Diversity and evolution of 11 innate immune genes in Bos taurus taurus and Bos taurus indicus cattle

The Toll-like receptor (TLR) and peptidoglycan recognition protein 1 (PGLYRP1) genes play key roles in the innate immune systems of mammals. While the TLRs recognize a variety of invading pathogens and induce innate immune responses, PGLYRP1 is directly microbicidal. We used custom allele-specific assays to genotype and validate 220 diallelic variants, including 54 nonsynonymous SNPs in 11 bovine innate immune genes (TLR1-TLR10, PGLYRP1) for 37 cattle breeds. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and we were unable to differentiate between the specialized B. t. taurus beef and dairy breeds, despite an average polymorphism density of one locus per 219 bp. Ninety-nine tagSNPs and one tag insertion-deletion polymorphism were sufficient to predict 100% of the variation at all 11 innate immune loci in both subspecies and their hybrids, whereas 58 tagSNPs captured 100% of the variation at 172 loci in B. t. taurus. PolyPhen and SIFT analyses of nonsynonymous SNPs encoding amino acid replacements indicated that the majority of these substitutions were benign, but up to 31% were expected to potentially impact protein function. Several diversity-based tests provided support for strong purifying selection acting on TLR10 in B. t. taurus cattle. These results will broadly impact efforts related to bovine translational genomics

Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several avenues of bovine translational genomics, and the potential for marker-assisted vaccination

Genome-Wide Polymorphism and Comparative Analyses in the White-Tailed Deer (Odocoileus virginianus): A Model for Conservation Genomics

The white-tailed deer (Odocoileus virginianus) represents one of the most successful and widely distributed large mammal species within North America, yet very little nucleotide sequence information is available. We utilized massively parallel pyrosequencing of a reduced representation library (RRL) and a random shotgun library (RSL) to generate a complete mitochondrial genome sequence and identify a large number of putative single nucleotide polymorphisms (SNPs) distributed throughout the white-tailed deer nuclear and mitochondrial genomes. A SNP validation study designed to test specific classes of putative SNPs provides evidence for as many as 10,476 genome-wide SNPs in the current dataset. Based on cytogenetic evidence for homology between cow (Bos taurus) and white-tailed deer chromosomes, we demonstrate that a divergent genome may be used for estimating the relative distribution and density of de novo sequence contigs as well as putative SNPs for species without draft genome assemblies. Our approach demonstrates that bioinformatic tools developed for model or agriculturally important species may be leveraged to support next-generation research programs for species of biological, ecological and evolutionary importance. We also provide a functional annotation analysis for the de novo sequence contigs assembled from white-tailed deer pyrosequencing reads, a mitochondrial phylogeny involving 13,722 nucleotide positions for 10 unique species of Cervidae, and a median joining haplotype network as a putative representation of mitochondrial evolution in O. virginianus. The results of this study are expected to provide a detailed template enabling genome-wide sequence-based studies of threatened, endangered or conservationally important non-model organisms

A Multi-Platform Draft <i>de novo</i> Genome Assembly and Comparative Analysis for the Scarlet Macaw (<i>Ara macao</i>)

<div><p>Data deposition to NCBI Genomes</p><p>This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMXX00000000 (SMACv1.0, unscaffolded genome assembly). The version described in this paper is the first version (AMXX01000000). The scaffolded assembly (SMACv1.1) has been deposited at DDBJ/EMBL/GenBank under the accession AOUJ00000000, and is also the first version (AOUJ01000000). Strong biological interest in traits such as the acquisition and utilization of speech, cognitive abilities, and longevity catalyzed the utilization of two next-generation sequencing platforms to provide the first-draft <i>de novo</i> genome assembly for the large, new world parrot <i>Ara macao</i> (Scarlet Macaw). Despite the challenges associated with genome assembly for an outbred avian species, including 951,507 high-quality putative single nucleotide polymorphisms, the final genome assembly (>1.035 Gb) includes more than 997 Mb of unambiguous sequence data (excluding N’s). Cytogenetic analyses including ZooFISH revealed complex rearrangements associated with two scarlet macaw macrochromosomes (AMA6, AMA7), which supports the hypothesis that translocations, fusions, and intragenomic rearrangements are key factors associated with karyotype evolution among parrots. <i>In silico</i> annotation of the scarlet macaw genome provided robust evidence for 14,405 nuclear gene annotation models, their predicted transcripts and proteins, and a complete mitochondrial genome. Comparative analyses involving the scarlet macaw, chicken, and zebra finch genomes revealed high levels of nucleotide-based conservation as well as evidence for overall genome stability among the three highly divergent species. Application of a new whole-genome analysis of divergence involving all three species yielded prioritized candidate genes and noncoding regions for parrot traits of interest (i.e., speech, intelligence, longevity) which were independently supported by the results of previous human GWAS studies. We also observed evidence for genes and noncoding loci that displayed extreme conservation across the three avian lineages, thereby reflecting their likely biological and developmental importance among birds.</p></div

