Sharon M. Hickey

Background: Rumen microbes break down complex dietary carbohydrates into energy sources for the host and are increasingly shown to be a key aspect of animal performance. Host genotypes can be combined with microbial DNA sequencing to predict performance traits or traits related to environmental impact, such as enteric methane emissions. Metagenome profiles were generated from 3139 rumen samples, collected from 1200 dual purpose ewes, using restriction enzyme-reduced representation sequencing (RE-RRS). Phenotypes were available for methane (CH4) and carbon dioxide (CO2) emissions, the ratio of CH4 to CH4 plus CO2 (CH4Ratio), feed efficiency (residual feed intake: RFI), liveweight at the time of methane collection (LW), liveweight at 8 months (LW8), fleece weight at 12 months (FW12) and parasite resistance measured by faecal egg count (FEC1). We estimated the proportion of phenotypic variance explained by host genetics and the rumen microbiome, as well as prediction accuracies for each of these traits. Results: Incorporating metagenome profiles increased the variance explained and prediction accuracy compared to fitting only genomics for all traits except for CO2 emissions when animals were on a grass diet. Combining the metagenome profile with host genotype from lambs explained more than 70% of the variation in methane emissions and residual feed intake. Predictions were generally more accurate when incorporating metagenome profiles compared to genetics alone, even when considering profiles collected at different ages (lamb vs adult), or on different feeds (grass vs lucerne pellet). A reference-free approach to metagenome profiling performed better than metagenome profiles that were restricted to capturing genera from a reference database. We hypothesise that our reference-free approach is likely to outperform other reference-based approaches such as 16S rRNA gene sequencing for use in prediction of individual animal performance. Conclusions: This paper shows the potential of using RE-RRS as a low-cost, high-throughput approach for generating metagenome profiles on thousands of animals for improved prediction of economically and environmentally important traits. A reference-free approach using a microbial relationship matrix from log ₁₀ proportions of each tag normalized within cohort (i.e., the group of animals sampled at the same time) is recommended for future predictions using RE-RRS metagenome profiles.

The greatest source of global anthropogenic methane (CH₄) emissions is from ruminant livestock. Multiple mitigation strategies in livestock are currently being explored. Of these breeding for lower CH₄ emitting ruminants has the advantage of being permanent and cumulative and universally applicable to all classes of livestock. Here, we show that methane emissions can be predicted by the complex community of microbiota sampled from rumens enabling evaluation of systems and individuals. Furthermore, there is evidence that the microbial community is controlled not only be the feed substrate but also by the host itself and that selecting hosts that favour a microbial fermentation with lowered methane emissions changes the energy source to the animal, and in turn both rumen physiology and body composition. Current methods for obtaining microbial DNA and subsequent sequencing of an animal’s microbiome, however, are too expensive to implement in commercial selection programs. A methodology that offers fast, low-cost, high throughput profiling of rumen microbiomes using Genotyping-by-sequencing (GBS) has been developed using an unbiased reference free approach to group microbiota. To date, this has been applied to over 4000 sheep samples and validated in cattle. Results show that microbial profiles are heritable and correlated with methane emissions and feed intake. This research is part of a flagship program funded by the global research alliance to disseminate global access to technologies that lower greenhouse gas emissions in ruminant livestock.

Microbial community profiles have been associated with a variety of traits, including methane emissions in livestock, however, these profiles can be difficult and expensive to obtain for thousands of samples. The objective of this work was to develop a low-cost, high-throughput approach to capture the diversity of the rumen microbiome. Restriction enzyme reduced representation sequencing (RE-RRS) using ApeK I or Pst I, and two bioinformatic pipelines (reference-based and reference-free) were compared to 16S rRNA gene sequencing using repeated samples collected two weeks apart from 118 sheep that were phenotypically extreme (60 high and 58 low) for methane emitted per kg dry matter intake (n=236). DNA was extracted from freeze-dried rumen samples using a phenol chloroform and bead-beating protocol prior to sequencing. The resulting sequences were used to investigate the repeatability of the rumen microbial community profiles, the effect of host genetics, laboratory and analytical method, and the genetic and phenotypic correlations with methane production. The results suggested that the best method was Pst I RE-RRS analyzed with the reference-free approach via a correspondence analysis, with estimates for repeatability of 0.62±0.06, heritability 0.31±0.29, and genetic and phenotypic correlation with methane emissions of 0.88±0.25 and 0.64±0.05 respectively for the first component of correspondence analysis. The reference-free approach assigned 62.0±5.7% of reads to common 65 bp tags, much higher than the reference-based approach of 6.8±1.8% of reads assigned. Sensitivity studies suggested approximately 2000 samples could be sequenced in a single lane on an Illumina HiSeq 2500, therefore the current work of 118 samples/lane and future proposed 384 samples/lane are well within that threshold. Our approach is now being used to investigate host factors affecting the rumen and its association with a variety of production and environmental traits. With minor adaptations, our approach could be used to obtain microbial profiles from other metagenomic samples.