Powerful detection of polygenic selection and evidence of environmental adaptation in US beef cattle.

Selection on complex traits can rapidly drive evolution, especially in stressful environments. This polygenic selection does not leave intense sweep signatures on the genome, rather many loci experience small allele frequency shifts, resulting in large cumulative phenotypic changes. Directional selection and local adaptation are changing populations; but, identifying loci underlying polygenic or environmental selection has been difficult. We use genomic data on tens of thousands of cattle from three populations, distributed over time and landscapes, in linear mixed models with novel dependent variables to map signatures of selection on complex traits and local adaptation. We identify 207 genomic loci associated with an animal's birth date, representing ongoing selection for monogenic and polygenic traits. Additionally, hundreds of additional loci are associated with continuous and discrete environments, providing evidence for historical local adaptation. These candidate loci highlight the nervous system's possible role in local adaptation. While advanced technologies have increased the rate of directional selection in cattle, it has likely been at the expense of historically generated local adaptation, which is especially problematic in changing climates. When applied to large, diverse cattle datasets, these selection mapping methods provide an insight into how selection on complex traits continually shapes the genome. Further, understanding the genomic loci implicated in adaptation may help us breed more adapted and efficient cattle, and begin to understand the basis for mammalian adaptation, especially in changing climates. These selection mapping approaches help clarify selective forces and loci in evolutionary, model, and agricultural contexts.

Genome-wide association and genotype by environment interactions for growth traits in U.S. Red Angus cattle.

BACKGROUND: Genotypic information produced from single nucleotide polymorphism (SNP) arrays has routinely been used to identify genomic regions associated with complex traits in beef and dairy cattle. Herein, we assembled a dataset consisting of 15,815 Red Angus beef cattle distributed across the continental U.S. and a union set of 836,118 imputed SNPs to conduct genome-wide association analyses (GWAA) for growth traits using univariate linear mixed models (LMM); including birth weight, weaning weight, and yearling weight. Genomic relationship matrix heritability estimates were produced for all growth traits, and genotype-by-environment (GxE) interactions were investigated. RESULTS: Moderate to high heritabilities with small standard errors were estimated for birth weight (0.51 ± 0.01), weaning weight (0.25 ± 0.01), and yearling weight (0.42 ± 0.01). GWAA revealed 12 pleiotropic QTL (BTA6, BTA14, BTA20) influencing Red Angus birth weight, weaning weight, and yearling weight which met a nominal significance threshold (P ≤ 1e-05) for polygenic traits using 836K imputed SNPs. Moreover, positional candidate genes associated with Red Angus growth traits in this study (i.e., LCORL, LOC782905, NCAPG, HERC6, FAM184B, SLIT2, MMRN1, KCNIP4, CCSER1, GRID2, ARRDC3, PLAG1, IMPAD1, NSMAF, PENK, LOC112449660, MOS, SH3PXD2B, STC2, CPEB4) were also previously associated with feed efficiency, growth, and carcass traits in beef cattle. Collectively, 14 significant GxE interactions were also detected, but were less consistent among the investigated traits at a nominal significance threshold (P ≤ 1e-05); with one pleiotropic GxE interaction detected on BTA28 (24 Mb) for Red Angus weaning weight and yearling weight. CONCLUSIONS: Sixteen well-supported QTL regions detected from the GWAA and GxE GWAA for growth traits (birth weight, weaning weight, yearling weight) in U.S. Red Angus cattle were found to be pleiotropic. Twelve of these pleiotropic QTL were also identified in previous studies focusing on feed efficiency and growth traits in multiple beef breeds and/or their composites. In agreement with other beef cattle GxE studies our results implicate the role of vasodilation, metabolism, and the nervous system in the genetic sensitivity to environmental stress.

Evidence of rare misassemblies in the bovine reference genome revealed by population genetic metrics.

