简单重复序列区间(ISSR)引物反应条件优化与筛选

Optimization of Experiment Conditions and PrimerScreening with ISSR Markers

摘要:

以豆科沙冬青(Ammopiptanthusmongolicus)的基因组DNA制备ISSR-PCR模板。通过对聚合酶链式反应(PCR)体系中模板DNA浓度、Mg²⁺浓度、dNTP的用量、Taq酶的含量以及退火温度梯度进行试验,探讨筛选出清晰、多态性高、可重复性好的ISSR引物的PCR反应条件。用100个ISSR引物进行了PCR扩增,筛选出效果较好的15个引物,得到121个位点,其中43个多态位点,多态位点比例为36%。

Abstract:

A mmopiptanthus mongolicus (Leguminosae) were subjected to ISSR (Inter-simple sequence repeat)-PCR analysis in order to investigate the genetic diversity within and among populations. Genomic DNA of A. mongolicus was extracted as the ISSR template, and the influencing factors of ISSR were studied and the experiment parameters were optimized. By adjusting template DNA concentration, Mg(supetscript 2+) concentration, dNTP and Taq polymerase contents, and annealing temperature, the PCR amplification conditions were optimized. The optimal experiment conditions were as follow: 20μ1 system containing 20 ngμ1^-1 template, 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 0.1%Triton × 100, 2.75 mmol/L MgCl₂, 0.15 mmol/L dNTP, 2% formamide, 200 nmol/L primer, 1.5 U Taq polymerase. With a MJ thermal cycle, optimal amplification program was 1 cycle of 5 min at 4℃; 35 cycles of 30 s at 94℃, 45 sat 46-56℃, and 1.5 min at 72℃; 1 cycle of 7 min at 72℃; 30 min at 4℃, using block control style. One hundred ISSR primers were used to screen the suitable primers for assessing the genetic diversity of A. mongolicus, of which 15 ISSR primers with high resolution and multiple polymorphic bands were screened. The total 121 ISSR bands were amplified with 15 primers, and produced 43 polymorphic bands.