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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Publications at this Location » Publication #412508

Research Project: Developing Strategies to Improve Dairy Cow Performance and Nutrient Use Efficiency with Nutrition, Genetics, and Microbiology

Location: Cell Wall Biology and Utilization Research

Title: Whole genome sequences of two nitrate-reducing strains of Selenomonas ruminantium isolated from the rumen

item COSTELLO, MARGARET - University Of Wisconsin
item RICKE, STEVEN - University Of Wisconsin
item McClure, Jennifer
item Anderson, Robin

Submitted to: Midwestern Section of the American Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: 12/13/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Selenomonas ruminantium is a functionally diverse, non-fibrolytic member of the rumen microbial community with two subspecies: lactilytica and ruminantium. Some strains of S. ruminantium can reduce nitrate and nitrite, which may be associated with ruminal fiber degradation and, thus, higher forage diets. Most work done with S. ruminantium has been culture-based, with less genomic and functional data. Here, we performed whole genome sequencing with an Oxford Nanopore GridION on two isolated, nitrate-reducing strains of S. ruminantium to provide complete genomic data and determine these novel strains' nitrogen usage functional capacities. The two isolated strains, 223 and 231, were shipped on dry ice to the University of Wisconsin-Madison, where they were grown, pelleted, and observed under a microscope to determine purity. The samples were processed with a three-minute bead-beating step before extraction with the Zymo Quick-DNA High Molecular Weight MagBead Kit. Subsequently, the extracted samples underwent a quality assessment step before moving into library preparation for Oxford Nanopore sequencing. The two libraries were then loaded onto two R9.4.1 flow cells per library and were sequenced on an Oxford Nanopore GridION for 72 hours. The data were extracted from the GridION, and the combined passed reads from each of the two flow cells per library were assembled with Canu and Flye, long-read de novo genome assemblers designed for noisy, single-molecule sequences. The assembled genomes were compared to a reference S.ruminantium genome with Quast, a quality assessment tool that evaluates assembly accuracy. Finally, the assembled genomes with the highest accuracy were analyzed with prodigal to generate protein-coding gene predictions and then with BLAST to assign functional predictions. For each strain, a different assembly was used. The Canu assembly of strain 223 had higher accuracy rates at 3205.23 mismatches per 100 kbp, while the Flye assembly of strain 231 had 2494.40 mismatches per 100 kbp. The two strains had similar genome sizes: 3,098,553 base pairs for strain 223 and 3,138,614 base pairs for strain 231. Strain 223 had a lower GC content than strain 231 of 51.02%, while strain 231 had a GC content of 52.80%, which were both lower than the 50.68% GC content of the reference genome. These genomes provide a resource for understanding nitrate reduction in the rumen and work to build a better database for analyzing the rumen microbiome.