| Cordyceps militaris has similar medicinal value as Cordyceps sinensis, even more active constituents such as cordycepin are much more than that of C. sinensis. As the substitutes of C.sinensis, C. militaris is overexploited and its natural resource was exhausted. The genetic affinity was roughly observed by inhibitory assay in this study, and then their genetic relationship and genetic diversity were studied by SRAP, ISSR, SCoT and ITS markers. It provides molecular foundation and theoretical direction for resources protection, artificial introduction cultivation, breeding and gene excavation of C.militaris. The results are as followed:1,210 tests were set in inhibitory assay. There were 82 tests obviously inhibiting,9 tests barely inhibiting. The results showed that inhibitory assay could reveal the genetic affinity to some extent. However, its inhibitory extent was influenced by subjective factor of experimenter and medium component. The inhibitory assay could only be a kind of assistant method of genetic differences identification between strains. Therefore, molecular markers which tested more accurately and reliably were used to strains identification and genetic diversity study in this study.2,Analyzing the genetic affinity and diversity of C.militaris by SRAP and ISSR molecular markers, the results that the polymorphism reached 100% showed that the C.militaris strain samples exhibit abundant genetic diversity. Under the optimized SRAP and SRAP reaction conditions, when the similarity coefficient is 0.7040 and 0.6560, the strain samples were clustered into 4 groups. In SRAP results, the No.1 and 2,3 and 8 clustered into groupâ… , No.7 and No.16 were groupâ…¢andâ…£, respectively, the others were clustered into groupâ…¡. The cluster results of ISSR marker were similar with that of SRAP. No.7 and No.16 was groupâ…¢andâ…£, respectively, while No.11,12,13 were added into groupâ… compared with the results of SRAP. The genetic similarity coefficient between No.16 and others in SRAP and ISSR result was 0.59 and 0.48, respectively. It illustrated that the genetic relationship between No.16 and others is far, which is consistent with the result of inhibitory assay. The genetic similarity coefficient between No.1 and No.2, No.4 and No.5 is highest which is 0.95 and 0.94 in SRAP and ISSR results, respectively. This means that they have close relationship. According to their origins, they may be obtained by tissue isolation.3,There is no primer to reveal differences in SCoT marker which illustrated that SCoT may be not suitable for C.militaris strains identification.4,By sequencing the ITS sequence, the results that No.16 is Tolypocladium inflatum and others are Cordyceps militaris demonstrated that ITS sequencing could be effective method to identify different strains. While the ITS sequence of No.4, No.5, No.6, No.7, No.15, No.18 and No.20 are the same, so are the sequence of No.3, No.8, No.12 and No.13. It revealed that the ITS sequence of C.militaris is conserved and it is not suitable for intraspecific identification.5,In this study, the polymorphism of SRAP,ISSR markers reached 100%, all of the strains could be effective discriminated and the cluster results were almost coincident. All these showed that SRAP and ISSR markers could be effective method to identify strains between and within the species and detect genetic relationship. The ITS sequencing could only discriminate the strains between species but not within species. The SCoT marker is not suitable for C. militaris identification. |
