Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with high-altitude pulmonary hypertension

Abstract

The availability of whole genome sequence (WGS) data has made it possible to discover protein variants in silico. However, existing bovine WGS databases do not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in U.S. beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene ( EPAS1), a gene associated with high-altitude pulmonary hypertension in Angus cattle. The identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded by EPAS1 (E270Q, P362L, A671G, and L701F) and confirm two variants previously associated with disease (A606T and G610S). The six EPAS1 missense mutations were verified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact of EPAS1 alleles on the adaptive response to chronic hypoxia in U.S. cattle. This public, whole genome resource facilitates in silico identification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.

Using sheep genomes from diverse U.S. breeds to identify missense variants in genes affecting fecundity

“…DNA was extracted from whole blood with a typical phenol:chloroform method and stored at 4°C in 10 mM TrisCl, 1 mM EDTA (pH 8.0) as previously described 16 . Library preparation for DNA sequencing was also accomplished as previously described 9 . Briefly, 2 μg of ovine genomic DNA was fragmented and used to make indexed, 500 bp, paired-end libraries.…”

“…Pooled libraries with compatible indexes were repeatedly sequenced until 40 GB of data with greater than Q20 quality was collected for each ram, thereby producing at least 10-fold mapped read coverage for each index. This level of coverage provides scoring rates and accuracies that exceed 99% 9 , 18 . The DNA sequence alignment process was similar to that previously reported 18 .…”

“…The nucleotide variation in the exon regions of GDF9, BMP15, and BMPR1B was visualized through the public access portal at ARS USMARC with open source software installed on a laptop computer. Variants were recorded manually in a spreadsheet as previously described 9 . Briefly, a Java Runtime Environment version 8, update 131 (Oracle Corporation, Redwood Shores, CA) was first installed on the computer.…”

“…We previously showed in cattle that protein variants for a gene of interest may be identified in silico with the appropriate population sample and 14x WGS datasets 9 . To that end, we created a similar publicly accessible, 16x WGS resource of 96 rams, that is viewable online with IGV.…”

Haplotype-Resolved Cattle Genomes Provide Insights Into Structural Variation and Adaptation

“…38 individuals with ~10x WGS short read Illumina data representing seven breeds were selected from the USMARC Beef Diversity Panel version 2.9 (MBCDPv2.9) 49 . The individuals selected for the panel were bulls with minimal pedigree relationships to maximize sampling of diverse alleles suitable for population genetics studies.…”