A Draft <i>De Novo</i> Genome Assembly for the Northern Bobwhite (<i>Colinus virginianus</i>) Reveals Evidence for a Rapid Decline in Effective Population Size Beginning in the Late Pleistocene

<div><p>Wild populations of northern bobwhites (<i>Colinus virginianus</i>; hereafter bobwhite) have declined across nearly all of their U.S. range, and despite their importance as an experimental wildlife model for ecotoxicology studies, no bobwhite draft genome assembly currently exists. Herein, we present a bobwhite draft <i>de novo</i> genome assembly with annotation, comparative analyses including genome-wide analyses of divergence with the chicken (<i>Gallus gallus</i>) and zebra finch (<i>Taeniopygia guttata</i>) genomes, and coalescent modeling to reconstruct the demographic history of the bobwhite for comparison to other birds currently in decline (i.e., scarlet macaw; <i>Ara macao</i>). More than 90% of the assembled bobwhite genome was captured within <40,000 final scaffolds (N50 = 45.4 Kb) despite evidence for approximately 3.22 heterozygous polymorphisms per Kb, and three annotation analyses produced evidence for >14,000 unique genes and proteins. Bobwhite analyses of divergence with the chicken and zebra finch genomes revealed many extremely conserved gene sequences, and evidence for lineage-specific divergence of noncoding regions. Coalescent models for reconstructing the demographic history of the bobwhite and the scarlet macaw provided evidence for population bottlenecks which were temporally coincident with human colonization of the New World, the late Pleistocene collapse of the megafauna, and the last glacial maximum. Demographic trends predicted for the bobwhite and the scarlet macaw also were concordant with how opposing natural selection strategies (i.e., skewness in the <i>r</i>-/<i>K</i>-selection continuum) would be expected to shape genome diversity and the effective population sizes in these species, which is directly relevant to future conservation efforts.</p></div

Chicken-Scarlet Macaw (<i>Ara macao</i>) Comparative Chromosome Painting (ZooFISH).

<p>Using chicken flow sorted macrochromosomes (GGA1-GGA9) as well as GGAZ and GGAW, the homologous chromosome segments of the scarlet macaw were established via fluorescent <i>in situ</i> hybridization. All flow sorted probes were validated via hybridization to chicken metaphase spreads (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062415#pone.0062415.s001" target="_blank">Figure S1</a>).</p

Relationship Between Total Contig Length (Kbp) and Total Contig Number for the Scaffolded Scarlet Macaw (<i>Ara macao</i>) Genome (SMACv1.1).

<p>The y-axis represents total contig length, expressed in kilobase pairs (Kbp), whereas the x-axis represents the total number of scaffolded contigs. Based on the estimated size of the scarlet macaw genome (1.11–1.16 Gbp), ≥90% of the assembled genome was captured within approximately 95,000 contigs.</p

Whole Genome Analysis of Divergence.

<p><b>(A)</b> Genome-wide nucleotide-based divergence (CorrectedForAL) between the scarlet macaw (<i>Ara macao</i>; simple <i>de novo</i> assembly) and chicken genomes (<i>Gallus gallus</i> 2.1). <b>(B)</b> Genome-wide nucleotide-based divergence (CorrectedForAL) between the scarlet macaw (<i>Ara macao</i>; simple <i>de novo</i> assembly) and zebra finch genomes (<i>Taeniopygia guttata</i> 1.1, 3.2.4). Each histogram represents the full, ordered distribution of the composite variable defined as: . The observed ranges of the composite variable for pane <b>(A)</b> and pane <b>(B)</b> were 3.89591E-05–0.052631579, and 3.33792E-05–0.052631579, respectively. The left edges of the distributions represent extreme conservation, whereas the right edges indicate extreme putative divergence. Distributional outliers were predicted using a percentile-based approach (99.98^th and 0.02^th) to construct interval bounds capturing >99.9% of the total data points in each ordered distribution.</p

SMACv1.0 simple <i>de novo</i> outlier contigs from a genome-wide analysis of divergence with the chicken.

<p>SMACv1.0 simple <i>de novo</i> outlier contigs from a genome-wide analysis of divergence with the chicken.</p

Summary data for the scarlet macaw first-generation draft <i>de novo</i> genome assembly with comparison to the initial turkey and chicken genome assemblies.

<p>Summary data for the scarlet macaw first-generation draft <i>de novo</i> genome assembly with comparison to the initial turkey and chicken genome assemblies.</p