Development Of EST-SSR Primer And Genetic Diversity Study Of Grifola Frondosa

Grifola frondosa(Dicks.Fr) S.F.Gray is a kind of important rare edible and medicinal fungus with many kinds of medicinal value, such as anti-tumor, enhancing immunity, lowering blood pressure and anti virus. In this study, high throughput sequencing was used to search for the SSR locus, and the genetic diversity of the 32 G. frondosa strains was evaluated by using the polymorphic EST-SSR markers. The following results were obtained:ITS rDNA of G. frondosa from different regions was analysed.The results showed that based on the phylogenetic tree the strains from Asia could be seperated from those in the United States, indicating that there is a certain correlation between geographical distance and genetic distance of G. Frondosa.Transcripnomes from mycelium of G. frondosa were sequenced based on Illumina HiSeq 2500. After DE NOVO splicing, 14347 unigenes were obtained. In the transcript of gene function annotation and classification, 9433 unigenes matched the NCBI-Nr database and accounted for a proportion 65.74%; among GO functional classification, 7366 unigenes from 54 categories of annotation were defined specific functions; a total of 4347 unigenes were annotated in KOG database; 2799 unigenes matched the KO database,255 pathways were orgnized into KEGG.Using MISA, a total of 515 SSR loci were found in the unigenes. 395 pairs of primers were designed with Primer3.0. Randomly 50 pairs of primers were chosen and designed, and 42 of them amplified bands. There were 12 pairs of EST-SSR primers with obvious polymorphism.Using fluorescence labeled capillary electrophoresis detection method, the PCR amplification products of 12 pairs of polymorphic EST-SSR primers on the 32 DNA were detected, the statistical sizes of amplified fragments were analysed using Popgene and NTSYS. The results showed that the range of PIC value was 0.16-0.67, with an average of 0.52 of 12 pairs of primers. UPGMA cluster analysis showed that 32 strains of G. frondosa were divided into 9 types at the similarity coefficient 0.62. The H5 from the United States was grouped into a branch alone, with the genetic similarity coefficient 0.24 from the strains from Asia.