Abstract: Alfalfa is an important forage and have been planted for more than 2000 years in China.Carrying a lot of genetic variation,alfalfa is an excellent plant for genetic diversity research,but the breeding rate and level of research of alfalfa is behind of other crops.In the research of the genetic diversity of alfalfa germplasm by using molecular markers,a good PCR amplification system is essential.The orthogonal design was used to optimize SRAP-PCR amplification system on Medicago varia Martin at four levels of five factors(Taq DNA polymerase,Mg²⁺,DNA template,dNTP and primer)respectively.This optimized system for SRAP marker would become one of the effective protocols for further research.The different factors have a significant impact on the PCR reaction.The quantity of dNTP was the most important affected factor of PCR,and the concentration of template DNA had the minimal impact.Various factors and their changes in the level had the impact on PCR reaction,in which descending order is dNTP,Taq DNA polymerase,Mg²⁺,primer and DNA template.One of the most suitable 25 μL SRAP-PCR systems for Medicago varia Martin.containing dNTP 2 μL(0.20 mmol·L^-1),Taq DNA polymerase 0.3 μL(1.50U),Mg²⁺ 3 μL(1.50 mmol·L^-1),each primer 1 μL(0.40 μmol·L^-1),50 ng DNA template 1 μL and 10×PCR buffer 2.5 μL(exclusion Mg2+) was established.This optimized SRAP-PCR system would play an important role in Medicago map construction,genetic diversity analysis,germplasm identification and so on.

Key words: Medicago varia Martin., SRAP, PCR, Orthogonal design, Optimization

摘要： 利用正交设计L₁₆(4⁵)对杂花苜蓿(Medicago varia Martin.)SRAP-PCR反应体系的5因素(Taq酶、Mg₂⁺、模板DNA、dNTP、引物)在4个水平上进行优化试验,旨在建立一套适用于苜蓿属(Medicago L.)种质资源的SRAP标记技术体系。结果表明:各因素对PCR反应结果都有显著影响,由F值可知,dNTP的量对反应结果影响最大,其次是Taq酶的量,模版DNA浓度的影响最小,各因素水平的变化对PCR反应的影响依次为:dNTP>Taq酶>Mg₂⁺>引物>模板DNA;筛选出各反应因素的最佳水平,建立杂花苜蓿SRAP-PCR反应的最佳体系(25μL)为:dNTP 2μL(0.20 mmol·L^-1),TaqDNA聚合酶0.3μL(1.50 U),Mg₂⁺3μL(1.50mmol·L^-1),上、下游引物各1μL(0.40μmol·L^-1),50ng模板DNA1μL,10×PCR buffer 2.5μL(不含Mg₂⁺)。这一优化体系的建立,为今后利用SRAP标记技术进行苜蓿属植物遗传图谱构建、遗传多样性分析及种质资源鉴定等研究奠定了技术基础。

关键词: 杂花苜蓿, SRAP, PCR, 正交设计, 优化