Optimization of inter simple sequence repeats (ISSR) analysis as applied to preliminary study of genetic variation in Abies beshanzuensis M. H. Wu
2005
AI Jian-guo | QIU Ying-xiong | YU Jiu-hua | CHEN Xiao-rong | DING Bing-yang
Factors which affect the ISSR analysis of Abies beshanzuensis, such as annealing temperature and concentrations of template DNA, Taq DNA polymerase, Mg<sup>2+</sup> 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 MgCl<sub>2</sub> 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.
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