利用全基因组关联分析挖掘抗大豆孢囊线虫4号生理小种的新基因

The soybean cyst nematode (SCN) was an important plant nematode in China, and causes serious yield losses and reduces quality during soybean production. In our previous study, the resistant or susceptible phenotypes of 750 soybean lines, which genomes were sequenced, were assayed by infected with both SCN Race 3 (SCN 3) and Race 4 (SCN 4), respectively. The genome-wide associated analysis (GWAS) between the genotypes of these 750 soybean lines and their phenotypes to SCN 3 and SCN 4 were performed by using EMMAX software, and two corresponding models GWAS-SCN 3 and GWAS-SCN 4 were established. The GWAS-SCN 3 model was preliminary evaluated with reported resistant genes to SCN, and the results indicated that both models of GWAS-SCN 3 and GWAS-SCN 4 were highly accurate and dependable. In the GWAS-SCN 4 model, there exist two significant peak regions. Moreover, the corresponding candidate genes contained in the peak regions were not yet reported. Therefore, in this study, we aim to identify the novel genes of soybean resistant to SCN 4. The novel resistant genes to SCN 4 will be identified according to the GWAS data combined with transcriptomic sequencing, qRT-PCR and functional annotations. Functional characterizations of the candidate resistant genes will be carried out by analysis the phenotypes of the transgenic soybean lines generated by overexpression and RNAi strategies. The resistant mechanisms will be uncovered on the cellular, physiological and chemical levels. These results will not only identify the novel resistant genes to SCN, but also lay a solid foundation for theoretical and technical support for soybean breeding resistant to SCN.

大豆孢囊线虫（SCN）是我国农业生产上的重要病原线虫，严重影响大豆产量和品质。本项目前期测定了750份已知基因组的大豆品种对我国SCN 3号和4号生理小种的抗感表型，用EMMAX软件进行抗感表型与基因型的全基因组关联分析（GWAS），初步建立了抗SCN 3号和4号的GWAS-SCN 3和GWAS-SCN 4模型，在此基础上，用已报道的抗SCN基因对该模型的准确性进行初步评估，鉴于GWAS-SCN 4模型中2处最明显峰的关联区域内的基因未被报道，本研究旨在鉴定抗SCN 4号的关键新基因，通过GWAS数据结合转录组测序、qRT-PCR和同源基因注释等确定抗SCN 4号的候选基因，通过超表达和RNAi技术验证抗性候选基因的功能，从细胞组织学、生理生化途径解析抗性机制。研究结果为抗SCN聚合育种提供强有力的理论基础和技术支持。

大豆孢囊线虫（SCN）是大豆生产上的重要病原线虫，严重影响大豆产量和品质。本研究前期测定了750份已知基因组的大豆品种对SCN两个生理小种（SCN3和SCN4）的抗感表型，用EMMAX软件进行抗感表型与基因型的全基因组关联分析（GWAS），初步建立了抗GWAS-SCN模型。通过扩增已报道抗病基因确定了候选抗病品种，并结合转录组分析结果确定GWAS候选区间内的新的抗病候选基因，利用农杆菌诱导的大豆发根转化体系初步验证了候选抗病基因的功能。.经初步表型鉴定从750份大豆品种鉴定出21份抗病大豆，表型重复鉴定确定了8株抗大豆孢囊线虫的大豆，对这些大豆的已知抗病基因进行序列分析，结果编号为TZX-1011的大豆的已知抗病基因为感病型，推断该抗性大豆可能存在未被挖掘的新抗病基因。TZX-1011接种线虫5d后取样进行转录组测定，共分析得到12个差异表达基因，经qPCR检测确定了4个抗病候选基因，分别为Glyma.04G199400、Glyma.06G319700、Glyma.13G206300、Glyma.13G207100。对这4个抗病候选基因分别构建了超表达、RNAi以及CRISPR载体，利用发根农杆菌诱导的大豆发根转化体系进行功能鉴定，结果表明分别过表达Glyma.04G199400、Glyma.13G207100时，能够显著减少大豆根部大豆孢囊的数量；而在将Glyma.06G319700进行RNAi沉默后，与对照相比，大豆根部孢囊数量显著增加。.本研究通过全基因组关联分析、转录组分析以及发根农杆菌诱导的大豆发根体系初步鉴定到抗病大豆品种TZX-1011中基因Glyma.06G319700为抗大豆孢囊线虫新基因，为大豆孢囊线虫与大豆互作机制的研究以及大豆孢囊线虫的绿色防治提供一定的理论基础及依据。