Transcript Linkage group on OL

ISSAG
Viterbo - 22 / 26 August 2005
CONTRIBUTION TO FINE
MAPPING OF OL-2 LOCUS IN
TOMATO
MINOIA 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. esculentum
cv.SuperMarmande: SM
Susceptible
L. esculentum var. cerasiforme: R28
Resistant
Susceptible
Resistant
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.
SM R28
PS
PR
F2 Resistant
F2 Susceptible
Piante F2 bulk suscettibile
M
270bp
F2 Resistant
F2 Susceptible
F1
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 4
IL4-1-1
+GP180
4-A
GP180
3.7
4-B
-TG15
+CT229A
IL4-1
TG15,CT229A
12.1
IL4-2
4.6
(CT269B,TG123)
TG483
3.4
Tpi-2
4.3
-CT175
+TG182
1.1
0.8
+TG182
-CT157
IL4-3-2
5.5
4-E
4-F
(CD49A)
TG370
4-C
4-D
(CT122C,CT63C,TG413B,TG49, 6Pgdh-1)
CD59
2.9
3.9
2.4
+TG208
-TG75A
TG474,TG339
CT175
TG182,CT192
(CD70,TG146)
CT157
(TG609,CT162,PC1, Pgm-2, Got-1)
TG506,TG2
(GP221,TG316,CT261,TG268,CT181,CT145B)
TG652,CT259
3.6
(CT97,TG516)
1.2
2.1
0.8
TG287
(TG633,PC3,CT178)
TG208
(TG272B,TG95, Adh-1)
TG75A
CD55
+CD55
-TG519
6.0
(CT286)
TG519,TG264,TG635,CT194
4.6
(CT185)
IL4-3
TG62,TG427,CT161
8.9
(CT188)
TG65
4-G
8.0
(TG120)
3.5
TG574
(TG305,CT132A,
CT264)
TG555
2.1
TG155
9.4
-TG155
+CT50
CT287B
5.6
1.0
1.0
4-H
3.5
(TG34)
CT50
TG500
CT133
(TG443)
CD39,CT73
IL4-4
4.9
+CP57
-CT173
2.3
(TG260)
CP57
1.6
CT173
TG22,CT126B
5.8
(CT253,CT239A,TG37X)
4-I
TG163,CT224B,CT199
3.8
(CT61)
TG464,TG498,TG587
+TG464
Our second step was to identify AFLP
markers always associated at the ol-2
gene.
We found 8 new molecular markers.
E39/M37(290)
Linkage group
on Ol-2 locus
3.2
E32/M47(174)
E32/M50(174)
0.4
E40/M56(100)
E34/M61(270)
E42/M45(248)
0.2
E41/M32(390)
0.5
OPU3
0.3
ol-2
CAPS/OPU3
cM
E32/M53(280)
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).
RESEARCH AIMS
To identify new PCR-based molecular markers
linked to ol-2 gene
To establish a linkage group for Ol-2 locus
To obtain co-dominant markers useful to perform
MAS in tomato: in particular to converted the 8
AFLP marker in SCAR or CAPS markers.
3
2
1
EcoRI 41/MseI 32
EcoRI 34/MseI 61
4
3
2
1
4
3
2
DNA Marker
EcoRI 32/MseI 53
Lambda DNA
ng/µl
90
60 30
1
EcoRI 34/MseI 61 (2)
4
3 2
1
4
SCAR MARKERS
SCAR markers obtained from conversion of new AFLP polymorphic
markers
Legend:
SM= 1; R28=2; BLKS=3; BLKR=4
DNA 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