辣椒果肉厚度性状基因的精细定位与候选基因的功能验证

QH1-1 and QH3-1 were the high generation inbred lines with obvious difference in the pericarp thickness of the pepper fruit. Fine mapping and candidate gene analysis of its related genes were great significance to know the mechanism of pericarp thickness formation and quality breeding. In this project, QTLs, controlling the fruit pericarp thickness related genes in pepper were identified by using the QTL-Seq which takes advantage of Bulked-segregant analysis (BSA) and next-generation sequencing (NGS).The QTL identified from QTL-seq were confirmed and fine mapping by conventional genetic linkage analysis. Based on the genome sequencing, gene specific and polymorphic Indel and SSR markers located in the.candidate region was designed and used to genotyping F2 plants and linkage analysis. The genes of related candidate regions were identified by comparison with the reference genome. The function of candidate genes will be predicted based on sequence alignment. According to the enzyme digestion information of the SNP locus of candidate genes, the CAPs markers closely linked to the target traits were developed. We investigated the expression pattern of candidate genes using RT-PCR, what is more, the function of main genes in the candidate regions were identified through VIGS and genetic transformation technology. The results of this project will be primary elucidate the molecular regulation mechanism of pepper fruit pericarp thickness formation. At the same time, the developed CAPs markers can be applied to molecular marker assisted selection(MAS).

QH1-1和QH3-1是辣椒果肉厚度性状差异明显的高代自交材料，对其相关基因的精细定位和分析对解析辣椒果肉厚度形成机制以及品质育种具有重要的意义。本项目采用基于第二代测序技术（NGS）和混合分离分析法（BSA）相结合的QTL-Seq技术，对辣椒果肉厚度相关的QTLs进行定位。同时，根据测序结果，在候选区域开发多态性的Indel和SSR标记，用F2群体单株进行连锁分析，完成对QTL-Seq定位QTLs的验证和主效QTL的精细定位。应用生物信息学技术，预测区域内的候选基因并进行功能注释；根据候选基因SNP位点的酶切信息，开发与目标性状紧密连锁的CAPs标记；采用实时定量PCR方法，分析候选基因的时空表达情况,并对主要候选基因进行VIGS及遗传转化功能验证；本研究结果将初步阐释辣椒果肉厚度形成的的分子机制，同时开发的CAPs标记可应用于分子标记辅助选择。

辣椒果肉厚度是辣椒重要的农艺性状之一。本研究以辣椒高代自交系QH1-1(薄果肉)和 QH3-1（厚果肉）构建遗传分离群体，并对辣椒果肉厚度及其他重要弄异性状开展遗传与基因的定位工作。研究结果表明：辣椒果肉厚度性状的遗传符合2对加性-显性上位基因+加性-显性上位性模型（E-0），BC1、BC2、F2群体的主基因遗传率分别为55.56%、56.70%和42.46%，多基因遗传率分别为23.47%、27.60%、17.68%；辣椒果实横径的遗传符合2对加性-显性-上位性主基因+加性-显性-上位性多基因模型(MX2-ADI-ADI)，主基因的遗传率为88.67%。辣椒果长性状的遗传符合两对等加性主基因+加性-显性-多基因混合遗传模型(MX2-AED-AD)，主基因遗传率为80.29%，多基因遗传率为19.71%；辣椒叶长和叶宽均为2对加性-显性-上位性主基因+加性-显性上位性多基因混合模型(MX2-ADI-ADI)，主基因遗传率分别为59.91%和46.06%。利用QTL-seq方法，将辣椒果肉厚度性状定位在辣椒第10号染色体196 950 001-218 750 000bp的区间内，通过区间内Kasp标记的开发和连锁分析，将辣椒果肉厚度位点进一步缩小至1.8Mb区间内；通过候选区域基因的功能注释，初步预测了2个候选基因并进行了功能分析；同时结合项目研究，初步将辣椒果实横径性状定位到辣椒3号染色体194700001-237100000bp区间。相关研究结果为辣椒不同农艺性状辣椒新品种选育及相关功能基因的挖掘提供了理论依据。