Creation of the bovine reference assembly paved the way to develop the high-throughput genotyping arrays of the single nucleotide polymorphisms (SNPs) based on the available map coordinates that facilitated major advances in gene mapping and selection programs. The assembly flaws, however, may cause false results in the downstream gene mapping studies. The most recent bovine reference genome (ARS-UCD1.2) is built on the long-read sequences that provides improved quality and continuity. By applying population genetic metrics in this study, we aimed to evaluate the map coordinates to which SNP markers were assigned. We employed a three-step approach by combining the recombination and linkage disequilibrium analyses to test if the markers fit into the assigned physical map coordinates. We applied the method to the bovine 50k array in a large pedigree of Holstein cattle and revealed a panel of 65 candidate markers, most of which were re-located either on a different chromosome or re-mapped as far as several million base pairs away on the same chromosome. This list of candidates accounts for 0.1% of the SNPs in the widely used 50k genotyping array and we foresee a reasonably larger set of markers being misplaced in the BovineHD 700K BeadChip. We suggest pre-removal of the candidate misplaced markers to reduce false signals in association mapping studies.

Introgression, admixture, and selection facilitate genetic adaptation to high-altitude environments in cattle.

Domestication and subsequent selection of cattle to form breeds and biological types that can adapt to different environments partitioned ancestral genetic diversity into distinct modern lineages. Genome-wide selection particularly for adaptation to extreme environments left detectable signatures genome-wide. We used high-density genotype data for 42 cattle breeds and identified the influence of Bos grunniens and Bos javanicus on the formation of Chinese indicine breeds that led to their divergence from India-origin zebu. We also found evidence for introgression, admixture, and migration in most of the Chinese breeds. Selection signature analyses between high-altitude (≥1800 m) and low-altitude adapted breeds (<1500 m) revealed candidate genes (ACSS2, ALDOC, EPAS1, EGLN1, NUCB2) and pathways that are putatively involved in hypoxia adaptation. Immunohistochemical, real-time PCR and CRISPR/cas9 ACSS2-knockout analyses suggest that the up-regulation of ACSS2 expression in the liver promotes the metabolic adaptation of cells to hypoxia via the hypoxia-inducible factor pathway. High altitude adaptation involved the introgression of alleles from high-altitude adapted yaks into Chinese Bos taurus taurus prior to their formation into recognized breeds and followed by selection. In addition to selection, adaptation to high altitude environments has been facilitated by admixture and introgression with locally adapted cattle populations.

QTL-mapping and genomic prediction for bovine respiratory disease in U.S. Holsteins using sequence imputation and feature selection.

BACKGROUND: National genetic evaluations for disease resistance do not exist, precluding the genetic improvement of cattle for these traits. We imputed BovineHD genotypes to whole genome sequence for 2703 Holsteins that were cases or controls for Bovine Respiratory Disease and sampled from either California or New Mexico to construct and compare genomic prediction models. The sequence variation reference dataset comprised variants called for 1578 animals from Run 5 of the 1000 Bull Genomes Project, including 450 Holsteins and 29 animals sequenced from this study population. Genotypes for 9,282,726 variants with minor allele frequencies ≥5% were imputed and used to obtain genomic predictions in GEMMA using a Bayesian Sparse Linear Mixed Model. RESULTS: Variation explained by markers increased from 13.6% using BovineHD data to 14.4% using imputed whole genome sequence data and the resolution of genomic regions detected as harbouring QTL substantially increased. Explained variation in the analysis of the combined California and New Mexico data was less than when data for each state were separately analysed and the estimated genetic correlation between risk of Bovine Respiratory Disease in California and New Mexico Holsteins was - 0.36. Consequently, genomic predictions trained using the data from one state did not accurately predict disease risk in the other state. To determine if a prediction model could be developed with utility in both states, we selected variants within genomic regions harbouring: 1) genes involved in the normal immune response to infection by pathogens responsible for Bovine Respiratory Disease detected by RNA-Seq analysis, and/or 2) QTL identified in the association analysis of the imputed sequence variants. The model based on QTL selected variants is biased but when trained in one state generated BRD risk predictions with positive accuracies in the other state. CONCLUSIONS: We demonstrate the utility of sequence-based and biology-driven model development for genomic selection. Disease phenotypes cannot be routinely recorded in most livestock species and the observed phenotypes may vary in their genomic architecture due to variation in the pathogen composition across environments. Elucidation of trait biology and genetic architecture may guide the development of prediction models with utility across breeds and environments.

