2n=28
[14]
28LnarbonenseL
IPK
LIN1653
France
2n=28
[14]
29Ldecumbens
Desf
IPK
LIN1754
Italy
2n=16
[14]
30Ldecumbens
Desf
IPK
LIN1913
Italy
2n=16
[14]
BioMed Research International 5
Table2Con
tinued
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)31
Lgrandiflorum
Desf
IPK
LIN2000
Unk
nown
2n=16
[14]
32Lgrandiflorum
Desf
IPK
LIN974
Unk
nown
2n=16
[14]
33Langustifoliu
mHud
sIPK
LIN1692
France
2n=30
(present
study)
34Langustifoliu
mHud
sVNIIL
K5695
Unk
nown
2n=30
(present
study)
35Langustifoliu
mHud
sVNIIL
K3108
Belgium
2n=30
(present
study)
36Langustifoliu
mHud
sIG
C15
Belarus
2n=30
[14]
37Langustifoliu
mHud
sVNIIL
K4731
EastGermany
2n=30
(present
study)
38Lbienne
Mill
(syn
Langustifolium
Hud
s)IG
C14
Hun
gary
2n=30
[14]
39Lusita
tissim
umLsubspbienne
Mill
Snankev
VIR
u-303794
Unk
nown
winterfl
ax2n
=30
(present
study)
40Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IGC
260
Belarus
dehiscentfl
ax2n
=30
(present
study)
41Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IPK
LIN119
Portugal
dehiscentfl
ax2n
=30
(present
study)
42Lusita
tissim
umLvarie
tyGiza
IPK
LIN277
Egypt
larges
eededflax
2n=30
(present
study)
43Lusita
tissim
umLG12Ru
zokvety
IPK
LIN633
Czecho
slovakia
dual-purpo
seflax
2n=30
(present
study)
44Lusita
tissim
umLvarietyColchidsky1
VIR
u099845
Abkh
azia
winterfl
ax2n
=30
(present
study)
45Lusita
tissim
umL
VNIIL
k7131
Morocco
larges
eededflax
2n=30
(present
study)
SectStellerolinum
46Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
TelyakovskyInlet
2n=20
(present
study)
47Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
near
Kraskino
settlem
ent
2n=20
(present
study)
6 BioMed Research International
was treated with 10U of TaqI restriction enzyme (ThermoScientific USA) for 3 h at 65∘b Ligation was performedby adding 25U of T4 DNA-ligase (Thermo ScientificUSA) 5mM ATP and 50 pmol of double-strand adapter[51015840-ACTCGATTCTCAACCCGAAAGTATAGATCCCA 51015840-PO4-CGTGGGATCTATACTT-(C6linker)-NH
2] and incu-
bation for 7 h at 37∘b The product was diluted twice withdeionized water The DNA sequence between the LTR (longterminal repeat) region of retrotransposons and the 119879119886119902120572119868restriction site was amplified using the adapter primer (51015840-GTTTACTCGATTCTCAACCCGAAAG 31015840) and primers tothe LTR regions of FL1a FL1b FL4 Cassandra FL10 FL8FL7 FL12 and FL9 retrotransposons [32]
1826 51015840-ACCCCTTGAGCTAACTTTTGGGGTAAG-31015840 (FL1a FL1b)1833 51015840-CTTGCTGGAAAGTGTGTGAGAGG-31015840(FL4)1838 51015840-TGTTAATCGCGCTCGGGTGGGAGCA-31015840(FL1a FL1b Cassandra)1845 51015840-AGCCTGAAAGTGTTGGGTTGTCG-31015840(FL11)1846 51015840-CTGGCATTTCCATTGTCGTCGATGC-31015840(FL10)1854 51015840-GCATCAGCCTGGACCAGTCCTCGTCC-31015840 (FL8)1868 51015840-CACTTCAAATTTTGGCAGCAGCGGATC-31015840 (FL1a FL1b)1881 51015840-TCGAGGTACACCTCGACTCAGG-31015840 (FL7)1886 51015840-ATTCTCGTCCGCTGCGCCCCTACA-31015840(FL12)1899 51015840-TGAGTTGCAGGTCCAGGCATCA-31015840(FL9)
Amplification was performed in two stages At the firststage only the primer to LTRof the retrotransposonwas usedAmplificationwas carried out in 25120583L of PCRmix containing5 120583L of ligation mix 1 U of TrueStart Hot Start Taq DNApolymerase (Thermo Scientific USA) TrueStart Taq DNApolymerase buffer 05mM MgCl
2 20120583M dNTP (Thermo
Scientific) and 5 pmol of the LTR primer The program foramplification for the first stage was 95∘C for 15min 30 cycles(95∘C for 30 s 62∘C for 1min 72∘C for 2min) and 72∘Cfor 10min At the second stage of amplification the adapterprimer 51015840-GTTTACTCGATTCTCAACCCGA-31015840 and one ofthe primers to the LTR region of retrotransposons wereused Amplification was carried out in 25 120583L of PCR mixcontaining 12 120583L of the first-stage PCR product 1 U ofTaq DNA polymerase (Thermo Scientific USA) Taq DNApolymerase buffer 15mM MgCl
2 200120583M dNTP (Thermo
Scientific USA) 25 pmol of LTR primer and 25 pmol of theadapter primerThe program for amplification for the secondstage was 95∘C for 15min 35 cycles (95∘C for 30 s 62∘C for1min 72∘C for 2min) and 72∘C for 10minThePCRproductswere separated in 25 agarose gel using fj buffer andthen stained with ethidium bromide Ten PCR products were
excised from agarose gel and characterized by sequencingon Applied Biosystems 3730 DNA Analyzer to confirm thespecificity of SSAP reaction The Bio-Rad Gel Doc systemwas used for gel documentation and photography as well asfor visual detection of presence or absence of polymorphicfragments in the samples from different accessions Thesedata were recorded in the form of a binary matrix in whichthe presence of a fragment was coded as 1 and its absence as0
