花吊丝竹ISSR反应体系的建立与优化

建立关于花吊丝竹的ISSR-PCR反应体系,为花吊丝竹种质资源鉴定提供理论基础。采用单因素试验法,对影响PCR扩增效果的Mg~(2+)浓度、d NTPs浓度、Taq DNA聚合酶用量、引物浓度及模板DNA用量等5个PCR反应体系主要成分以及退火温度和循环次数进行分析,并利用建立的优化反应体系和扩增程序对100条候选引物进行筛选。结果表明,适合花吊丝竹的ISSR-PCR反应体系为:20μL的反应液中含3.0 mmol/L Mg~(2+)、0.20 mmol/L d NTPs、1.25 U Taq DNA聚合酶、0.6μmol/L引物、10 ng模板DNA、2μL 10×Buffer、8.55μL dd H_2O。扩增程序为:94℃预变性5 min;94℃变性45 s,52.7℃退火30 s,72℃延伸90 s,40个循环;72℃延伸10 min,4℃保存。建立的花吊丝竹的ISSR-PCR反应体系能够扩增出清晰、多态性较高的条带,且筛选出的16条引物具有高度多态性。表明该体系具有较高的稳定性、重现性和适用性。

Establishment and Optimization of ISSR-PCR System for Dendrocalamus minor var.amoenus

The purpose of this study was to establish optimized ISSR system for Dendrocalamus minor var.amoenus.The single factor design was applied for optimizing five factors in ISSR amplification system,such as Mg2 +,dNTPs,Taq DNA polymerase,primer and template DNA concentration.The results showed that the optimal 20 μL ISSR-PCR reaction system for Dendrocalamus minor var.amoenus contained 3.0 mmol/L Mg2+,0.20 mmol/L dNTPs,1.25 U Taq DNA polymerase,0.6 μmol/L primer,10 ng DNA template,2 μL 10 × Buffer,8.55 μL ddH20.The amplification procedure was as follows:94 ℃ 5 min;94 ℃ 45 s,52.7 ℃30 s,72 ℃90 s,40 cycles;72 ℃ 10 main,4 ℃ for storage.Under this optimal ISSR amplification system,16 primers were screened with high polymorphism from 100 primers.The test showed that the system was stable,reliable and applicative.