Introgression and disruption of migration routes have shaped the genetic integrity of wildebeest populations.

The blue wildebeest (Connochaetes taurinus) is a keystone species in savanna ecosystems from southern to eastern Africa, and is well known for its spectacular migrations and locally extreme abundance. In contrast, the black wildebeest (C. gnou) is endemic to southern Africa, barely escaped extinction in the 1900s and is feared to be in danger of genetic swamping from the blue wildebeest. Despite the ecological importance of the wildebeest, there is a lack of understanding of how its unique migratory ecology has affected its gene flow, genetic structure and phylogeography. Here, we analyze whole genomes from 121 blue and 22 black wildebeest across the genus' range. We find discrete genetic structure consistent with the morphologically defined subspecies. Unexpectedly, our analyses reveal no signs of recent interspecific admixture, but rather a late Pleistocene introgression of black wildebeest into the southern blue wildebeest populations. Finally, we find that migratory blue wildebeest populations exhibit a combination of long-range panmixia, higher genetic diversity and lower inbreeding levels compared to neighboring populations whose migration has recently been disrupted. These findings provide crucial insights into the evolutionary history of the wildebeest, and tangible genetic evidence for the negative effects of anthropogenic activities on highly migratory ungulates.

African bushpigs exhibit porous species boundaries and appeared in Madagascar concurrently with human arrival.

Several African mammals exhibit a phylogeographic pattern where closely related taxa are split between West/Central and East/Southern Africa, but their evolutionary relationships and histories remain controversial. Bushpigs (Potamochoerus larvatus) and red river hogs (P. porcus) are recognised as separate species due to morphological distinctions, a perceived lack of interbreeding at contact, and putatively old divergence times, but historically, they were considered conspecific. Moreover, the presence of Malagasy bushpigs as the sole large terrestrial mammal shared with the African mainland raises intriguing questions about its origin and arrival in Madagascar. Analyses of 67 whole genomes revealed a genetic continuum between the two species, with putative signatures of historical gene flow, variable FST values, and a recent divergence time (<500,000 years). Thus, our study challenges key arguments for splitting Potamochoerus into two species and suggests their speciation might be incomplete. Our findings also indicate that Malagasy bushpigs diverged from southern African populations and underwent a limited bottleneck 1000-5000 years ago, concurrent with human arrival in Madagascar. These results shed light on the evolutionary history of an iconic and widespread African mammal and provide insight into the longstanding biogeographic puzzle surrounding the bushpig's presence in Madagascar.

First gene-edited calf with reduced susceptibility to a major viral pathogen.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Colonialism in South Africa leaves a lasting legacy of reduced genetic diversity in Cape buffalo.

The iconic Cape buffalo has experienced several documented population declines in recent history. These declines have been largely attributed to the late 19th century rinderpest pandemic. However, the effect of the rinderpest pandemic on their genetic diversity remains contentious, and other factors that have potentially affected this diversity include environmental changes during the Pleistocene, range expansions and recent human activity. Motivated by this, we present analyses of whole genome sequencing data from 59 individuals from across the Cape buffalo range to assess present-day levels of genome-wide genetic diversity and what factors have influenced these levels. We found that the Cape buffalo has high average heterozygosity overall (0.40%), with the two southernmost populations having significantly lower heterozygosity levels (0.33% and 0.29%) on par with that of the domesticated water buffalo (0.29%). Interestingly, we found that these lower levels are probably due to recent inbreeding (average fraction of runs of homozygosity 23.7% and 19.9%) rather than factors further back in time during the Pleistocene. Moreover, detailed investigations of recent demographic history show that events across the past three centuries were the main drivers of the exceptional loss of genetic diversity in the southernmost populations, coincident with the onset of colonialism in the southern extreme of the Cape buffalo range. Hence, our results add to the growing body of studies suggesting that multiple recent human-mediated impacts during the colonial period caused massive losses of large mammal abundance in southern Africa.

Impact of Four Ovine TMEM154 Haplotypes on Ewes during Multiyear Lentivirus Exposure.

