1. 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)

2. Powdery mildew caused by Oidium lycopersici on tomato’s leaves

3. L. esculentum var. cerasiforme: R28 L. esculentum cv.SuperMarmande: SM Resistant Susceptible Resistant Susceptible

4. 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.

5. Our first step was isolated a RAPD marker (OPU3 1500 ) linked to ol-2 gene to using a “Bulked Segregant Analysis” (BSA): it was detected in the susceptible bulk. The OPU3 1500 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 F 2 population.

6. R28SMF 2 ResistantF 2 Susceptible F 2 Resistant F 2 Susceptible F1F1 BLKR BLKS R28 SM

7. Electrophoretic patterns of PCR-amplified DNA products obtained with OPU3 primer 5'-CTATGCCGA-3' from genomic DNA of parents (SM susceptible; R28 resistant), susceptible F 1 plants, bulks (BLKS, bulk of F 2 susceptible plants; BLKR, bulk of F 2 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 OPU3 1500, is associated with resistance. M 1 and M 2, DNA molecular weight markers (1kb and 100bp Ladders, respectively)

9. Our second step was to identify AFLP markers always associated at the ol-2 gene. We found 8 new molecular markers.

10. Linkage group E39/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) OPU3 ol-2 on Ol-2 locus

11. 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).

12. 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. RESEARCH AIMS

13. SCAR MARKERS 2 2 2 4 3 2 1 1 11 Lambda DNA ng/µl 30 60 90 DNA Marker EcoRI 41/MseI 32 EcoRI 34/MseI 61 EcoRI 32/MseI 53 EcoRI 34/MseI 61 (2) 4 4 4 3 3 3 SCAR markers obtained from conversion of new AFLP polymorphic markers Legend: SM= 1; R28=2; BLKS=3; BLKR=4 DNA Marker= 25bp

14. 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.

15. 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.

16. ‘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.

17. Our work is in progress… …

18. THANK’S FOR YOUR ATTENTION

19. 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.

20. Steps of LMS-PCR technique: to digest genomic DNA to attach adaptors to amplify it using primers of known sequence and adaptor primers