| In this paper, genetic diversity and population genetic structure of Pinus massoniana Lamb, seed orchard in Chengbu and Guiyang, Hunan Provence were studied by ISSR (inter-simple sequence repeat) marks. The main methods and results are as follows:1,The genome DNA was extracted with CTAB method, and the quality of the isolated DNA was high, the DNA was suitable for ISSR reaction by detection.2,The optimal ISSR reaction system suitable for Pinus massoniana Lamb.was found, which was:20μL reaction volume, containing 2μL 15 ng/μL template DNA,2.0μL 10×PCR buffer,0.6μL primer,1.5μL 2.5 mmol/L dNTPs,0.2μL 5 U/μL Taq DNA polymerase,1.5μL 2 mmol/L MgCl2. Amplification program was:pre-denature at 94℃for 5min; denature at 94℃for 30s; annealing at 56℃for 45s; extention at 72℃for 2 min,35 cycles; last extention at 72℃for 7min; preservation at 4℃.3,Genetic diversity of 117 clones in 4 populations of Pinus massoniana Lamb.seed orchard in Chengbu, Hunan Provence was studied using ISSR markers. Ten ISSR primers, selected from 100 UBC primers, were applied to amplify all clones and a total of 218 markers were obtained. Among them,210 are polymorphic loci and the rate of polymorphic loci is 96.33%. The percentage of polymorphic loci between populations shared a range of 88.99%-90.37% and the highest (90.37%) was between Guangdong and Guangxi populations.which explained its ability adapts to circumstances or environment strongly and the hereditary change level is the highest among the populations. The Shannon diversity index and the Nei's diversity index were 0.5030 and 0.3347. The percentage of genetic identity of the 4 populations ranged from 0.9689 to 0.9812 and the coefficient of genetic differentiation and gene flow were 0.0393 and 12.2219, respectively. The dendrogram based Nei's genetic distance showed that a high similarity was between Xuefeng and Guizhou populations with the distance of 0.0190. But there is a lower similarity between Guizhou and Guangdong-Guangxi populations with the distance of 0.0316. The results revealed their geographic origination, which Xuefeng,Nanling,Guizhou populations are adjacent to each others, while Guangdong-Guangxi population is farther away from the above 3 populations. This result provides a scientific evendience for the management and choosing in Pinus massoniana Lamb.seed orchard.4,Seed orchard of Pinus massoniana Lamb was established in Guiyang (Hunan) using the 2nd generation seedlings. To better understand their genetic diversity and further utilize them for breeding,87 samples were collected and analyzed using 11 screened ISSR primers. A total of 249 markers from 200 to 2000 bp were generated and 246 of them (98.80%) were polymorphic markers. Two geographic seed origins were clearly defined and they were Chengbu and Guangxi groups. Within Chengbu group, seeds collected from Hunan and adjacent Guizhou were also revealed. Results from POPGEN32 analysis showed that the Shannon information index (I) was 0.5837 and Nei's gene diversity index (h) 0.4003.Within Chengbu and Guangxi, the polymorphic loci were 93.57% and 98.80%, respectively. The coefficient of gene differentiation among populations was 0.0363 and gen flow was 13.2586. These results indicated that Guiyang (Hunan) Seed Orchard of Pinus massoniana had high levels of genetic diversity and great individual variations. More similar seed orchard should be set up in the future.5,The anlysis of the genetic distance of the progeny and parentage populations showed that it correlated with the geographic location. The genetic diversity of Parent-child generation of seed orchard are relatively abundant. Although the sources of the progeny seed orchard are best of the best individuals, the result showed that its genetic diversity is not lower than the parentage seed orchard, indicating that parent-child generation seed orchard garden construction approach is worthy of reference.
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