栽培绿豆V1128抗豆象新基因Br3的精细定位

Mungbean (Vigna ridiata) is one of the most important traditional legumes in China. Bruchid beetles (Callosobruchus spp.Coleoptera, Bruchidae), also known as seed weevils are major post-harvest insects of it which cause considerable damage to the storage mungbean. Resistant resources discovered by now include wild species and cultivated species. Wild bruchids resistant resources have many imperfections when used as mungbean breeding materials and food security problems as well. However, genetic and breeding research of cultivated resistant resources is greatly lagged up to now. In our previous studies, a novel bruchids resistant gene Br3 identified from a cultivated mungbean variety V1128 was mapped to linkage group and was located in a 13.5 cM interval between two SSR markers by using F2:3 populations derived from V1128 and a susceptible cultivar Jilv No. 7. In this study, two near-isogenic lines (NILs) were constructed firstly by using continuous backcrossing and remaining hybrid line (RHL).Then new molecular markers will be developed from mungbean genome database to fine mapping of Br3 gene by using recessive extreme individual analysis. And the related genes of bruchids resistance will be predicted by comparing the differences between two parents and between opposite resistant NILs. All research findings of this study will rich the knowledge of genetic mechanism of bruchid resistant gene of mungbean in theory, and facilitate the molecular marker assisted breeding, gene cloning and gene-transfer of mungbean bruchids resistance in practice.

绿豆是我国传统的食用豆类，市场消费需求旺盛。豆象是绿豆主要的仓储害虫，对绿豆品质和产业发展影响巨大。研究发现，绿豆野生种和栽培种中均有抗豆象资源，但野生种在育种应用中比较困难。我们前期研究发现，栽培绿豆种质V1128中含有1个显性抗性基因Br3，并结合F2:3群体的抗豆象鉴定数据，将Br3基因初步定位在2个SSR标记之间，两侧标记遗传距离分别为4.00cM和9.54cM。本项目拟在前期研究的基础上，通过利用连续回交和剩余杂合系（RHL）形成的两套近等基因系，进一步从绿豆基因组数据库中开发验证新的分子标记，采用隐性极端个体分析法确定交换单株，实现对Br3基因的精细定位，并通过抗感材料间的核酸序列比对，预测抗豆象候选基因。研究结果理论上将丰富对绿豆抗豆象基因遗传机制的认识，实践上将为在绿豆中开展分子标记辅助育种提供可靠分子标记，并为Br3基因的图位克隆和转基因应用奠定基础。

豆象是危害绿豆等食用豆类作物的严重仓储害虫，是影响我国绿豆生产的重要威胁。本研究以抗豆象栽培绿豆资源V1128为研究对象，旨在对其抗豆象基因进行精细定位，以促进绿豆抗豆象分子标记辅助育种和抗豆象基因克隆。遗传分析表明，V1128的豆象抗性由具有主效作用的显性单基因控制。按照显性单基因进行连锁定位分析或以种子被害率进行QTL定位分析，均将其抗豆象基因Br3定位于标记DMB158和VRBR-SSR032之间，两标记之间的物理距离为288 kb。利用连续回交和从重组自交系中筛选剩余杂合系的方法构建了2套近等基因系，进一步在定位区间内开发分子标记，并从近等基因系形成的次级作图群体中筛选交换单株。利用筛选出的8个交换单株可以将抗豆象基因Br3定位在标记VRID1和标记Mchr5-138之间，两个标记之间的物理距离为95.6 kb。分析来源于不同染色体的分子标记之间的连锁关系表明，原绿豆基因组在抗豆象基因位点附近存在组装错误。为了提高Br3基因定位的准确度及辅助分析定位区间内的基因信息，我们采用三代测序（Pacbio）结合Hi-C的方法对本研究的亲本之一“冀绿7号”进行了基因组测序组装。新绿豆基因组的组装质量与原绿豆基因组相比有了显著提升。利用新的绿豆基因组的序列信息进行引物设计，对抗豆象基因Br3精细定位区间内及附近的12个注释基因进行基因扩增及测序分析，除基因M3.2135在亲本冀绿7号、V1128及近等基因系RIL155-3-8及RIL155-9-9间不存在差异外，其他基因在抗豆象材料和感豆象亲本间均存在差异。后续需要进一步在定位区间内寻找新的交换单株来减少候选基因数目，并通过转基因验证的手段验证基因功能。