西瓜籽粒大小一因多效主效QTL的精细定位及关键等位变异挖掘

Seed size is an important trait in watermelon breeding, with large seeds preferred for planting and edible seeds but small seeds preferred in fruit sold for consumption. Our study has identified a pleiotropic major QTL for seed size (included seed weight, seed length and seed width) on LG6. Moreover, the target QTL has been fine-mapped to a physical distance of 320kb. In order to positive clone the pleiotropic major QTL for seed size, the BCF2 populations derived from certain RILs will be used to map-based cloning. Corresponding markers will be developed from the genetic variation information (InDel, SNP and SV) on the target QTL region. The F2 and BCF2 populations will be genotyped by the co-dominant markers (InDel, CAPS/dCAPS) of seed size. Combined the phenotype and genotype of the BCF2 population, the confidence interval of target QTL will be narrowed down step by step. Finally, the candidate genes and key alleles will be validated by gene annotation, gene expression analysis and genetic variation (InDel, SNP and SV) analysis. The project will provide key alleles as well as functional markers for its genetic improvement. The result of present study will also provide important theoretical basis on gene cloning and its regulation mechanism that responsible for the formation of seed size in watermelon.

本项目在利用西瓜F2群体完成籽粒大小QTL初定位的基础上，在6号连锁群确定了一个籽粒大小主效QTL，并通过开发QTL区域的InDel标记，将QTL的物理区间缩减至320kb。以该主效QTL为目标，结合分子标记辅助选择，本项目利用筛选得到的重组自交系回交再自交构建大的回交分离群体（BCF2）；利用两个亲本的深度重测序检测InDel、SNP和SV等DNA水平的遗传差异，继续开发标记加密目标QTL区域；利用开发的标记在F2和BCF2群体的基因型鉴定结合表型数据逐步缩小目标QTL区间，实现对目标QTL的精细定位。精细定位对应的物理图谱结合基因注释、基因表达和InDel和SNP等变异分析确定候选基因和关键的等位变异。研究结果可用于分子标记辅助育种和西瓜品种籽粒大小的定向改良，也可为进一步的目标QTL克隆、基因功能验证和籽粒大小调控的机理研究奠定基础。

项目前期完成了QTL的初定位，在6号染色体确定了一个籽粒大小主效QTL，并通过开发标记将其缩减至320kb的物理区间。本项目利用双亲进行了50X的深度重测序，挖掘到了193395个SNPs和45065个indels。利用QTL区域内的遗传变异开发标记在F2、RIL和BCF2群体中进行精细定位，最终将目标QTL缩减至55.5kb的物理区间，通过RT-PCR和目标QTL区域预测基因的分析，最终获得3个潜在的候选基因，并开发了可用于辅助筛选的分子标记。通过分析结构变异（SV），发现一个约1.2kb的缺失可能是导致候选基因Cla009291表达显著差异的主要原因，且西瓜核心种质的GWAS分析也发现这个SV与籽粒大小显著关联，因此确定为关键的遗传变异。我们的结果为西瓜籽粒大小形成的分子机理研究奠定了坚实的理论和实验基础，获得的分子标记可用于籽粒大小定向遗传改良和辅助育种。