Optimization of Experiment Conditions and PrimerScreening with ISSR Markers

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.