bassilnv10_microsatellite markers distinguish

8/11/2019 BassilNV10_Microsatellite Markers Distinguish

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Microsatellite Markers Distinguish Hawaiian Ohelo from OtherVacciniumL. SectionMyrtillusSpecies

N.V. Bassil1,a, T. Bunch1, A. Nyberg1, K. Hummer1 and F.T. Zee21USDA-ARS, National Clonal Germplasm Repository (NCGR), 33447 Peoria Road,

Corvallis, Oregon 97333, USA2USDA-ARS, Pacific Basin Agricultural Research Center (PBARC), Tropical PlantGenetic Resource and Disease Research Unit (TPGRDR), Hilo, Hawaii 96720, USA

Keywords:Vaccinium reticulatum, Vaccinium calycinum, Vaccinium myrtillus, bilberry,ohelo berry, simple sequence repeat (SSR) markers

AbstractThe US Department of Agriculture, Agricultural Research Service, National

Clonal Germplasm Repository, Corvallis, collection of Vaccinium L. contains >1700accessions representing 66 species from 33 countries. About 377 accessions belong to10 species of Vaccinium section Myrtillus. This section is restricted to the NorthernHemisphere where its primary center of diversity occurs around the Pacific Rim inhigh elevations from Alaska to Guatemala and in mountains of Japan, Kurile,Sakhalin, and Hawaiian Islands, and Russian Siberia. The first objective of thisstudy was to use microsatellite or simple sequence repeat markers (SSR) previouslyisolated from domestic blueberry, to uniquely identify three clones of Ohelo berry,V. reticulatum,selected for their ornamental qualities. The second objective was toevaluate the SSR markers for cross-transference to other species within Section

Myrtillus including: two genotypes from another endemic Hawaiian species, V.calycinum; 17 representatives from seven North American species (V. caespitosum,V. deliciosum, V. membranaceum, V. myrtillus, V. ovalifolium, V. parvifolium, and V.scoparium), and in two accessions of V. praestans from Sakahlin Island, RussianFederation and one genotype from Hokaido, Japan. As many as eighteenmicrosatellite markers cross-amplified and appeared polymorphic in most of the

genotypes evaluated. The three V. reticulatum genotypes were uniquely identifiedwith eight SSR primer pairs. UPGMA clustering based on nine microsatellite primerpairs clearly separated the Hawaiian taxa and grouped V. praestanstogether. Twoaccessions of V. myrtillus(V myrt 1684-1, V myrt 1684-3) collected from within theKalmiopsis Wilderness of southern Oregon had identical fingerprints indicating aclonal relationship. Conserved primers that amplify what appear to be single lociSSRs are recommended for population genetic analyses in diploid Vacciniumsection

Myrtillustaxa including V. calycinum, V. reticulatum, V. scopariumand V. praestans.SSR-based analysis indicated high diversity in other taxa of sectionMyrtillusand theneed for a larger amount of accessions from each species, additional markers orsequence-based markers, for taxonomic resolution.

INTRODUCTIONThe genus VacciniumL. contains more than 400 species of terrestrial or epiphyticshrubs or small trees (Van der Kloet, 1988) and is traditionally divided into 33 sections(Sleumer, 1941). In their morphologic treatment of the taxonomy of section Myrtillus

based on phenetic analyses, Van der Kloet and Dickinson (1999) described five clustersthat contain seven species (V. caespitosum Michaux, V. deliciosum Piper, V.membranaceumDouglas ex Torrey, V. myrtillusL., V. ovalifoliumSmith, V. parvifoliumJ.E. Smith, and V. scopariumLeiberg). These species share a number of morphologicalfeatures including, most distinctively, flowers that are borne singly in the axils of thelowermost leaves, and a pedicel that is continuous with the calyx tubes. Phylogeneticanalysis based on molecular data from the nuclear ribosomal internal transcribed spacer

[email protected]

Proc. IS on Molecular Markers in HorticultureEds.: N.V. Bassil and R. Martin

Acta Hort. 859, ISHS 2010


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(nrITS) and the chloroplast genes matK and ndhF resulted in the identification of threeHawaian species (V. dentatum Smith, V. calycinum Smith and V. reticulatum Smith)

previously placed in Macropelma as a monophyletic group that is derived from withinsectionMyrtillus (Powell and Kron, 2002). Furthermore, theMyrtillusclade was sister toa clade that includes sections Oxycoccoides, Praestantia(V. praestansLambert) and the

two Asian representatives (V. hirtum Thunb. and V. smallii A. Gray) of sectionHemimyrtillus. Species from these sections share the character of perennating bud scaleswith two prophylls and appear to form a monophyletic Pacific Rim clade (Powel andKron, 2002). V. praestansis considered in sectionMyrtillus sensu latoin this study.

