香蕉ISSR反应体系的建立

Establishment of an Inter-Simple Sequence Repeats Reaction System (ISSR) for Banana (Musa AAA)

以35份香蕉品种为材料,采用正交设计L25(56)对PCR反应中的DNA质量浓度、TaqDNA聚合酶用量、Mg2+浓度、引物浓度、退火温度及甲酰胺浓度等6个因素在5个水平上进行了优化试验.建立了稳定的、可重复的香蕉ISSR最佳反应体系和PCR扩增参数.确定在25μL的反应体系中,DNA模板质量浓度为0.8 ng/μL,Mg2+浓度为1.5 mmol/L,dNTPs浓度为0.3 mmol/L,引物浓度为0.2μmol/L,Taq酶为1.25 U,退火温度为52℃.

A suitable ISSR-PCR amplification system of banana was established using thirty five banana eultivars as materials. Orthogonal design was used to optimize the reaction condition of ISSR experiment in six factors （ DNA, Taq DNA polymerase, Mg^2＋ , primer, formamide and annealing temperature） at five levels respectively. A stable and repeatable reaction system of ISSR for banana was established. PCR reaction volume of 25 uL included 0.8 ng/p,L DNA template,1.5 mmol/L Mg^2＋ ,0. 3 mmol/L dNTPs,0. 2 umol/L primer concentration and 1.25 U Taq DNA polymerase dosage, and proper annealing temperature was 52℃.