菜用豌豆籽粒大小重要调控基因鉴定与分子机制解析

Seed size is not only a major component of yield, but aslo a key determinant of market values in the vegetable pea. Identification and characterization of key regulatory related to seed size remains a challenge in the current breeding program. In this project,we will employ the composite gene trapping strategy to discovery the key genes associated with seed size. Frist, we will conduct RAD-seq technology to identify SNPs in the natural populations of vegetable pea, and use GWAS to discover the seed size-related genes by relating SNPs as well as our developed EST-SSR markers to phenotypic data. Meanwhile, the transcriptome sequencing will be constructed and applied for screening differential expression of genes and finding their genetic metabolic pathway in vegetable pea cultivars with different seed size. As an important supplement, we will clone the seed size-related genes which reported in other plants by homologous sequence analysis. Subsequently, in order to explore the seed size-related gene from the post-transcriptional regulation level, a combined approach of high-throughput sequencing and degradome analysis will be applied to reveal the different expression miRNA and their target genes. The spatial-temporal dynamic expressions and metabolic pathway of all the candidate genes∕miRNAs will be further analyzed by qRT-PCR and bioinformatic analyses. Finally, integrating all the results of mining genes methods, we will explore the key genes related to seed size from the gene expression and post-transcriptional regulation levels, and establish molecular regulation mechanism for seed size in the vegetable pea, which will be helpful for the molecular breeding of the vegetable pea.

籽粒大小不仅是菜用豌豆产量构成的重要因素，而且直接影响其商品性。因此鉴定大粒相关优质基因已成为菜用豌豆优质高产育种的关键问题之一。本项目拟采用复合型基因捕获策略高效鉴定菜用豌豆大粒相关优质基因。以菜用豌豆自然群体为材料，利用RAD-seq技术获取SNP，整合本项目组已开发的分子标记，与籽粒表型数据结合进行全基因组关联分析，筛选籽粒大小相关候选基因∕区域；选择粒型差异显著材料开展高通量转录组测序，筛选差异表达基因；同时通过同源克隆获取已报道的籽粒大小相关基因，作为基因发掘的重要补充；采用高通量小RNA测序与降解组分析，鉴定差异表达miRNA及其靶基因，从转录后水平发掘籽粒大小相关基因。最后，综合所有候选基因分析其结构、时空表达模式及代谢途径，广泛全面地从基因表达与转录后调控水平获得菜用豌豆籽粒大小重要调控基因，进而解析籽粒大小分子调控机制，为菜用豌豆大粒型分子设计育种奠定基础。

籽粒大小不仅直接影响作物产量，也直接关系作物的品质和商品性。挖掘和鉴定调控菜用豌豆籽粒大小的功能基因，并明确其分子调控机理，是当前通过分子设计育种对菜用豌豆粒型改良研究的重点内容之一。通过本项目的实施，共搜集到菜用豌豆种质资源948份，并调查了9个农艺性状，根据LDSS方法结合欧式距离在25%取样比例下构建了包含289份种质材料的菜用豌豆核心种质库，极差符合率在95%以上。通过连续两年测定核心种质的鲜百粒重表型值，基于RAD-seq获得的82798个高质量SNP标记进行全基因组关联分析，检测到10个与鲜百粒重显著关联的SNP，其中3个SNP位于7号染色体且具有较高表型变异贡献率。分别以大粒型和小粒型种质为材料开展转录组测序，发现籽粒发育后期较前期基因表达更活跃，其中发育25天的籽粒有801个unigenes差异表达，而发育10天的籽粒仅有459个unigenes差异表达。这些差异基因包括：转录因子、信号转导蛋白、激素合成相关基因等。构建不同籽粒大小种质材料的miRNA文库及降解组文库，共鉴定到35个miRNA家族在内的198条miRNA差异表达，这些miRNA的靶基因包括：转录因子、激酶等，参与激素、代谢等多种生物学过程。经过上述联合分析，对3个与籽粒大小关联的候选基因：ADP-葡萄糖焦磷酸化酶（ADP-glucose pyrophosphorylase，AGP）、ASR（abscisic acid，stress and ripening-induced）和Teosinte branched, Cycloidea, Proliferating cell factor（TCP），进行了同源克隆及进化分析，通过转基因技术，将候选基因在拟南芥中过表达，粒重显著提高，结果与其他作物中籽粒大小相关基因功能研究吻合。通过本研究，我们分别从基因、转录、转录后水平获得了菜用豌豆籽粒大小调控关键功能基因，为今后开展菜用豌豆籽粒大小分子育种奠定了理论依据。