Development And Utilization Of Functional Marker For The Fusarium Wilt Resistance Gene Fom-2 In Melon (Cucumis Melo L.) Based On Single Nucleotide Polymorphism

Fusarium wilt of melon (Fusarium oxysporum f. sp. Melonis, Fom) caused by the fungus Fusarium oxysporum f. sp. melonis Snyder and Hansen, has become one of the most destructive diseases of melon. It severely limited the melon yeild production and quality. This research focused on the development and utilization of the SNP (single nucleotide polymorphism)-based FM associated with resistance of fusarium wilt in melon. The DNA moleculer marker, CAPS (Cleaved amplified polymorphic sequences, CAPS) and AS-PCR (Allele-specific PCR, AS-PCR), were employed to validate the effectiveness and reliability of the markers in F₁ and F₂ population, which derived from a cross between Cantaloupe and xianguo 027-5. Then the candidate markers were converted into the functional marker and used in the selection of cultivars.A 1311bp leucine-rich repeat (LRR) region of the Fom-2 gene was cloned. Three single nucleotide polymorphism (SNP) sites were found and located at 281st, 1076th and 1216th of the cloned LRR region, respectively. These three single nucleotide polymorphism sites were analysed by CAPS method in F₁ (30 plants) and F₂ populations (100 plants) derived from cross between wilt resistant and susceptible germplasms. According to CAPS method, we used CAPS-2F and CAPS-2R, CAPS-3F and CAPS-3R as two pairs of specific primers to amplify fragments of interest, then the PCR products were digested by EcoR I and Xba I. The result showed the 1:2:1 Mendelian single-dominant segregation pattern for site 2 and 3 in the F₂ population. The CAPS makers were sucessfully converted into the fuctional markers. As for the AS-PCR method, the specific primers 2-3F1 and 2-3R1 were used to amplify fragments of interest. When the annealing tempreture was 62℃and cycle number was 35, it could be easily distinguished the resistant and susceptible plants. The genetic segregation ratio in F₂ population was 3:1 among the resistant and susceptible plants. The results of CAPS and AS-PCR methods showed that site 2 and 3 were reliable and effective.The further screening of 34 cultivars were conducted by CAPS and AS-PCR markers. Among the 34 tested varieties, 11 were identified as resistant genotypes including 2 homozygous and 9 heterozygous genotypes. These resistant plants can be used as good germplasm resources in resistant breeding.