The center of diversity of species in section Myrtillus is found along the PacificRim from Japan, arching through Alaska, southerly to Guatemala (Van der Kloet andDickinson, 1999). The mountains of Honshu, Japan, form a secondary center of diversity.Fruits of these wild plants are highly aromatic and harvested mostly for home use as freshor processed products. The fruit pulp in addition to the skin is intensely colored in

Myrtillus, as opposed to green or white in blueberries (section Cyanococcus). Differencesin anthocyanin content or in the aglycone profiles identify fruits of V. parvifolium, V.deliciosum, V. membranaceumand V. ovalifoliumof the Pacific Northwest (Ballington etal., 1988). Interspecific hybridization between cultivated V. corymbosum species from

section Cyanococcus and V. membranaceum species were attempted to introgress thearoma of fruits from these species into the cultivated blueberry but did not result in anyreleases (Chad Finn, pers. commun.). The USDA-ARS Pacific Basin AgriculturalResearch Center (PBARC) identified and selected three clones of ohelo (Vacciniumreticulatum Smith) for their highly attractive foliage and berries. On the Islands of Hawaiiand Maui, residents gather berries in summer from the wild in disturbed, open sites at 640to 3700 m elevation for use in jam, jelly and pie filings. A long term goal of this projectconsists of developing sustainable ohelo berry production in Hawaii for culinary andvalue added product use; and to determine nutraceutical components and value-added

products for consumers.The USDA-ARS National Clonal Germplasm Repository in Corvallis, Oregon,

preserves a collection of >1700 Vaccinium accessions representing 66 species from 33

countries. Ten species in section Myrtillus are represented. The first objective of thisstudy was to use microsatellite markers previously developed in blueberry, Vacciniumcorymbosum L., to identify the three Hawaiian ohelo, V. reticulatum, selections. Thesecond objective was to evaluate these SSR primer pairs for cross-transference to otherspecies within section Myrtillus including: two genotypes of the another Hawaiianendemic, V. calycinum; 17 representatives from seven North American species [V.caespitosum (dwarf bilberry), V. deliciosum, V. membranaceum, V. myrtillus (NorthAmerican and Eurasian: bilberry, whinberry, whortleberry, European blueberry), V.ovalifolium(Alaska blueberry), V. parvifolium(red huckleberry), and V. scoparium; andin three accessions of V. praestansfrom Sakhalin Russian Federation and Hokaido, Japan.

MATERIALS AND METHODSDNA was extracted from actively-growing leaves of thirty-two accessions (Table

1) of Vacciniumsection Myrtillusplants growing in screenhouses at the National ClonalGermplasm Repository (NCGR) in Corvallis, Oregon. Tissue samples were homogenizedwith an MM 301 Mixer Mill (Retsch International, Haan, Germany) and DNA wasextracted using a modified Puregene (Gentra Systems Inc. Minneapolis, Minn) protocolused routinely in the NCGR lab.

Twenty-three SSR primer pairs previously developed in blueberry were reportedto amplify in V. parvifolium(Boches et al., 2005). These primer pairs were evaluated foramplification of a product in 24 accessions (Table 1) representing ten Vacciniumsection

Myrtillusspecies by 2% agarose gel electrophoresis.PCRs were performed in 10 l volume containing 1 reaction buffer, 2 mM

MgCl2, 0.2 mM dNTPs, 0.3 M fluorescent WellRed forward primer, 0.3 M reverseprimer, 0.25 U of Biolase TaqDNA polymerase (Bioline), and 2.5 ng genomic DNA. The


