Studies On Protoplast Preparation And Genetic Diversity Of Populations Of The Anamorph Of Ophiocordyceps Sinensis

Ophiocordyceps sinensis is a precious and traditional Chinese herbal medicine. Atpresent, the production of the O. sinensis declines sharply, and the price soars. To changethe status quo, artificial culture of O. sinensis has become a focus of study. To study thegenetic diversity of O. sinensis and to carry out protoplast fusion breeding will provide thebasis and the premise for reaching these goals. In this paper, We obtained the dozens ofthe anamorphic isolates of O. sinensis by different isolation methods and by separationfrom different parts of tissue. We should study the optimum conditions for protoplastpreparation of these isolates and use the ISSR molecular technology and the mating typegene mat1-2-1to obtain information about the genetic diversity within species of O.sinensis. The results are as follows.We cultured the mycelium of Hirsutella sinensis, the anamorph of O.sinensis, inliquid flasks. In the paper, the different conditions for protoplast preparation of H.sinensiswas investigated including the different enzyme and its concentration, stabilizer of osmoticpressure and its concentration, pH value of the enzyme and the digesting times. The resultsshowed that protoplasts were obtained for the highest yield of protoplast under theconditions as follows: digested by Yatalase enzyme of4mg/ml and Glucanex enzyme of3mg/ml,1.0mol/L KCl as osmotic stabilizer, pH value of enzyme of6.0and enzymatichydrolysis of3hours.s.The strip amplified by7ISSR primers, which is selected from the60ISSR primers,was clear and good polymorphisms. We employed the appropriate conditions for the PCRreaction of40isolates. The7ISSR primers produced a total of62scorable amplifiedfragments, in which38(63.15%) showed polymorphism. The40isolates had a Jaccardsimilarity coefficient at0.78-1.00. UPGMA cluster analysis showed that40isolates did notcluster into3groups according to different isolation methods. The strains, isolated by themethod of the single ascospore isolation, were clustered into one group alone. The otherisolates mixed cluster. So, ISSR fingerprinting of the single ascospore isolate wassignificantly different from the other isolate. In general, the isolates from same place hadsome genetic differentiation, while the genetic differentiation did not show significantly.For this study, MAT1-2-1sequences of40isolates were amplified with the primersmat1-2-1. Evolutionary phylogenetic analyses were performed with sequences of themat1-2-1gene, of which7sequences of MAT1-2-1came from this experiment and4sequences of MAT1-2-1cited from the Genbank data. The result showed that7isolates collected from Qinghai, together with the isolates from Qinghai in Genbank make up alarge group, the bootstrap value is96%. While the isolates from the other area clusteredinto the other part. Mat1-2-1gene sequence can distinguish the genetic diversity of isolatesfrom different areas, but it failed to distinguish the genetic diversity of the isolates in thesame area. The fact that all of single ascospore isolates contain the mat1-2-1gene suggeststhat mat1-1: mat1-2of O. sinensis is not separated by4:4. So O. sinensis might not beheterothallism, to a large extent, it is homothallic.