Genome-wide association and genotype by environment interactions for growth traits in U.S. Gelbvieh cattle.

BACKGROUND: Single nucleotide polymorphism (SNP) arrays have facilitated discovery of genetic markers associated with complex traits in domestic cattle; thereby enabling modern breeding and selection programs. Genome-wide association analyses (GWAA) for growth traits were conducted on 10,837 geographically diverse U.S. Gelbvieh cattle using a union set of 856,527 imputed SNPs. Birth weight (BW), weaning weight (WW), and yearling weight (YW) were analyzed using GEMMA and EMMAX (via imputed genotypes). Genotype-by-environment (GxE) interactions were also investigated. RESULTS: GEMMA and EMMAX produced moderate marker-based heritability estimates that were similar for BW (0.36-0.37, SE = 0.02-0.06), WW (0.27-0.29, SE = 0.01), and YW (0.39-0.41, SE = 0.01-0.02). GWAA using 856K imputed SNPs (GEMMA; EMMAX) revealed common positional candidate genes underlying pleiotropic QTL for Gelbvieh growth traits on BTA6, BTA7, BTA14, and BTA20. The estimated proportion of phenotypic variance explained (PVE) by the lead SNP defining these QTL (EMMAX) was larger and most similar for BW and YW, and smaller for WW. Collectively, GWAAs (GEMMA; EMMAX) produced a highly concordant set of BW, WW, and YW QTL that met a nominal significance level (P ≤ 1e-05), with prioritization of common positional candidate genes; including genes previously associated with stature, feed efficiency, and growth traits (i.e., PLAG1, NCAPG, LCORL, ARRDC3, STC2). Genotype-by-environment QTL were not consistent among traits at the nominal significance threshold (P ≤ 1e-05); although some shared QTL were apparent at less stringent significance thresholds (i.e., P ≤ 2e-05). CONCLUSIONS: Pleiotropic QTL for growth traits were detected on BTA6, BTA7, BTA14, and BTA20 for U.S. Gelbvieh beef cattle. Seven QTL detected for Gelbvieh growth traits were also recently detected for feed efficiency and growth traits in U.S. Angus, SimAngus, and Hereford cattle. Marker-based heritability estimates and the detection of pleiotropic QTL segregating in multiple breeds support the implementation of multiple-breed genomic selection.

A multi-breed reference panel and additional rare variants maximize imputation accuracy in cattle.

BACKGROUND: During the last decade, the use of common-variant array-based single nucleotide polymorphism (SNP) genotyping in the beef and dairy industries has produced an astounding amount of medium-to-low density genomic data. Although low-density assays work well in the context of genomic prediction, they are less useful for detecting and mapping causal variants and the effects of rare variants are not captured. The objective of this project was to maximize the accuracies of genotype imputation from medium- and low-density assays to the marker set obtained by combining two high-density research assays (~ 850,000 SNPs), the Illumina BovineHD and the GGP-F250 assays, which contains a large proportion of rare and potentially functional variants and for which the assay design is described here. This 850 K SNP set is useful for both imputation to sequence-level genotypes and direct downstream analysis. RESULTS: We found that a large multi-breed composite imputation reference panel that includes 36,131 samples with either BovineHD and/or GGP-F250 genotypes significantly increased imputation accuracy compared with a within-breed reference panel, particularly at variants with low minor allele frequencies. Individual animal imputation accuracies were maximized when more genetically similar animals were represented in the composite reference panel, particularly with complete 850 K genotypes. The addition of rare variants from the GGP-F250 assay to our composite reference panel significantly increased the imputation accuracy of rare variants that are exclusively present on the BovineHD assay. In addition, we show that an assay marker density of 50 K SNPs balances cost and accuracy for imputation to 850 K. CONCLUSIONS: Using high-density genotypes on all available individuals in a multi-breed reference panel maximized imputation accuracy for tested cattle populations. Admixed animals or those from breeds with a limited representation in the composite reference panel were still imputed at high accuracy, which is expected to further increase as the reference panel expands. We anticipate that the addition of rare variants from the GGP-F250 assay will increase the accuracy of imputation to sequence level.