Polypeptide variation encoded by the ovine transmembrane protein 154 gene (TMEM154) is associated with susceptibility to ovine lentivirus, the causative agent of Ovine Progressive Pneumonia (OPP) and Visna/Maedi. Our aim was to compare the four most prevalent TMEM154 haplotypes on the incidence of infection and ewe productivity during natural multiyear virus exposure. Prospective cohort studies were designed to test gene action and estimate effects of TMEM154 haplotypes encoding distinctive variant residues: K35 ("1"), I70 ("2"), ancestral ("3"), and A4del/M44 ("4"). Exposure consisted of co-mingling infected ewes at a rate greater than 30% with serological status evaluated every four months. For ewes with one or two copies of the highly susceptible haplotypes "2" and "3", the infection prevalence steadily increased to nearly 100% at 55 months. Haplotypes "2" and "3" were equally susceptible and dominant to haplotype "1". A difference was not detected (p < 0.53) in the magnitude of effect with haplotype combinations of "1" and "4". The ewe infection prevalence with "1,1"; "1,4"; and "4,4" was 10% to 40% at 55 months. The latter suggested that two copies of the K35 amino acid substitution ("1") were as effective as a homozygous TMEM154 "knockout" with the frame-shift deletion mutation ("4") in reducing infection susceptibility. When considering ewe reproductive performance, a difference was not detected when comparing haplotypes "2", and "3" to each other, or "1" and "4" to each other. Our study indicated that ewes with two copies of the severely truncated versions of TMEM154 ("4,4") had normal lamb productivity. Without complete understanding of the natural function of TMEM154 our recommendations to producers interested in using TMEM154 selection to reduce their flock's genetic predisposition to OPP are encouraged to increase the frequency of TMEM154 haplotype K35 ("1") since it encodes a full-length protein with minimal difference to the ancestral polypeptide.

Structural variant-based pangenome construction has low sensitivity to variability of haplotype-resolved bovine assemblies.

Advantages of pangenomes over linear reference assemblies for genome research have recently been established. However, potential effects of sequence platform and assembly approach, or of combining assemblies created by different approaches, on pangenome construction have not been investigated. Here we generate haplotype-resolved assemblies from the offspring of three bovine trios representing increasing levels of heterozygosity that each demonstrate a substantial improvement in contiguity, completeness, and accuracy over the current Bos taurus reference genome. Diploid coverage as low as 20x for HiFi or 60x for ONT is sufficient to produce two haplotype-resolved assemblies meeting standards set by the Vertebrate Genomes Project. Structural variant-based pangenomes created from the haplotype-resolved assemblies demonstrate significant consensus regardless of sequence platform, assembler algorithm, or coverage. Inspecting pangenome topologies identifies 90 thousand structural variants including 931 overlapping with coding sequences; this approach reveals variants affecting QRICH2, PRDM9, HSPA1A, TAS2R46, and GC that have potential to affect phenotype.

Gaur genome reveals expansion of sperm odorant receptors in domesticated cattle.

BACKGROUND: The gaur (Bos gaurus) is the largest extant wild bovine species, native to South and Southeast Asia, with unique traits, and is listed as vulnerable by the International Union for Conservation of Nature (IUCN). RESULTS: We report the first gaur reference genome and identify three biological pathways including lysozyme activity, proton transmembrane transporter activity, and oxygen transport with significant changes in gene copy number in gaur compared to other mammals. These may reflect adaptation to challenges related to climate and nutrition. Comparative analyses with domesticated indicine (Bos indicus) and taurine (Bos taurus) cattle revealed genomic signatures of artificial selection, including the expansion of sperm odorant receptor genes in domesticated cattle, which may have important implications for understanding selection for male fertility. CONCLUSIONS: Apart from aiding dissection of economically important traits, the gaur genome will also provide the foundation to conserve the species.

An improved ovine reference genome assembly to facilitate in-depth functional annotation of the sheep genome.

BACKGROUND: The domestic sheep (Ovis aries) is an important agricultural species raised for meat, wool, and milk across the world. A high-quality reference genome for this species enhances the ability to discover genetic mechanisms influencing biological traits. Furthermore, a high-quality reference genome allows for precise functional annotation of gene regulatory elements. The rapid advances in genome assembly algorithms and emergence of sequencing technologies with increasingly long reads provide the opportunity for an improved de novo assembly of the sheep reference genome. FINDINGS: Short-read Illumina (55× coverage), long-read Pacific Biosciences (75× coverage), and Hi-C data from this ewe retrieved from public databases were combined with an additional 50× coverage of Oxford Nanopore data and assembled with canu v1.9. The assembled contigs were scaffolded using Hi-C data with Salsa v2.2, gaps filled with PBsuitev15.8.24, and polished with Nanopolish v0.12.5. After duplicate contig removal with PurgeDups v1.0.1, chromosomes were oriented and polished with 2 rounds of a pipeline that consisted of freebayes v1.3.1 to call variants, Merfin to validate them, and BCFtools to generate the consensus fasta. The ARS-UI_Ramb_v2.0 assembly is 2.63 Gb in length and has improved continuity (contig NG50 of 43.18 Mb), with a 19- and 38-fold decrease in the number of scaffolds compared with Oar_rambouillet_v1.0 and Oar_v4.0. ARS-UI_Ramb_v2.0 has greater per-base accuracy and fewer insertions and deletions identified from mapped RNA sequence than previous assemblies. CONCLUSIONS: The ARS-UI_Ramb_v2.0 assembly is a substantial improvement in contiguity that will optimize the functional annotation of the sheep genome and facilitate improved mapping accuracy of genetic variant and expression data for traits in sheep.

