八角ISSR-PCR反应体系的建立及引物筛选研究

Establishment of ISSR-PCR Reaction System of Illicium verumand Selection of Primers

以文山、广西2个居群6个单株的八角嫩叶为研究材料,以八角叶片基因组DNA为模板,在分析归纳相关树种ISSR-PCR体系的基础上,确定一个反应体系并对ISSR引物库进行筛选,同时结合正交设计针对具体引物进行体系优化。结果表明,八角的ISSR-PCR反应体系(25μL)为DNA模板0.003～0.016 ng,Taq DNA聚合酶0.5～1.0 U,dNTPs 0.2 mmol.L-1,Mg2+浓度2.5～3.0 mmol.L-1,引物浓度0.6～0.8μmol.L-1。扩增程序为,94℃预变性5 min;94℃变性30 s,引物特异温度退火45 s,72℃延伸1 min,40个循环;72℃最后延伸7min;4℃保存。获得效果稳定并具有多态性的ISSR引物13条。研究结果可为八角遗传多样性分析和种质资源鉴定等研究奠定基础。

Based on summarizing related researches of ISSR - PCR systems of other tree species, experiments were designed and conducted to establish the ISSR -PCR reaction systems and to screen for primers. The experi- ment took leaves of Illicium verum from 6 individuals of 2 populations from Yunnan Wenshan and Guangxi as materials and genomic DNA of I. verum leaf as the template. A reaction system was established to screen ISSR primer library and optimize specific primers combining with orthogonal design. The results indicated that the optimized reaction sys- tem of 25 μL included 0. 003 - 0. 016 ng template DNA, 0. 5 - 1.0 U Taq DNA polymerase, 0. 2 mmol L-l dNTPs, 2. 5 -3.0 mmol L-1 MgC12, 0. 6 -0. 8 μmol 1 L-1 primer. Amplifications were performed with the fol- lowing programs ： 94℃ for 5 min ; 40 cycles of 94℃ for 30 s, primer-dependent annealing temperature for 45 s, 72℃ for 1 rain; a 7 -rain extension at 72℃; 4℃ conservation. Thirteen stable polymorphism primers were selected out. The research result would play an important role in further research of genetic diversity and identification of germ- plasm of I. verum.