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

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%。

Ammopiptanthus mongolicus (Leguminosae) were s ubjected to ISSR (Inter-simple sequence repeat)-PCR analysis in order to inv esti gate the genetic diversity within and among populations. Genomic DNA of A . m ongolicus was extracted as the ISSR template, and the influencing facto rs of ISSR were studied and the experiment parameters were optimized. By adjusti ng template DNA concentration, Mg2+ concentration,dNTP and Taq polymera se conte nts, and annealing temperature, the PCR amplification conditions were o ptimized. The optimal experiment conditions were as follow: 20 μl system conta ining 20 n g μl-1 template, 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 0.1%Tri ton × 100, 2.75 mmol/L MgCl2, 0.15 mmol/L dNTP, 2% formamide, 200 nmol/L prime r, 1.5 U Taqpolymerase. With a MJ thermal cycle, optimal amplification program was 1 cycle o f 5 min at 94℃; 35 cycles of30 s at 94℃, 45 s at 46-56℃, and 1.5 min at 72℃; 1 cycle of 7 min at 72℃; 3 0 min at 4℃, using block control style. One hundred ISSR primers were used to s creen the suitable primers for assessing the genetic diversity of A. mon g olicus, of which 15 ISSR primers with high resolution and multiple poly mor phic bands were screened. The total 121 ISSR bands were amplified with 15 pr imer s, and produced 43 polymorphic bands.