Hindawi Publishing Corporation Genetics Research International Volume 2013, Article ID 980191, 7 pages http://dx.doi.org/10.1155/2013/980191 Research Article Molecular Genetic Diversity and Quantitation of Methanogen in Ruminal Fluid of Buffalo (Bubalus bubalis) Fed Ration (Wheat Straw and Concentrate Mixture Diet) K. M. Singh, 1,2 A. K. Tripathi, 2 P. R. Pandya, 3 S. Parnerkar, 3 R. K. Kothari, 4 and C. G. Joshi 2 1 P. G. Department of Genetics, ARIBAS, New V V Nagar, Anand, Gujarat 388121, India 2 Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat 388 001, India 3 Animal Nutrition Research Station, AAU, Anand, Gujarat 388001, India 4 Department of Microbiology, Christ College, Rajkot, Gujarat 360 005, India Correspondence should be addressed to K. M. Singh; kmsingh18@gmail.com Received 27 February 2013; Revised 13 May 2013; Accepted 16 May 2013 Academic Editor: Jorge H. Leit˜ ao Copyright © 2013 K. M. Singh et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. High roughage diet causes more methane emissions; however, the total methanogen abundance is not infuenced by roughage proportion. Technologies to reduce methane emissions are lacking, and development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on present knowledge of the methanogens. In this work, we have investigated molecular diversity of rumen methanogens of Surti bufalo. DNA from rumen fuid was extracted, and 16S rRNA encoding genes were amplifed using methanogen specifc primer to generate 16S rDNA clone libraries. Seventy-six clones were randomly selected and analysed by RFLP resulting in 21 operational taxonomic units (OTUs). BLAST analysis with available sequences in database revealed sequences of 13 OTUs (55 clones) showing similarity with Methanomicrobium sp, 3 OTUs (15 clones) with Methanobrevibacter sp. Te remaining 5 OTUs (6 clones) belonged to uncultured archaea. Te phylogenetic analysis indicated that methanogenic communities found in the library were clustered in the order of Methanomicrobiales (18 OTUs) and Methanobacteriales (3 OTUs). Te population of Methanomicrobiales, Methanobacteriales, and Methanococcales were also observed, accounting for 1.94%, 0.72%, and 0.47% of total archaea, respectively. 1. Introduction Methanogens, members of the domain Archaea, fall within the kingdom euryarchaeota [1]. Tey are obligate anaerobes and can be unmistakably diferentiated from other organisms since they all produce methane as a major catabolic end- product [2]. Te most common species of methanogens isolated from the rumen are Methanobrevibacter ruminan- tium, Methanomicrobium mobile, Methanobacterium formici- cum, Methanobacterium smithii, Methanobacterium olleyae, Methanobacterium bryantii, Methanosarcina barkeri, and Methanoculleus olentangyi [3–9]. Te diversity of archaea found in the rumen has been recently reviewed [10–12]. Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that enteric methane emission is a major source of greenhouse gas in agriculture sector. Currently, India possesses the world’s largest livestock population of 485 million, which accounts for 13% of the global livestock population (Intergovernmental Panel on Climate Change, 2001). It has 57% of the world’s bufalo and 16% of the cattle population. Bufalo contributes to 42% of the total methane emission by livestock in India [13]. Reducing enteric methane emissions has been identifed as one way of lowering global methane emissions. However, the efectiveness of any strategy that reduces greenhouse gas emissions and also increases production or nutritional efciency will likely depend upon having an understanding of the numbers and/or distribution of methanogen species among ruminant livestock. In the present study, diversity analysis of methanogen consortium