Establishment and optimization of ISSR system in Crataegus spp.
DAI Hong-yan,ZHANG Zhi-hong,ZHOU Chuan-sheng,LI He,GUO Xiu-wu (College of Horticulture,Shenyang Agricultural University,Shenyang,Liaoning 110161 China)
Hawthorn (Crataegus spp.) has been cultivated as a fruit crop in China for a long history. The effects of annealing temperature,the quality of DNA template,the concentration of Mg2+ and the source and concentration of Taq DNA polymerase on ISSR analysis in hawthorn were studied,and the optimized ISSR system for hawthorn was established. The optimum ISSR system for hawthorn was as follows: total DNA extracted with modified CTAB method or QIAGEN Plant DNeasy Kit as template; in 20 μL volumes containing 1×PCR buffer (Promega),3.0 mmol/L of MgCl2,0.2 mmol/L of dNTP,0.3 μmol/L of ISSR primer,0.5 U Taq DNA polymerase (Tiangen) and 50 ng of total DNA; the cycling conditions of PCR consisted of an initial denaturation step for 3 min at 94 ℃,followed by 38 cycles of 30 s at 94 ℃,1 min at annealing temperature,2 min at 72 ℃,and then a final elongation step for 7 min at 72 ℃. Ten ISSR primers that are suitable for hawthorn were screened. This research laid a sound foundation for hawthorn germplasm analysis at DNA level.