荞麦种质资源遗传多样性及繁殖遗传完整性的研究
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摘要
荞麦(Fagopyrum)属于双子叶蓼科(Polygonaceae)荞麦属(Fagopyrum Mill),是一种粮药兼备的重要作物。荞麦有苦荞和甜荞两个栽培种。本研究就苦荞种质资源的遗传多样性和甜荞种质资源的繁种更新技术两个方面分别开展了一定的研究,旨在为我国荞麦种质资源的保护、研究和利用提供一定的依据。
本研究利用AFLP分子标记技术对14个不同地理来源的165份苦荞种质进行了遗传多样性分析,从分子水平研究苦荞种质资源的遗传多样性,为综合评价苦荞种质资源提供了依据。筛选出的20对AFLP引物共扩增出938条清晰的条带,其中314(33.48%)条呈多态性,平均每对引物组合的条带数和多态性带数分别为46.9个和15.7个。不同地理来源苦荞种质的Shannon-Weaver多样性指数为0.1093～0.2661,四川资源群最高,青海、云南和甘肃／宁夏等资源群次之,湖南资源群最低。利用Popgen Ver.1.32软件,依不同地理来源苦荞资源群间Nei's遗传一致度可聚类成5个组,聚类结果与苦荞地理分布相关。基于Structure 2.2软件分析,165份苦荞资源分为5大组群,并与Popgen Ver.1.32聚类结果呼应得较好,其中云南和四川资源的群体结构最复杂,最为多样化,分别被聚到了5个组群中。结果表明,苦荞类群的亲缘关系以及遗传多样性与其地理分布有一定相关性,并认同中国西南部四川、云南、西藏一带是栽培苦荞的起源中心和遗传多样性中心的观点和苦荞从中国西藏_不丹→尼泊尔→克什米尔→波兰的传播路线。
甜荞是典型的异花授粉作物,异交率达100%,保持甜荞繁殖更新前后最大的遗传相似性和遗传完整性,是目前荞麦繁种工作的重要课题。根据甜荞的授粉方法(虫媒和风媒)及繁殖生物学特性,本研究设计了空间隔离、纱网隔离和无隔离的繁殖方式,并以种质库取出的原种作为对照,分别从形态标记和分子标记两个方面对甜荞种质繁殖更新前后的遗传完整性进行了研究。观察记载主要农艺性状,并对其进行差异显著性分析；基于AFLP标记数据计算原种与不同隔离方法繁殖后代的遗传相似系数；根据SSR结果分析比较不同隔离处理与原种群体间等位基因频率的变异,并对其进行遗传一致度聚类分析。结果表明,甜荞繁殖更新时不设置隔离,繁殖后代与原种遗传差异显著；纱网隔离能够有效地防止外来花粉混杂,对保持甜荞种质遗传完整性效果最佳；而空间隔离则以500米以上间隔距离最为有效。
The buckwheat is a multipurpose plant which is belongs to the family Polygonaceae, Genus fagopyrum Mill.The buckwheat has two cultivated species, tartary buckwheat and common buckwheat.In this study, the genetic diversity of tartary buckwheat germplasm resources was assessed and the genetic integrity of common buckwheat germplasm resources regenerated with different methods was studied in order to obtain useful information for conservation and use of buckwheat genetic resources.
This work analyzed the genetic diversity of 165 accessions of tartary buckwheat from 14 different geographical regions using 20 informative primer pairs of AFLP markers,and further obtained the helpful information for breeding and germplasm evaluation.Totally 938 loci were detected among which 314 (33.48%) were polymorphic.The number of amplified fragments and polymorphic fragments per primer combination were 46.9 and 15.7,respectively. Shannon's information index of different geographical groups was 0.1093-0.2661.The group from Sichuan possessed the highest level of genetic diversity, followed by those from Qinghai,Yunnan and Gansu/Ningxia;The group from Hunan was the lowest in genetic diversity. Five cluster groups were identified based on the dendrogram of pairwise Nei's genetic identity. The clustering results revealed that the genetic diversity of accessions of tartary buckwheat closely related to their origins. Five types of population structure within 165 tartary buckwheat accessions were inferred by Structure analysis,which also correlated to their geographic origins.The population structure of accessions from Yunnan and Sichuan provinces were the most complicated and multiplex.The result showed that the genetic relationship and diversity of tartary buckwheat populations correlated to their geographic distribution to a certain extent. Moreover, the current study supported that southwest in china is the original birthplace and the center of genetic diversity of cultivated tartary buckwheat. And we supported the spreading route from the origin of tartary buckwheat in China: Tibet in China-Bhutan-Nepal-Kashmir-Karakoram Hindukush route.
Common buckwheat is a typical cross-pollinated species with the outcrossing rate of 100%.Maintaining the genetic integrity of germplasm resources during regeneration is an important subject for buckwheat multiplication. According to the method of pollination (insect and wind) and the reproductive biology of common buckwheat, spatial isolation, gauze net isolation and no isolation was designed for this reseach, with the genebank seeds as a control.In this study, we compared the impact of different isolation methods on genetic integrity changes in common buckwheat using morphological markers and molecular markers.The t-test was used to compare the agronomic traits of common buckwheat between the genebank seeds and their offsprings regenerated with different isolation methods. Genetic similarity index between the genebank seeds and their offsprings regenerated with different isolation methods was calculated based on the data of AFLP markers.The allele frequency and dendrogram comparison between the genebank seeds and their offsprings regenerated with different isolation methods was analyzed using SSR markers.The results indicated that the difference between genebank seeds and their offsprings was significant when isolation was not set during the regeneration of common buckwheat germplasm resources. Gauze net isolation could avoid pollen adulteration effectively. The offsprings regenerated by gauze net isolation possessed the highest level of genetic identity and genetic resemblance with the genebank seeds.For the spatial isolation, the internal distance more than 500 meters was the most effective.
