小麦小穗数主效基因位点QTss-7A候选基因克隆与单倍型分析

QTss-7A is a major stable QTL for total spikelet number per spike (TSS) in common wheat (Triticum aestivum L.). It was detected to be significant in five environments using a recombinant inbred line (RIL) population derived from the cross between Xiaoyan 54 and Jing 411; and further confirmed to be effective in improving TSS and kernel number per spike (KNS) by backcrossing. In this project, we proposed to do fine mapping, candidate gene cloning and haplotype analysis of QTss-7A. Near-isogenic lines (NILs) and advanced backcrossing segregating populations will be constructed. Tightly-linked and co-segregated markers will be exploited by comparing the results of SNP array, BSR-Seq and reduced-representation sequencing with the reference sequence of the bread wheat variety Chinese Spring (IWGSC RefSeq v1.0). The high-density fine genetic linkage map of QTss-7A will be constructed by combining the molecular markers and the phenotypic performance of the recombinants. The tightly-linked and co-segregated markers will be used to conduct sequence analysis of the IWGSC RefSeq v1.0 physical map and reference sequences to construct a physical map of QTss-7A. Sequence annotation will be done and candidate genes will be speculated with the aid of the differently-expressed genes that detected by BSR-Seq. Multi-environment phenotyping of the NILs will be conducted to verify the effects of QTss-7A on yield-related traits. The haplotypes and allelic variation will be studied using natural population that existed wide range of genetic diversity. Candidate gene association mapping analysis will be conducted to detect the favorable alleles and haplotypes of QTss-7A; and molecular markers that can be used in wheat molecular breeding programs to improve wheat spikelet number per spike and kernel number per spike will be developed.

利用“小偃54×京411”重组自交系群体在7A染色体检测到一个控制小穗数的稳定表达主效QTL——QTss-7A。通过回交转育发现该位点能显著增加小穗数和穗粒数。本项目拟以QTss-7A为对象，培育近等基因系并构建次级分离群体。利用SNP芯片、BSR-Seq、简化基因组测序等手段开发多态性分子标记；结合次级分离大群体，构建目标区段饱和遗传图谱，实现QTss-7A精细作图。根据QTss-7A紧密连锁或共分离分子标记的物理位置，确定其对应中国春的物理图谱和序列区间，并对序列区间进行基因注释；结合转录组测序获得的表达差异基因确定QTss-7A候选基因。对QTss-7A的近等基因系进行多环境表型鉴定，解析其对小穗数、穗粒数等产量性状的影响。对遗传多样性广泛的自然群体进行多环境表型鉴定，分析QTss-7A的单倍型变异及其遗传效应，通过候选基因关联分析明确其优异等位基因，并开发育种上可用的分子标记。

小麦(Triticum aestivum L.)是我国最重要的粮食作物之一。高产是小麦育种的最基本目标。穗粒数和粒重均属于小麦的产量三因素。通过选育穗粒数多、粒重高的大穗品种，促进穗粒数和粒重等产量性状协调改良，持续提高产量潜力，是开展小麦高产育种的有效途径。.在前期的研究中，我们利用“小偃54×京411”和“Ning7840 × Clark”两个重组自交系(Recombinant inbred lines，RIL)群体在7A染色体长臂上定位到1个同时控制小穗数/穗粒数和粒长/粒重的多环境稳定表达主效QTL，并依托两个RIL群体构建了7A产量QTL的次级分离群体。本研究在此基础上开展工作，主要取得了以下结果：.利用分子标记扫描“小偃54×京411”群体衍生杂合自交家系的5699个单株和“Ning7840 × Clark”群体衍生杂合自交家系的8167个单株，通过多轮染色体步移，构建了小麦7A产量QTL目标区段的饱和遗传图谱和物理图谱，将7A产量QTL调控小穗数的关键基因定位到AX-111159341 (7A: 674019191 bp)与AX-109360122 (7A：674,106,327 bp)之间87 kb物理区间内。.综合两个群体的定位结果，结合基因功能注释、单倍型分析和Tilling突变体表型鉴定，确定了7A产量QTL控制小穗数的候选基因，命名为WAPO1。通过基因结构分析，发现WAPO1共有3个不同的单倍型。通过单倍型分析，明确了WAPO1的优异等位基因。.根据关键重组体的基因型和表型数据，发现小穗数基因WAPO1并非调控粒长和粒重的关键基因，将7A产量QTL置信区间中的粒重QTL与小穗数基因WAPO1分离，并发现了1种结合了7A产量QTL位点上的多小穗数/穗粒数和长粒/大粒基因型的重组体。该重组体为通过分子设计育种培育穗粒数和粒重协同改良的大穗型小麦新品种提供了可能性。.提出了一种有效克隆数量性状基因的策略。本策略基于高代分离材料连续自交构建杂合自交家系，有助于更高效地实现目标基因的精细定位和图位克隆。