莲SRAP-PCR反应体系的优化与建立

Optimization and Establishment of SRAP-PCR in Lotus

以中国古代莲(Nelumbo nucifera)和美洲黄莲(N.lutea)为材料,利用单因素分析法对影响莲SRAP-PCR扩增效果的模板、Mg2+、dNTP、Taq DNA聚合酶、引物浓度进行了探索和研究。获得了能稳定扩增莲基因组的SRAP-PCR最佳10μL反应体系:模板DNA浓度为50 ng、1μL 10×Buffer、MgCl2浓度为2 mmol/L、dNTPs浓度为0.2 mmol/L、Taq DNA聚合酶浓度为0.75 U、正反向引物浓度均为0.8μmol/L。为检测该优化体系的稳定性,进一步选取16对引物组合对88份花莲核心种质进行PCR扩增,获得了183条清晰的谱带,其中165条具有多态性,比率为90%,说明建立的莲SRAP反应体系是稳定可靠的。莲SRAP-PCR反应体系的优化和建立,为利用SRAP标记技术深入开展莲的遗传多样性评价、遗传连锁图构建和分子辅助育种等研究提供成熟的技术体系支撑。

We established an advanced SRAP-PCR reaction system for Chinese Antique Lotus and American Lotus using a single factor experiment with five impact factors,including DNA template,Mg2＋,dNTP mixture,Taq DNA polymerase and primer.The 10 μL reaction mixture contained 50 ng of genomic DNA template,1 μL of 10×Buffer,2 mmol/L of MgCl2,0.20 mmol/L of dNTP,0.75 U of Taq DNA polymerase,and 0.8 μmol/L of primers.To test the stability of the optimized SRAP-PCR system,the PCR was further amplified in the core-collection of flower lotus with 88 cultivars using 16 primer pairs.A total of 183 clear bands were obtained throughout all materials,of which 165（90%） bands were polymorphic.Therefore,the established SRAP reaction system for lotus was reliable.The results provided technical support for evaluating genetic diversity as well as constructing genetic linkage maps and molecular marker-assisted breeding of lotus.