The genetic distances between varieties were calculatedbased on the binary matrix of amplified fragments usingDicersquos formula [41]The dendrogramswere constructed usingSplitsTree 410 software [42] Cluster analysis was performedusing neighbor-joining method [43] and bootstrap valueswere determined based on 5000 permutations
24 Preparation of Cassandra Retrotransposon DNA ProbePCR primers were designed for amplification of Cassandraretrotransposon We amplified an internal domain (primersIntDom-F AGTGGTATCCGAGCCTCT and IntDom-R CCCATAGGACTCAACGTC) and the LTR with theexception of the 5S rDNA region of this retrotransposon(primers LTR-1-86-F TGTAATGTAACACGTTAGGCA andLTR-1-86-R TTAGTTAGGGACGGATTGTT LTR-206-279-F AAATAAATCTGTGAGGGATTAGT and LTR-206-279-RACTTGTAACACCCCGTACT) The amplification wascarried out in 20 120583L of PCR mixture that contained 1Uof TaqF DNA polymerase (Amplisens Russia) 1x TaqFbuffer 25 pmol of the forward and reverse primer 200120583MdNTP (Amplisens Russia) and 10 ng of genomic DNA Theprogram for amplification was 95∘C for 15min 40 cycles(95∘C for 10 s 62∘C for 20 s 72∘C for 30 s) and 72∘C for10min The amplicons were analyzed in 2 agarose gel andthen used as a template for biotin PCR labeling to obtainbiotin-labeled probes for FISH PCR labeling was carriedout using Biotin PCR Labeling Core Kit (Jena BioscienceGermany) according to the manufacturersquos protocol LabeledPCR products were precipitated with ethanol
25 FISH with Cassandra Retrotransposon DNA ProbeChromosome preparation was carried out according to thetechnique developed earlier for plants having small-sizedchromosomes [14] The hybridization mixture contained 2xSSC 50 formamide 10 dextran sulphate and 2 ng120583L ofa biotinylated DNA probe of Cassandra retrotransposonTheprobe was hybridized overnight at 31∘C After hybridizationthe slides were washed twice with 01x SSC at 38∘C for10min followed by two washes with 2x SSC at 44∘C for5min and a final 5min wash in 2x SSC at room temperatureThe biotin-labeled DNA probe was detected using a highlysensitive Alexa Fluor 488 Tyramide Signal Amplificationsystem (Invitrogen) according tomanufacturerrsquos instructions
3 Results
31 Analysis of SSAP Fingerprints For analyzing of thereproducibility of the SSAP method DNA of flax variety
BioMed Research International 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder
ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod
All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons
32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties
33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)
All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed
34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 BioMed Research InternationalTa
ble2Th
eaccessio
nsof
studied
specieso
fgenus
Linu
m
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)SectA
denolin
um1
Lperenn
eL
IPK
LIN1807
RussianFederatio
n2n
=18
[14]
2Lperenn
eLsub
spextraaxillare(Kit)
Nym
anIPK
LIN1651
Poland
2n=36
[17]
3Laltaicu
mLedebex
Juz
IPK
LIN1632
Unk
nown
2n=18
[17]
4LkomaroviiJuz
IPK
LIN1716
Unk
nown
2n=18
[17]
5Lperenn
eLsub
spperenne
IPK
LIN1521
Slovakia
2n=18
[17]
6Lperenn
eLsub
spalpinum
(Jacq)
Stojand
Stef
IPK
LIN1905
Austr
ia2n
=18
[17]
7Lperenn
eLsub
spanglicum
(Mill)
Ockendo
nIPK
LIN1524
GB
2n=36
[17]
8Lperenn
eL
VNIIL
K5500
Unk
nown
2n=36
[17]
9Lleo
niiFW
Schultz
IPK
LIN1672
Germany
2n=18
[14]
10LpallescensB
unge
IPK
LIN1645
Tajik
istan
2n=18
[17]
11LpallescensB
unge
Wild
popu
latio
ncollected
byNLB
olsheva
RussianFederatio
nAltai
2n=18
(present
study)
12Lmesostylum
Juz
IPK
LIN1774
Tajik
istan
2n=18
[17]
13Lmesostylum
Juz
IPK
LIN1662
Tajik
istan
2n=18
[17]
14Llew
isiiP
ursh
IPK
LIN1648
USA
2n=18
[17]
15Llew
isiiP
ursh
IPK
LIN1550
USA
2n=18
[17]
16Lau
striacum
LIPK
LIN1608
Germany
2n=18
[17]
17Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaRu
ssianFederatio
nRo
stov
2n=18
(present
study)
18Lau
striacum
Lsubspeuxinu
m(Ju
z)Ockendo
nIPK
LIN1546
Ukraine
2n=18
[17]
19Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaCr
imea
2n=18
(present
study)
20Lau
striacum
LWild
popu
latio
ncollected
byAA
Svetlo
vaUkraine
2n=18
(present
study)
21Lam
urenseAlef
BGI
outdoo
rscultivatedcollection
RussianFederatio
nFar
East
near
Pokrovka
settlem
ent
2n=18
[17]
SectD
asylinu
m22
Lhirsutum
Lsubsphirsutum
LIPK
LIN1676
Hun
gary
2n=16
[35]
23Lhirsutum
subsphirsutum
LIPK
LIN1649
Romania
2n=16
[36]
