大豆百粒重QTL上位性效应分析及关键基因精细定位与功能验证

100-seed weight is an important agronomic trait and a restrictive factor underlying soybean yield. The chromosome segment substitution lines (CSSLs) covering the whole genome of wild soybean parent ZYD00006 was constructed by crossing and backcrossing of ZYD00006 (donor parent) with cultivar Suinong 14 (recurrent parent). There is significant difference in 100-seed weight between parents. Three QTL underlying 100-seed weight were identified, including qSW-B1-1, qSW-F-1 and qSW-D1a-1. Then qSW-B1-1 and qSW-F-1 could explain more than 12 % variation in 100-seed weight. According to three candidate loci, the chromosome segment substitution lines including candidate segments were selected in advanced generation. Residual heterozygous line (RHL), single segment substitution lines (SSSLs) and double segment substitution lines were constructed by target fragments which was heterozygous or homozygous in CSSLs, respectively (after sequencing). Fine mapping for QTL will be realized by secondary (RHL and SSSLs) populations and GWAS of CSSLs. Candidate genes will be predicted by combining with the genome information of parents. Genes underlying 100-seed weight will be cloned, the Real-time PCR technology would be used to analyze the expression of candidate genes and gene function verification was completed by transgenic soybean (CRISPR/Cas9). Epistatic effect Analysis of QTL will be evaluated by integration of single segment substitution lines and double segment substitution lines. The results of this study would provide the theoretical foundation and material for the study of 100-seed weight in soybean.

百粒重是大豆产量重要构成因子，是影响大豆单产的重要农艺性状。本研究以百粒重性状差异显著的栽培大豆绥农14与野生大豆ZYD00006经杂交、回交构建了一套覆盖野生大豆全基因组的染色体片段代换系CSSLs（两亲本及CSSLs均已完成重测序）。利用这套材料定位获得3个表现稳定的百粒重QTL: qSW-B1-1，qSW-F-1和qSW-D1a-1，其中qSW-B1-1和qSW-F-1对表型贡献率大于12%。在高世代CSSLs中选择含有目标QTL片段的个体，分别构建RHL群体、单片段与双片段代换系。利用次级分离群体结合CSSLs的GWAS分析完成精细定位；预测候选基因，Real-time PCR分析候选基因表达情况，确定候选基因并克隆；转基因验证（基因编辑技术突变目标基因）；结合单片段与双片段代换系研究候选基因（QTL）间的上位性效应，本项目研究结果将为大豆百粒重性状的研究提供理论基础与材料保障。

百粒重是大豆产量重要构成因子，是影响大豆单产的重要农艺性状。本研究利用栽培大豆绥农14与野生大豆ZYD00006经杂交、回交构建了一套覆盖野生大豆全基因组的染色体片段代换系。在其中筛选百粒重极端小的材料C1536，与绥农14回交构建C1536 F2群体，利用此群体在2号、3号、11号和12号染色体分别定位到百粒重QTL，挑选表型贡献率最高的两个位点qHSW-11-1和qHSW-12-1作进一步精细定位。通过构建剩余杂合系群体，分别将qHSW-11-1和qHSW-12-1区间缩小到0.19Mb和0.07Mb。qHSW-12-1区间内包含5个基因，通过基因表达量分析和单倍型分析，预测Glyma.12G088900为影响大豆百粒重的功能基因。qHSW-11-1区间内包含25个基因，通过基因表达量分析和单倍型分析，预测Glyma.11G106800和Glyma.11G106900为影响大豆百粒重的功能基因。拟南芥及大豆的遗传转化实验表明Glyma.11G106900负调控粒重，因此将Glyma.11G106900作为影响大豆百粒重的功能基因，计划开展进一步研究。此外，还研究了上位性效应对大豆百粒重的影响，利用染色体片段代换系4年的百粒重数据，结合单片段、双片段代换系进行上位性QTL分析，共发现了1160对上位性互作对。说明百粒重QTL调控网络非常复杂，除主效QTL的调控外，非主效QTL的上位性作用也具有十分重要的作用。本项目研究结果将为揭示大豆百粒重形成机理提供重要的理论基础与材料保障。