小麦基部小穗结实性QTL QGNBS-5A的精细定位及候选基因分析

Grain numbers in basal spikelets of wheat directly affect kernel number per spike, and the basal spikelet sterility is considered as a limiting factor for further improvement of grain yield. To date, it has been hardly addressed in genetic studies. Based on a natural population of Chinese wheat varieties and a recombinant inbred line (RIL) population from a cross between varieties Yangmai 18 and Yangmai 17 with similar agronomic characteristics, but different grain numbers in the basal spikelets, a QTL (QGNBS-5A) for grain numbers in basal spikelets was mapped on chromosome 5A by combining data from a genome-wide association study and linkage mapping. Following the genotypic data of the RILs analyzed by the Wheat 90K SNP chip, we selected individual lines with different homozygous alleles at the QGNBS-5A locus and significantly different grain numbers in basal spikelets to build secondary segregating populations (the parents showed only allelic variations at QGNBS-5A locus). Based on the above research, this project is aimed to further characterize the basal spikelet sterility of the secondary segregating F2 and F2:3 populations. QTL fine mapping will be performed using the polymorphic markers in the target region between two parents screened with the Wheat 660K chip and BSR-Seq. Differentially expressed genes involved in determination of grain-setting rate will be identified in the target region by BSR-Seq and gene annotations of wheat genome sequences. The candidate genes responsible for fertility differences between Yangmai 18 and Yangmai 17 will be analyzed and validated, providing a good basis for further gene functional analysis.

小麦基部小穗结实性直接影响穗粒数，是产量进一步提升的限制因子。然而，小麦穗基部结实性的遗传研究鲜有报道。申请人前期利用育成品种组成的自然群体，以及农艺性状相似但基部小穗结实粒数存在显著差异的扬麦18和扬麦17构建的RIL群体，联合全基因组关联分析和连锁分析，在5A染色体上发现一个控制基部小穗结实性的稳定QTL QGNBS-5A。进一步选择QGNBS-5A位点基因型不同，且基部结实粒数显著差异的家系（双亲只在QGNBS-5A有差异），构建了次级分离群体。在此基础上，本项目将进一步鉴定F2和F2:3次级分离群体基部小穗结实性，利用Wheat 660K SNP芯片筛选双亲得到的在该QTL区段的多态性标记，结合BSR-Seq分析，对QGNBS-5A精细定位，鉴定QGNBS-5A区间的育性差异表达相关基因，并结合小麦基因组序列信息筛选候选基因，利用自然群体分析其遗传效应，为该基因的功能研究奠定基础。

穗基部结实性差是禾谷类作物中常见的生物学现象。根据申请者前期对小麦育成品种的调查发现，在生产实践中，若其它产量要素不变，穗基部小穗结实粒数每增加1粒，每公顷小麦产量就能提高约180千克。因此，适当增加基部小穗的结实粒数，进而增加穗粒数，是实现小麦高产再高产的途径之一。目前关于小麦穗基部结实性遗传研究鲜有报道，这在一定程度上说明穗基部结实性遗传机理较为复杂且研究难度大。针对小麦生产中存在的这一瓶颈问题，本项目在前期研究基础上，鉴定出6个与小麦穗基部结实粒数显著相关的QTLs，分别为qGNBS-4D、qGNBS-5A-1、qGNBS-5A-2、qGNBS-5D、qGNBS-6D和qGNBS-7D。在6个QTLs区段内中共检测到368个候选基因，为缩小候选基因的范围，对6个QTLs分别进行了序列分析、GO富集分析、连锁不平衡分析、表达量分析以及基因注释，最终挖掘到两个控制小麦穗基部结实粒数的关键候选基因，TaGNBS-5A和TaGNBS-6D。通过对两个基因的单倍型分析，确定了两个基因各自的优异单倍型（频率）分别为TaGNBS-5A-Hap2（59.6%）和TaGNBS-6D-Hap2（67.7%），同时开发了相应的KASP分子标记。为更进一步验证TaGNBS-5A在控制小麦穗基部结实粒数过程中发挥的功能，分别对野生型矮抗58（AK58W）和基于该基因筛选到的突变型AK58M植株在目标基因进行测序比对，结果表明，AK58W和AK58M植株在GNBS1和GNBS2上存在极显著差异（P<0.01），初步验证了TaGNBS-5A是控制小麦穗基部结实粒数的关键功能基因。另外，通过进化树分析和酵母双杂实验初步探明TaGNBS-5A具有与E类基因相近的小麦花穗发育的调控模式。上述研究结果将有助于进一步解析小麦穗基部结实粒数的遗传机制，并为小麦高产分子育种提供重要信息。