云南地区荷花种质资源收集及重要花莲观赏性状的GWAS解析

Nelumbo nucifera Gaertn is only aquatic plant among ten traditional flowers of China. whether its plant size, flower type, flower color or florescence, showed extremely plentiful changes. As a region with lotus of the most abundant genetic diversity, there are a lot of unique germplasms in Yunnan, such as the ‘Yiliang Qianban’, ‘Puzhehei Honghe’ and ‘Puzhehei Baihe’. For many years scientists have studied on the molecular mechanism of its agronomic and ornamental traits formation, and a number of genes were cloned. However because these traits showed quantitative characters controlled by multiple genes, cloning the gene by homologous sequence is not satisfactory with low efficiency and pertinence. With the completion of the whole genome sequencing of the lotus, it provided us a new vision. In this project the lotus germplasm resources of Yunnan Province will be collected, the genomes of all the core germplasms early selected, will be re-sequenced. Then the SNPs loci of genome for main ornamental traits will be scanned , and haplotype map will be constructed. Through association analysis of main ornamental traits, such as plant size, number of petals and florescence, will be carried out with the genome SNPs, the candidate genes will be screened out. By RT-PCR expression analysis and gene transformation their function will be identified then. Furthermore the molecular mechanism of formation and regulation for the main ornamental traits will be probed and revealed. This studies will provide the basis for the molecular breeding and directive improvement of lotus.

荷花（Nelumbo nucifera Gaertn）为我国传统名花中唯一的水生花卉，无论是株型、花型、花色或是花期，均表现出极为丰富的变化，云南作为荷花遗传多样性最为丰富的地区之一，有很多独特的种质，如‘宜良千瓣’、‘普者黑红荷’与‘普者黑白荷’等。多年来科学家们对其农艺与观赏性状形成的分子机制展开了研究，克隆了一批基因，但由于这些性状表现出多基因控制的数量性状的特点，靠传统的同源序列克隆，基因捕获的效率以及针对性难以令人满意。荷花全基因组测序的完成，给我们提供了新的视野。本项目系统收集云南省的荷花种质资源。对前期筛选出的花莲核心种质，进行基因组重测序，扫描SNPs位点，构建单倍型图谱，重点对株型、花瓣数、花期等重要观赏性状进行关联分析，寻找相关的候选基因，通过表达分析、基因转化等验证其功能，进而探寻其形成与调控的分子机制，为荷花定向改良的分子育种提供依据。

于云南昆明地区引种荷花种质资源200余份并建立了西南林业大学国家荷花种质资源库，对其进行了适应性观测并筛选了44个表现良好的种质资源。采用InDel和SRAP标记对174个荷花品种构建了指纹图谱。分析了荷花生物活性成分-总生物碱、总多酚含量及其主要成分。证明低聚原花青素对DPPH、ABTS自由基具有清除效果；通过建立高脂血症小鼠实验模型发现，荷叶生物碱对于降低小鼠的总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白（LDL）效果明显。.通过基因组学结合转录组与成分分析，挖掘出荷叶碱异喹啉类生物碱合成途径的关键候选基因7个，分别为NnCYP80G、Nn6OMT、Nn7OMT、NnTYDC、NnNCS、NnRAV和NnERF，挖掘了10个与原花青素合成途径相关的基因和9个促进原花青素聚合反应的候选基因。对301 份核心种质进行基因组重测序，检测到2747598个优质SNPs和356474个InDels，分布在荷花的8条染色体上，密度分别为3.44和0.45个/kb。利用GWAS和RNA-seq整合分析筛选重要性状关键候选基因及利用qRT-PCR 重验证，筛选出与株型相关性状显著关联的 SNP/InDel 标记附近的候选基因，发现了大量与植物细胞壁相关基因，挖掘出8个与植物株型相关的关键候选基因，分别是NuXTH22 (LOC104599112)，NuXTH23 (LOC104599114、LOC104599115、LOC104599118、LOC104599117、LOC104599116)，Nu4CL2 (LOC104599133)和NuWAKL2(LOC104590721)。并发现大株型植株细胞壁纤维素骨架类物质更多，支撑机械力更强，而小株型则果胶填充类物质更多，这与筛选出的细胞壁重塑类基因的功能吻合，间接证明了细胞壁重塑类基因在株型建成中的功能。对荷花花型进行重点研究，通过 GWAS、RNA-seq 及生物信息学整合分析，挖掘出了 7 个荷花花型发育（瓣化）的关键候选基因—MADS-box 基因家族基因 【NnMADS-box2，36，19，15（AGL9），11（AGL104），13（AGL82），38（MADS6）】，并进行了初步的候选基因验证。初步建立了荷花器官再生与体胚再生体系。上述研究为进一步揭示荷花重要性状形成的分子机制奠定了基础。