Analysis On Genetic Diversity Of Germplasm Resources For Male Plant Of Ginkgo Biloba L.

Gingko(Ginkgo biloba L.), a plant of dioecism,is native to China in which its germplsm resources are rich, and has a different gene variation during a long evolution. The germplsm resources of gingko are the important matter support for its researches, development and utilization. The male plant of gingko not only plays an important role in ecological construction and beautification, but also maintains its special position in the seed- and leaf-purpose as a pollinator and resources with higher active ingredients. The germplsm types directly influence its character expression of resources, so research on its genetic structures and diversity has an important theoretical and practical significance for development and utilization of gingko's male plant. In this study, 100 pieces of tender leaf at top of one-year-old shoots from 91 male plants of gingko distributing in Yangzhou city, Xuzhou urban area and gingko orchard of Yangzhou University were respectively collected at 30 days after germination for successive two years. ISSR markers were used to analyze its genetic diversity and the results achieved are showed below:1. The genomic DNA bands extracted from the male plant of gingko by modified CTAB, the mobilities of which were equaral to that ofλDNA, were clear, complete and bright. The minimum concentration of DNA was 652 ng/μl, and the minimum and maximum of A260/A280 was 1.75 and 1.94 respectively. The purity of DNA was suitable to do ISSR-PCR.2. Four factors including amount of Taq DNA polymerase, Mg2+, dNTP and primers, four treatments each factor, was used to select the optimized system for ISSR-PCR by orthogonal design [L16( 44)], the results showed that the optimized system should consist of template DNA of 50 ng, 10×buffer 2μl, Taq DNA polymerase of 1.0 U, Mg2+ of 2.5 mmol·L-1, dNTP of 0.20 mmol·L-1 and primers of 0.4μmol·L-1etc.3. Out of 100 ISSR primers, 12 primers which amplified more clear polymorphic sites were selected, the sequences of which were (AC)8CG,(CA)8AG,(AC)8SG,(AC)8GA,(AC)8GG,(GA)8A,(GA)8C,(CA)8GT,(GACA)4,(AC)8CA,(CT)8G,(AC)8 YG, respectively.4. 94 bands were amplified from 12 primers, among which 56 were polymorphic bands, percentage of polymorphic loci was 59.57 %, with an average being 5-11 bands per primer and the segment size ranging from 200 to 2000 bp.5. Average effective number of alleles (Ne), average gene diversity (H), and average Shannon's information index (I) of the 91 plants were 1.7149, 0.3966 and 0.5771 respectively, which showed the genetic diversity on male plant of Ginkgo biloba L. was quite rich.6. The Nei's distance ranged from 0.0443 to 0.9667. Clustering analysis by furthest-neighbor method showed the male plant of gingko were classified into two groups (GroupⅠandⅡ) with 37 and 54 plants respectively when based on the threshold value of 0.9434, while five groups (Group 1, 2, 3, 4 and 5) were gained when the threshold value equaled 0.6842. In summary, Group 1 and Group2 were subgroups affiliated with GroupⅠ, including 6 and 31 plants respectively. While Group 3, 4 and 5 were subgroups affiliated with GroupⅡ, including 48, 2 and 4 plants respectively.7. The total genetic diversity for species(Ht)of three populations from male plant of gingko in Yangzhou city, Xuzhou urban area and gingko orchard of Yangzhou University was 0.3876, the mean heterozygosity within populations was 0.3470, and the coefficient of gene differentiation(Gst)was 0.1048. The results showed that 10.48% out of the total genetic variation exists among populations, while the genetic variation within populations was 89.52%, much higher than that among populations.8. Average effective numbers of alleles (Ne) of the male plant of gingko in Yangzhou city, Xuzhou urban area and gingko orchard of Yangzhou University was 1.7199, 1.5916 and 1.5520 respectively. The average gene diversity (H) was 0.3964, 0.3380 and 0.3066 respectively. The average Shannon's information index(I)was 0.5760, 0.4964 and 0.4473 respectively. And the Ne, H and I had the same size orders, that is the male plant of gingko in Yangzhou city >Xuzhou urban area> gingko orchard of Yangzhou University.9. The gene flow (Nm) of the male plant of Ginkgo biloba L. populations in Yangzhou city, Xuzhou urban area and gingko orchard of Yangzhou University were 4.2710. Moreover, genetic identity was high. These indicated that extensive gene exchange exists in different populations.The results of the study have great importance for the classification, evaluation and utilization for male plant of Ginkgo biloba L. germplasm resources.