issag viterbo - 22 / 26 august 2005 contribution to fine mapping of ol-2 locus in tomato minoia...
Post on 18-Dec-2015
217 Views
Preview:
TRANSCRIPT
ISSAG ISSAG Viterbo - 22 / 26 August 2005Viterbo - 22 / 26 August 2005
CONTRIBUTION TO FINE CONTRIBUTION TO FINE MAPPING OF MAPPING OF OL-2 LOCUSOL-2 LOCUS IN IN
TOMATOTOMATO
MINOIA SILVIAMINOIA SILVIA
Department of Agro-forestry and Environmental Biology and Chemistry Sect. Genetics and Plant Breeding, University of Bari (Italy)
Powdery mildew caused by Oidium lycopersici on tomato’s leaves
L. esculentumL. esculentum var. var. cerasiforme: cerasiforme: R28R28
L. esculentumL. esculentum cv.SuperMarmande: SMcv.SuperMarmande: SM
Resistant Resistant
SusceptibleSusceptible
Resistant Resistant SusceptibleSusceptible
Sources of resistance to the fungus are available in wild material and in particular in the accession of L.
esculentum var. cerasiforme,we have selected a line (LC-95) resulting in
complete resistance to powdery mildew, and also showed that a single recessive gene, named ol-2, was responsible for
desease control.
Our first step was isolated a RAPD marker (OPU31500) linked to ol-2 gene to using a “Bulked Segregant Analysis” (BSA): it
was detected in the susceptible bulk.
The OPU31500 was converted to a CAPS marker and the estimation of the distance between the marker and the ol-2 gene
was identified by linkage analysis in the F2 population.
R28SM F2 Resistant F2 Susceptible
F2 Resistant F2 SusceptibleF1B
LK
RB
LK
SR
28S
M
Electrophoretic patterns of PCR-amplified DNA products obtained with OPU3 primer 5'-CTATGCCGA-3' from genomic DNA of parents (SM susceptible; R28 resistant), susceptible F1 plants, bulks (BLKS, bulk of F2 susceptible plants; BLKR, bulk of F2 resistant plants) and all the individuals included in the bulks (lanes 1-9 susceptible; lanes 10-19 resistant): the absence of the 1.5 kb band, indicated with the arrow and designated OPU31500, is associated with resistance. M1 and M2, DNA molecular weight markers (1kb and 100bp Ladders, respectively)
chromosome 4GP180
CD59
TG15,CT229A
TG370
TG483
Tpi-2
TG474,TG339 CT175 TG182,CT192
CT157
TG506,TG2
TG652,CT259
TG287
TG208
TG75ACD55
TG519,TG264,TG635,CT194
TG62,TG427,CT161
TG65
TG574
TG555
TG155
CT287B
CT50
CT133
CD39,CT73
CP57
CT173 TG22,CT126B
TG163,CT224B,CT199
TG464,TG498,TG587
3.8
5.8
1.6
2.3
4.9
3.5
1.0
5.6
9.4
2.1
3.5
8.0
8.9
4.6
6.0
2.1 0.8
3.6
3.9
2.4
5.5
0.8 1.1
4.3
3.4
4.6
12.1
2.9
3.7
1.2
(CT122C,CT63C,TG413B,TG49, 6Pgdh-1)
(CD49A)
(CT269B,TG123)
(CD70,TG146)
(TG609,CT162,PC1, Pgm-2, Got-1)
(CT97,TG516)
(GP221,TG316,CT261,TG268,CT181,CT145B)
(TG633,PC3,CT178)
(TG272B,TG95, Adh-1)
(CT286)
(CT185)
(CT188)
(TG120)
(TG305,CT132A,CT264)
(TG34)
(TG443)
(CT253,CT239A,TG37X)
(TG260)
(CT61)
1.0 TG500
IL4-1
IL4-1-1
IL4-2
IL4-3
IL4-3-2
IL4-4
+TG182-CT157
-TG15
+TG208-TG75A
+CT229A
+TG182
+CP57
-CT173
-CT175
+CD55
-TG519
-TG155
+CT50
4-A
4-B
4-C
4-D
4-E
4-F
4-G
4-H
4-I
+GP180
+TG464
Our second step was to identify AFLP markers always associated at the ol-2
gene.
We found 8 new molecular markers.
Linkage groupLinkage groupE39/M37(290)
3.2
E32/M47(174)
E32/M50(174)
CAPS/OPU3
E32/M53(280)
0.2
0.5
0.4
0.3
cM
E40/M56(100)E34/M61(270)
E42/M45(248)
E41/M32(390)
OPU3ol-2
on on Ol-2Ol-2 locuslocus
The finale purpose of our investigation is to identify molecular markers linked to resistance and to use these markers for MAS (marker
assistent selection), to set up improved lines of tomato resistant to
powdery mildew (Oidium lycopersicum).
To identify new PCR-based molecular markers To identify new PCR-based molecular markers linked to linked to ol-2ol-2 gene gene
To establish a linkage group for To establish a linkage group for Ol-2 locusOl-2 locus
To obtain co-dominant markers useful to perform To obtain co-dominant markers useful to perform MAS in tomato: in particular to converted the 8 MAS in tomato: in particular to converted the 8 AFLP marker in SCAR or CAPS markers.AFLP marker in SCAR or CAPS markers.
RESEARCH AIMSRESEARCH AIMS
SCAR MARKERSSCAR MARKERS
22
22
22 44 33 22
11
11 1111
Lambda DNA ng/µl
306090DN
A M
ark
er
EcoRI 41/MseI 32EcoRI 34/MseI 61EcoRI 32/MseI 53
EcoRI 34/MseI 61 (2)
44 44
44
33
33 33
SCAR markers obtained from conversion of new AFLP polymorphic SCAR markers obtained from conversion of new AFLP polymorphic
markersmarkers Legend: Legend: SM= 1; R28=2; BLKS=3; BLKR=4SM= 1; R28=2; BLKS=3; BLKR=4DNA Marker= 25bpDNA Marker= 25bp
When we begins work with this CAPS markers started our problems:
• the fragments that we amplified were small (between 100-300 bp)
• when we cutted with restiction enzymes we obteined smaller fragments and we
lost the polymorphism.
LMS-PCR technique (Schupp et al., 1999)
(Ligation-Mediated Suppression PCR)
this is an systematic approach to obtain, from AFLP markers, information about
internal and flanking sequence.
‘Wolking’ on the genome, we arrive to amplify unknown regions flanking known AFLP regions and then to
lengthen our fragments.
Starting from 100-300 bp fragments we obteined fragments of 1000 bp.
Our work is in progress…
…
THANK’S FOR YOUR ATTENTION
Michelmore RW, Paran RV, Kesseli, Identification of marker linked to desease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating population, Proc. Natl. Acad. Sci. USA 88 (1991) 9828-9833.
Schupp JM, Price LB, Klevytska A and Keim P, 1999. Internal and flanking sequence from AFLP fragment using ligation-mediated suppression PCR. BioTechniques 26, 905-912.
Steps of LMS-PCR technique:
• to digest genomic DNA
• to attach adaptors
• to amplify it using primers of known sequence and adaptor primers
top related