24Lhirsutum
Lsubsppseudoanatolicu
mPH
Davis
Wild
popu
latio
ncollected
byMPavelka
TurkeyK
aram
an2n
=32
(present
study)
25Lhirsutum
Lsubspan
atolicu
m(Boiss)Hayek
Wild
popu
latio
ncollected
byMPavelka
TurkeyA
ksaray
2n=32
(present
study)
SectLinum
26Lmargina
leACun
nex
Planch
IPK
LIN1920
Austr
alia
2n=84
(present
study)
27LnarbonenseL
IPK
LIN2002
Unk
nown
2n=28
[14]
28LnarbonenseL
IPK
LIN1653
France
2n=28
[14]
29Ldecumbens
Desf
IPK
LIN1754
Italy
2n=16
[14]
30Ldecumbens
Desf
IPK
LIN1913
Italy
2n=16
[14]
BioMed Research International 5
Table2Con
tinued
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)31
Lgrandiflorum
Desf
IPK
LIN2000
Unk
nown
2n=16
[14]
32Lgrandiflorum
Desf
IPK
LIN974
Unk
nown
2n=16
[14]
33Langustifoliu
mHud
sIPK
LIN1692
France
2n=30
(present
study)
34Langustifoliu
mHud
sVNIIL
K5695
Unk
nown
2n=30
(present
study)
35Langustifoliu
mHud
sVNIIL
K3108
Belgium
2n=30
(present
study)
36Langustifoliu
mHud
sIG
C15
Belarus
2n=30
[14]
37Langustifoliu
mHud
sVNIIL
K4731
EastGermany
2n=30
(present
study)
38Lbienne
Mill
(syn
Langustifolium
Hud
s)IG
C14
Hun
gary
2n=30
[14]
39Lusita
tissim
umLsubspbienne
Mill
Snankev
VIR
u-303794
Unk
nown
winterfl
ax2n
=30
(present
study)
40Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IGC
260
Belarus
dehiscentfl
ax2n
=30
(present
study)
41Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IPK
LIN119
Portugal
dehiscentfl
ax2n
=30
(present
study)
42Lusita
tissim
umLvarie
tyGiza
IPK
LIN277
Egypt
larges
eededflax
2n=30
(present
study)
43Lusita
tissim
umLG12Ru
zokvety
IPK
LIN633
Czecho
slovakia
dual-purpo
seflax
2n=30
(present
study)
44Lusita
tissim
umLvarietyColchidsky1
VIR
u099845
Abkh
azia
winterfl
ax2n
=30
(present
study)
45Lusita
tissim
umL
VNIIL
k7131
Morocco
larges
eededflax
2n=30
(present
study)
SectStellerolinum
46Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
TelyakovskyInlet
2n=20
(present
study)
47Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
near
Kraskino
settlem
ent
2n=20
(present
study)
6 BioMed Research International
was treated with 10U of TaqI restriction enzyme (ThermoScientific USA) for 3 h at 65∘b Ligation was performedby adding 25U of T4 DNA-ligase (Thermo ScientificUSA) 5mM ATP and 50 pmol of double-strand adapter[51015840-ACTCGATTCTCAACCCGAAAGTATAGATCCCA 51015840-PO4-CGTGGGATCTATACTT-(C6linker)-NH
2] and incu-
bation for 7 h at 37∘b The product was diluted twice withdeionized water The DNA sequence between the LTR (longterminal repeat) region of retrotransposons and the 119879119886119902120572119868restriction site was amplified using the adapter primer (51015840-GTTTACTCGATTCTCAACCCGAAAG 31015840) and primers tothe LTR regions of FL1a FL1b FL4 Cassandra FL10 FL8FL7 FL12 and FL9 retrotransposons [32]
1826 51015840-ACCCCTTGAGCTAACTTTTGGGGTAAG-31015840 (FL1a FL1b)1833 51015840-CTTGCTGGAAAGTGTGTGAGAGG-31015840(FL4)1838 51015840-TGTTAATCGCGCTCGGGTGGGAGCA-31015840(FL1a FL1b Cassandra)1845 51015840-AGCCTGAAAGTGTTGGGTTGTCG-31015840(FL11)1846 51015840-CTGGCATTTCCATTGTCGTCGATGC-31015840(FL10)1854 51015840-GCATCAGCCTGGACCAGTCCTCGTCC-31015840 (FL8)1868 51015840-CACTTCAAATTTTGGCAGCAGCGGATC-31015840 (FL1a FL1b)1881 51015840-TCGAGGTACACCTCGACTCAGG-31015840 (FL7)1886 51015840-ATTCTCGTCCGCTGCGCCCCTACA-31015840(FL12)1899 51015840-TGAGTTGCAGGTCCAGGCATCA-31015840(FL9)
Amplification was performed in two stages At the firststage only the primer to LTRof the retrotransposonwas usedAmplificationwas carried out in 25120583L of PCRmix containing5 120583L of ligation mix 1 U of TrueStart Hot Start Taq DNApolymerase (Thermo Scientific USA) TrueStart Taq DNApolymerase buffer 05mM MgCl
2 20120583M dNTP (Thermo
Scientific) and 5 pmol of the LTR primer The program foramplification for the first stage was 95∘C for 15min 30 cycles(95∘C for 30 s 62∘C for 1min 72∘C for 2min) and 72∘Cfor 10min At the second stage of amplification the adapterprimer 51015840-GTTTACTCGATTCTCAACCCGA-31015840 and one ofthe primers to the LTR region of retrotransposons wereused Amplification was carried out in 25 120583L of PCR mixcontaining 12 120583L of the first-stage PCR product 1 U ofTaq DNA polymerase (Thermo Scientific USA) Taq DNApolymerase buffer 15mM MgCl
2 200120583M dNTP (Thermo
Scientific USA) 25 pmol of LTR primer and 25 pmol of theadapter primerThe program for amplification for the secondstage was 95∘C for 15min 35 cycles (95∘C for 30 s 62∘C for1min 72∘C for 2min) and 72∘C for 10minThePCRproductswere separated in 25 agarose gel using fj buffer andthen stained with ethidium bromide Ten PCR products were
