Identification of Candidate Resistance Genes against Squash leaf curl Philippine virus in Pumpkin (Cucurbita moschata) by Bulked Segregant Analysis and Massive Analysis of cDNA Ends

The whitefly-transmitted squash leaf curl viruses (Geminiviridae) cause significant yield losses in Cucurbita species in many regions. Resistance against Squash leaf curl Philippine virus-Taiwan strain (SLCuPV) has been identified in AVRDC – The World Vegetable Center C. moschata accession TOT8101A. Parents and F₂ plants of a cross between TOT8101A and a SLCuPV susceptible parent were submitted to bulked segregant analysis using massive analysis of cDNA ends (MACE) of cDNA pools to identify resistance genes. The resistant pool consisted of cDNA from 7 immune plants that remained PCR negative for SLCuPV DNA after one week of exposure to viruliferous whiteflies; the susceptible pool was made up from cDNA of 7 highly susceptible plants. The sequencing reads obtained by MACE were assembled and submitted to variant analysis, which resulted in a single nucleotide polymorphism catalog for the resistant and susceptible pools as well as the two parents. The segregation ratio in the population suggested that resistance was based on two recessive genes. From 11,164 assembled cDNAs with sequence polymorphisms, 9 showed a polymorphism distribution in the pools and the parents as expected for recessive resistance genes: homozygous in the resistant pool and heterozygous in the susceptible pool, and the corresponding different homozygous alleles in the two parents. These sequence variants were converted to PCR-based markers and 8 of these sequences were successfully amplified from C. moschata DNA. Validation of these candidate markers in SLCuPV resistant and susceptible C. moschata breeding material is under way.