Rapid PCR-based method for detection and differentiation of Didymiaceae and Physaraceae (myxomycetes) in environmental samples.
Ecological studies of myxomycetes have been limited by the absence of universal cultivation techniques and the lack of life stage independent identification methods. We designed a novel PCR primer pair for the specific amplification of small subunit ribosomal RNA gene of Didymiaceae and Physaraceae. The primers produced amplicons from 192 fruiting body samples belonging to 10 genera. Twenty-four samples yielded longer fragments and sequence analysis revealed the presence of intron(s). As for the exonic regions, while sequence heterogeneities within a single species/varietas/forma were frequently observed, identical sequences were obtained only from identical species/varietas. The effectiveness of this primer pair in the analysis of morphologically unidentifiable samples was confirmed with the applications to samples of environmental plasmodium/sclerotium and soil. Denaturing gradient gel electrophoresis analysis was also tested with the soil samples. The results presented here demonstrate this PCR-based method can facilitate further ecological studies of Physaraceae and Didymiaceae in the environment.
Kamono
Akiko
A
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, Hachioji, Tokyo 192-0397, Japan. kamono@lowtem.hokudai.ac.jp <kamono@lowtem.hokudai.ac.jp>
Fukui
Manabu
M
D000818:Animals; D017931:DNA Primers; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D004783:Environmental Microbiology; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009235:Myxomycetes / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S