UK survey demonstrates a wide range of impacts attributable to clinical librarian services.

OBJECTIVE: To understand the impact of the UK Clinical Librarian (CL) workforce and benchmark the results against a study undertaken in the North West region of the English National Health Service (NHS). METHODS: An online survey was distributed by CLs to their service users regarding literature searches that had been carried out on their behalf in the 6 months from April to October 2017. Interviews were later carried out in person with selected respondents to the questionnaires. RESULTS: CLs across the UK contribute to a wide range of outcomes, with 41% of search requests contributing to the choice of intervention, and 41% also to the advice offered by the clinician requester to a patient or their carer. These results are in line with the previous work undertaken in the North West. DISCUSSION: CLs provide diverse services to clinical teams. They support the continuing professional development and personal research needs of team members, service development needs of organisations, and the information provided contributes to improved quality and safety of patient care. CONCLUSION: The survey confirms the findings of the earlier NW study. It demonstrates the impact of services based around literature searching on patient care.

Using whole genome sequence to compare variant callers and breed differences of US sheep.

As whole genome sequence (WGS) data sets have become abundant and widely available, so has the need for variant detection and scoring. The aim of this study was to compare the accuracy of commonly used variant calling programs, Freebayes and GATK HaplotypeCaller (GATK-HC), and to use U.S. sheep WGS data sets to identify novel breed-associated SNPs. Sequence data from 145 sheep consisting of 14 U.S. breeds were filtered and biallelic single nucleotide polymorphisms (SNPs) were retained for genotyping analyses. Genotypes from both programs were compared to each other and to genotypes from bead arrays. The SNPs from WGS were compared to the bead array data with breed heterozygosity, principal component analysis and identifying breed associated SNPs to analyze genetic diversity. The average sequence read depth was 2.78 reads greater with 6.11% more SNPs being identified in Freebayes compared to GATK-HC. The genotype concordance of the variant callers to bead array data was 96.0% and 95.5% for Freebayes and GATK-HC, respectively. Genotyping with WGS identified 10.5 million SNPs from all 145 sheep. This resulted in an 8% increase in measured heterozygosity and greater breed separation in the principal component analysis compared to the bead array analysis. There were 1,849 SNPs identified in only the Romanov sheep where all 10 rams were homozygous for one allele and the remaining 135 sheep from 13 breeds were homozygous for the opposite allele. Both variant calling programs had greater than 95% concordance of SNPs with bead array data, and either was suitably accurate for ovine WGS data sets. The use of WGS SNPs improved the resolution of PCA analysis and was critical for identifying Romanov breed-associated SNPs. Subsets of such SNPs could be used to estimate germplasm composition in animals without pedigree information.

Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle.

Background: Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. Methods: A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar's test. Results: Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene ( ARRDC3), and the nuclear factor IA gene ( NFIA, mid- p-values, 1x10 -8 and 2x10 -7, respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI 95 = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others ( p-value = 1×10 -7, CI 95 = 4-206). A missense variant in ARRDC3 (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Conclusions: Although pathogenic roles for variants in the ARRDC3 and NFIA genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.

Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance.

Bovine viral diarrhea virus's (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Association of TLR2 haplotypes encoding Q650 with reduced susceptibility to ovine Johne's disease in Turkish sheep.