excised from agarose gel and characterized by sequencingon Applied Biosystems 3730 DNA Analyzer to confirm thespecificity of SSAP reaction The Bio-Rad Gel Doc systemwas used for gel documentation and photography as well asfor visual detection of presence or absence of polymorphicfragments in the samples from different accessions Thesedata were recorded in the form of a binary matrix in whichthe presence of a fragment was coded as 1 and its absence as0
The genetic distances between varieties were calculatedbased on the binary matrix of amplified fragments usingDicersquos formula [41]The dendrogramswere constructed usingSplitsTree 410 software [42] Cluster analysis was performedusing neighbor-joining method [43] and bootstrap valueswere determined based on 5000 permutations
24 Preparation of Cassandra Retrotransposon DNA ProbePCR primers were designed for amplification of Cassandraretrotransposon We amplified an internal domain (primersIntDom-F AGTGGTATCCGAGCCTCT and IntDom-R CCCATAGGACTCAACGTC) and the LTR with theexception of the 5S rDNA region of this retrotransposon(primers LTR-1-86-F TGTAATGTAACACGTTAGGCA andLTR-1-86-R TTAGTTAGGGACGGATTGTT LTR-206-279-F AAATAAATCTGTGAGGGATTAGT and LTR-206-279-RACTTGTAACACCCCGTACT) The amplification wascarried out in 20 120583L of PCR mixture that contained 1Uof TaqF DNA polymerase (Amplisens Russia) 1x TaqFbuffer 25 pmol of the forward and reverse primer 200120583MdNTP (Amplisens Russia) and 10 ng of genomic DNA Theprogram for amplification was 95∘C for 15min 40 cycles(95∘C for 10 s 62∘C for 20 s 72∘C for 30 s) and 72∘C for10min The amplicons were analyzed in 2 agarose gel andthen used as a template for biotin PCR labeling to obtainbiotin-labeled probes for FISH PCR labeling was carriedout using Biotin PCR Labeling Core Kit (Jena BioscienceGermany) according to the manufacturersquos protocol LabeledPCR products were precipitated with ethanol
25 FISH with Cassandra Retrotransposon DNA ProbeChromosome preparation was carried out according to thetechnique developed earlier for plants having small-sizedchromosomes [14] The hybridization mixture contained 2xSSC 50 formamide 10 dextran sulphate and 2 ng120583L ofa biotinylated DNA probe of Cassandra retrotransposonTheprobe was hybridized overnight at 31∘C After hybridizationthe slides were washed twice with 01x SSC at 38∘C for10min followed by two washes with 2x SSC at 44∘C for5min and a final 5min wash in 2x SSC at room temperatureThe biotin-labeled DNA probe was detected using a highlysensitive Alexa Fluor 488 Tyramide Signal Amplificationsystem (Invitrogen) according tomanufacturerrsquos instructions
3 Results
31 Analysis of SSAP Fingerprints For analyzing of thereproducibility of the SSAP method DNA of flax variety
BioMed Research International 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder
ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod
All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons
32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties
33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)
All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed
34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 5
Table2Con
tinued
Num
berAc
cessionname
Source
Genebanknu
mber
Orig
inCh
romosom
enum
ber(2n
)31
Lgrandiflorum
Desf
IPK
LIN2000
Unk
nown
2n=16
[14]
32Lgrandiflorum
Desf
IPK
LIN974
Unk
nown
2n=16
[14]
33Langustifoliu
mHud
sIPK
LIN1692
France
2n=30
(present
study)
34Langustifoliu
mHud
sVNIIL
K5695
Unk
nown
2n=30
(present
study)
35Langustifoliu
mHud
sVNIIL
K3108
Belgium
2n=30
(present
study)
36Langustifoliu
mHud
sIG
C15
Belarus
2n=30
[14]
37Langustifoliu
mHud
sVNIIL
K4731
EastGermany
2n=30
(present
study)
38Lbienne
Mill
(syn
Langustifolium
Hud
s)IG
C14
Hun
gary
2n=30
[14]
39Lusita
tissim
umLsubspbienne
Mill
Snankev
VIR
u-303794
Unk
nown
winterfl
ax2n
=30
(present
study)
40Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IGC
260
Belarus
dehiscentfl
ax2n
=30
(present
study)
41Lusita
tissim
umconvarcrepita
ns[Boenn
ingh
]Ku
lpae
tDanert
IPK
LIN119
Portugal
dehiscentfl
ax2n
=30
(present
study)
42Lusita
tissim
umLvarie
tyGiza
IPK
LIN277
Egypt
larges
eededflax
2n=30
(present
study)
43Lusita
tissim
umLG12Ru
zokvety
IPK
LIN633
Czecho
slovakia
dual-purpo
seflax
2n=30
(present
study)
44Lusita
tissim
umLvarietyColchidsky1
VIR
u099845
Abkh
azia
winterfl
ax2n
=30
(present
study)
45Lusita
tissim
umL
VNIIL
k7131
Morocco
larges
eededflax
2n=30
(present
study)
SectStellerolinum
46Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
