Application of Nested PCR-DGGE (Denaturing Gradient Gel Electrophoresis) for the Analysis of Ciliate Communities in Soils
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概要
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Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha-1 year-1 of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allowed for the phylogenetic identification of these ciliates at the class level.
- 日本微生物生態学会・日本土壌微生物学会の論文
著者
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KASAHARA YASUHIRO
Institute of Low Temperature Science, Hokkaido University
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Kasahara Yasuhiro
Institute Of Genetic Ecology Tohoku University
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Shimano Satoshi
Miyagi University of Education
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Sambe Mitsuo
Miyagi University of Education
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Shimano Satoshi
Miyagi Univ. Of Education
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Shimano Satoshi
Institute of Low Temperature Science, Hokkaido University
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Sambe Mitsuo
Institute of Low Temperature Science, Hokkaido University
関連論文
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- Detection of Effects of a High Trophic Level Predator, Sorex unguiculatus (Soricidae, Mammalia), on a Soil Microbial Community in a Cool Temperate Forest in Hokkaido, Using the ARISA Method
- Variability of sexual organ possession rates and phylogenetic analyses of a parthenogenetic Japanese earthworm, Amynthas vittatus (Oligochaeta: Megascolecidae)
- Earthworm Fauna of Daikokujima Island, North Japan, with a Redescription of Amynthas yunoshimensis (Hatai, 1930) : Annelida, Clitellata, Megascolecidae
- Linkage between Light Microscopic Observations and Molecular Analysis by Single-Cell PCR for Ciliates
- PB-05 Analysis of soil ciliates community using 18S rDNA(MICROBL COMMUNITY ANALYSIS,Session B,(1) Poster presentations)
- Effects of the Agar Concentration on Colony Formation of a Soil Bacterium
- Variability of sexual organ possession rates and phylogenetic analyses of a parthenogenetic Japanese earthworm, Amynthas vittatus (Oligochaeta : Megascolecidae)
- Application of Nested PCR-DGGE (Denaturing Gradient Gel Electrophoresis) for the Analysis of Ciliate Communities in Soils
- The Oligomeric States of the Photosystems and the Light-Harvesting Complexes in the Chl b-Less Mutant
- Application of Nested PCR-DGGE (Denaturing Gradient Gel Electrophoresis) for the Analysis of Ciliate Communities in Soils
- Corrigendum: Variability of sexual organ possession rates and phylogenetic analyses of a parthenogenetic Japanese earthworm, Amynthas vittatus (Oligochaeta: Megascolecidae) [Genes Genet. Syst. (2011) 86, p. 27-35]