Optimization of chloroplast SSR-PCR reaction system for cucumber and its application | 黄瓜cpSSR―PCR反应体系优化及应用

Китайский. 叶绿体微卫星（chloroplast microsatellite，cpSSR）是近几年发展起来的新型高效分子标记，在植物系统发育和基因漂移研究中有广泛应用。在前期开发黄瓜cpSSR引物的研究基础上，本文研究了砌酶量、MgCl2浓度、dNTPs浓度、模板DNA质量和浓度、循环次数等参数对cpSSR―PCR扩增效果的影响，获得优化的反应体系，即15μL体积包括：1×Taq酶缓冲液，Q5UTaq聚合酶，0．2μmol／L引物，0．2mmogLdNTPs，1．5mmol／LMgCl2，约10ng模板DNA，循环25次。采用该优化体系，以已开发的黄瓜cpSSR引物，对12份黄瓜材料进行了重复性PCR扩增。结果发现国外种质中存在3种单倍型，而在国内种质中只检测到2种单倍型，说明在本研究所用的材料中，国外种质叶绿体遗传差异明显大于国内栽培种。此结果说明优化的cpSSR反应体系具有稳定性和可靠性。[著者文摘]

Английский. Chloroplast microsatellite （cpSSR）, a newly developed efficient molecular marker, has been widely used in the field of plant phylogenetic development and gene flow. Based on the previous research on development of cucumber cpSSR primers, this article documented the effect of influencing factors, i.e., Taq polymerase dosage, MgCl2 concentration, dNTPs concentration, template DNA quality and concentration and cycle number, on cpSSR-PCR amplification; the optimized cpSSR reaction system was obtained as follows： 15 μL reaction volume containing 1 × Taq polymerase buffer, 0.5 U Taq polymerase, 0.2 μmol/L of each primer pairs, 0.2 mmol/L of each dNTPs, 1.5 mmol/L MgCl2, about 10 ng template DNA and 25 times of cycles. Using the optimized system as well as newly developed cucumber cpSSR markers, 12 cucumber accessions were used for PCR amplification. The resuits showed＇that three haplotypes existed among exotic cucumber accessions but only two were detected in domestic accessions. That indicated that among the accessions used in this study, exotic accessions had higherrequency variance in chloroplast DNA than domestic cultivated accessions. Also, this finding revealed stability and reliability of the optimized cpSSR reaction system.