TelyakovskyInlet
2n=20
(present
study)
47Lste
lleroidesPlanchon
BGI
Outdo
orsc
ultiv
ated
collection
RussianFederatio
nFar
East
near
Kraskino
settlem
ent
2n=20
(present
study)
6 BioMed Research International
was treated with 10U of TaqI restriction enzyme (ThermoScientific USA) for 3 h at 65∘b Ligation was performedby adding 25U of T4 DNA-ligase (Thermo ScientificUSA) 5mM ATP and 50 pmol of double-strand adapter[51015840-ACTCGATTCTCAACCCGAAAGTATAGATCCCA 51015840-PO4-CGTGGGATCTATACTT-(C6linker)-NH
2] and incu-
bation for 7 h at 37∘b The product was diluted twice withdeionized water The DNA sequence between the LTR (longterminal repeat) region of retrotransposons and the 119879119886119902120572119868restriction site was amplified using the adapter primer (51015840-GTTTACTCGATTCTCAACCCGAAAG 31015840) and primers tothe LTR regions of FL1a FL1b FL4 Cassandra FL10 FL8FL7 FL12 and FL9 retrotransposons [32]
1826 51015840-ACCCCTTGAGCTAACTTTTGGGGTAAG-31015840 (FL1a FL1b)1833 51015840-CTTGCTGGAAAGTGTGTGAGAGG-31015840(FL4)1838 51015840-TGTTAATCGCGCTCGGGTGGGAGCA-31015840(FL1a FL1b Cassandra)1845 51015840-AGCCTGAAAGTGTTGGGTTGTCG-31015840(FL11)1846 51015840-CTGGCATTTCCATTGTCGTCGATGC-31015840(FL10)1854 51015840-GCATCAGCCTGGACCAGTCCTCGTCC-31015840 (FL8)1868 51015840-CACTTCAAATTTTGGCAGCAGCGGATC-31015840 (FL1a FL1b)1881 51015840-TCGAGGTACACCTCGACTCAGG-31015840 (FL7)1886 51015840-ATTCTCGTCCGCTGCGCCCCTACA-31015840(FL12)1899 51015840-TGAGTTGCAGGTCCAGGCATCA-31015840(FL9)
Amplification was performed in two stages At the firststage only the primer to LTRof the retrotransposonwas usedAmplificationwas carried out in 25120583L of PCRmix containing5 120583L of ligation mix 1 U of TrueStart Hot Start Taq DNApolymerase (Thermo Scientific USA) TrueStart Taq DNApolymerase buffer 05mM MgCl
2 20120583M dNTP (Thermo
Scientific) and 5 pmol of the LTR primer The program foramplification for the first stage was 95∘C for 15min 30 cycles(95∘C for 30 s 62∘C for 1min 72∘C for 2min) and 72∘Cfor 10min At the second stage of amplification the adapterprimer 51015840-GTTTACTCGATTCTCAACCCGA-31015840 and one ofthe primers to the LTR region of retrotransposons wereused Amplification was carried out in 25 120583L of PCR mixcontaining 12 120583L of the first-stage PCR product 1 U ofTaq DNA polymerase (Thermo Scientific USA) Taq DNApolymerase buffer 15mM MgCl
2 200120583M dNTP (Thermo
Scientific USA) 25 pmol of LTR primer and 25 pmol of theadapter primerThe program for amplification for the secondstage was 95∘C for 15min 35 cycles (95∘C for 30 s 62∘C for1min 72∘C for 2min) and 72∘C for 10minThePCRproductswere separated in 25 agarose gel using fj buffer andthen stained with ethidium bromide Ten PCR products were
excised from agarose gel and characterized by sequencingon Applied Biosystems 3730 DNA Analyzer to confirm thespecificity of SSAP reaction The Bio-Rad Gel Doc systemwas used for gel documentation and photography as well asfor visual detection of presence or absence of polymorphicfragments in the samples from different accessions Thesedata were recorded in the form of a binary matrix in whichthe presence of a fragment was coded as 1 and its absence as0
The genetic distances between varieties were calculatedbased on the binary matrix of amplified fragments usingDicersquos formula [41]The dendrogramswere constructed usingSplitsTree 410 software [42] Cluster analysis was performedusing neighbor-joining method [43] and bootstrap valueswere determined based on 5000 permutations
24 Preparation of Cassandra Retrotransposon DNA ProbePCR primers were designed for amplification of Cassandraretrotransposon We amplified an internal domain (primersIntDom-F AGTGGTATCCGAGCCTCT and IntDom-R CCCATAGGACTCAACGTC) and the LTR with theexception of the 5S rDNA region of this retrotransposon(primers LTR-1-86-F TGTAATGTAACACGTTAGGCA andLTR-1-86-R TTAGTTAGGGACGGATTGTT LTR-206-279-F AAATAAATCTGTGAGGGATTAGT and LTR-206-279-RACTTGTAACACCCCGTACT) The amplification wascarried out in 20 120583L of PCR mixture that contained 1Uof TaqF DNA polymerase (Amplisens Russia) 1x TaqFbuffer 25 pmol of the forward and reverse primer 200120583MdNTP (Amplisens Russia) and 10 ng of genomic DNA Theprogram for amplification was 95∘C for 15min 40 cycles(95∘C for 10 s 62∘C for 20 s 72∘C for 30 s) and 72∘C for10min The amplicons were analyzed in 2 agarose gel andthen used as a template for biotin PCR labeling to obtainbiotin-labeled probes for FISH PCR labeling was carriedout using Biotin PCR Labeling Core Kit (Jena BioscienceGermany) according to the manufacturersquos protocol LabeledPCR products were precipitated with ethanol
