Analysis And Evaluation On The Diversity Of Male Plant Resources Of Ginkgo Biloba L.

Ginkgo (Ginkgo biloba L.) is a dioecious, perennial tree. The male plant has important economic, ecological and ornamental values. Male plant resources is quite abundant in China, and it is important to study the diversity of male ginkgo resources and evaluate the palynology characterization and secondary metabolite levels of such as flavonoids. It may provide vital theoretical and practical significance for classification of ginkgo male plants, breeding of excellent male lines and exploitation of leaves and pollens Based on many years of research,86male plants of ginkgo distributed at Yangzhou, Xuzhou and Taizhou were used as materials in this study. The diversities of ginkgo male plant resources were investigated by techniques palynology and ISSR molecular marker techniques. The contents of flavonoids in leaves and pollens were determined by high performance liquid chromatography (HPLC). The ginkgolic acid levels were analzed with UV spectrophotometry. The comprehensive evaluation of clustering relations of different male ginkgo types was carried out. The excellent strains were screened on the basis of the set values of flavonoids aglycone total flavonoids. The main results were as followings:(1) The pollens were observed with optical microscope, scanning electron microscope and transmission electron microscope. The fresh pollen was spherical or elliptical, its polar axis and equatorial axis were19.93μm-25.63μm and27.65μm-33.97μm in length, respectivly, the ratio of polar axis to equatorial axis (P/E) was0.64-0.86, their CV is4.87%,6.37%and6.72%respectivly; The dry pollen shows as the shape of a seed stone of ginkgo in equatorial view, its polar axis and equatorial axis were12.05μm-20.29μm and26.03μm-40.78μm in length, respectivly, the index of pollen shape (P/E) was0.43-0.56, their CV is13.75%,13.26%and4.99%, respectivly; The external veins of pollen could be classified into four types:mosaic spherical pearl-like, mosaic conch-like, mosaic lineolatus-like and mosaic curve-like. Thickness of pollen wall, in which the thickness of exine was2-6times as more as that of intine, was0.861μm-1.076μm. The differences of above characters were significant or much significant among the male plants. After3-variate cluster analysis on the indexes of polar axis, equatorial axis and P/E value, the experimental male plants could be divided into four types, in which the first, second, third and foruth type included4,34,44and4strains, respectivly.(2) The genomic DNA was extracted from the leaves of male ginkgo plant by modified CTAB method, the bands were clear, complete and bright, and the mobilities of which were equaral to that of λDNA. The optimized system was selected for ISSR-PCR analysis by L16(44) orthogonal design. The results showed that the optimized system should consist of template DNA of50ng,10×buffer2μl, Taq DNA polymerase of1.0U, Mg2+of2.5mmol·L-1, dNTP of0.20mmol·L-1and primers of0.4μmol·L-1. Among100ISSR primers,12primers which can amplify more clear polymorphic bands were selected. Using these12selected primers,94bands were amplified, among which56were polymorphic bands, percentage of polymorphic loci was59.57%, with an average being5-11bands per primer and the segment size ranging from200to2000bp. The86male ginkgo plants used in this study showed abundant genetic diversities, with average effective number of alleles (Ne), average gene diversity (H), and average Shannon’s information index (Ⅰ) of1.7149,0.3966and0.5771, respectively. The Nei’s distance between individual male plant ranged from0.0443to0.9667. Clustering analysis by furthest-neighbor method showed the male plant of ginkgo were classified into two groups when based on the threshold value of0.8234, while five groups were gained when the threshold value equaled to0.6342.(3) The results showed that10.48%out of the total genetic variation exists among populations, while the genetic variation within populations was89.52%, which was much higher than that among populations. For the three male ginkgo populations, the average effective numbers of alleles (Ne) was1.7199,1.5916and1.5520, respectively. The average gene diversity (H) was0.3964,0.3380and0.3066, respectively. The average Shannon’s information index (Ⅰ) was0.5760,0.4964and0.4473, respectively. The values of Ne, H and I were in the same orders for these three ginkgo populations, that is, Yangzhou>Xuzhou> Taizhou. The gene flow (Nm) of the male plant of Ginkgo biloba L. were4.2710. Moreover, genetic identity was high. These indicated that extensive gene exchange exists in different populations.(4) The flavonoid aglycone content in the leaves of different male strains were determined by HPLC. The flavonoid glycoside content of the samples were calculated according to the three-factor method. The results showed that the optimal extraction combinations of flavonoids in male ginkgo plant leaves were material liquid ratio1:15, ethanol concentration70%, extraction time60min, extraction temperature80℃, extraction2times.there were significant differences between contens of each flavonoid aglycone and the total flavonoid in the leaves of male ginkgo plants in this experiment. The average content of quercetin, kaempferol and isorhamnetin in leaf were2.381mg·g-1DW,2.155mg·g-1DW and1.8515mg·g-1DW, respectively. There was little difference in the content of individual flavonoid aglycones in leaves, with the total flavonoid of15.99mg·g-1DW. There was a significantly positive correlation between the content of flavonoid glycosides in leaf with leaf thickness and specific leaf weight (SLW). Therefore, the leaf thickness and SLW could be used to evaluate the content of flavonoids in leaf as an important index.Five strains from Yangzhou (04,05,12,39and49), four strains from Taizhou (59,66,68and74) and two strains from Xuzhou (80and85) met with the standard of leaf utilization by setting the choice threshold of flavonoid aglycone and total flavonoids content.(5) The flavonoid aglycone content in the pollens of different male strains were determined by HPLC. The flavonoid glycoside content of the samples were calculated according to the three-factor method. The average content of quercetin, kaempferol and sorhamnetin in pollens was0.327mg·g-1DW,7.891mg·g-1DW and0.254mg·g-1DW, respectively. The content of kaempferol in pollens was higher than the other two flavone aglycones. The flavonoid glycoside content of pollens was22.240mg·g-1DW, which was higher than that of leaves. There was a positive relationship between the flavonoid glycoside content of leaves and pollens with correlation coefficient0.9270*. Fourteen strains from Yangzhou (02,04,05,08,10,11,12,16,18,34,39,44,46and49), five strains from Taizhou (59,63,66,68and70) met with the standard of pollen utilization by setting the choice threshold of flavonoid aglycone and total flavonoids content.(6) In this study, the leaves on different part of branch, sampled from9male ginkgo plants growing uniformly in Ginkgo germplasm garden of Yangzhou University at different seasons in2008and2009, were used to determine the ginkgolic acid contents of ginkgo leaves with different treatments by UV spectrophotometric method, in order to clarify extraction technology and determination method of phenolic acids, and select male ginkgo plants which contain low phenolic acid. The results demonstrated that ginkgolic acid content in the leaf of male ginkgo plant varied from June15to November15. The average content of Ginkgo acid in Ginkgo leaves were1.372%.The ginkgolic acid contents on September30and October15were higher, and those on July30and August15were lower. The ginkgolic acid contents in the leaves of long branch was lower than that of short branch. The leave-collecting time at early in August or in mid-August in Yangzhou was recommended, at these time, the ginkgolic acid content was1.361%and1.384%, respectively. There existed significant difference between the ginkgolic acid contents in leaves of9tested male ginkgo plant. The ginkgolic acid content of58was lower than average level, with the value of1.404%, respectively. This male ginkgo plant can provide materials for further experiment research for low-acid male plant selection. The above results may provide important theoretical and material basis for screening low phenolic acid ginkgo male plant germplasm resources.