Ovine Johne's disease (OJD) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and carries a potential zoonotic risk for humans. Selective breeding strategies for reduced OJD susceptibility would be welcome tools in disease eradication efforts, if available. The Toll-like receptor 2 gene (TLR2) plays an important signaling role in immune response to MAP, and missense variants are associated with mycobacterial infections in mammals. Our aim was to identify and evaluate ovine TLR2 missense variants for association with OJD in Turkish sheep. Eleven TLR2 missense variants and 17 haplotype configurations were identified in genomic sequences of 221 sheep from 61 globally-distributed breeds. The five most frequent haplotypes were tested for OJD association in 102 matched pairs of infected and uninfected ewes identified in 2257 Turkish sheep. Ewes with one or two copies of TLR2 haplotypes encoding glutamine (Q) at position 650 (Q650) in the Tir domain were 6.6-fold more likely to be uninfected compared to ewes with arginine (R650) at that position (CI95 = 2.6 to 16.9, p-value = 3.7 × 10-6). The protective TLR2 Q650 allele was present in at least 25% of breeds tested and thus may facilitate selective breeding for sheep with reduced susceptibility to OJD.

Evaluation of CD109, PCP4 and SEMA3D genes for their association with Ovine Johne's disease in Turkish sheep.

Johne's disease is a chronic, contagious, zoonotic disease that affects numerous species including livestock and sometimes humans. The disease is globally distributed in sheep populations and caused by Mycobacterium avium Subsp. paratuberculosis (MAP). A previous genome-wide association study identified single nucleotide polymorphism (SNP) markers associated with OJD serostatus in CD109, PCP4, and SEMA3D genes. Our aim was to evaluate the same markers for association with OJD seroprevalence in Turkish sheep in a retrospective matched case-control study. The serological status for OJD in 1801 sheep was determined for four native and four composite breeds from three research flocks. One hundred eleven matched case-control pairs were constructed according to breed type and age from 1750 comingled ewes reared in the same environment. A Single Nucleotide Primer Extension (SNuPE) assay was designed to genotype PCP4-Intron 1, PCP4-3'UTR, SEMA3D, CD109-intron 2 and CD109-intron 8 markers and a McNemar's test was performed on the matched pairs. An association with these five markers was not detected with the OJD serostatus in Turkish sheep (power of detection, 0.95; odds ratio >3; McNemar's p < .05). Thus, a wider search may be needed to identify any major underlying genetic risk factors for OJD in Turkish sheep.

A Reference Genome Assembly of American Bison, Bison bison bison.

Bison are an icon of the American West and an ecologically, commercially, and culturally important species. Despite numbering in the hundreds of thousands today, conservation concerns remain for the species, including the impact on genetic diversity of a severe bottleneck around the turn of the 20th century and genetic introgression from domestic cattle. Genetic diversity and admixture are best evaluated at genome-wide scale, for which a high-quality reference is necessary. Here, we use trio binning of long reads from a bison-Simmental cattle (Bos taurus taurus) male F1 hybrid to sequence and assemble the genome of the American plains bison (Bison bison bison). The male haplotype genome is chromosome-scale, with a total length of 2.65 Gb across 775 scaffolds (839 contigs) and a scaffold N50 of 87.8 Mb. Our bison genome is ~13× more contiguous overall and ~3400× more contiguous at the contig level than the current bison reference genome. The bison genome sequence presented here (ARS-UCSC_bison1.0) will enable new research into the evolutionary history of this iconic megafauna species and provide a new tool for the management of bison populations in federal and commercial herds.

A Reference Genome Assembly of Simmental Cattle, Bos taurus taurus.

Genomics research has relied principally on the establishment and curation of a reference genome for the species. However, it is increasingly recognized that a single reference genome cannot fully describe the extent of genetic variation within many widely distributed species. Pangenome representations are based on high-quality genome assemblies of multiple individuals and intended to represent the broadest possible diversity within a species. A Bovine Pangenome Consortium (BPC) has recently been established to begin assembling genomes from more than 600 recognized breeds of cattle, together with other related species to provide information on ancestral alleles and haplotypes. Previously reported de novo genome assemblies for Angus, Brahman, Hereford, and Highland breeds of cattle are part of the initial BPC effort. The present report describes a complete single haplotype assembly at chromosome-scale for a fullblood Simmental cow from an F1 bison-cattle hybrid fetus by trio binning. Simmental cattle, also known as Fleckvieh due to their red and white spots, originated in central Europe in the 1830s as a triple-purpose breed selected for draught, meat, and dairy production. There are over 50 million Simmental cattle in the world, known today for their fast growth and beef yields. This assembly (ARS_Simm1.0) is similar in length to the other bovine assemblies at 2.86 Gb, with a scaffold N50 of 102 Mb (max scaffold 156.8 Mb) and meets or exceeds the continuity of the best Bos taurus reference assemblies to date.