Bottlenecks and selective sweeps during domestication have increased deleterious genetic variation in dogs.

Population bottlenecks, inbreeding, and artificial selection can all, in principle, influence levels of deleterious genetic variation. However, the relative importance of each of these effects on genome-wide patterns of deleterious variation remains controversial. Domestic and wild canids offer a powerful system to address the role of these factors in influencing deleterious variation because their history is dominated by known bottlenecks and intense artificial selection. Here, we assess genome-wide patterns of deleterious variation in 90 whole-genome sequences from breed dogs, village dogs, and gray wolves. We find that the ratio of amino acid changing heterozygosity to silent heterozygosity is higher in dogs than in wolves and, on average, dogs have 2-3% higher genetic load than gray wolves. Multiple lines of evidence indicate this pattern is driven by less efficient natural selection due to bottlenecks associated with domestication and breed formation, rather than recent inbreeding. Further, we find regions of the genome implicated in selective sweeps are enriched for amino acid changing variants and Mendelian disease genes. To our knowledge, these results provide the first quantitative estimates of the increased burden of deleterious variants directly associated with domestication and have important implications for selective breeding programs and the conservation of rare and endangered species. Specifically, they highlight the costs associated with selective breeding and question the practice favoring the breeding of individuals that best fit breed standards. Our results also suggest that maintaining a large population size, rather than just avoiding inbreeding, is a critical factor for preventing the accumulation of deleterious variants.

Candidate lethal haplotypes and causal mutations in Angus cattle.

BACKGROUND: If unmanaged, high rates of inbreeding in livestock populations adversely impact their reproductive fitness. In beef cattle, historical selection strategies have increased the frequency of several segregating fatal autosomal recessive polymorphisms. Selective breeding has also decreased the extent of haplotypic diversity genome-wide. By identifying haplotypes for which homozygotes are not observed but would be expected based on their frequency, candidates for developmentally lethal recessive loci can be localized. This analysis comes without the need for observation of the loss-associated phenotype (e.g., failure to implant, first trimester abortion, deformity at birth). In this study, haplotypes were estimated for 3961 registered Angus individuals using 52,545 SNP loci using findhap v2, which exploited the complex pedigree among the individuals in this population. RESULTS: Seven loci were detected to possess haplotypes that were not observed in homozygous form despite a sufficiently high frequency and pedigree-based expectation of homozygote occurrence. These haplotypes were identified as candidates for harboring autosomal recessive lethal alleles. Of the genotyped individuals, 109 were resequenced to an average 27X depth of coverage to identify putative loss-of-function alleles genome-wide and had variants called using a custom in-house developed pipeline. For the candidate lethal-harboring haplotypes present in these bulls, sequence-called genotypes were used to identify concordant variants. In addition, whole-genome sequence imputation of variants was performed into the set of 3961 genotyped animals using the 109 resequenced animals to identify candidate lethal recessive variants at the seven loci. Following the imputation, no variants were identified that were fully concordant with the marker-based diplotypes. CONCLUSIONS: Selective breeding programs could utilize the predicted lethal haplotypes associated with SNP genotypes. Sequencing and other methods for identifying the causal variants underlying these haplotypes can allow for more efficient methods of management such as gene editing. These two methods in total will reduce the negative impacts of inbreeding on fertility and maximize overall genetic gains.

Genome-wide association study for feed efficiency and growth traits in U.S. beef cattle.

