绿豆遗传连锁图谱构建及抗豆象基因定位
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摘要
绿豆是我国传统的食用豆类作物，也是我国传统出口创汇产品，具有良好的食用和药用价值。随着人们生活水平提高和膳食结构的改变，绿豆的市场需求也日益增加。然而目前绿豆遗传育种研究相对落后，现代分子标记技术在育种中还鲜有应用。加强绿豆重要性状的遗传和现代分子生物学研究，对高产高抗优质新品种的培育具有重要指导意义。本研究以绿豆基因组SSR标记开发与评价为基础，利用野生绿豆和栽培绿豆杂交衍生的RIL群体，开展了绿豆遗传连锁图谱的构建，并进一步对抗豆象基因进行了精细定位，同时开展了绿豆重要农艺性状的QTL发掘，为进一步开展基因克隆等后续研究奠定了基础。
主要研究结果如下：
1.新开发的2240对绿豆基因组SSR引物中有1205对在绿豆材料中能有效扩增，选取绿豆、小豆、豇豆及饭豆材料各3份，分析1205对新开发的绿豆基因组SSR引物在这些材料中的扩增效果，结果显示绿豆基因组SSR引物在豇豆、小豆和饭豆中的通用性比率分别为50.0%、73.3%和81.6%；多态性比率分别为4.1%、1.7%和1.5%；有469对引物在4个种间均可通用。
2.以高抗豆象澳大利亚野生绿豆ACC41和高感豆象美国栽培绿豆Berken为亲本构建的重组自交系F10为实验群体，利用264对在亲本间表现多态的SSR和STS标记对群体进行分析，结合前人的199个分子数据，构建了一张含有419个标记（333个SSR标记、74个RFLP标记、9个STS标记和3个RAPD标记）和一个抗豆象基因位点Br1的绿豆遗传图谱，图谱总长735.0cM，含11个连锁群，标记平均间距1.75cM。各连锁群长度在17.5cM～120.3cM之间，标记位点数9～76个之间，各连锁群标记分布较均匀，是迄今为止国内外标记最多、密度最高的绿豆遗传图谱。
3.利用上述图谱将抗豆象基因Br1基因定位于第9连锁群上2.6cM的区间内，距离其两侧的SSR标记P2-627和C220均只有1.3cM。
4.基于上述图谱，在4个环境下，对绿豆的株高、主茎节数、荚长、荚宽、单荚粒数和百粒重进行QTL定位，6个产量相关性状共检测到了62个QTL，分布在除第3连锁群以外的10个连锁群上， QTL贡献率在2.89%-30.19%之间，6个性状均检测到5-18个QTL。有7个QTL在2个或2个以上不同环境中都检测到，具有环境稳定性。
Mungbean [Vigna radiata (L.) Wilczek] is an important legume crop in China, which serve theirroles as cash crops for farmers and as protein sources for consumers. It is a traditional crop in China andhas a long history of cultivation. China is the number one export country of mungbean in the world.Bruchid (Callosobruchus spp.) is a serious pest during storage of seeds of mungbean and other Vignaspecies. Breeding for bruchid-resistance is a major goal in mungbean improvement. In order to set upthe foundation of the molecular marker assisted breeding of bruchid-resistance and molecular designbreeding, we need to initiate the construction of a high-density genetic linkage map, genetic research ofobjective traits and gene location. At present, SSR markers for genetic analysis of these legumes aremuch limited. Transferability analysis of primers has the vital significance to reduce their developmentcost and improve their development efficiency. In this study, we evaluated the transferability ofmungbean Genomic-SSR markers in other Vigna species and constructed a high-density genetic linkagemap of mungbean using SSR and STS markers we developed. The bruchid-resistance gene Br1we finemapped using a Recombine-inbred-lines (RIL) population from the cross between Berken and ACC41.We analyzed the QTLs for Plant height, Number of nod, Pod length, Pod width, Seed per pod and100-seed weight yield-related traits. The results are as follow:
1.1205SSRs primers were tested for their transferability and polymorphism by PCRamplification with the genomic DNA of four Vigna species, cowpea, adzuki bean, mungbean and ricebean. The results indicated that the transferability rate of mungbean genomic-SSR in cowpea, adzukibean and rice bean was50.0%,73.3%, and81.6%, and the ratio of polymorphism SSR primers in thesecrops was4.1%,1.7%, and1.5%, whereas32.0%in mungbean. A total of469mungbean genomic-SSRprimers were detected to be highly transferable among different species of Vigna. The transferability ofmungbean genomic-SSR was higher in adzuki bean and rice bean than in cowpea. These transferablemarkers are useful for further genetic and breeding studies in these species.
2. A molecular genetic map of mungbean was constructed with a190F10recombinant inbred linepopulation from a cross of a highly bruchid-susceptible cultivar Berken and a highly bruchid-resistancewild mungbean ACC41, by using SSR, STS, RFLP and RAPD markers. The total length of the map,which comprised11linkage groups, spanned735cM with an average distance between markers of1.75cM and a maximum distance between linked markers of9.7cM. The new genetic linkage map wasconstructed with419genetic markers(included333SSR markers,74RFLP markers,9STS markers and3RAPD markers). The lengths of the linkage groups ranged from17.5cM to120.3cM, and the numberof loci varied from9to76per linkage group. This genetic map is fundamental to gene localization，comparative genomics and QTL mapping of important agronomic traits.