Using triallelic SNPs for determining parentage in North American yak ( Bos grunniens) and estimating cattle ( B. taurus) introgression.

Background: Genetic testing for pedigree accuracy is critical for managing genetic diversity in North American (NA) yak ( Bos grunniens), a population expanded mostly from imported zoological park specimens.  DNA testing also enhances species conservation by identifying recent B. taurus F1 hybrid ancestors (within three generations).  Biallelic single nucleotide polymorphisms (SNPs) can accomplish either task, but increases the marker count and costs necessary to achieve both.  Our aim was to identify novel, multifunctional, triallelic yak SNPs (tySNPs), with each having two alleles for yak parentage testing, and a third allele for identifying recent cattle introgression.  Methods:  Genome sequences were aligned to the cattle UMD3.1 assembly and SNPs were screened for 1) heterozygosity in a NA and a Chinese yak, 2) a third allele at high frequency in cattle, and 3) flanking sequences conserved in both species.  Subsequently, tySNPs were filtered for unique alignment to the haplotype-resolved F1 yak assembly.  Allele frequencies were estimated in a subset of 87 tySNPs by genotyping 170 NA yak. Results:  We identified 610 autosomal tySNPs, distributed in 441 clusters with 5 Mb average genome spacing.  The average NA yak minor allele frequency was high (0.296), while average introgressed cattle alleles were low (0.004).  In simulations with tySNPs, 28 were sufficient for globally-unique animal identification (P I=5.81x10 -12), 87 were able to exclude 19 random bulls from parentage at the 99% level without using the dam's genotype (P E=5.3x10 -4), and 87 were able to detect F1 hybridization events after three generations of yak backcrosses (1/16th B. taurus germplasm). Conclusions:  Identifying animals, determining parentage and detecting recent hybridization events was efficient with as few as 87 tySNPs.  A similar triallelic approach could be used with other bottlenecked Bos species that hybridize with cattle, such as NA plains bison ( B. bison).

Haplotype-resolved genomes provide insights into structural variation and gene content in Angus and Brahman cattle.

Inbred animals were historically chosen for genome analysis to circumvent assembly issues caused by haplotype variation but this resulted in a composite of the two genomes. Here we report a haplotype-aware scaffolding and polishing pipeline which was used to create haplotype-resolved, chromosome-level genome assemblies of Angus (taurine) and Brahman (indicine) cattle subspecies from contigs generated by the trio binning method. These assemblies reveal structural and copy number variants that differentiate the subspecies and that variant detection is sensitive to the specific reference genome chosen. Six genes with immune related functions have additional copies in the indicine compared with taurine lineage and an indicus-specific extra copy of fatty acid desaturase is under positive selection. The haplotyped genomes also enable transcripts to be phased to detect allele-specific expression. This work exemplifies the value of haplotype-resolved genomes to better explore evolutionary and functional variations.

Continuous chromosome-scale haplotypes assembled from a single interspecies F1 hybrid of yak and cattle.

BACKGROUND: The development of trio binning as an approach for assembling diploid genomes has enabled the creation of fully haplotype-resolved reference genomes. Unlike other methods of assembly for diploid genomes, this approach is enhanced, rather than hindered, by the heterozygosity of the individual sequenced. To maximize heterozygosity and simultaneously assemble reference genomes for 2 species, we applied trio binning to an interspecies F1 hybrid of yak (Bos grunniens) and cattle (Bos taurus), 2 species that diverged nearly 5 million years ago. The genomes of both of these species are composed of acrocentric autosomes. RESULTS: We produced the most continuous haplotype-resolved assemblies for a diploid animal yet reported. Both the maternal (yak) and paternal (cattle) assemblies have the largest 2 chromosomes in single haplotigs, and more than one-third of the autosomes similarly lack gaps. The maximum length haplotig produced was 153 Mb without any scaffolding or gap-filling steps and represents the longest haplotig reported for any species. The assemblies are also more complete and accurate than those reported for most other vertebrates, with 97% of mammalian universal single-copy orthologs present. CONCLUSIONS: The high heterozygosity inherent to interspecies crosses maximizes the effectiveness of the trio binning method. The interspecies trio binning approach we describe is likely to provide the highest-quality assemblies for any pair of species that can interbreed to produce hybrid offspring that develop to sufficient cell numbers for DNA extraction.