不同秋眠性苜蓿SRAP体系优化及遗传多样性分析

用改良CTAB(十六烷基三甲基溴化铵)法提取苜蓿基因组DNA,用L₁₆(4⁵)正交设计,研究了模板DNA、dNTPs、Mg²⁺、Taq酶和引物的浓度对扩增迁移率重现性的影响,结果表明,在20 μL体系中含有50 ng DNA、1.4 mmol/L Mg²⁺、1.2 U Taq酶、0.4 mmol/L dNTPs和0.25 μmol/L浓度的引物最为稳定,重现率达到100%。利用最佳体系从100对引物中筛选出8对在34个苜蓿上扩增效果好的引物,共扩增出87个SRAP标记,有84个多态性位点。聚类分析结果表明,来自安徽和江苏两地的野生南苜蓿和其他栽培苜蓿的遗传差异最大,单独聚为一类;其他32个苜蓿品种在相似系数0.68附近聚为3个亚群,秋眠型苜蓿品种主要分布在第2个亚群中,半秋眠和非秋眠型苜蓿品种分布散乱,并不表现出成族分布,表明秋眠性与苜蓿的亲缘关系不完全一致。

A study on optimization and primer screening of a SRAP reaction system and genetic diversity of different fall dormancy alfalfas

Genomic DNA was extracted by advanced CTAB methods from Medicago sativa.The L16(45) orthogonal diagram was applied to study the effects of mobility reproducible in different concentrations of template DNA,dNTPs,Mg2+,Taq polymerase,and primer on M.sativa.A suitable SRAP reaction system had 50 ng DNA,1.4 mmol/L Mg2+,1.2 U Taq polymerase,0.4 mmol/L dNTPs and 0.25 μmol/L in a 20 μL reaction system.Better primers were screened out from 100 pairs of primers using optimizated systems,that could amplify clear fragments.A total of 87 amplicons were resolved by 8 pairs of well-chosen primers,of which 84 were polymorphic.These varieties had high genetic polymorphisms.A dendrogram from the analysis clustered two Medicago hispida cultivars from Anhui and Jiangsu into one group.Thirty two other cultivars were classified into sub-groups with a similarity coefficient of 0.68.Fall dormancy alfalfa cultivars were mostly distributed in the second sub-group.Semi-fall dormancy and non-fall dormancy alfalfa cultivars did not fall in the same sub-group.There was no absolute distinction of fall dormancy of alfalfa.