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PCR protocol consisted of one cycle of initial denaturation at 94C for 4 min, followed bya touchdown PCR program consisting of10 cycles of 40 s at 94C; 40 s at 60C; and 40s at 72C. Annealing temperatures were decreased 1C each cycle to 50C. PCRcontinued for 25 additional cyclesof 40 s at 94C; 40 s at 50C; and 40 s at 72C with afinal elongation step of 72C for 10 min. DNA was amplified in an Eppendorf Gradient

thermocycler (Eppendorf, Westbury, NY) or an MJ Research Tetrad thermocycler(BioRad, Hercules, CA). The success of the PCR was evaluated by 2% agarose gelelectrophoresis. PCR products were pooled for fragment analysis for separation on aBeckman CEQ 8000 genetic analyzer (Beckman Coulter Inc., Fullerton, CA). Allelesizing and visualization were performed using the fragment analysis module of the CEQ8000 software. Due to the multiple ploidy levels of the accessions, individuals werescored for the presence or absence of each allele and each allele was treated as a separatelocus. PowerMarker (Liu and Muse, 2004) was used for Unweighted Pair Group Methodwith Arithmatic Mean (UPGMA) clustering and bootstrap analysis.

RESULTS AND DISCUSSIONEighteen of 23 SSRs previously reported to amplify in V. parvifoliumgenerated a

PCR product in each of 24 accessions (Table 1) tested and were further evaluated by

capillary electrophoresis in 32 accessions listed in Table 1. SSR primer pairs that weredifficult to score in all the accessions included: NA800, VCC_J9, VCC_I8, VCC_S10 andwere excluded. NA824 appeared to amplify multiple loci in each of the species tested. Inaddition to NA800, seven of the remaining fourteen SSR primer pairs amplified differentsize products in the three ohelo berry accessions and identified each accession (Table 2).Each of these SSR primer pairs, except for VCC_I2, generated unique genetic profiles foreach of the ohelo clones. These SSRs can be used by the nascent genotyping industry toenforce intellectual property and certify identity of these ohelo accessions.

Data for four primer pairs (CA421, NA172, NA961 and NA1040) that generatedpolymorphic fragments was incomplete and they were not included in cluster analysis.UPGMA clustering (Fig. 1) based on the remaining nine SSRs (Table 2) identified aHawaiian group that consisted of the V. reticulatum and V. calycinum accessions,

supporting Van der Kloets hypothesis of the Hawaiian taxa as a coherent evolutionaryunit (Van der Kloet, 1996) . V. praestans from Sakhalin and from Hokkaido were alsogrouped together. Based on these 9 SSRs, each of the 32 accessions had a unique genetic

profile except for two accessions of V. myrtillus (V myrt 1684-1, V myrt 1684-3)collected from within the Kalmiopsis Wilderness of southern Oregon. Up to 100%

bootstrap support was obtained for these identical genotypes. Other relationships thatwere strongly supported by bootstrap analysis (70) consisted of: the V. calycinumaccessions (71%); the V. praestansaccessions (96%); the two Hawaiian ohelo selections,V ret Ohelo 6 and V ret Ohelo 9, (83%); and between V memb 141 collected from anunknown location and V memb 1213 obtained from Idaho (100%).

Based on amplification of up to two allelic variants in diploid accessions, possiblyindicating single loci, we recommend testing the indicated SSR primer pairs for

population genetic analyses (Table 2). Four SSRs that appear to amplify single loci in atleast one of the diploid species tested are not listed in Table 2. They include: CA421(recommended for each of the four species); NA172 and NA961 (recommended for all

but V. praestans); and NA1040 (in V. calycinum and V. reticulatum). According toSunnucks (2000), single locus microsatellites are the mainstay of modern populationgenetics other than systematics. Single loci that appear highly conserved in Vacciniuminclude CA23, CA421, CA787, CA794 and CA855. In addition to generating products insection Cyanococcus from which they were isolated, they amplified PCR fragments ineach of the Myrtillus species evaluated in this study, and in representatives from sevenadditional sections (Boches et al., 2005). Each of these SSRs were isolated fromexpressed sequence tags (ESTs), and thus are believed to be more conserved thangenomic SSRs. High Basic Local Alignment Search Tool (BLAST) nucleotide similaritywas reported for CA421, CA787, CA794 and CA855 (Boches et al., 2005). The


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Tables

Table 1. Thirty-two accessions representing 10 species of Vaccinium section Myrtillusevaluated in this study. Plant Introduction Number, origin, and ploidy are listed. Suspected ploidy of 2x, 4x indicates species that have diploid and tetraploidaccessions.

Name Accessionno.

