基于转录组测序的绿豆抗豆象特异新标记开发及新基因Br2精细定位

Mungbean is one of the important food legume crops, and the bruchid (Coleoptera: Bruchidae) is the most devastating stored pests of grain legumes causing considerable loss to mungbean worldwide. Although the study of bruchid resistance for many years, the progress has been very slow mainly due to the lack of sufficient numbers of microsatellite markers and bruchid resistance resource, and it has not been reported that bruchid resistance gene is cloned. The wild species Z5 high-resistance to bruchid was identified. Genetic analysis indicates that the bruchid resistance of Z5 caused by a single dominant nuclear gene mutation and the bruchid resistance gene (Br2) was preliminarily located at approximately 9 cM on linkage group (LG) 6 by using F2 preliminary population. The bruchid resistance cultivar V6 have been obtained by successive backcrossing to the recurrent parent ZL1. The mungbean near-isogenic line (NIL) pairs carrying bruchid resistant and susceptible alleles, sequenced by high-throughput RNA-seq, novel EST-SSR and bruchid resistance-specific molecular markers will be developed according to the sequences of Unigene and differential expression genes in the transcriptome data. Br2 will be fine mapped by using F2 large segregation population (Z5×ZL1) and EST-SSR markers tightly linked. The candidate genes information will be obtained in refined interval compared to other legumes using comparative genomics. The bruchid resistance-related genes will be identified by analyzing of differential expression genes in the transcriptome data. In summary, this study will contribute to the molecular mechanism of bruchid resistance and facilitate the marker-assisted selection for mungbean bruchid resistance in breeding programs.

绿豆是我国重要的食用豆类作物，而豆象是危害绿豆最严重的世界性仓储害虫。由于抗豆象材料稀缺和SSR标记匮乏，至今尚未有抗豆象基因被克隆。前期筛选到绿豆高抗豆象新材料Z5，研究发现Z5含单个显性核抗豆象基因（Br2），并已将其粗定位于第6连锁群约9 cM范围内。用感豆象材料ZL1作轮回亲本，获得含Br2基因的近等基因系材料V6。在前期工作基础上，本项目基于高通量测序技术（RNA-seq）对ZL1和V6进行转录组测序，拟根据Unigene序列及其差异表达序列分别开发EST-SSR和抗豆象特异标记。利用Z5与ZL1杂交构建的F2分离群体和筛选的多态性标记对Br2进行精细定位。借助比较基因组方法分析绿豆Br2定位区段与其它豆科植物基因组的共线性区段，结合从转录组数据中挖掘的抗豆象相关基因，获取候选基因信息。该项目有助于从分子水平上揭示绿豆抗豆象的作用机制，促进绿豆抗豆象分子标记辅助育种的发展。

绿豆是我国重要的食用豆类作物，而豆象是危害绿豆最严重的世界性仓储害虫。本项目首先通过对抗豆象亲本V6、感豆象亲本Z5及其杂交获得的F2群体进行简化基因组测序，构建了含4180个SNP的绿豆高密度遗传图谱。将抗豆象和不抗豆象的后代各取30株进行BSA-seq分析，对获得的多态性SLAF标签利用SNP-index的方法进行关联分析，将抗豆象基因初步定位于绿豆第5号染色体约230kb区间。对抗、感近等基因系ZL1和V6进行转录组测序，根据差异Unigene序列开发了13134个EST-SSR和3325个InDel标记，进一步用转录组测序开发的分子标记筛选并获得V6与ZL1间的多态性标记634个，在限定区段内筛选出与Br2基因紧密连锁的标记，将Br2基因限定在Marker36246和Marker36414两个标记之间约120kb范围内。结合转录组数据分析差异表达基因，通过RT-PCR方法验证在Z5与V6之间差异基因转录水平的表达情况，结果显示Rse1，RD22和WRKY36等在抗豆象亲本Z5中表达量明显增加，推测这些成员可能参与抗豆象代谢过程，定位区间的抗豆象候选基因分析工作正在进行。综合上述结果，本研究为图位克隆抗豆象基因并揭示抗豆象的分子机制奠定了坚实的工作基础。