突托蜡梅ISSR引物反应条件的优化与筛选

Optimization of Experiment Conditions and Primer Screening with ISSR Markers for Chimonanthus grammatus

为从突托蜡梅(Chimonanthus grammatus)叶片中提取高质量的总DNA,比较了CTAB法、改良的CTAB法、SDS法、高盐低pH值法、SDS-CTAB法、SDS-蛋白酶K法。结果表明:改良的CTAB是提取突托蜡梅基因组DNA的有效方法。以U811(GA)8C为引物,确立了适合突托蜡梅的ISSR反应体系:在20μL反应体系(50 ng DNA、0.6μmol/L引物、1.5 mmol/L Mg2+、0.15 mmol/L dNTPs、2.0 UTaqDNA聚合酶)中,反应程序为:94℃预变性5 min、94℃变性1 min、57.2℃退火45 s、72℃延伸2 min,循环40次,最后于72℃延伸5 min。用来自不同居群的7个个体,以100个ISSR引物进行PCR扩增,筛选出了扩增效果较好的10个引物,得到了74个位点,其中39个为多态位点,多态性位点比例为52.7%。

The methods of CTAB, modified CTAB, SDS,High salt low pH, SDS-CTAB and SDS-Proteinase K were used to be compared in order to isolate high-quality genomic DNA from leaves of Chimonanthus grammatus. The results showed that the modified CTAB was the best one. By applying the single factor experiment with primer U811, a suitable ISSR reaction system for the analysis of genetic diversity of C. grammatus was established, namely 20μL reaction system containing 1 x PCR buffer,50 ng template DNA, 0.6μmol/L primer,1.5 mmol/L Mg^2＋ ,0.15 mmol/L dNTPs and 2.0 U Taq DNA polymerase. The reaction program was devised for 5 minutes of predenaturalization at 94℃, 1 min of denaturalization at 94℃,45 seconds of anneal at 57.2℃ ,2 minutes of extension at 72℃,40 cycles,and 5 minutes of extension at 72℃ in the final cycle. One hundred ISSR primers were used to screen the suitable primers with 7 samples from different populations for assessing the genetic diversity of C. grammatus, of which 10 ISSR primers with high resolution and multiple polymorphic bands were screened. The total 74 ISSR bands were amplified with 10 primers, and produced 39 polymorphic bands. The percentage of polym-orphic bands was 52.7%.