Abstract
Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6–V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to16S rRNA gene references produced taxonomic identification to Order level including 2–3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19–35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10–18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6–V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.
Highlights
Methanogenic archaea are part of the anaerobic microbial community of the rumen
The production of methane by ruminants represents a loss of energy to the animal [2], and with 3.6 billion domestic ruminants and 1.1 billion sheep globally enteric methane is believed to be a significant contributor to anthropogenic global greenhouse gas (GHG) emissions [3]
The assembly of the Sanger amplicon sequenced 16S rRNA amplicon (SA rrn) sequences produced 203 contigs of sequence length .1000 nt after vector removal, and clustering produced a set of 21 operational taxonomic units (OTUs) (RINH01RINH21; Table 1)
Summary
Methanogenic archaea are part of the anaerobic microbial community of the rumen. Though less abundant than the ruminal bacteria they have received a great deal of attention due to their ability to synthesise methane. The production of methane by ruminants represents a loss of energy to the animal [2], and with 3.6 billion domestic ruminants and 1.1 billion sheep globally enteric methane is believed to be a significant contributor to anthropogenic global greenhouse gas (GHG) emissions [3]. Even at a national level, methane production from sheep has been recognised as a significant challenge to meeting proposed targets of lowering GHG emissions by 20% by 2020 [5]. Assessment of the archaeal community is a prerequisite for rational manipulation of the ruminal microbiota to lower methane emissions
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