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Previous shotgun metagenomic analyses of ruminal digesta identified some microbial information that might be useful as biomarkers to select cattle that emit less methane (CH 4 ), which is a potent greenhouse gas. It is known that methane production (g/kgDMI) and to an extent the microbial community is heritable and therefore biomarkers can offer a method of selecting cattle for low methane emitting phenotypes. In this study a wider range of  Bos Taurus  cattle, varying in breed and diet, was investigated to determine microbial communities and genetic markers associated with high/low CH 4  emissions. Digesta samples were taken from 50 beef cattle, comprising four cattle breeds, receiving two basal diets containing different proportions of concentrate and also including feed additives (nitrate or lipid), that may influence methane emissions. A combination of partial least square analysis and network analysis enabled the identification of the most significant and robust biomarkers of CH 4  emissions (VIP > 0.8) across diets and breeds when comparing all potential biomarkers together. Genes associated with the hydrogenotrophic methanogenesis pathway converting carbon dioxide to methane, provided the dominant biomarkers of CH 4  emissions and methanogens were the microbial populations most closely correlated with CH 4  emissions and identified by metagenomics. Moreover, these genes grouped together as confirmed by network analysis for each independent experiment and when combined. Finally, the genes involved in the methane synthesis pathway explained a higher proportion of variation in CH 4  emissions by PLS analysis compared to phylogenetic parameters or functional genes. These results confirmed the reproducibility of the analysis and the advantage to use these genes as robust biomarkers of CH 4  emissions. Volatile fatty acid concentrations and ratios were significantly correlated with CH 4 , but these factors were not identified as robust enough for predictive purposes. Moreover, the methanotrophic  Methylomonas  genus was found to be negatively correlated with CH 4 . Finally, this study confirmed the importance of using robust and applicable biomarkers from the microbiome as a proxy of CH 4  emissions across diverse production systems and environments.

Identification, Comparison, and Validation of Robust Rumen Microbial Biomarkers for Methane Emissions Using Diverse Bos Taurus Breeds and Basal Diets