葡萄果实早熟性状QTL定位及候选基因分析

Selection and breeding of early-ripening grape cultivar is an important task of present grape industry development.The project team has explored an early-ripening bud sport of 'Kyoho', 98-2, which matured 30 days earlier than 'Kyoho' and the variation of early-ripening trait is extremely notable. This project was firstly to conduct QTL mapping of grape early-ripening trait using three methods (Linkage map analysis, association analysis and transmission analysis of breeding) in hybrid population (F1 population crossed between 'Kyoho' and 98-2), natural population (grape core collections constructed in the last NSFC project) and breeding pedigree population (early-ripening grape varieties derived from 'Pearl of Csaba'),respectively, based on the observation of grape berry phenological period, Vitis9KSNP and SSR marker analyses in order to study in depth the genetic mechanism of this trait; Then the candidate genes distributed in the major mapped QTL intervals were analyzed by PICARA method based on the whole genome sequences and gene expression network (VitisNet)of the grape; At last, the candidate genes were validated by RT-PCR analysis. The mapping of the grape berry early-ripening trait and exploration of corresponding genes would be significance to the study of molecular mechanisms of grape berry early-ripening trait formation and genetic improvement of grape by genetic engineering. It would provide important theoretical basis for molecular design of grape varieties and cloning of corresponding genes and scientific theoretical instructions for grape early-ripening grape cultivar breeding in future.

早熟葡萄品种选育已成为当前葡萄产业发展的重要任务。项目组前期发掘了一个比'巨峰'葡萄成熟早30d左右的早熟芽变，98-2，其早熟性状变异极其显著。为准确定位控制该性状的遗传基因位点，本项目拟基于果实发育物候期观测，Vitis9KSNP和SSR分析；采用遗传连锁分析，关联分析和育种传递分析方法分别在杂交群体（'巨峰'和98-2的F1代群体），自然群体（上一基金项目构建的葡萄核心种质）和育种系谱群体（'莎巴珍珠'系早熟葡萄品种群）中综合多方面信息对葡萄果实早熟性状QTL进行定位；然后参照葡萄全基因组序列和葡萄基因表达的分子网络（VitisNet），用PICARA方法在定位的QTL区进行候选基因分析；并对筛选出的候选基因进行RT-PCR验证。本项目定位葡萄果实早熟性状QTL位点并发掘相关候选基因，将为早熟葡萄品种分子设计育种和相关基因的克隆奠定重要基础，为今后早熟葡萄品种改良提供科学的理论指导。

葡萄果实的成熟期是影响葡萄种植者经济效益的重要影响因子之一。本项目先采用均匀分布于葡萄不同染色体的SSR标记，对早熟葡萄骨干亲本‘莎巴珍珠’及其32个早熟葡萄衍生品种群进行了分析，表明1号染色体上的VMC4F8，17号染色体上的SCU06标记和16号染色体上VVMD5标记，具有较高的遗传频率，是与葡萄早熟性状有关的染色体区段；对包含葡萄初级核心种质在内的179份葡萄种质，采用GBS简化基因组测序技术，基于全基因组关联研究方法进行葡萄果实发育相关性状定位，16号染色体上发现三个与果实发育期相关的强关联区段，其中chr 16:13403379到chr 16:14301597区段与前人定位结果一致，基于葡萄基因组注释信息，此区段候选基因主要是类受体蛋白激酶。同时对果穗大小，果粒重量，果肉质地，果实香型等性状也进行了关联分析，与前人结果吻合度较高。对'巨峰'及其葡萄早熟芽变果实发育特性和转录组对比分析，荧光定量PCR结果表明，SOD和TMV基因表达差异显著，葡萄果实的早熟与活性氧代谢有关，外施活性氧产生物质，核黄素，确实促进了‘巨峰’果实早熟近20天。项目研究结果基于基因组，转录组方法，结合自然群体和源于骨干亲本的早熟葡萄品种群的分析，参考前人基于不同杂交群体定位结果，将葡萄果实早熟性状定位于16号染色体13M到15M的区间，揭示了葡萄果实早熟性状的遗传控制位点，提高了对葡萄果实早熟性状遗传的认识，为进一步解析葡萄早熟性状遗传机理提供了理论基础。本项目成果中的活性氧能促进葡萄果实提早成熟的发现，理论上揭示了果实早熟的新机理，为果实发育研究提供了新思路，实践中提供了一种简单，高效，健康的促进果实成熟的新方法。