调控菜豆生育期主效基因的克隆与功能解析

Common bean (Phaseolus vulgaris) , one of the economically important crops, is widely cultivated in China with multifarious cultivars and great variations in traits. Although it is classified as a short-day plant, there are quite differences in maturity and photoperiodic response among cultivars. Furthermore, the molecular bases of genes underlying maturity and photoperiodic response and their regulatory networks are largely unknown. In this project, on the basis of accurate phenotyping of the maturity and photoperiodic response, a F2 mapping population will be derived from a cross between two short-plant-type cultivars, Huangjingou of Northern cultivar and Dedoujia of Southen cultivar. With the use of developed genechip (BARCBean6_3 BeadChip), we quickly build linkage maps and identify QTL for maturity. On the basis of deep resequencing of both parental cultivars, we will identify all potential molecular markers. The tested polymorphic markers will be used to identify the recombinants whose recombination occurred within major QTL region. After accurate phenotyping of these recombinants and their selfed progenies, fine-mapping will be performed to narrow down the QTL region at a resolution of one to several genes. Meanwhile, we will perform deep-resequencing of two extreme early and late maturity pools, candidate gene or region will be identified using the own bioinformatics pipeline developed by the applicant. Taken together with the results obtained via two parallel mapping strategies, deliberate candidate gene will be identified. Thereafter, gene knockout using gene adding (CRISPR/Cas9) technology and/or overexpression will be performed to verify the physiological function of the candidate gene. With other functional evidences, the regulatory mechanism of the major gene in control of flowering time/maturity and photoperiodic response in common bean will be elucidated.

菜豆为重要的经济作物，在我国种植的品种繁多，具有丰富的基因资源。虽属于短日照植物，但品种间生育期与光周期反应的差别较大，且相关的分子基础与调控机制并不明确。本申请拟在对生育期与光周期反应精准表型鉴定的基础上，选择北方矮杆品种"黄金钩"系列与的南方品种"地角豆"配制F2分离群体，利用成熟的芯片技术(BARCBean6_3 BeadChip)构建遗传图谱与定位调控菜豆生育期QTL。通过对父母本进行深度重测序，并利用多态性分子标记大量鉴定主要生育期QTL区域的重组体，并对重组体及后代进行精准表型鉴定，将主效QTL进一步精细定位至数个基因至单个基因水平；同时对极早熟与极迟熟混池深度重测序，并利用自行研制的生物信息学技术流程分析并鉴定出候选基因或区间。根据两项定位技术综合确定主效QTL的候选基因，通过基因编辑(CRISPR/Cas9)定向敲除或过量表达等验证功能，明晰菜豆中生育期基因调控网络。

制备了菜豆黄金勾珈玛射线突变体库，获得了一系列变异的突变体类型，包括开花期与株型变异材料，为进一步研究菜豆光周期反应的调控机理奠定了基础。在此基础上，配制了菜豆品种黄金勾类型的品种大龙1号与油豆角类型的菜豆品种PI60234的杂交组合，并利用芯片技术进行了控制开花期的基因进行QTL定位。同时通过高通量基因组与转录组混池测序技术，利用自行研发的基因定位技术，快速确定锁定候选基因。研发了一套实用的INDEL引物，能有效地对大龙1号XPI60234的杂交群体进行基因型鉴定，一方面可以确认混池测序的鉴定结果，同时还可以利用之进行QTL定位，并结合父母本的高通量测序，对确定的QTL进行精细定位与图位克隆。在大龙1号XPI60234的遗传群体中，于1号染色体中鉴定了一个控制开花期的QTL，在油豆角PI60234和黄金勾中PHYA3（Phvul.001G221100）基因分别是G1066>A1066使功能丧失的点突和在534bp插入一个C使蛋白质翻译提前终止的移码突变，PHYA3基因的突变使得菜豆的光周期敏感性降低。PHYA3基因型鉴定结果说明油豆角PI60234和黄金勾属于Andas驯化型中光周期不敏感的分类亚群具有重要的分子基础。在油豆角PI60234和黄金勾中PvTFL1y（Phvul.001G189200）基因型分别是在第一个外显子处缺少2bp的移码突变和在第四个外显子处插入逆转座子使得基因表达量急剧下降的突变，这两种突变都导致PvTFL1y基因功能丧失。油豆角PI60234中鉴定的PvTFL1y基因型为一个全新的等位变异。分析了开花期基因在菜豆基因组中的分布，并配制了相关的遗传图谱。利用黄金勾（架豆）品种进行不同光照时数处理，转录组分析与开花相关基因的表达特征。同时，对菜豆的其他农艺性状如调控豆荚大小的基因也进行了定位与克隆。培育了相关的菜豆品种翠冠，在生产上进行了推广应用。