结合GWAS与RNA-seq技术挖掘油菜耐铝相关SNP位点及重要候选基因

Aluminum (Al) toxicity stress of acidic soil has become a highly limiting factor for the growth and seed yield of Brassica napus in southern China. It would be extremely useful to detect SNP loci associated with Al tolerance and identify candidate genes for tolerance across the whole genome for use in genetic improvement and breeding of Al tolerance in rapeseed. In this study, it is proposed that a set of 300 inbred lines of B. napus with large genetic variation and Al-tolerance differences would be utilised. This study would take advantage of the 201,817 high-quality SNPs developed by SLAF-seq (specific-locus amplified fragment sequencing) technology to carry out a genome-wide association study (GWAS) to detect SNPs linked to the Al-tolerance character, Loci of elite allelic variation and lines carrying elite alleles will be identified based on phenotypic effect values. In addition, the roots and leaves of two highly tolerant and susceptible rapeseed lines treated with Al ion stress will be used for identifying differentially expressed genes (DEGs) related to Al tolerance using RNA sequencing. Combining the analysis of GWAS and RNA-seq, DEGs for Al tolerance within in the LD intervals containing significant SNP markers will be selected as Al tolerance candidate genes. Finally, to confirm the contribution of candidate genes to Al tolerance, a transformation test will be carried out, genetically modifying susceptible lines. The results in this research will make for revealing the molecular mechanism of Al tolerance in B. napus, meanwhile the developed SNP makers and genes will provide the help for genetic improvement and variety breeding of Al tolerance in B. napus.

酸性土壤的铝毒胁迫是限制我国南方油菜生长和产量提升的重要障碍因子之一。挖掘油菜耐铝相关基因位点及候选基因对改良油菜耐铝性和加快耐铝油菜品种选育具有重要意义。本项目拟利用前期构建的300份甘蓝型油菜关联分析群体和针对该群体开发的201,817个SNP标记，对油菜耐铝相关性状进行全基因组关联分析（GWAS），探测油菜耐铝显著关联SNP位点，鉴别耐铝优异等位变异及其典型载体材料，搜寻显著SNP位点所在LD区间的耐铝候选基因。同时，利用RNA-seq技术分析鉴定铝胁迫下耐铝和铝敏感油菜植株根系及叶片中耐铝相关差异表达基因。结合GWAS与RNA-seq分析结果，选择SNP位点所在LD区间内的耐铝相关差异表达基因作为重要候选基因，通过转基因功能验证明确油菜耐铝基因。研究结果有利于从全基因组水平揭示油菜耐铝分子机制，同时发掘的耐铝SNP位点和筛选的耐铝基因可为油菜耐铝性遗传改良及新品种选育提供帮助。

油菜是我国重要的油料作物，主要种植于土壤酸化较重的南方地区。酸性土壤的铝毒胁迫成为影响我国油菜生长和产量提升的重要限制因素之一。挖掘油菜耐铝相关基因位点及候选基因对改良油菜耐铝性和加快耐铝油菜品种选育具有重要意义。植物耐铝性是受多基因调控的复杂的数量性状，近年来基于GWAS与RNA-seq联合分析方法成为解析油菜复杂数量性状的有效途径。本项目以254份甘蓝型油菜为关联分析群体，考察了主根相对伸长率、相对根鲜重、相对总根表面积、相对地上部鲜重等8个油菜耐铝相关性状的表型值，结合前期开发的200,510个群体SNP标记，开展了油菜耐铝相关性状GWAS分析，探测到分布于14条油菜染色体上的43个与8个性状显著关联的重要SNP位点，筛选到14个携带耐铝优异等位基因的典型载体材料，在SNP位点上下游100kb序列上搜寻到了777个相关候选基因。同时，对耐铝性与铝敏感油菜进行苗期铝胁迫（0h、6h、24h）处理，RNA-seq分析获得了在6h vs 0h与24h vs 0h共同存在的铝胁迫响应差异表达基因151个。联合分析GWAS与RNA-seq分析结果，在分布于13条染色体上的显著关联SNP位点所在LD区间内筛选到64个油菜耐铝相关差异表达基因，其中4个基因（BnaA03g30320D、BnaA10g11500D、BnaC03g38360D和BnaC06g30030D）在耐铝和铝敏感材料中不同处理时间点下均表现为差异表达。因此，后续对4个重要候选基因进行了克隆和转基因验证，结果表明过表达BnaA03g30320D和BnaA10g11500D能显著提高转基因植株的耐铝性，采用酵母单杂交筛选获得了可能调控BnaA03g30320D表达的6个bHLH转录因子。此外，在A03号染色体上开发了1个与耐铝性表现呈负相关性的CAPS标记（CAPS-A03-16212704），可用于耐铝油菜品种资源的筛选。研究结果为揭示油菜耐铝性的分子机制和耐铝油菜的新品种选育提供了理论依据和基因资源。