| Hepialus gonggaensis is one of the worm formed Cordyceps,it is easily infected by Paecilomyces farinosus leading to Paecilomyces disease in its breeding course. In order to study the genetic diversity of pathogens, A L12 (41×33) mixed-level orthogonal design was used to optimize SCoT-PCR and ISSR-PCR reaction system and to select the best reaction cycles, correspond primer, and annealing temperature on this basis, using two molecular markers SCoT and ISSR for 80 Paecilomyces farinosus genomic DNA clustered analysis and comparative study. The results are as follows:1.SCoT markers for Genetic diversity of Paecilomyces farinosusThe optimized SCoT-PCR reaction system:20?L PCR reaction system including template DNA 45 ng, primer 0.7 ?mol/L, MIX mixture 7 ?L, Taq enzyme 1 U; The optimal number of cycles of the reaction system is 40. Ten primers that was clear and high polymorphism were selected from 15 SCoT universal primers using this reaction system. These 10 primers were S12, S13, S14, S15, S19, S28, S29, S31, S32 and S34, respectively. With Paecilomyces farinosus genomic DNA as a template, with the above PCR reaction conditions conducted SCoT-PCR. This 10 SCoT primers total of 104 amplified loci in which 69 polymorphic loci, and polymorphism ratio was 66.35%. SCoT-PCR cluster analysis showed that 80 strains have an abundant genetic polymorphism, genetic similarity coefficient between 0.66-0.95, and when the coefficient is 0.751 can be divided into six groups.2.ISSR markers for Genetic diversity of Paecilomyces farinosuThe optimized ISSR-PCR reaction system:20?L PCR reaction system including template DNA 60 ng, primer 0.6 ?mol/L, MIX mixture 6 ?L, Taq enzyme 0.8U; The optimal number of cycles of the reaction system is 40. Using this reaction system,10 primers which clear and high polymorphism were selected from 15 ISSR universal primers. These 10 primers were 701, 702,703,807,808,809,810,835,855,873. These 10 ISSR primers total of 96 amplified loci in which 56 polymorphic loci, and polymorphism ratio was 58.33%. ISSR-PCR results of cluster analysis results:genetic similarity coefficient of 80 strains between 0.70-0.96, and when the coefficient is 0.756 can be divided into five groups.3.SCoT and ISSR markers compared and analyzed genetic diversity of Paecilomyces farinosusAverage numbers of bands, polymorphic loci and percentage of polymorphism were slightly higher than those of using ISSR markers, which indicated that SCoT markers can detect more polymorphic genetic loci and be slightly better than ISSR markers; Their average genetic similarity coefficient range of SCoT markers, and genetic similarity coefficient of strain classification are less than those of ISSR markers, which explained that SCoT markers are more recognizable than ISSR markers.The results could be obtained by clustering of SCoT and ISSR markers as the following: the 65 strain isolated from the wood which is south of the buildings were clustered into a single class because of its farthest genetic distance; the strains isolated from the same geographical origin were not all clustered into the same group, which was in same as that the 12 and 26strains isolated from south of external walls of buildings, the 20 and 61 strains isolated from north external walls of buildings were clustered into two groups respectively. The strains isolated from No.8,9,10,11,14 laboratory room on the second floor and central hall were divided into different groups. The most of strains from the same parasite or insect were divided into different groups. However, some strains could be classified into the same category. For example, the 16 and 46 strains from gonggaensis pupa were classfied into two different groups; the 37 and 62 strains from 3-4 ages larvae gonggaensis body were classified into different groups; the 48 and 78 strains from 5-6 ages gonggaensis larvae body belonged to the same group; the 1 strain from Linzhi in Tibet, the 14 strain from 6-8 ages gonggaensis larvae and the 16 strain from gonggaensis pupa were classified into the I(1) sub-category.In the test results, isolated from artificial breeding base in different environments or instars and collected Paecilomyces farinosus which isolated from geographical distantly Linzhi in Tibet, Maqu in Gansu, Xiaojin in Sichuan, Laji snow mountain in Qinghai,which could be clustered into the same or different groups.The results fully illustrated that Paecilomyces farinosus which located in Cordyceps artificial breeding base in Kangding Zheduo mountain showed abundant genetic diversity. |
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