梅REMAP分子标记技术体系的优化及其应用

Optimization and Application of REMAP Molecular Marker on Prunus mume Sieb. et Zucc.

通过分析构成梅REMAP分子标记技术体系的主要成分适宜浓度、SSR引物选择性碱基的添加以及RE-MAP-PCR扩增产物检测的非变性PAGE最佳浓度,建立了适合梅REMAP分子标记的技术体系,并对24个梅品种的遗传多样性进行了分析。结果表明:(1)适宜梅REMAP-PCR的反应体系为:25.0μL总体积中,基因组DNA40.0 ng2、.5 mmol/L MgCl21.5μL、2.5 mmol/L dNTPs 2.0μL、10×PCR Buffer(TaKaRa)2.5μL、TaqDNA聚合酶1.5 U、10μmol/L LTR引物1.0μL、10μmol/L SSR引物1.0μL,且SSR引物的3′端加碱基可明显提高RE-MAP的扩增效果,但碱基数量无明显影响。(2)5.0%非变性PAGE电泳条带数量多,扩增范围广(300～2 000bp),且各品种扩增产物均可显示,能够清晰地反映品种间的多态性。(3)24个梅品种的遗传多样性分析显示,花梅的绿萼型及朱砂型分别聚在一起,这与形态学分类的结果一致,证明建立的REMAP技术体系的有效性和可行性。

The optimal system of REMAP molecular marker was constructed through studying the condign concentration of main components of PCR amplification,adding of SSR primer pairs selected base pair,and the concentration of PAGE gel used for detection of REMAP amplification products.The genetic diversity of 24 mei cultivars were analyzed according to this method.The results showed as the following：（1） The final total 25.0 μL solution was composed of template DNA 40.0 ng,2.5 mmol/L MgCl2 1.5 μL,2.5 mmol/L dNTPs 2.0 μL,10×PCR buffer 2.5 μL,rTaq DNA polymerase 1.5 U,10 μmol/L LTR primer 1.0 μL,and 10 μmol/L SSR primer 1.0 μL.The effect of PCR amplification in REMAP was improved significantly through adding base to the 3′ end of SSR primer pairs.But no significant difference was observed with the various base numbers.（2）The best concentration of non-denaturing PAGE gel was 5.0% with the most numbers,extensive amplification size（300~2 000 bp）,and the clearest electrophoresis bands,showing that it could open out polymorphism among the different varieties.（3）The analyses of the genetic diversity of 24 mei based on REMAP showed that green calyx form and cinnabar form were clustered together,respectively.This result was the same as that of the classified of morphologic characters,showing the optimized system of REMAP was available and feasible.