长豇豆荚长主效QTL Qpl-3图位克隆、功能验证及自然变异分析

Pod length is an important trait of asparagus bean, yet the mechanism controlling this trait remains unknown. Using a RIL population derived from the cross between a long-podded and a short-podded accessions, we have revealed that pod length was genetically controlled by one major QTL and one minor QTL in that population. The major QTL, Qpl-3 accounting for ~ 40% of the phenotypic variance, has been mapped to a 0.44 cM interval on LG3. In this project, we are planned to create a long-podded and short-podded RIL pools that harbor the single Qpl-3 and no pod length QTl, respectively. Comparative transcriptomic analysis will be conducted in pods between the two pools. Consensus genetic mapping and in-depth re-sequencing of the parental lines will be conducted to develop new DNA markers in the Qpl-3 interval. Fine mapping of Qpl-3 will be achieved by using the enlarged RIL population as well as a large F2 population, in combination with genotypic and phenotypic analysis of the recombinants. Through screening a BAC library , the Qpl-3 is expected to be confined to a genome region harboring no more than 10 predicted genes. To help determine most possible candidate genes, real-time RT-PCR　will be conducted for each predicted gene between the long-podded and short-podded pools. This information, together with the annotation of predicted genes and the transcriptomic data, will lead to the identification of one or a few more candidate genes. Transgenic approaches will be used to verify candidate gene functions using cowpea callus and Arabidopsis. Finally, natural variations of the cloned pod length gene will be investigated across the Chinese asparagus bean mini core to reveal the impact of human selection on the domestication of pod length in this species. A comparative analysis will also be performed for the pod length genes/QTLs between asparagus bean and the broader plant species.

荚长是豇豆重要性状，其调控机制未明。申请人前期利用一个长荚×短荚品系衍生的RIL群体明确该群体荚长受1主效+1微效QTL控制，主效QTL Qpl-3解释的表型变异~40%，并将该QTL定位在一个0.44 cM的区间。本项目在此基础上1)从RIL群体筛选只携单个主效QTL（长荚)与不携长荚QTL（短荚)的典型植株，混成长、短荚两池进行比较转录组分析;2)构建整合遗传图谱、双亲深度重测序等在Qpl-3区间开发新标记，对扩大的RIL和F2大群体进行基因型和表型分析，结合重组体分析实现精细定位;3)筛选BAC文库将Qpl-3限定于含预测基因<10个的基因组区段;4)综合各预测基因在长、短荚间的表达水平、功能注释、转录组信息推定候选基因，利用转基因豇豆愈伤组织和拟南芥验证基因功能;5)分析该基因在豇豆自然群体中的序列变异，揭示其在育种驯化中的作用，探明其与其他物种已知荚长QTL或基因的关系。

豇豆是世界范围内重要的豆类作物之一，超长豆荚是长豇豆最显著、最重要、最独特的生物学和农艺特征，但目前对荚长发育的调控机制尚不清楚。项目组前期利用一个长荚栽培品种“之豇282”(荚长～62cm)和一个短荚地方品种“ZN016”(荚长～34cm)构建的重组自交系群体，从中鉴定到一个控制荚长的主效QTL Qpl-3。为了克隆和验证Qpl-3，项目组首先利用“ZN016”和“之豇282”构建了一个含2156个单株的F2群体，通过向目标区段添加新标记、筛选重组单株的方法将Qpl-3精细定位至0.094cM的遗传距离内，对应3号染色体上139kb的区间，在参考基因组上该区段内含有8个候选基因。通过对“ZN016”和“之豇282”开花后不同发育时期的嫩荚转录组测序分析，发现有2395个基因上调表达，1202个基因下调表达，这些基因包括生长素响应相关蛋白、乙烯反应转录因子等与植物生长发育相关的基因。“之豇282”对应的的Qpl-3候选基因表达量在荚长发育前期低于或接近“ZN016”的表达量，但在荚长发育的后期显著高于“ZN016”，说明“之豇282”候选基因的表达在荚长发育过程中更稳定、更持久。项目组检测了“ZN016”和“之豇282”不同时期嫩荚中的内源激素含量，发现二者在IAA(吲哚乙酸)、IPR(异戊烯基腺嘌呤核甘)、ABA(脱落酸)上含量具有显著的差异。为了验证Qpl-3的功能，项目组克隆了“之豇282”中Vigun03g155600的CDS序列，构建了该基因的过表达载体并转入拟南芥，结果显示转基因单株的角果长度明显大于野生型。Qpl-3在自然群体中存在18种单倍型，且在不同遗传背景下的效应不同；比较基因组学分析发现Qpl-3与已经报道的豆类荚长QTL无共线性关系，且该基因与拟南芥已知的荚果发育基因无同源关系。本项目为研究豇豆荚长发育机制奠定了基础，并将促进长豇豆荚长分子育种。