25 FISH with Cassandra Retrotransposon DNA ProbeChromosome preparation was carried out according to thetechnique developed earlier for plants having small-sizedchromosomes [14] The hybridization mixture contained 2xSSC 50 formamide 10 dextran sulphate and 2 ng120583L ofa biotinylated DNA probe of Cassandra retrotransposonTheprobe was hybridized overnight at 31∘C After hybridizationthe slides were washed twice with 01x SSC at 38∘C for10min followed by two washes with 2x SSC at 44∘C for5min and a final 5min wash in 2x SSC at room temperatureThe biotin-labeled DNA probe was detected using a highlysensitive Alexa Fluor 488 Tyramide Signal Amplificationsystem (Invitrogen) according tomanufacturerrsquos instructions
3 Results
31 Analysis of SSAP Fingerprints For analyzing of thereproducibility of the SSAP method DNA of flax variety
BioMed Research International 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder
ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod
All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons
32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties
33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)
All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed
34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
was treated with 10U of TaqI restriction enzyme (ThermoScientific USA) for 3 h at 65∘b Ligation was performedby adding 25U of T4 DNA-ligase (Thermo ScientificUSA) 5mM ATP and 50 pmol of double-strand adapter[51015840-ACTCGATTCTCAACCCGAAAGTATAGATCCCA 51015840-PO4-CGTGGGATCTATACTT-(C6linker)-NH
2] and incu-
bation for 7 h at 37∘b The product was diluted twice withdeionized water The DNA sequence between the LTR (longterminal repeat) region of retrotransposons and the 119879119886119902120572119868restriction site was amplified using the adapter primer (51015840-GTTTACTCGATTCTCAACCCGAAAG 31015840) and primers tothe LTR regions of FL1a FL1b FL4 Cassandra FL10 FL8FL7 FL12 and FL9 retrotransposons [32]
1826 51015840-ACCCCTTGAGCTAACTTTTGGGGTAAG-31015840 (FL1a FL1b)1833 51015840-CTTGCTGGAAAGTGTGTGAGAGG-31015840(FL4)1838 51015840-TGTTAATCGCGCTCGGGTGGGAGCA-31015840(FL1a FL1b Cassandra)1845 51015840-AGCCTGAAAGTGTTGGGTTGTCG-31015840(FL11)1846 51015840-CTGGCATTTCCATTGTCGTCGATGC-31015840(FL10)1854 51015840-GCATCAGCCTGGACCAGTCCTCGTCC-31015840 (FL8)1868 51015840-CACTTCAAATTTTGGCAGCAGCGGATC-31015840 (FL1a FL1b)1881 51015840-TCGAGGTACACCTCGACTCAGG-31015840 (FL7)1886 51015840-ATTCTCGTCCGCTGCGCCCCTACA-31015840(FL12)1899 51015840-TGAGTTGCAGGTCCAGGCATCA-31015840(FL9)
Amplification was performed in two stages At the firststage only the primer to LTRof the retrotransposonwas usedAmplificationwas carried out in 25120583L of PCRmix containing5 120583L of ligation mix 1 U of TrueStart Hot Start Taq DNApolymerase (Thermo Scientific USA) TrueStart Taq DNApolymerase buffer 05mM MgCl
2 20120583M dNTP (Thermo
Scientific) and 5 pmol of the LTR primer The program foramplification for the first stage was 95∘C for 15min 30 cycles(95∘C for 30 s 62∘C for 1min 72∘C for 2min) and 72∘Cfor 10min At the second stage of amplification the adapterprimer 51015840-GTTTACTCGATTCTCAACCCGA-31015840 and one ofthe primers to the LTR region of retrotransposons wereused Amplification was carried out in 25 120583L of PCR mixcontaining 12 120583L of the first-stage PCR product 1 U ofTaq DNA polymerase (Thermo Scientific USA) Taq DNApolymerase buffer 15mM MgCl
2 200120583M dNTP (Thermo
Scientific USA) 25 pmol of LTR primer and 25 pmol of theadapter primerThe program for amplification for the secondstage was 95∘C for 15min 35 cycles (95∘C for 30 s 62∘C for1min 72∘C for 2min) and 72∘C for 10minThePCRproductswere separated in 25 agarose gel using fj buffer andthen stained with ethidium bromide Ten PCR products were
excised from agarose gel and characterized by sequencingon Applied Biosystems 3730 DNA Analyzer to confirm thespecificity of SSAP reaction The Bio-Rad Gel Doc systemwas used for gel documentation and photography as well asfor visual detection of presence or absence of polymorphicfragments in the samples from different accessions Thesedata were recorded in the form of a binary matrix in whichthe presence of a fragment was coded as 1 and its absence as0
The genetic distances between varieties were calculatedbased on the binary matrix of amplified fragments usingDicersquos formula [41]The dendrogramswere constructed usingSplitsTree 410 software [42] Cluster analysis was performedusing neighbor-joining method [43] and bootstrap valueswere determined based on 5000 permutations
