美花石斛SSR-PCR体系的优化研究
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摘要
采用正交设计法对影响美花石斛SSR-PCR体系的5个主要因素进行了优化,建立适宜的美花石斛SSR-PCR的反应体系,并利用黔产5个不同的美花石斛居群共计48株进行了验证。结果表明:在10μL的美花石斛的反应体系中各因素的最佳浓度分别为2.0mmol/L Mg~(2+),0.4mmol/L dNTP,1.5UTaq酶,0.4μmol/L引物,40ng模板DNA,建立的SSR-PCR体系可用于美花石斛的遗传多样性研究。
In order to establish the best SSR-PCR reaction system,by the method of the orthogonal design affect on D.loddigesii SSR-PCR system of 5 elements(Mg~(2+),dNTP,Taq enzyme,primers and DNA)was optimized,which established suitable D.loddigesii SSR-PCR reaction system.And a total of 48 species of D.loddigesii Rolfe from 5 group in guizhou were tested.The results showed the optimum concentration of each factor in the reaction system of D.loddigesii in 10μL volumes was 2.0 mmol/L Mg~(2+),0.4 mmol/L dNTP,1.5 UTaq DNA polymerase,0.4μmol/L primer and 40 ng template DNA.The established SSR-PCR system can be used for analyzing genetic diversity of D.loddigesii.
In order to establish the best SSR-PCR reaction system,by the method of the orthogonal design affect on D.loddigesii SSR-PCR system of 5 elements(Mg~(2+),dNTP,Taq enzyme,primers and DNA)was optimized,which established suitable D.loddigesii SSR-PCR reaction system.And a total of 48 species of D.loddigesii Rolfe from 5 group in guizhou were tested.The results showed the optimum concentration of each factor in the reaction system of D.loddigesii in 10μL volumes was 2.0 mmol/L Mg~(2+),0.4 mmol/L dNTP,1.5 UTaq DNA polymerase,0.4μmol/L primer and 40 ng template DNA.The established SSR-PCR system can be used for analyzing genetic diversity of D.loddigesii.
引文
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[8]何仁锋,冯尚国,陈喆,等.药用菊花SSR-PCR反应体系优化及引物筛选[J].分子植物育种,2015,13(2):367-378.
[9]杨妮,苏伟敏,靳雅慧,等.广西莪术SSR-PCR反应体系优化和引物筛选研究[J].湖北农业科学,2016,55(23):6 271-6 275.
[2]Schemeske DW,Husband BC,Ruckelhaus MH.Evaluating approaches to the conservation of rare and endangered plants.Ecology,1994,75:584-606.
[3]Saghai-Maroof MA,Soliman KM,Jorgensen RA et al.Ribosomal DNA spacer-length polymorphisms in barly:Mendelian inheritance,chromosomal location and population dynamics[J].Proceedings of the National Academy of Sciences,USA,1984,81:8 014-8 018.
[4]赵树进.中药材生物多样性及核酸分析技术[M].北京:科学出版社,2009:64-70.
[5]王伟,杨文鹏,关琦,等.玉米SSR分子标记技术操作规程的优化[J].安徽农业科学,2008,36(11):4 459-4 464.
[6]段永红,渠云芳,王长彪,等.药用植物苦参SSR-PCR体系的优化与验证[J].中国农业大学学报,2014,19(5):95-100.
[7]刘亚令,宋美玲,候俊玲,等.药用甘草SSR-PCR反应体系的优化与引物筛选[J].时珍国医国药,2017,28(3):740-744.
[8]何仁锋,冯尚国,陈喆,等.药用菊花SSR-PCR反应体系优化及引物筛选[J].分子植物育种,2015,13(2):367-378.
[9]杨妮,苏伟敏,靳雅慧,等.广西莪术SSR-PCR反应体系优化和引物筛选研究[J].湖北农业科学,2016,55(23):6 271-6 275.