柱花草SRAP-PCR体系优化及其遗传多样性分析

草业学报 ›› 2011, Vol. 20 ›› Issue (4): 159-168.

柱花草SRAP-PCR体系优化及其遗传多样性分析

张伟丽¹,刘凤民²,刘艾^3*

1.仲恺农业工程学院生命科学学院,广东广州 510225;
2.仲恺农业工程学院教学科研基地,广东广州510225;
3.仲恺农业工程学院园艺园林学院,广东广州510225

收稿日期:2011-04-15 出版日期:2011-04-25 发布日期:2011-08-20
通讯作者: E-mail:liuai268@163.com
作者简介:张伟丽(1969-),女,河北乐亭人,副教授,硕士生导师,博士。E-mail:zhangweili7218@163.com
基金资助:
广东省科技计划项目(2010B020305011)资助。

Optimization of SRAP-PCR system and its application in genetic diversity analysis of Stylosanthes

ZHANG Wei-li¹, LIU Feng-min², LIU Ai³

1.College of Life Sciences, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China;
2.Teaching and Science Reserch Base, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China;
3.College of Horticulture and Landscape Architecture, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China

Received:2011-04-15 Online:2011-04-25 Published:2011-08-20

摘要/Abstract

摘要： 以热研2号、热研13号柱花草为试验材料,对影响SRAP标记PCR反应的模板、Mg²⁺、dNTPs、酶及引物浓度进行了优化,建立了适合于柱花草SRAP标记的扩增体系。反应体系具体为:模板DNA 40 ng, Mg²⁺ 2.5 mmol/L,dNTPs 0.2 mmol/L,Taq酶1.0 U,引物0.3 μmol/L,反应总体积为25 μL。采用优化的扩增体系,对9份柱花草种质材料进行了遗传多样性分析。从48对SRAP引物中筛选出14对扩增清晰且多态性高的引物,共扩增出154条谱带, 其中有150条多态性谱带。多态性比率(PPB)达97.36%。应用NTSYSpc 2.1数据分析软件计算各品种间的遗传相似系数(GS),结果表明这些材料间的遗传相似系数的变化范围为0.386~0.882,平均遗传相似系数为0.631,平均遗传距离(GD)为0.369。通过UPGMA分子系统聚类法,将9份柱花草种质分为3个类群,有钩柱花草是第Ⅰ类,西卡柱花草是第Ⅱ类,热研5号、热研10号、热研13号、白花库克、热研2号、格拉姆、Tardio为第Ⅲ类。从遗传聚类图可以很明确地看出9份种源间的遗传距离及亲缘关系。对聚类结果分析显示,大部分具有亲缘关系的品种及形态学、生物学特征相近的品种聚在一类,说明聚类分析结果与系谱及生物学特征具有一定的相符性。

Abstract: Two different genotypes of Stylosanthes germplasm, S. guianensis cv. Reyan No.2 and S. guianensis cv. Reyan No.13 were used as material for studying the effects of concentrations of Mg²⁺, dNTPs, DNA polymerase, primers and DNA template on the SRAP-PCR reactions and optimizing the establishment of SRAP molecular marker system in Stylosanthes. The optimum system was established as follows: template DNA 40 ng, Mg²⁺ 2.5 mmol/L, dNTPs 0.2 mmol/L, polymerase 1 U, primer 0.3 μmol/L, the total reaction volume was 25 μL. The genetic diversity and relationships of nine Stylosanthes germplasms were analyzed by using this optimum system. Forty-eight primers were studied for an analysis of genetic diversity by SRAP in Stylosanthes germplasms. Fourteen effective primers selected from 48 primers combination were used for SRAP-PCR, and 150 of the 154 DNA fragments amplified, showed polymorphisms. The average binds from each primer was 11.0 and the average percentage of polymorphic bands was 97.36%. The Nei’s genetic similarity coefficient of the tested accessions ranged from 0.386 to 0.882 by software NTSYSpc 2.1 based on SRAP results, and the average Nei’s coefficient was 0.631, average the genetic distance(GD) was 0.369. Based on the presence of bands, nine Stylosanthes germplasm were classified into there major groups by UPGMA cluster analysis, group Ⅰ, group Ⅱ and group Ⅲ. Group Ⅰ included S. hamata cv. Verano, and group Ⅱ included S. scabra cv. Seca. Group Ⅲ included S. guianensis cv. Reyan No.5, S. guianensis cv. Reyan No.10, S. guianensis cv. Reyan No.13, S. guianensis cv. White cook, S. guianensis cv. Reyan No.2, S. guianensis cv. Graham and S. guianensis tardio CIAT1283. This research should provide a scientific basis at the molecular level for further study and the application of nine Stylosanthes germplasms. The most of the varieties with relative relationship in their pedigrees and similar biological characteristics were clustered into the same group.

中图分类号:

张伟丽,刘凤民,刘艾. 柱花草SRAP-PCR体系优化及其遗传多样性分析[J]. 草业学报, 2011, 20(4): 159-168.

ZHANG Wei-li, LIU Feng-min, LIU Ai. Optimization of SRAP-PCR system and its application in genetic diversity analysis of Stylosanthes[J]. Acta Prataculturae Sinica, 2011, 20(4): 159-168.

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