Construction Of A High-Density Genetic Map And Fine Mapping Of The Candidate Genes For Three Fruit Traits In Watermelon (Citrullus Lanatus)

Watermelon(Citrullus lanatus)is an important horticulture crop that is grown worldwide and China is the largest producer and consumer of watermelon.Although the genetic breeding research of watermelon have great significance,the density of genetic map is not enough for QTL/gene mapping.Most of the mapping population is temporary separation population.In present study,we constructed a high-density genetic map using bin markers based on resequencing RILs population.Based on this genetic map and the phenotype of the RILs population,we performed QTL/gene mapping and fine mapping to find out the candidate genes for watermelon rind color,seed coat color and fruit bitterness.The main results are as follow:1.In this study,a RIL population containing 126 individuals were developed from a cross between the inbred lines 9904 and Handel.We have constructed a high-density genetic map based on resequencing both parental lines and RILs.2132 recombination bin markers were mapped onto 11 linkage group.The total length of the linkage map was 1508.94 cM,with an average distance of 0.74 cM between adjacent bin markers.One locus for rind color,one locus for fruit bitterness,and one locus for seed coat color with high LOD scores were identified on chromosome 1,chromosome 8,chromosome 3,respectively.2.We also performed fine mapping for watermelon rind color(dark green vs light green)by using CAPS and SNP markers and we narrowed down the candidate region to 31.4 kb.The sequence alignment of the candidate genes with the two parental lines suggested that a nonsynonymous SNP mutation in the CDS of ClCG08G017810,converting an arginine(R)to glycine(G).A CAPS marker based on the SNP mutation was co-segregated with the phenotype in 103 watermelon germplasms.The qRT-PCR analysis revealed that higher expression of ClCG08G017810 in dark green rind than in light green rind.Therefore,ClCG08G017810 is likely to be a candidate gene controlling the watermelon rind color.3.Based on the initial mapping region with a genetic map,we conduct fine mapping for seed coat color(black vs light yellow)and the candidate region was narrowed down to 70.2 kb.Finally,we detected a candidate Cla019481 gene that encodes a polyphenol oxidase controlling black seed coat color in watermelon.The sequence alignment of the candidate gene with the two parental lines revealed a base insertion in the CDS resulting a frameshift mutation and leading to early termination of translation in 9904.Hence,we speculate that the formation of the difference in seed coat color is due to the different in expression of Cla019481 resulting in difference for PPO activity,which influenced the formation of melanin.4.In addition to this we also performed fine mapping for fruit bitterness and the candidate gene was delimited to a 116.7 kb region.The sequence alignment of the two parental lines suggested a single base pair mutation at CDS region in ClCG08G017810 resulting in a premature protein in Handel.This gene is homologous to Bl which is the transcription factor regulating bitterness compound biosynthesis in cucumber.Therefore,we speculated that ClCG08G017810 is the candidate gene responsible for watermelon fruit bitterness.