Application Of ISSR Marker In The Genetics Analysis Of Sanghuang And Auricularia Heimuer

DNA-based molecular markers can reflect the difference of the DNA fragment. Compared to morphological markers and biochemical markers that are commonly used, DNA-based molecular markers are stable, accurate and easy to operate. The ISSR marker which was based on SSR marker is one of the most widely used DNA-based molecular markers, and it was widely used in molecular identification and genetic diversity research.The sanghaung mushroom is a popular medicinal polypore in traditional Chinese medicine,was famous for its pharmacological activities, such as anti-tumor, anti-oxidant and enhancing immune.In the sanghuang scale, the true sanghuang is difficult to depart it from other related species, and the phenomenon of species confusion is increasingly, so it has greatly restricted the sustainable development of sanghuang.Auricularia heimuer artificial cultivation has been two thousand years in China, and the phenomenon of the same strain with different names and different strains with the same name,aged and degenerate strains were introduced to the production. It's time for selecting fine strains.Annlysis of genetic diversity on cultivated strains of sanghang, main cultivated strain of A heimuer and momokaryons from A.heimuer were studied. The genetics diversity was evaluated and methods of distinguishing different cultivated strains were found out. Those were the basis of strains identification, protecting and utilizing germplasm, breeding of sanghuang and A.heimuer.Five cultivated strains and one wild strain were selected in this study, their molecular identification and genetic similarities were analyzed by ISSR and ITS. Thirty-four mainly strains of A.heimuer and one hundred Flmomokaryons from five A.heimuer strains which were cultivated were selected in this study. Their molecular finger prints and genetic similarities were analyzed by ISSR. The main results were as following:The reaction system and amplified procedure of ISSR-PCR suitable for sanghuang were established. The main elements include, 20?L amplification reaction mixture containing 1U Taq DNA polymerase, 0.5?mol/L Primer, 0.15 mmol/L d NTPs, 50ng template DNA. Optimal annealing temperature was higher that theoretic Tm. Optimal cycling times were thirty-five.Twelve ISSR primers selected were used to amplify genomes of five sanghaung strains tested.A total of one hundred and ninety-four bands were amplified. The ISSR dendrogram based on the similarity coefficient matrix was constructed, Sang NO.2, Sang NO.4 and Sang NO.5 fell into same group, Sang NO.1 and Sang NO.3 fell into groups alone. Model figure of ISSR finger printing were constructed by better twenty-nine ISSR bands selected.The r DNA ITS regions of six sanghaung strains were cloned and sequenced, and the characteristics of ITS sequences were compared and analyzed. The ITS sequence were BLAST matched with GenBank database on homology and were used to construct a phylogenic tree. The results demonstrated that Sang NO.1 was Inonotus linteus, Sang NO.2, Sang NO.4 and Sang NO.5 was Inonotus vaninill, Sang NO.3 was Inonotus obliquus, Sang NO.6 was Inonotus sanghuang.The results of ITS and ISSR were consistent, the two molecular marker technique can be incorporated in strain identification.Ten ISSR primers selected were used to amplify genomes of thirty-four A.heimuer strains tested. A total of one hundred and sixty-seven bands were amplified. The ISSR dendrogram based on the similarity coefficient matrix was constructed. The analysis result showed the tested strains could be classified into five major groups, but were not classified strictly based on region, the six strains from Northeast were classified into the same sub group, Heijin and Heiyuan were the same strain but with different names. It showed that cultivated strains of A.heimuer were import frequently, moreover, the background of strains of Northeast were relatively singularity, the phenomenon of the same strain with different names was exist.At the population lever, thirty-four A.heimuer strains' Nei's gene diversity (H) =0.2865,Shannon information index=0.4420, Nm=2.0850. It showed that the genetic diversity among the tested strains is high, the resources are quite rich and diverse.Twenty-two ISSR primers were screened and eleven to thirteen primers were selected to amplify strain NK-5,strain SX-2,strain DDB, strain Hei 7,strain Li 5' F1 sporulated monokaryons.A total of one hundred and eighteen to one hundred and ninety-seven fragments were amplified,among them above eighty percent were polymorphic. The ISSR dendrogram based on the similarity coefficient matrix was constructed. Tested monokaryons were not classified strictly based on mating types. The ISSR dendrogram combined with traditional mating type gene identification could provide the selection of monokaryons in crossbreeding.