Diversity analysis of methanogens in rumen of Bubalus bubalis by 16S riboprinting and sequence analysis

The molecular diversity of rumen methanogens was investigated by 16S rDNA gene library prepared from the rumen contents obtained from Murrah buffaloes in India. Genomic DNA was isolated from adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the positive clones were selected assuming based on blue–white screening and sequenced. Positive clones were reamplified and the resulting PCR products were further subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) by using HaeIII enzyme. A total of 108 clones were examined, and the analysis revealed 16 phylotypes. Out of sixteen phylotypes, nine phylotypes belong to the uncultured group of methanogens, and the rest of seven phylotypes belong to the order Methanomicrobiales, Methanococcales and Methanobacteriales. Out of the 108 rDNA clones, 66 clones which constitute 61.1% of the total clone representing 9 phylotypes, show less than 97% sequence similarity with any of the cultured strain of methanogens. The second largest group of clones (24 clones) represented by four phylotypes show a sequence similarity ranging from 91% to 99% with Methanomicrobium mobile strain of methanogens. The third group of 16S rDNA clones clustered along with M. burtonii strain of methanogens. This group consists of 6 clones and constitutes about 5.5% of the total clones and represented by only single phylotype. Fourth and fifth clusters of 16S rDNA clones consist of 5 and 7 clones respectively, and these were matched with Methanobrevibacter gottschalkii and Methanobrevibacter rumanatium strain of methanogens and constitute about 4.6% and 6.4% of the total clones.

► We have investigated diversity of rumen methanogens in Murrah buffalo.
► A total of 108 clones were examined for diversity.
► Analysis revealed 16 phylotypes of methanogens.
► The largest group matched with uncultured methanogens.
► Others group present were M. mobile, M. burtonii, M. gottschalkii and M. rumanatium.