ISSR Analysis On Genetic Diversity Of The Ancient Male Ginkgo Trees And The Preliminary Study Of Selecting Superior Individuals For Pollen

The ancient male Ginkgo trees which came from main distribution areas ofGinkgo Boliba L were chosen as the study objects in this paper, their geneticdiversity were investigated, the different analysis were performed for biologicalcharacteristics of Ginkgo male flower, the ultrastructure and many kinds nutrientingredients of Ginkgo pollen. The results were as follows:1, The influencing factors including concentration of template DNA, Taq DNApolymerase, primer concentration, dNTPs and the annealing temperature upon theamplification effect with PCR technique were studied for optimizing the ISSRreaction conditions of Ginkgo. The results showed that the optimum reactionconditions for ISSR PCR of Ginkgo were as follows: 0.3U Taq DNA, 2μL 10×Taqbuffer (including 15mMMgCl₂), 40ng template DNA, 0.2mmol/LdNTP, 0.5μmol/Lprimer must be consisted in a total volume of 20μL. The amplification was 1 cycleafter initial denaturation at 94℃for 5 min, followed by 38cycles of 30s at 94℃, 30sannealing at 48～53℃(depending on different primers), 2 min at 72℃, and a final 5min extension at 72℃, after which the samples were held at 4℃.2, 13 primers with high staility and good repeat ability were selected from 60ISSR primers and produced 114 bands, of which 83 were polymorphic, across a totalof 97 individuals, the polymorphic bands percentage were 72.8%, the averagenumber of bands per prime was 8.76. Average effective number of alleles, averageNei's gene diversity, average Shannon's information index were 1.8117, 0.4374 and0.6253 respectively. The cluster analysis showed the relative relationship amongmale Ginkgo trees was correlated with geographical distribution. The results showedthe genetic diversity of male Ginkgo trees was quite rich.3, The pollen of 33 ancient male Ginkgo trees were observed through electronmicroscope. The results showed that the pollen outside configuration of differentindividuals were approximately similar, and they were all olive shaped and had abourgeon channels which was as long as pollen length. There was significantdifference in smooth type, ektexine ornamentation, and pores condition of pollen.The results showed that the ultrastructure of Ginkgo pollen was complex anddiverse.4, There were significant differences in the number of spica on short shoot,length of spica, width of spica, anther number of spica, length of anther, width ofanther, length of pollen, width of pollen and the absolute ratio of pollen productivityamong 33 ancient male Ginkgo trees.5, With the help of factor analysis and cluster analysis, 12 individuals including Fenghua-01, Nanxiong-01, Nanxiong-02, Tongjiang02, Xixia-02, Taixing-01,Guangde-01, Nanjing-01, Tianmu-01, Changxing-04, Beijing-01 and Taian-02 wereselected as the preparing-selected individuals with high pollen production.6, The pollen vigor of ginkgo was average 73.42%, the pollen vigor ofTaixing-02, Tancheng-02, Nanxiong-02, Taixing-01 and Tianmu-01 was quite high,and can be treated as pollination tree in production practice.7, There were significant differences in the contents of total flavonoid, totalterpene lactone, vitamin B₁, vitamin B₂, vitamin C and amino acid. The lactoneingredient was examined in plant pollen for the first time in this study. The contentsof flavonoid, lactone, and total flavonoid and lactone of 8 individuals includingLueyang-01, Chongqing-01, Jinzhai-01, Nanxiong-01, Songxian-01, Anhua-01,Kangxian-01 and Fenghua-01 were quite high, and they were selected as thepreparing-selected superior individuals in flavonoid and lactone productdevelopment aspect; Guilin-09, Lueyang-01, Pizhou-01, Guilin-06 and Nanxiong-02were selected as the preparing-selected superior individuals with high vitamin B₁contents; Guilin-09, Pizhou-01, Fenghua-01, Doujiangyan-01, Taian01 were selectedas the preparing-selected superior individuals with high vitamin B₂ contents;Taixing-01, Chongqing-01, Nanjing-01, Taixing-02, Lueyang-01 were selected asthe preparing-selected superior individuals with high vitamin C contents; Taixing-02,Guilin-09, Changxing-04, Guilin-06, Xixia-02, Tancheng-01, Chongqing-01,Tongjiang-02 were selected as the preparing-selected superior individuals with highamino acid contents.