中国苦荞SSR分子标记体系构建及其在遗传多样性分析中的应用

Construction and Application of SSR Molecular Markers System for Genetic Diversity Analysis of Chinese Tartary Buckwheat Germplasm Resources

【目的】从分子水平优化并构建用于中国苦荞种质资源遗传多样性分析的SSR分子标记体系,为综合评价中国苦荞种质资源提供依据。【方法】以50份苦荞种质为试验材料,用正交设计法[L16(45)]筛选适用于苦荞SSR标记分析的PCR反应体系,浓度梯度检测最佳胶分离效果,并从250对不同科属作物SSR引物中筛选出19对引物进行苦荞遗传多样性分析。【结果】优化的苦荞SSR反应体系为DNA模板30 ng,Taq酶2.0 U.L-1,dNTP、引物和Mg2+终浓度分别为150μmol.L-1、0.1μmol.L-1、2.0 mmol.L-1,总体积为25μL,6%聚丙烯酰胺凝胶电泳检测。SSR引物筛选率为7.6%,蓼科同属甜荞的SSR引物适用于苦荞SSR扩增。19对引物共检测到157个等位变异,每对SSR引物检测到的等位变异2—11个,平均等位变异(NA)7.42个,平均多态性信息量(PIC)0.888,平均鉴定力(DP)5.684,2对为SSR骨干引物。利用Popgen Ver.1.31软件,当遗传相似度(GS)为0.578时,50份苦荞材料被分为5个组群,聚类结果与苦荞地理分布相关性不大。四川苦荞资源组群各遗传多样性参数均最高,该区域苦荞种质资源多样性最丰富。利用骨干引物可鉴定部分近缘苦荞品种。【结论】构建的SSR分子标记体系适用于中国苦荞种质资源遗传多样性分析,甜荞SSR引物可用于苦荞SSR标记分析,TBP5和Fes2695为苦荞SSR骨干引物,50份苦荞材料遗传多样性丰富,可划分为5个组群。

【Objective】 The SSR molecular markers system was optimized and constructed for genetic diversity analyses of Chinese tartary buckwheat germplasm resources,which is helpful for evaluating Chinese tartary buckwheat collections.【Method】 The SSR-PCR system was optimized by [L16（45）]orthogonal design,the optimized gel concentration of PAGE was confirmed,and the genetic diversity of 50 tartary buckwheat accessions was analyzed by 19 SSR primer pairs screened from 250 ones of different crops.【Result】The optimized SSR-PCR system was as follows： 30 ng DNA template,150 μmol？L-1 dNTP,0.1 μmol？L-1 primer,2.0 U？L-1 TaqDNA polymerase,2.0 mmol？L-1 Mg2＋,1×Taq buffer and ddH2O then added up to terminal volume of 25 μL with 6% PAGE for testing.The primers screening efficiency was 7.6%,and the primers from common buckwheat were applicable.A total of 157 alleles were detected by 19 primers,with 2-11 alleles for each primer pair,and the average was 7.42.Moreover,the averaged PIC and DP values were 0.8881 and 5.684,respectively.Using Popgen Ver.1.31,50 accessions were clustered into 5 groups at GS 0.578.The clustering results revealed that the genetic diversity of accessions of tartary buckwheat was not correlated to their geographic origins.The genetic diversity of tartary buckwheat from Sichuan was very rich as genetic parameters were the highest.The core primers could be used to identify the similar accessions.【Conclusion】 The SSR molecular markers system was effective for assessment of genetic diversity of Chinese tartary buckwheat germplasm resources.SSR primers of common buckwheat could be used in tartary buckwheat.TBP5 and Fes2695 were SSR core primers.It showed a high genetic diversity in 50 Chinese tartary buckwheat accessions which could be classified into 5 groups.