板栗ISSR反应体系的优化及河北省板栗生态型的分析

Optimization of Chestnut Inter-simple Sequence Repeat Reaction System and its Ecotypes in Hebei Province

以早丰和燕明2个板栗品种为试材,应用编号为ISSR52的引物,对ISSR反应体系中的dNTPs、Taq DNA聚合酶、引物、模板和甲酰胺等主要影响因子进行了优化筛选。结果表明:20μL ISSR反应体系各组分的最适浓度分别为1×Buffer(含2.0 mmol/L的Mg2+),dNTPs 0.2 mmol/L,Taq DNA聚合酶1.0 U,引物0.2μmol/L,模板25 ng。加人2.0%的甲酰胺有利于减轻背景的干扰。利用该体系对9个河北省板栗品种(系)和1个山东省板栗品种进行了分析,以明确河北省板栗居群的遗传多样性。通过对53个ISSR引物的重复筛选,获得了19个稳定的ISSR引物,在10个品种(系)中共产生93条带,33.3%为多态性条带。通过聚类分析进一步明确河北省板栗可分为燕山和太行山2个生态型,并且太行山地区的板栗具有较高的遗传多样性。

Taking two chestnut varieties （Zaofeng and Yanming2） as materials, the optimization of main influential factors of ISSR reaction system were studied. The results showed that the optimum concentrations of components such as Buffer, dNTPs, Taq DNA polymerase, primer, template DNA in 20 μL reaction system were 1 x Buffer （ with 2.0 mmol/L Mg^2＋ ）, 0. 2 mmol/L, 1.0U, 0. 2 μmol/L and 25 ng, respectively. Furthermore, the addition of 2. 0% formamide was propitious to reduce the disturbance. The genetic diversity of 9 chestnut cultivars in Hebei Province and 1 chestnut cultivar in Shandong Province was studied by ISSR method. Nineteen stable ISSR primers were obtained from 53 tested primers. Ninety three bands were produced in 10 cuhivars with 33.3 percent polymorphic. The result of cluster analysis showed that Heibei Province chestnut could be divided into Yan mountain ecotypes and Taihang mountain ecotypes. More over, the chestnut cultivars or clones in Taihangshan Mountain were more genetic diversity.