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