BACKGROUND: Single nucleotide polymorphism (SNP) arrays for domestic cattle have catalyzed the identification of genetic markers associated with complex traits for inclusion in modern breeding and selection programs. Using actual and imputed Illumina 778K genotypes for 3887 U.S. beef cattle from 3 populations (Angus, Hereford, SimAngus), we performed genome-wide association analyses for feed efficiency and growth traits including average daily gain (ADG), dry matter intake (DMI), mid-test metabolic weight (MMWT), and residual feed intake (RFI), with marker-based heritability estimates produced for all traits and populations. RESULTS: Moderate and/or large-effect QTL were detected for all traits in all populations, as jointly defined by the estimated proportion of variance explained (PVE) by marker effects (PVE ≥ 1.0%) and a nominal P-value threshold (P ≤ 5e-05). Lead SNPs with PVE ≥ 2.0% were considered putative evidence of large-effect QTL (n = 52), whereas those with PVE ≥ 1.0% but < 2.0% were considered putative evidence for moderate-effect QTL (n = 35). Identical or proximal lead SNPs associated with ADG, DMI, MMWT, and RFI collectively supported the potential for either pleiotropic QTL, or independent but proximal causal mutations for multiple traits within and between the analyzed populations. Marker-based heritability estimates for all investigated traits ranged from 0.18 to 0.60 using 778K genotypes, or from 0.17 to 0.57 using 50K genotypes (reduced from Illumina 778K HD to Illumina Bovine SNP50). An investigation to determine if QTL detected by 778K analysis could also be detected using 50K genotypes produced variable results, suggesting that 50K analyses were generally insufficient for QTL detection in these populations, and that relevant breeding or selection programs should be based on higher density analyses (imputed or directly ascertained). CONCLUSIONS: Fourteen moderate to large-effect QTL regions which ranged from being physically proximal (lead SNPs ≤ 3Mb) to fully overlapping for RFI, DMI, ADG, and MMWT were detected within and between populations, and included evidence for pleiotropy, proximal but independent causal mutations, and multi-breed QTL. Bovine positional candidate genes for these traits were functionally conserved across vertebrate species.

A homozygous PIGN missense mutation in Soft-Coated Wheaten Terriers with a canine paroxysmal dyskinesia.

Hereditary paroxysmal dyskinesias (PxD) are a heterogeneous group of movement disorders classified by frequency, duration, and triggers of the episodes. A young-adult onset canine PxD has segregated as an autosomal recessive trait in Soft-Coated Wheaten Terriers. The medical records and videos of episodes from 25 affected dogs were reviewed. The episodes of hyperkinesia and dystonia lasted from several minutes to several hours and could occur as often as >10/day. They were not associated with strenuous exercise or fasting but were sometimes triggered by excitement. The canine PxD phenotype most closely resembled paroxysmal non-kinesigenic dyskinesia (PNKD) of humans. Whole genome sequences were generated with DNA from 2 affected dogs and analyzed in comparison to 100 control canid whole genome sequences. The two whole genome sequences from dogs with PxD had a rare homozygous PIGN:c.398C > T transition, which predicted the substitution of an isoleucine for a highly conserved threonine in the encoded enzyme. All 25 PxD-affected dogs were PIGN:c.398T allele homozygotes, whereas there were no c.398T homozygotes among 1185 genotyped dogs without known histories of PxD. PIGN encodes an enzyme involved in the biosynthesis of glycosylphosphatidylinositol (GPI), which anchors a variety of proteins including CD59 to the cell surface. Flow cytometry of PIGN-knockout HEK239 cells expressing recombinant human PIGN with the c.398T variant showed reduced CD59 expression. Mutations in human PIGN have been associated with multiple congenital anomalies-hypotonia-seizures syndrome-1 (MCAHS1). Movement disorders can be a part of MCAHS1, but this is the first PxD associated with altered GPI anchor function.

The players may change but the game remains: network analyses of ruminal microbiomes suggest taxonomic differences mask functional similarity.

