Optimization of inter simple sequence repeats (ISSR) analysis as applied to preliminary study of genetic variation in Abies beshanzuensis M. H. Wu

Abstract

Factors which affect the ISSR analysis of Abies beshanzuensis, such as annealing temperature and concentrations of template DNA, Taq DNA polymerase, Mg2+ and dNTP, etc. were examined. The optimal ISSR-PCR reaction system (25 μL) in our experiments was as the following: 1 × Taq buffer, 40 ng template DNA, 1.2 U Taq DNA polymerase, 0.4 μmol/L primer, 2.0 mmol/L MgCl2 0.2 mmol/L dNTPs and 2% formamide. Of the 100 primers subjected to screening, 13 produced highly reproducible and polymorphic bands. Using these primers, 91 discernible DNA fragments were generated with 39(42.86%) being polymorphic bands, indicating the existence of low genetic variation in the species. The level of genetic variation in population originated from seedlings (PPB=37.36%) was higher than that in population of old trees (PPB=16.48%) and population of graft-originated (PPB=20.88%), which suggested that cross-pollination by human beings was helpful to increasing genetic diversity of A. beshanzuensis.

Keywords

• ISSR
• optimization
• genetic variation
• <italic>Abies beshanzuensis</italic>