Species Diversity Of Rhytismataceae And RAPD Analysis Of Coccomyces And Hypoderma

Some important members of 8 species of 4 genera,which were Coccomyces, Hypoderma,Lophodermium and Hypohelion of Rytismataceae were systematically studied from morphology,ontogeny,ecology and distribution and so on according to the more advanced taxonomic system.Among them,Coccomyces castaneae sp.nov.,Hypoderma cunninghamiicola sp.nov.,Lophodermium sabiae sp.nov.and Lophodermiun trachelospesperi sp.nov.were new species,and 4 known species of China were supplied and studied in new hosts and new geography distribution.The living materials of fungi of Rytismataceae from Tiantangzhai National Forest Park were separated and cultivated by tissue isolation techniques.Sixteen strains of Coccomyces spp.and Hypoderma rubi were obtained.The strains were cultured on MA culture medium, and then regularly observed and noted the shape,size,luster,structure,mycelium,the rate of growth,and if which produce pigment and conidiomata.The strains of Hypoderma rubi were classified to 8 biology(culture)types based on the characteristics of culture.The DNA of 12 Coccomyces strains and 21Hypoderma ones were respectively extracted.The RAPD-PCR reaction system was optimized,and the DNA from intraspecies and interspecies were amplified.It is necessary to find a RAPD reaction system suitable for this experiment in order to promote the stability and repetition because of their more lacking stability and repetition.The reaction system of Coccomyces and Hypoderma were optimized in the quantity of primers,template,Mg²⁺,dNTPs,Taq DNA polymerase and annealing temperature,annealing time,elongation and cycle numbers.The optimal RAPD-PCR reaction system included 16.3μl ddH₂O,2.5μl 10×Buffer,2μl Mg²⁺ (25mmol/L),2μl dNTPs(10mmol/L),1μl(100ng/μl)template,0.2μl Taq DNA polymerase (5U/μl),1μl random primers(12.5μmol/L)in the system of 25μl.The amplification process as follow:95℃beforehand denaturalization for 5 minutes,50 cycles;94℃denaturalization for one minute,36℃annealing for 100 seconds,72℃elongation for 140 seconds,after the last circulation,then 72℃elongation for 10 minutes.The DAN of 12 strains of Coccomyces were amplified with 14 collected random primers by the optimal process,140 fragments were obtained,133 polymorphic ones among,accounting for 95%.The most sites,20 ones,were amplified by Primer S11,the second ones were Primer S404,the least,three ones,were amplified by Primer S7 and Primer S220.The length of most amplified fragments ranged from 250bp to 2000bp.The phenotype was well consistent with the results of RAPD based on the clustering tree map which were established by the method of UPGMA.The DAN of 21 strains of Hypoderma were amplified with 14 collected random primers using the reaction process,132 fragments were obtained,120 polymorphic ones among them,accounting for 91%.The most sites,17 ones,were amplified by S130,the second ones were 16.Four sites were the least,amplified by Primers S7.The length of most fragments ranged from 250bp to 2000bp,too.From the clustering tree map which were established by the method of UPGMA,21 strains were classified into 7 sorts at the genetic distance of 4.10,the results was basically consistent with the phenotype,host and location.