本研究利用AFLP分子标记技术对14个不同地理来源的165份苦荞种质进行了遗传多样性分析,从分子水平研究苦荞种质资源的遗传多样性,为综合评价苦荞种质资源提供了依据。筛选出的20对AFLP引物共扩增出938条清晰的条带,其中314(33.48%)条呈多态性,平均每对引物组合的条带数和多态性带数分别为46.9个和15.7个。不同地理来源苦荞种质的Shannon-Weaver多样性指数为0.1093～0.2661,四川资源群最高,青海、云南和甘肃／宁夏等资源群次之,湖南资源群最低。利用Popgen Ver.1.32软件,依不同地理来源苦荞资源群间Nei's遗传一致度可聚类成5个组,聚类结果与苦荞地理分布相关。基于Structure 2.2软件分析,165份苦荞资源分为5大组群,并与Popgen Ver.1.32聚类结果呼应得较好,其中云南和四川资源的群体结构最复杂,最为多样化,分别被聚到了5个组群中。结果表明,苦荞类群的亲缘关系以及遗传多样性与其地理分布有一定相关性,并认同中国西南部四川、云南、西藏一带是栽培苦荞的起源中心和遗传多样性中心的观点和苦荞从中国西藏_不丹→尼泊尔→克什米尔→波兰的传播路线。
甜荞是典型的异花授粉作物,异交率达100%,保持甜荞繁殖更新前后最大的遗传相似性和遗传完整性,是目前荞麦繁种工作的重要课题。根据甜荞的授粉方法(虫媒和风媒)及繁殖生物学特性,本研究设计了空间隔离、纱网隔离和无隔离的繁殖方式,并以种质库取出的原种作为对照,分别从形态标记和分子标记两个方面对甜荞种质繁殖更新前后的遗传完整性进行了研究。观察记载主要农艺性状,并对其进行差异显著性分析；基于AFLP标记数据计算原种与不同隔离方法繁殖后代的遗传相似系数；根据SSR结果分析比较不同隔离处理与原种群体间等位基因频率的变异,并对其进行遗传一致度聚类分析。结果表明,甜荞繁殖更新时不设置隔离,繁殖后代与原种遗传差异显著；纱网隔离能够有效地防止外来花粉混杂,对保持甜荞种质遗传完整性效果最佳；而空间隔离则以500米以上间隔距离最为有效。
The buckwheat is a multipurpose plant which is belongs to the family Polygonaceae, Genus fagopyrum Mill.The buckwheat has two cultivated species, tartary buckwheat and common buckwheat.In this study, the genetic diversity of tartary buckwheat germplasm resources was assessed and the genetic integrity of common buckwheat germplasm resources regenerated with different methods was studied in order to obtain useful information for conservation and use of buckwheat genetic resources.
This work analyzed the genetic diversity of 165 accessions of tartary buckwheat from 14 different geographical regions using 20 informative primer pairs of AFLP markers,and further obtained the helpful information for breeding and germplasm evaluation.Totally 938 loci were detected among which 314 (33.48%) were polymorphic.The number of amplified fragments and polymorphic fragments per primer combination were 46.9 and 15.7,respectively. Shannon's information index of different geographical groups was 0.1093-0.2661.The group from Sichuan possessed the highest level of genetic diversity, followed by those from Qinghai,Yunnan and Gansu/Ningxia;The group from Hunan was the lowest in genetic diversity. Five cluster groups were identified based on the dendrogram of pairwise Nei's genetic identity. The clustering results revealed that the genetic diversity of accessions of tartary buckwheat closely related to their origins. Five types of population structure within 165 tartary buckwheat accessions were inferred by Structure analysis,which also correlated to their geographic origins.The population structure of accessions from Yunnan and Sichuan provinces were the most complicated and multiplex.The result showed that the genetic relationship and diversity of tartary buckwheat populations correlated to their geographic distribution to a certain extent. Moreover, the current study supported that southwest in china is the original birthplace and the center of genetic diversity of cultivated tartary buckwheat. And we supported the spreading route from the origin of tartary buckwheat in China: Tibet in China-Bhutan-Nepal-Kashmir-Karakoram Hindukush route.
Common buckwheat is a typical cross-pollinated species with the outcrossing rate of 100%.Maintaining the genetic integrity of germplasm resources during regeneration is an important subject for buckwheat multiplication. According to the method of pollination (insect and wind) and the reproductive biology of common buckwheat, spatial isolation, gauze net isolation and no isolation was designed for this reseach, with the genebank seeds as a control.In this study, we compared the impact of different isolation methods on genetic integrity changes in common buckwheat using morphological markers and molecular markers.The t-test was used to compare the agronomic traits of common buckwheat between the genebank seeds and their offsprings regenerated with different isolation methods. Genetic similarity index between the genebank seeds and their offsprings regenerated with different isolation methods was calculated based on the data of AFLP markers.The allele frequency and dendrogram comparison between the genebank seeds and their offsprings regenerated with different isolation methods was analyzed using SSR markers.The results indicated that the difference between genebank seeds and their offsprings was significant when isolation was not set during the regeneration of common buckwheat germplasm resources. Gauze net isolation could avoid pollen adulteration effectively. The offsprings regenerated by gauze net isolation possessed the highest level of genetic identity and genetic resemblance with the genebank seeds.For the spatial isolation, the internal distance more than 500 meters was the most effective.
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