Genetic Diversity Of Different Populations Of Wild Poa Pratensis In Shanxi Province Based On ISSR Analysis

Shanxi Province is located in the agriculture and animal husbandry ecotone, wild Poa pratensis resources are very abundant. Forty-nine wild Poa pratensis in Shanxi province were studied by comparing two kinds of improved genomic DNA extraction methods, establishment and optimization of ISSR-PCR amplification system and the genetic diversity of Poa pratensis. The purpose of this experiment was to provide a theoretical basis for utilizing native grasses and breding new varieties. The results showed as following:(1)DNA template was extracted from Poa pratensis leaf tissue with two improved methods, the results showed that this two methods(CTAB, SDS) are feasible for the extraction of Poa pratensis, the difference between these method is the purity and the stability, the methods of CTAB is better.(2)Base on the Poa pratensis genomic DNA, The orthogonal design was applied to optimize ISSR amplification system at five factors (Taq DNA polymerase, Mg2+, dNTP, DNA template and primer) respectively and hot cycle parameter(annealing temperature, cycles, denaturing time, annealing time and extension time)on ISSR-PCR. Optimal reaction system was established,10×PCR buffer 2.5 ?L, Mg2+ 2.0 mmol·L-1,dNTP 0.2 mmol·L-1,0.5 U Taq DNA polymerase, primer 0.8 ?mol·L-1 and 1 ng·?L-1 DNA templates with total 25 ?L reaction solution. Amplification program included pre-denaturation at 94? for 3 min; denaturation at 94? for 45 min; annealing at 53? for 1 min; extending at 72 "C for 2 min; after 35 cycles, the product was extended at 72? for 7 min and kept at 4?. Steady and higher polymorphic bands were obtained through this optimized system.(3) Ten primers with high polymorphism were screened out from 24 common ISSR primers to amplified 50 Poa pratensis. Total 175 locis were detected by 10 primers, polymorphic locis were 170,17.5 locis per primer on average,the fragment size were 150-3000 bp and the percentage of polymorphic locis (PPL) was 97.14%.(4) The genetic diversities of 50 Poa pratensis resources were analyzed using inter-simple sequence repeat(ISSR) techniques. The results showed that the Poa pratensis has abundant genetic diversity, the number of alleles(Na) was 1.9714±0.0126; the effective number of alleles(Ne) was 1.5700±0.0235; Nei's genetic diversities (H) was 0.3351±0.0109 and Shannon information index(I) was 0.5025±0.0139.(5) Cluster analysis of UPGMA showed that 50 population could be clustered into 3 groups at the level:the first group was made up of 19 materials from Huozhou, Jiangxian and Ningwu, the second group was made up of 30 materials could be divided into two subgroups(? and ?), Poa pratensis from xingzhou, datong, shuozhou and qinshui were mainly concentrated in the class i, Poa pratensis from ningwu and tunliu were mainly concentrated in the class ii, the third group was made up of one materials Aimyty.Through molecular marker method defined tthe genetic differences of wild Poa pratensis in Shanxi province, it's very important to choose Poa pratensis resources and breeding materials.