大豆蛋白质含量QTL的Meta分析、精细定位与候选基因挖掘鉴定

Soybean protein content is the core of soybean quality improvement research. A RIL population crossed by Charleston and Dongnong594 were performed to construct a high-density genetic map based on specific-locus amplified fragment sequencing (SLAF-seq) technology. A set of chromosome segment substitution lines (CSSLs) crossed by Suinong14 and ZYD00006 have also been constructed and the bin map were obtained by genome resequencing. The soybean protein content QTLs have been mapped in these two populations based on our previously study. Next step, the other published soybean protein QTLs are collected in recent 30 years, the protein QTLs are integrated onto Williams82 physical map, meta-analysis method was employed to get the main effect Meta-QTLs. Based on the Meta-QTL located on the “hot region” of Gm20, the residual heterozygous line (RHL) will be screened by the marker linked with Meta-QTL from CSSLs populations, which could construct the secondary population RHLs. The classical mapping methods of adding markers and the mapping method of bulked segregant analysis sequencing are conducted together for fine mapping the Meta-QTL. One or two candidate genes will be screened by gene function annotation and real-time PCR. Transformation validation experiment will be performed with soybean variety Tianlong1 (high protein content) and Dongnong50 (low protein content) based on agrobacterium mediated soybean regeneration system. The results of this study will provide the theoretical foundation for protein content QTL fine mapping and validation in soybean.

大豆蛋白质含量是大豆品质改良的核心。本研究以一套重组自交系群体（Charleston×东农594，SLAF测序建图）和一套野生大豆全基因组导入系群体（绥农14×ZYD00006，重测序建bin map）初步定位蛋白质含量QTL为基础，整合国内外近30年发表的大豆蛋白质含量QTL定位信息，利用Williams82物理图谱整合QTL，应用Meta分析获得主效Meta-QTLs。并针对Gm20染色体大豆蛋白质含量研究热点处的1个Meta-QTL为研究目标，筛选导入系群体中目标区段的剩余杂合体（RHL）构建次级群体，应用经典遗传加密标记定位和BSA混池重测序分析，联合对该Meta-QTL的精细定位。结合功能注释和实时定量PCR筛选确定1-2个候选基因，在天隆一号（高蛋白）和东农50（低蛋白）中应用农杆菌介导的大豆再生体系进行遗传转化验证功能。研究结果为大豆蛋白质含量QTL精细定位与验证奠定基础。

大豆蛋白含量是影响大豆品质的重要因素之一。本项目利用一套构建多年的导入系群体，对大豆蛋白质含量QTL进行初步定位，同时整合Soybase数据库已发表的QTL位点，通过筛选剩余杂合体个体，构建近等基因系群体，标记选择等方法，对Gm20染色体蛋白QTL SPCI_1进行精细定位。基于双亲本的重测序信息，对精细定位后的候选区间内基因进行分析注释，并在收集的近700份东北地区主栽品种和资源群体中进行单倍型验证，主要结果如下：.1）基于CSSL群体2年的蛋白含量数据，应用ANOVA，CIM等方法，完成蛋白含量QTL的初步定位。.2）根据初步定位的QTL结果，结合Soybase大豆蛋白QTL的整合数据，通过加密分子标记及衍生出的3套NIL群体，对Gm20染色体SPCI_1进行精细定位，最终该蛋白含量QTL定位到GM20的约9.13-9.98Mb处。.3）候选区间852Kb包括15个候选基因（V1）（20个V2版候选基因），结合2个亲本的重测序与拼接，分析了该15个（V2：20个）候选基因在2亲本间的序列差异，发现V1版本的Glyma20g06725（野生豆提前终止）和Glyma20g06970；V2版本的Glyma.20g060700，Glyma.20g061800和Glyma.20g061900均由于野生豆ZYD00006的碱基插入或缺失而导致提前终止或易位，使得蛋白结构差异很大，作为进一步验证的重要基因。.4）搜集了651份东北地区的主栽品种品系并进行重测序，结合材料2年的品质测量数据，在分析群体结构和变异基础上，分析了不同候选基因的单倍型差异。其中V1版本基因Glyma20g06725的1个优异单体型对大豆蛋白质含量的提高有极显著作用。V2版本基因Glyma.20G061900有1个优异单体型对大豆蛋白质含量的提高有极显著作用。.研究为候选基因的进一步验证和大豆品质分子育种提供基础。