棉籽油分含量位点 qOil-18-2的精细定位和候选基因功能验证

Cottonseed oil is a valuable co-product derived from the processing of cottonseed fiber, and it is one of top six oil resources in the world. The improvement of cottonseed oil content is also one of main goals in cotton breeding. In our previous study, qOil-18-2 was identified as a major QTL for oil content in cottonseed. In various environments, the embryo and maternal additive main effects of this QTL both exceed 5%, and it explained 10.36%-13% of the phenotype variance by additive main effect. These results implied qOil-18-2 was of important value for the molecular improvement of oil content in cottonseed. However, because of its large genetic distance ( 6cM-11.5cM) between two molecular markers in this locus, it is currently difficult to be directly used in genetic improvement of cottonseed oil content. Therefore, we plan to further carry out the following projects: (1) to develop enough amount of molecular markers in the target QTL region, and to finally fine map of qOil-18-2 based on near isogenic line (NIL) segregating populations constructed (2) to search all the open read frames (ORFs) in the qOil-18-2 region and predict the candidate genes (3) to find the most possible candidate gene for this QTL by differential analysis of the DNA and cDNA sequence of the candidate genes in the parents, and (4) to clone the candidate genes and perform the function analysis for these genes. The results of this study will provide the theoretical foundation and molecular marks tightly linkage to this QTL for the improvement of oil content in cottonseed. It could also lay basis for revealing the biosynthetic and metabolic mechanisms in cottonseed oil, as well as the development and qualitative formation of cotton fiber .

棉籽是全球六种主要油料资源之一，棉籽油分含量改良是棉花遗传改良的主要目标之一。qOil-18-2是申请人前期研究发现的棉籽油分含量主效数量遗传位点，在多种环境下都能检测到，解释了10.36%-13%的表型变异，其胚加性和母体加性主效应贡献率均超过5%。这表明该位点不仅对棉籽油分含量的分子改良具有重要作用，而且也有利于棉纤维研究。本项目通过携带qOil-18-2位点近等基因系的分离群体，开发目标区域新标记，实现qOil-18-2的精细定位。利用生物信息学方法预测并克隆qOil-18-2的候选基因，并对其进行功能验证。研究结果不仅为棉籽油分含量遗传改良提供理论依据和分子标记，也为揭示油分合成代谢机制以及纤维发育和品质的形成提供基因资源。

本项目以两亲本HS46（母本）和MARCABUCAG8US-1-88（父本，简称MAR-1-88）以及它们的188个重组近交系为研究材料，在QTL初定位的基础上，联合基因组BSA重测序及关联分析方法将qOil-18-2定位在棉花D05染色体的52788270-53954679区域。根据陆地棉参考基因组注释、qPCR、功能互补及Crispr/Cas9基因编辑技术，在目标区域发掘了3个新的油分含量相关基因，分别为Gh-D05G3254、Gh-D05G3310及Gh-D05G3315。通过克隆及序列比对，这三个基因在亲本HS46和MAR-1-88中存在显著的结构和功能差异。Gh-D05G3254基因在两亲本中的结构差异表现在基因调控区：共有3个位点，2个在上游，1个在下游区。该基因在HS46中降低棉籽油分含量；Gh-D05G3310在亲本HS46和MAR-1-88中的结构差异分布在8个位点，其中4个位点在外显子区域，均在第1个外显子上。其余4个分布在上游和下游的调控区，均为SNP变异。该基因的功能与油分含量及抗病性均相关，故将该基因命名为ghRPP13-L。该基因在亲本MAR-1-88中提高棉籽油分含量。Gh_D05G3315在亲本HS46和MAR-1-88之间的序列差异分布在外显子和内含子两个部分。外显子差异均为第4外显子区域的SNP变异，共有3个位置发生变化，但只有第667碱基由T（HS46）变成C（MAR-1-88），氨基酸由S变成L，为非同义编码变化，其余位置为同义编码变化。在内含子区域的差异为2处SNP变异。该基因具有磷酯酶结构域，故将该基因命名为ghPEARLI-4L，它在亲本HS46中降低棉籽油分含量。在执行本项目研究过程中，在A03染色体上，发现2个新的油分基因（Gh_A03G0701 和Gh_A03G0699）。同时也对3种重要的脂肪酸（油酸、亚油酸及棕榈酸）含量进行QTL定位。这些研究结果可以为揭示棉籽油脂形成的分子遗传机制及培育高油棉花新品种奠定了基础。

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