辣椒核雄性不育基因的图位克隆与功能分析

Exploration and research on genic male sterility (GMS) is one of the hot topics of heterosis utilization in crops. To date, there have been more than 20 resources of GMS were reported, whereas no main gene for GMS were successfully cloned in pepper. In this project, using a homemade GMS line ‘SC13AB’ and its fertility restorer inbred line ‘T154’ as materials, based on the accomplishment of pepper whole genome sequencing, genome-wide molecular marker development and the preliminary mapping of the GMS gene CaMSC13 into 1.82 Mb region on chromosome P2, we then intend to further map the CaMSC13 gene using a large F2:3 population (n=5000-10000). Then, we plan to validate CaMSC13 through sequence comparisons, expression analyses and CRISPR/Cas9-based knockout experiments of candidate genes. At last, to preliminarily explore the role of CaMSC13 in fertility regulation in pepper, the expression models of some genes that have been known being involved in plant GMS will be detected in sterile and fertile, as well as the knockout transgenic pepper plants. This project will be very useful for further exploration of the molecular mechanism underlying male sterility and overcoming the bottlenecks of GMS applications in pepper.

探究核雄性不育是当今作物杂种优势利用的热点课题之一。迄今，在辣椒中已有20多例核雄性不育相关报道，但尚无核雄性不育突变基因被克隆或验证。本项目以自主选育的辣椒核雄性不育两用系‘SC13AB’及育性恢复自交系‘T154’为材料，在完成辣椒全基因组测序和全基因组分子标记开发以及将辣椒核雄性不育基因CaMSC13初定位于2号染色体1.82Mb区段的基础上，拟利用超大F2:3群体（n=5000-10000）对CaMSC13基因进行精细定位。通过对候选基因序列比较分析、表达分析和CRISPR/Cas9编辑敲除，确定CaMSC13基因。再通过分析已知的核雄性不育相关基因在两用系‘SC13AB’中不育株和可育株以及经基因敲除的转化植株中的表达模式，探讨CaMSC13基因对辣椒雄性不育的调控机理。开展本研究从理论上为探究辣椒核雄性不育的分子机理，从应用上为克服辣椒核雄性不育利用的瓶颈均具有重要意义。

探究核雄性不育分子遗传机制是当今作物杂种优势利用的热点课题之一。本项目主要开展了辣椒核雄性不育的遗传分析、等位性测验、显微组织学观察、BSA-seq定位、重要候选基因的功能分析以及核雄性不育的分子调控机制研究。研究结果表明：遗传分析表明辣椒MSC3AB雄性不育受单隐性基因（msc3）控制。等位性测验表明msc3是一个新的辣椒核雄性不育基因。显微组织学观察表明MSC3AB不育株发生败育的开始时期主要是四分体到小孢子形成时期。BSA-seq定位和标记验证表明辣椒msc3基因位于辣椒10号染色体上3.38 Mb~3.55 Mb大约170 Kb的范围。序列比较和表达分析结果表明Capana10g000179、Capana10g000183、Capana10g000188、Capana10g000192和Capana10g000197是msc3的重要候选基因。目前基于本实验室已建立的成熟辣椒VIGS技术体系，重要候选基因沉默实验正在开展中。同时，我们已经成功构建了msc3重要候选基因的CRISPR/Cas9载体，且进行了遗传转化，初步检验结果显示辣椒遗传转化效率较低，目前正在进行T0代植株的观察鉴定。最后，在MSC3AB不育株中发现bHLH089和TDF1表达量明显上调，DYT1、AMS和CYP703A2表达量轻微上调，表明msc3可能位于bHLH089、TDF1、DYT1、AMS和CYP703A2的上游，主要对下游基因起负调控作用。本项目研究结果为探明辣椒核雄性不育的分子机理和杂种优势高效利用奠定了重要基础。