A Comparative Analysis Of Genetic Diversity In Different Strains Of Lentinula Edodes And Ganoderma And The Protoplast Fusion Of Ganoderma Lucidum

The genetic diversity of 23 Lentinula edodes strains was analyzed by RAPD, ISSR and SRAP markers, respectively, in this thesis. Also, the diversity of 16 strains from Ganoderma family was evaluated by rDNA-ITS sequence analysis.The fusants from protoplast fusion of Ganoderma strains of 05 and 06 were identified by SRAP markers. The main results are as follows:1. From 23 L. edodes strains, 138, 77 and 144 DNA bands were obtained by PCR amplication with 16 RAPD primers, 5 ISSR primers and 23 SRAP primer combinations, respectively. The bands with polymorphism were 81、56、81, and the rates were 58.8%, 73.5% and 56.3%, respectively.The rusults indicated that there were some genetic differences and diversity among the tested 23 L. edodes strains.The similarity coefficients between two strains of 23 L. edodes ranged from 0.57 to 1 according to UPGMA cluster analysis. No. 23 (Xiangjiu) showed the least similarity coefficient and firstly separate from other strains. The other 22 strains were classed into two groups according to the similarity coefficient (above 0.8). The first group was comprised of 17 strains, including No.1 (Shenxiang-2), No.2 (Shenxiang-4), No.3 (Shenxiang-6), No.4 (Shenxiang-9), No.5 (Shenxiang-7), No.6 (Shenxiang-8), No.7 (Shenxiang-10), No.8 (Shenxiang-12), No.9 (Suxiang), No.10 (Wuxiang-1), No.1 (L26), No.12 (L03), No.13 (L66), No.17 (Cr02), No.18 (Cr04), No.19 (Minfeng-1), and No.20 (Hunong-1), which are from Shanghai Academy of Agricultural Sciencesd and Jiangsu Institute of Microbiology and of early maturing and middle-high temperature. The second group consisted of 5 strains, including No.14 (241), No.15 (241-4),No.16 (Qingke-20), No.21 (Zhexiang-939-9)and No.22 (Zhexiang-939-6), which are from Qingyuan Edible Fungi Scientific Research Center and Lishui Edible Fungi Research and Development Center and of late maturing and middle-low temperature.The UPGMA dendrograms found on RAPD, ISSR, SRAP or comprehensive analysis of triplicity was consistent and had the similar pedigree. This suggested that the three marking techniques were all suitable for genetic diversity research. The facts that the strains from the same breeding institutions were gathered to the same group and had greater similarity coefficient, indicated that the 23 strains of L. edodes were of closer relationship and lower genetic diversity.2. The genetic diversity of 16 Ganoderma strains was investigated by rDNA-ITS sequencing. Alignment analysis showed that whole length of internal transcribed spacer(ITS1+ITS2) was 490bp and with 139 variation sites (accounting for 28.3%, ITS1 was 66 and ITS2 was 73), 351 conserved sites (accounting for 71.7%), 59 informative sites (accounting for 12.0%). There were more transition and transversion in the ITS sequence of 16 Ganoderma strains, including 86 conversion sites (G-A,C-T) , 13 transversion site(C-G,T-A). The ratio of transition and transversion in ITS1 was higher than that in ITS2, however, the variable sites of ITS2 were more than thoses of ITS1.Analysis of ITS sequences of 16 Ganoderma strains by DNAman indicated that the genetic distance among 16 strains is from 0.000 to 0.121. The genetic distance between G. lipsiense and F-1 was 0.000 and maybe synonyms.The genetic distance between Heizhi 02 and Huizhou, Jingda, G. capense was 0.121, 0.117 and 0.120, respectively.The 16 G. lucidum strains were classed into four groups by MEGA(version 2.1)software. The group I is comprised of 12 strains, including Xinzhou, Huizhou, Jingda, 902, F-1, xianzhi, Meiluo, Taishan, G. applanatum, 05, G. luteomarginatum. The G. atrum and G. sinense were clustered into one group. The G. capense and zhongzhi were independent group, respectively. These results showed that there were some genetic difference among the groups, however, there were lower genetic diversity and closer relationships between two strains in same groups.3. The breeding study was carried out by protoplast fusion with Ganoderma lucidum 05 and 06 as parents. 30 fusants were screened out by antagonistic tests. The further identification and screen were made by PCR amplication with 153 SRAP primer combinations. The 4 primer combinations were obtained and could be uesd to furtherly identfy fusants. Besides two common bands, the specific bands of Chizhi 06 appeared in the two fusants with antagonistic effects to parent Chizhi 05 and the specific bands of Chizhi 05 appeared in the two fusants with antagonistic effects to parent Chizhi 06. There were 7 fusants with two parent specific bands in 30 identified fusants. These results indicated that the PCR amplication with SRAP primers could be used to the screen of fusants.