The Study On Genetic Diversity Of Pinus Densiflora From Xianrendong Natural Reserves

Being an evergreen tree,Pinus densiflora Sieb.et Zucc,which belongs to Pinus of Pinaceae,has great ecological,ornamental and economic value.Aa a zonal native species in the State-level Natural Reserves of XIANRENDONG,Pinus densiflora is also the main object to protect.In this study,genetic diversity,population structure and genetic differentiation of8 populations of Pinus densiflora were analyzed using ISSR DNA markers,which aim to provide a theoretical basis for the effective protection and utilization the resources of Pinus densiflora.The main results are as follows:(1)The best reaction system of ISSR-PCR to Pinus densiflora was established as follows:3 mmol·L-1 dNTP,1.5 mmol·L-1 Mg2+,0.5 U Taq DNA Polymerase,0.3?mol·L-1 primer,9 ng DNA template and 2.5 ?L 10×PCR Buffer in 25 ?L reaction solution,and the augmentation procedure was pre-denaturation at 94 ? for 5 min,denaturation at 94 ?for 30 s,annealing at 52?~61?(depends on different primers)for 45 s,extension at 72 ?for 2 min,reaction with 35 cycles,and extension at 72 ?for 7 min.(2)Thirteen ISSR-PCR primers had been screened.182 loci were generated with13 arbitrary selected in the amplification within 8 natural populations.The total percentage of polymorphic loci reached 76.92%,the number of bands amplified by primers was 6~22,with an average of 14,and the band size was between250~4000bp.(3)The average number of alleles(Na)in the 8 population was 1.58512,the average effective number of allele(Ne)was 1.3606,the mean Nei's gene diversity index(H)and the Shannon's index(I)were 0.2093,0.3118,respectively.The richest genetic diversity of the 8 populations was in Jinyuanshanzhuang,the poorest in Lujuangou,the order is: Jinyuanshanzhunag > Shuijing > Tashigou >Miaozhenggou > Erdaogou > Beishantou > Xiaoyuhe > Lujuangou.(4)Future,the result of AMOVA showed that the genetic variation mainly existed within population 93% and only 7% among population.Population structure analysisshowed that the Gst was 0.1017,gene flow Nm was 4.4130,which clear that there was a high level of gene exchange among populations,and the degree of genetic variation was not high.(5)Based on the cluster analsis of Nei's genetic distances,we found that the 8populations mainly clustered into two groups.LJG?XYH?TSG and MZG clustered into one group,EDG?BST?JYSZ and SJ clustered into another one group.Genetic identity between EDG and BST was 0.9873 at the highest point,the two population clustered together and had a closer genetic relationship;Genetic identity between XYH and EDG was 0.9621 at the lowest point,which belonged two groups respectively and had a farthest genetic relationship.Although the 8 populations tested had a tendency to cluster into two distinct subgroups,all individuals of the 8population of the Pinus densiflora came from the same source.