菜用豌豆矮生形态建成重要功能基因发掘及分子机理研究

Dwarf vegetable pea has broad application prospects because of its compact plant type and needless for scaffolding. Identification and characterization of important functional genes for dwarf morphogenesis in pea becomes one of the main problems in the current breeding program. In present project, we plan first at all to construct high density genetic linkage map using RAD-seq technology based on the two parental materials, dwarf mutant type and trailing type, as well as their RIL groups. Then, the putative relative quantitative trait locus (QTLs) affecting the dwarf traits will be located by integrating the phenotypic data and the genetic linkage map. In parallel, we will identify the differential gene expression from the dwarf and trailing type by transcriptome sequencing and subsequently analysize their metabolic pathway by GO analysis. As an important supplement to dwarf genes discovery, the candidate genes will be identified both from the dwarf and trailing genotype by comparative analysis to the published dwarf genes. Moreover, in order to explore the dwarf gene from the post-transcriptional regulation level, a set of miRNA candidates and their potential target genes with different expression will be identified in vegetable pea using high throughput sequencing and bioinformatic analyses. Finally, the integration of the genetic information of the mining genes and their spatiotemporal expression level will be performed. The results will explore the important functional genes related to dwarf morphogenesis from the gene expression and post-transcriptional regulation levels, and to reveal the dwarf gene regulation network with vegetable pea in the whole genome, which will be helpful to the future development of dwarf vegetable pea breeding program.

矮生菜用豌豆株型紧凑，无需搭架，具有广阔的应用前景。发掘和鉴定矮生形态建成重要功能基因，已成为当前菜用豌豆矮生育种亟待解决的主要问题之一。本项目拟以豌豆矮生突变型、蔓生型及其RIL群体为材料，利用RAD-seq技术绘制高密度遗传图谱，并结合表型数据进行QTL定位矮化基因区域；在此基础上，对矮生型和蔓生型材料开展高通量转录组测序，筛选差异表达基因并分析其代谢途径；同时根据已公布的矮化基因序列，对豌豆进行同源序列分析并鉴定，作为矮化基因发掘的重要补充。此外，结合高通量miRNA测序与生物信息学分析，鉴定miRNA介导的矮生形态建成相关基因，从转录后调控角度发掘豌豆矮化基因。整合上述所挖掘的基因遗传、结构信息及时空表达水平，将使我们最大程度从基因表达与转录后调控水平获得菜用豌豆矮生形态建成重要功能基因，并揭示矮生基因在菜用豌豆全基因组中的调控网络，为今后开展矮生菜用豌豆分子标记育种奠定坚实基础。

矮生菜用豌豆株型紧凑，无需搭架，具有广阔的应用前景。发掘和鉴定矮生形态建成重要功能基因，已成为当前菜用豌豆矮生育种亟待解决的主要问题之一。本项目新搜集菜用豌豆种质资源212份，并完成植株形态鉴定；通过遗传距离、取样比例等参数研究，构建包括46份材料在内的菜用豌豆核心种质库。在此基础上，以代表性矮生型和半蔓生型种质为材料开展高通量转录组测序，获得8702条差异unigene；进一步分析，发现有21个unigene与植株高矮生表型相关，经荧光定量PCR验证表明，结果基本一致。为从转录后水平探究miRNA与豌豆矮生基因调控之间的关系，进一步发掘矮生相关基因，我们构建了不同植株类型小RNA文库并进行高通量测序，从中鉴定到包括18个家族在内的56条miRNA存在差异表达，利用荧光定量PCR进行验证，获得一致结果，这类miRNA靶向167个基因调控。同时，我们利用矮生及半蔓生材料构建杂交后代群体，开展简化基因组测序，构建高密度遗传图谱1份，主要包括7个连锁群，上图标签7148个，SNP标记12213个；经QTL分析获得9个包括植株类型、株高、节间距三个性状关联区域。经上述研究联合分析，共同指向5个与矮生性状关联基因，经同源分析，结果与其他作物中研究吻合。同时，我们利用转录组测序的差异表达片段，开发了160个SSR标记，其中21对表现出典型多态性；通过X2测验和F测验获得11个与矮生性状高度关联标记，明确鉴定了28份我国主要菜用豌豆类型。通过本研究，使我们最大程度从基因表达与转录后调控水平获得菜用豌豆矮生形态建成重要功能基因，并揭示矮生基因在菜用豌豆分子调控网络，为今后开展矮生菜用豌豆分子标记育种奠定坚实基础。