24 Preparation of Cassandra Retrotransposon DNA ProbePCR primers were designed for amplification of Cassandraretrotransposon We amplified an internal domain (primersIntDom-F AGTGGTATCCGAGCCTCT and IntDom-R CCCATAGGACTCAACGTC) and the LTR with theexception of the 5S rDNA region of this retrotransposon(primers LTR-1-86-F TGTAATGTAACACGTTAGGCA andLTR-1-86-R TTAGTTAGGGACGGATTGTT LTR-206-279-F AAATAAATCTGTGAGGGATTAGT and LTR-206-279-RACTTGTAACACCCCGTACT) The amplification wascarried out in 20 120583L of PCR mixture that contained 1Uof TaqF DNA polymerase (Amplisens Russia) 1x TaqFbuffer 25 pmol of the forward and reverse primer 200120583MdNTP (Amplisens Russia) and 10 ng of genomic DNA Theprogram for amplification was 95∘C for 15min 40 cycles(95∘C for 10 s 62∘C for 20 s 72∘C for 30 s) and 72∘C for10min The amplicons were analyzed in 2 agarose gel andthen used as a template for biotin PCR labeling to obtainbiotin-labeled probes for FISH PCR labeling was carriedout using Biotin PCR Labeling Core Kit (Jena BioscienceGermany) according to the manufacturersquos protocol LabeledPCR products were precipitated with ethanol
25 FISH with Cassandra Retrotransposon DNA ProbeChromosome preparation was carried out according to thetechnique developed earlier for plants having small-sizedchromosomes [14] The hybridization mixture contained 2xSSC 50 formamide 10 dextran sulphate and 2 ng120583L ofa biotinylated DNA probe of Cassandra retrotransposonTheprobe was hybridized overnight at 31∘C After hybridizationthe slides were washed twice with 01x SSC at 38∘C for10min followed by two washes with 2x SSC at 44∘C for5min and a final 5min wash in 2x SSC at room temperatureThe biotin-labeled DNA probe was detected using a highlysensitive Alexa Fluor 488 Tyramide Signal Amplificationsystem (Invitrogen) according tomanufacturerrsquos instructions
3 Results
31 Analysis of SSAP Fingerprints For analyzing of thereproducibility of the SSAP method DNA of flax variety
BioMed Research International 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder
ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod
All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons
32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties
33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)
All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed
34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
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[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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PeptidesInternational Journal of
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder
ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod
All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons
32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties
33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)
All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed
34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
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[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 BioMed Research International
Ant
Tor
Tve
VerTos
Mer
Pra
Dip
A93
Ale
Novk-1147
K6Ors
v29Vik
Imp
Lid
Tom
Vor Kro
GorVasLav
Ros
AlfLadSla
Don
Oce
Sve
LM98 SmolicTvers
Len
Soy
A29
Ki5BelFor
Smo
Tost5
001
MogRus
S108
TMR
Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
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[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M
Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder
L mesostylumL mesostylum
L austriacum
L hirsutumL hirsutum subsp an
L usitatissimum subsp bien
L bienne
L usitatissimum convarcrepL usitatissimum convarcrep
L usitatissimum
L grandiflorum L grandiflorum
L decumbens
L marginale
L decumbens
L usitatissimum L angustifoliumL usitatissimumL angustifolium
L angustifolium
L usitatissimum
L narbonenseL narbonense
L hirsutumL hirsutum subsp ps
L austriacumL altaicum
L perenne subspextrL perenne
L austriacumL austriacum subsp eux
L leonii
L pallescens
L amurenseL lewisii
L komarovii
L pallescens
L stelleroidesL stelleroides
L perenne L pallescensL perenne subsp angl
L perenne susp alp
L austriacum
A
D I
F
G
H
E
С B
32
29
26
30
4647
31
39
45414044
3837433433 35
2728
2524 23 22
20 18
17 161312
193
21
99 9802
99 9
99 9
97
95
100
100 100
100
100
100
99 8871732
81
97 811
6105
21
15
14
4
3642
Sect Stellerolinum
Sect
Lin
um
Sect Dasylinum
Sect
Ade
nolin
um
Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
10 BioMed Research International
(a) (b)
(c) (d)
1 2 3 4 5 6 7 8
(e)
Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m
possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular
4 Discussion
41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic
polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 11
In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important
The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]
Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions
42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)
43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum
(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study
44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]
45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum
SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum
46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]
Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]
The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers
It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
12 BioMed Research International
from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication
L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation
The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable
47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)
5 Conclusions
The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP
analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work
Acknowledgments
The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center
References
[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968
[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949
[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996
[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009
[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959
[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975
[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995
[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000
[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o
vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 13
[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007
[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002
[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006
[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009
[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010
[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011
[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013
[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000
[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007
[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002
[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009
[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011
[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997
[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998
[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004
[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic
Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011
[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012
[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000
[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003
[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007
[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008
[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011
[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011
[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012
[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005
[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005
[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010
[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010
[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012
[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
14 BioMed Research International
[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945
[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006
[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987
[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014
[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985
[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984
[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology