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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Research Project #439531

Research Project: Assembly and Annotation of Rumen Protist Genomes to Determine Their Metabolic Contribution and Environmental Presence

Location: Cell Wall Biology and Utilization Research

Project Number: 5090-31000-028-046-I
Project Type: Interagency Reimbursable Agreement

Start Date: Feb 1, 2021
End Date: Jan 31, 2025

1. Sequence enriched rumen protist samples from four cannulated Holstein cows with HiFi reads. Assemble sequence reads into reference quality contigs, and polish assemblies with short reads. Generate Hi-C sequence data to bin contigs into full genome assemblies. 2. Annotate protist reference genomes and perform functional genetic analysis. Use Hi-C data to associate protists with the bacteria that they consume. Perform functional gene analysis on bacteria associated with protist predation and identify the impacts that each bacterial taxon may have on rumen metabolic function. 3. Develop a high throughput assay for rumen protist species using the genome assemblies developed in aim 1. Apply this assay to eight cannulated cows to test its efficacy. Concurrently, test the ability of the assay to detect protist DNA in environmental samples to identify potential contact surfaces that may spread infection to other cattle.

The protist population of the cattle rumen has been implicated in the increase of methane emissions and the reduction in animal feed efficiency; however, these microorganisms lack the genetic resources needed to fully characterize their contribution to these two phenomena. Furthermore, there are no suitable assays to detect individual protist species in the cattle rumen and protist content must currently be assessed via light microscopy. Therefore, we will generate reference quality assemblies for the rumen protist populations to aid further research into their ecological significance. To reduce the difficulty in separating protist genomes from bacterial and archaeal genomes in silico, we have developed a method of low-g centrifugation that greatly enriches the protist composition of a rumen sample. We will then sequence existing protist enrichment samples to generate high fidelity (HiFi) reads with low error rates. We will then assemble HiFi reads into contigs and annotate those contigs with gene loci. We will then use Hi-C read data to bin protist contigs and to identify potential associations with bacteria that would suggest a predator-prey relationship. This data will be further explored to identify the metabolic impact of rumen protist presence in the rumen, by identifying the metabolic contribution of prey bacteria to the rumen ecosystem. Finally, we will develop markers that distinguish between rumen protist species and use them in amplicon sequencing experiments on eight cannulated Holstein cows. After validating these markers, we will then test environmental samples in the vicinity of these cows to identify potential surfaces that could spread protists to other animals.