| Rhytismataceae belong to Rhytismatales Discomycetes Ascomycota, is a importantgroup in fungi resources of the world. There were 500 species 74 genera reported. Butclassification of known groups was mainly based on phenotypic characters such asmorphology in the past. With the development of modern molecular biology theory andtechnology, the current developmental tendency is that establish a reasonable taxonomysystem according with natural phylogenetic relationship using molecular biologytechnique. Systematics was studied in some groups of Rhytismataceae from morphology,developmental biology, ecology, distribution, culture and RAPD (randomly amplifiedpolymorphic DNA) analysis etc. Phylogenetic relationship between intraspecies andsimilar interspecies of Lophodermium was studied firstly using RAPD-PCR technology. Itis clear further to the phylogenetic relationship among those groups. The thesis includedthree parts. The first part: The Rhytismataceae dry samples which came from all of China anddeposited in AAUFP (Anhui Agricultural University Forest Protection) were systematiclystudied from morphology, developmental biology, ecology and distribution etc. 10 speciesamong 6 genera were checked and their dichotomous keys were compiled. The resultincluded a new species——Lophodermium tortuosum sp. nov. and a China newrecord——Rhytisma umbonatum. The studies of 8 known-species include addingcharacters of morphology and developmental biology, the new hosts-record anddistributions. The 8 species' names are Coccomyces sichuanensis, Hypoderma commune,Lophodermium himalayense, L. minus, Ploioderma handelii, P. pini-armandii, Rhytismapunctatum and Soleella chinensis. The second part: Isolation, purification and cultivation of some Lophodermiumspecies. 59 fungi fresh materials collected from the mountains of South Anhui and Hefeicity were isolated and purified. Among them, 29 strains' cultivating characters werestudied. The cultures' characters such as colony shape, color, structure, growth rate, and thecapability of produce coloring matter and conidia etc. were observed and recordedperiodically. The chromophotographs of pure cultures were screened at the same time. Thecharacters of 16 represent strains cultured in single colony were recorded as data for thefurther classification research based on phenotypic-molecular characters. The third part: RAPD analysis of intraspecies and similar interspecies ofLophodermium, which included the construction and optimation of RAPD-PCR amplifiedsystem and RAPD analysis of 34 Lophodermium strains. The RAPD-PCR amplifiedsystem was optimized from aspects of the dosage of template DNA, prime, Mg2+, dNTPsand Taq enzyme, anneal temperature and time, elongation temperature and time, cyclenumber. The amplified diagram is quit well. The final reaction system (25μl): includedddH2O 14.2μl, 10×Buffer 2.5μl, Mg2+ (25mmol·L-1) 2μl, dNTPs (10mmol·L-1) 1.5μl, primer(12.5μmol·L-1) 0.6μl, Taq enzyme (5U·μl-1) 0.2μl, template DNA (100ng·μl-1) 1μl. Itsamplified procedure is: Pre-denature at 95℃ for 5 min. Cycle 50 times, every cycle include:denature at 94℃ for 1 min, anneal at 36℃ for 100s, elongation at 72℃ for 140s. And thelatest cycle elongation time is 10min. Template DNA extracted by Benzyl Chloride and its RAPD-PCR amplified by theoptimal procedure. 16 strains on conifers amplified by 9 primers were acquired 95 DNAgene fingerprint diagram. And 18 strains on broadleaf trees were amplified by 9 primersacquired 122 DNA gene fingerprint diagram. Its genetic distance cluster figuresconstructed by DPS Statistics software using UPGMA method. From the clustering figureswe can found: the strains of intraspecies and similar interspecies of Lophodermium cancluster very well, and the strains of different species also can distinct quite well. TheRAPD consequence can reflect the relationship between intraspecies and similarinterspecies of Lophodermium from the level of gene. From the results we can concludethat RAPD technique has good applicability in the study of systemati...
|
PDF Full Text Request