Application Of ISSR In Genetic Diversity Analysis For Mushroom Cultivars

ISSR analyses were investigated in 105 strains involved in eight edible fungi: Auicularia auricula,Pleurotus nebrodensis, Grifola frondosa, Flammulina velutipes, Pholiota nameko, Agrocybecylindracea, Pleurotus eryngii, Coprinus comatus. Since the abundant polysaccharide in the mycelium,the DNA extract methods were modified to get the high quality DNA. 20 primers were designed, andthe ingredients and programs of ISSR reactions were optimized. We formed a universal ISSR methodfor all cultivars' discrimination and genetic analysis.ISSR analysis to the eight edible fungi indicated that the genetic diversity was abundant for ediblemushroom cultivated in China. The genetic distance (D) was more than 0.4 among most strains in onespecies. For 21 strains of A. auricula, 10 primers amplified 185 loci, the Percentage of polymorphic loci(P) was 97.84%, the number of observed alleles (Na) was 1.9784, the number of effective alleles (Ne)was 1.2396, the Nei's gene diversity (H) was 0.2732, Shannon's information index (I) was 0.4278, thegene flow (Nm) was 2.7528; For 20 strains of P. nebrodensis, 11 primers amplified 70 loci, P was75.71%, Na was 1.7571, Ne was 1.5310, H was 0.2967,I was 0.4324; For 13 strains of G.frondosa, 12primers amplified 178 loci, P was 84.83%, Na was 1.8483, Ne was 1.3245, H was 0.1988, I was 0.3168;For 13 strains of F. velutipes, 12 primers amplified 124 loci, P was 91.94%, Na was 1.9194, Ne was1.4819, H was 0.2844, I was 0.4313; For 12 P. nameko, 16 primers amplified 146 loci, P was 86.99%,Na was 1.8699, Ne was 1.3880, H was 0.2395, I was 0.3733; For 10 strains of A. cylindracea, 13primers amplified 194 loci, P was 87.63%, Na was 1.8763, Ne was 1.4455, H was 0.2669, I was 0.4095;For 9 strains of P. eryngii, 13 primers amplified 250 loci, P was 90.80%, Na was 1.9080, Ne was 1.3722,H was 0.2388, 1 was 0.3803; For 7 strains of C. comatus, 14 primers amplified 141 loci, P was 70.21%,Na was 1.6474, Ne was 1.2931, H was 0.1803, I was 0.2828. The UPGMA cluster analysis based onNei's genetic distance of every species showed that the distribution of cultivated strains in China wasindependent of geography origin. This maybe indicated the adaptability of all strains was good, and thespawn exchange was frequent in China.Comparison analysis between ISSR and EST isozyme were carried out for P. nebrodensis and P.eryngii. The result showd they were both good methods to identify strains, but the result of geneticrelationship was not the same.24 sequences of ISSR loci in P. nebrodensis were analyzed, and the result showed: 1) 5' anchoredprimers were not specific enough, the silimar primers mismatched on the template; 2) there werenucleotides difference in the the same loci amplified in different strains; 3) the Blast result in GenBankshowed no homology to the sequences Accessed in GenBank. They were the noncoding regions in thegenome; 4) the abundant tandem repeats in the ISSR clone sequence proved that the ISSR amplifiedregions were the SSR activate region.