Species Origin Suspectedploidy

V caesp 16511 PI 638503 V. caespitosum Michaux Oregon, US 2x

V caesp 14891 PI 638359 V. caespitosum Michaux Washington, US 2x

V cal 18121 PI 657261 V. calycinum Smith Hawaii, US 2x

V cal 18141 PI 657263 V. calycinum Smith Hawaii, US 2x

Bluecrop PI 554885 V. corymbosumL. Cultivar, Maryland, US 4xV del 403-3

1 PI 554908 V. deliciosum Piper Hawaii, US 4x

V del 403-61 PI 554908 V. deliciosum Piper Hawaii, US 4xV memb 141

1 PI 555033 V. membranaceum

Douglas ex TorreyWashington, US 4x

V memb 12131 PI 613646 V. membranaceum

Douglas ex TorreyIdaho, US 4x

V memb 1372 PI 618219 V. membranaceumDouglas ex Torrey

Idaho, US 4x

V myrt 379 PI 555091 V. myrtillus L. Oregon, US 2x, 4xV myrt 464

1 PI 555097 V. myrtillus L. Finland 2x, 4x

V myrt 6271 PI 555107 V. myrtillus L. Colorado, US 2x, 4x

V myrt 16221 PI 638468 V. myrtillus L. Selected in Michigan, US

(from European seed)2x, 4x

V myrt 1684-11

PI 651624 V. myrtillus L. Oregon, US 2x, 4xV myrt 1684-3 PI 651624 V. myrtillus L. Oregon, US 2x, 4xV oval 1189

1 PI 618146 V. ovalifoliumSmith BC, Canada 2x, 4x

V oval 14211 PI 618266 V. ovalifoliumSmith Unknown 2x, 4x

V oval 1118 PI 613564 V. ovalifoliumSmith Alaska, US 2x, 4xV oval 1252 PI 613685 V. ovalifoliumSmith Alaska, US 2x, 4xV oval 1258

1 PI 613691 V. ovalifoliumSmith Alaska, US 2x, 4x

V oval 13751 PI 618222 V. ovalifoliumSmith Washington, US 2x, 4x

V oval 15461 PI 638403 V. ovalifoliumSmith Sakhalin, Russian Fed. 2x, 4x

V parv 4361 PI 555217 V. parvifolium J.E. Smith Oregon, US 2x, 4x

V praest 16261 PI 638482 V. praestansLambert Hokkaido, Japan 2x

V praest 15701 PI 638427 V. praestansLambert Sakhalin, Russian Fed. 2x

V praest 1569 PI 638426 V. praestansLambert Sakhalin, Russian Fed. 2xV ret 7801 PI 555197 V. reticulatum Sm. Oregon, US 2xV ret Ohelo 6

1 CVAC1819 V. reticulatum Sm. Hawaii, US 2x

V ret Ohelo 71 CVAC1817 V. reticulatum Sm. Hawaii, US 2x

V ret Ohelo 9 CVAC1818 V. reticulatum Sm. Hawaii, US 2xV scop

1 CVAC1826 V. scopariumLeiberg Oregon, US 2x

1Indicates samples that were tested for amplification by 23 SSR primer pairs that previously amplified in V.parvifolium.


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Table 3. SSR-based genetic profile of three clones of ohelo berry, Vaccinium reticulatum. SSRs thHawaiian clones included CA23, CA236, CA344, CA787, CA824, CA855 and NA961.

Name NA398 CA794 NA172 MEC1_I2 CA421 NA741 NH06-6 225, 229 238, 242 184 210, 212 191, 193 294, 308 NH06-7 217, 221 236, 238 186, 188 210 193, 217 294, 298

NH06-9 213, 229 238, 248 182, 184 210, 212 195, 207 294, 296

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Figures

Fig. 1. UPGMA cluster analysis of 32 accessions from sectionMyrtillusspecies based onmicrosatellite analysis using 9 SSR primer pairs. Bootstrap support at >70 isillustrated where found.

V ret Ohelo 6

V ret Ohelo 9

V ret 780

V ret Ohelo 7

V cal 1812

V cal 1814

Hawaiian Taxa

V praes 1626

V praes 1569

V praes 1570

V. praestans

V memb 1372

V memb 141

V memb 1213

V scop

V myrt 464

V myrt 1622

V myrt 379

V oval 1118

V oval 1189

V parv 436

V myrt 627

V oval 1421

V cesp 1489

Bluecrop

V cesp 1651

V oval 1546

V myrt 1684-1

V myrt 1684-3

V oval 1375

V del 403 3

V del 403 6

V oval 1252V oval 1258

0.05

97

96

99

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bassilnv10_microsatellite markers distinguish

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