3. Bruchid-resistant locus Br1was mapped to a2.6cM segment between two SSR markers onlinkage group9. Two markers P2-627and C220are closest to the bruchid-resistence gene,approximately1.3cM away.
4. A total of62QTLs were identified for yield-related traits under the4environments.Contribution of single QTL to phenotypic variation varied from2.89%to30.19%．Of these，7QTLswere common under2or more than2environments．21QTLs were up to10%of the phenotypicvariation．
主要研究结果如下：
1.新开发的2240对绿豆基因组SSR引物中有1205对在绿豆材料中能有效扩增，选取绿豆、小豆、豇豆及饭豆材料各3份，分析1205对新开发的绿豆基因组SSR引物在这些材料中的扩增效果，结果显示绿豆基因组SSR引物在豇豆、小豆和饭豆中的通用性比率分别为50.0%、73.3%和81.6%；多态性比率分别为4.1%、1.7%和1.5%；有469对引物在4个种间均可通用。
2.以高抗豆象澳大利亚野生绿豆ACC41和高感豆象美国栽培绿豆Berken为亲本构建的重组自交系F10为实验群体，利用264对在亲本间表现多态的SSR和STS标记对群体进行分析，结合前人的199个分子数据，构建了一张含有419个标记（333个SSR标记、74个RFLP标记、9个STS标记和3个RAPD标记）和一个抗豆象基因位点Br1的绿豆遗传图谱，图谱总长735.0cM，含11个连锁群，标记平均间距1.75cM。各连锁群长度在17.5cM～120.3cM之间，标记位点数9～76个之间，各连锁群标记分布较均匀，是迄今为止国内外标记最多、密度最高的绿豆遗传图谱。
3.利用上述图谱将抗豆象基因Br1基因定位于第9连锁群上2.6cM的区间内，距离其两侧的SSR标记P2-627和C220均只有1.3cM。
4.基于上述图谱，在4个环境下，对绿豆的株高、主茎节数、荚长、荚宽、单荚粒数和百粒重进行QTL定位，6个产量相关性状共检测到了62个QTL，分布在除第3连锁群以外的10个连锁群上， QTL贡献率在2.89%-30.19%之间，6个性状均检测到5-18个QTL。有7个QTL在2个或2个以上不同环境中都检测到，具有环境稳定性。
Mungbean [Vigna radiata (L.) Wilczek] is an important legume crop in China, which serve theirroles as cash crops for farmers and as protein sources for consumers. It is a traditional crop in China andhas a long history of cultivation. China is the number one export country of mungbean in the world.Bruchid (Callosobruchus spp.) is a serious pest during storage of seeds of mungbean and other Vignaspecies. Breeding for bruchid-resistance is a major goal in mungbean improvement. In order to set upthe foundation of the molecular marker assisted breeding of bruchid-resistance and molecular designbreeding, we need to initiate the construction of a high-density genetic linkage map, genetic research ofobjective traits and gene location. At present, SSR markers for genetic analysis of these legumes aremuch limited. Transferability analysis of primers has the vital significance to reduce their developmentcost and improve their development efficiency. In this study, we evaluated the transferability ofmungbean Genomic-SSR markers in other Vigna species and constructed a high-density genetic linkagemap of mungbean using SSR and STS markers we developed. The bruchid-resistance gene Br1we finemapped using a Recombine-inbred-lines (RIL) population from the cross between Berken and ACC41.We analyzed the QTLs for Plant height, Number of nod, Pod length, Pod width, Seed per pod and100-seed weight yield-related traits. The results are as follow:
1.1205SSRs primers were tested for their transferability and polymorphism by PCRamplification with the genomic DNA of four Vigna species, cowpea, adzuki bean, mungbean and ricebean. The results indicated that the transferability rate of mungbean genomic-SSR in cowpea, adzukibean and rice bean was50.0%,73.3%, and81.6%, and the ratio of polymorphism SSR primers in thesecrops was4.1%,1.7%, and1.5%, whereas32.0%in mungbean. A total of469mungbean genomic-SSRprimers were detected to be highly transferable among different species of Vigna. The transferability ofmungbean genomic-SSR was higher in adzuki bean and rice bean than in cowpea. These transferablemarkers are useful for further genetic and breeding studies in these species.
2. A molecular genetic map of mungbean was constructed with a190F10recombinant inbred linepopulation from a cross of a highly bruchid-susceptible cultivar Berken and a highly bruchid-resistancewild mungbean ACC41, by using SSR, STS, RFLP and RAPD markers. The total length of the map,which comprised11linkage groups, spanned735cM with an average distance between markers of1.75cM and a maximum distance between linked markers of9.7cM. The new genetic linkage map wasconstructed with419genetic markers(included333SSR markers,74RFLP markers,9STS markers and3RAPD markers). The lengths of the linkage groups ranged from17.5cM to120.3cM, and the numberof loci varied from9to76per linkage group. This genetic map is fundamental to gene localization，comparative genomics and QTL mapping of important agronomic traits.
3. Bruchid-resistant locus Br1was mapped to a2.6cM segment between two SSR markers onlinkage group9. Two markers P2-627and C220are closest to the bruchid-resistence gene,approximately1.3cM away.
4. A total of62QTLs were identified for yield-related traits under the4environments.Contribution of single QTL to phenotypic variation varied from2.89%to30.19%．Of these，7QTLswere common under2or more than2environments．21QTLs were up to10%of the phenotypicvariation．
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