By mapping translated metagenomic reads to a microbial metabolic network, we show that ruminal ecosystems that are rather dissimilar in their taxonomy can be considerably more similar at the metabolic network level. Using a new network bi-partition approach for linking the microbial network to a bovine metabolic network, we observe that these ruminal metabolic networks exhibit properties consistent with distinct metabolic communities producing similar outputs from common inputs. For instance, the closer in network space that a microbial reaction is to a reaction found in the host, the lower will be the variability of its enzyme copy number across hosts. Similarly, these microbial enzymes that are nearby to host nodes are also higher in copy number than are more distant enzymes. Collectively, these results demonstrate a widely expected pattern that, to our knowledge, has not been explicitly demonstrated in microbial communities: namely that there can exist different community metabolic networks that have the same metabolic inputs and outputs but differ in their internal structure.

Worldwide patterns of ancestry, divergence, and admixture in domesticated cattle.

The domestication and development of cattle has considerably impacted human societies, but the histories of cattle breeds and populations have been poorly understood especially for African, Asian, and American breeds. Using genotypes from 43,043 autosomal single nucleotide polymorphism markers scored in 1,543 animals, we evaluate the population structure of 134 domesticated bovid breeds. Regardless of the analytical method or sample subset, the three major groups of Asian indicine, Eurasian taurine, and African taurine were consistently observed. Patterns of geographic dispersal resulting from co-migration with humans and exportation are recognizable in phylogenetic networks. All analytical methods reveal patterns of hybridization which occurred after divergence. Using 19 breeds, we map the cline of indicine introgression into Africa. We infer that African taurine possess a large portion of wild African auroch ancestry, causing their divergence from Eurasian taurine. We detect exportation patterns in Asia and identify a cline of Eurasian taurine/indicine hybridization in Asia. We also identify the influence of species other than Bos taurus taurus and B. t. indicus in the formation of Asian breeds. We detect the pronounced influence of Shorthorn cattle in the formation of European breeds. Iberian and Italian cattle possess introgression from African taurine. American Criollo cattle originate from Iberia, and not directly from Africa with African ancestry inherited via Iberian ancestors. Indicine introgression into American cattle occurred in the Americas, and not Europe. We argue that cattle migration, movement and trading followed by admixture have been important forces in shaping modern bovine genomic variation.

Association of single nucleotide polymorphisms in candidate genes previously related to genetic variation in fertility with phenotypic measurements of reproductive function in Holstein cows.

Many genetic markers related to health or production traits are not evaluated in populations independent of the discovery population or related to phenotype. Here we evaluated 68 single nucleotide polymorphisms (SNP) in candidate genes previously associated with genetic merit for fertility and production traits for association with phenotypic measurements of fertility in a population of Holstein cows that was selected based on predicted transmitting ability (PTA) for daughter pregnancy rate (DPR; high, ≥1, n = 989; low, ≤ -1.0, n = 1,285). Cows with a high PTA for DPR had higher pregnancy rate at first service, fewer services per conception, and fewer days open than cows with a low PTA for DPR. Of the 68 SNP, 11 were associated with pregnancy rate at first service, 16 with services per conception, and 19 with days open. Single nucleotide polymorphisms in 12 genes (BDH2, BSP3, CAST, CD2, CD14, FUT1, FYB, GCNT3, HSD17B7, IBSP, OCLN, and PCCB) had significant associations with 2 fertility traits, and SNP in 4 genes (CSPP1, FCER1G, PMM2, and TBC1D24) had significant associations with each of the 3 traits. Results from this experiment were compared with results from 2 earlier studies in which the SNP were associated with genetic estimates of fertility. One study involved the same animals as used here, and the other study was of an independent population of bulls. A total of 13 SNP associated with 1 or more phenotypic estimates of fertility were directionally associated with genetic estimates of fertility in the same cow population. Moreover, 14 SNP associated with reproductive phenotype were directionally associated with genetic estimates of fertility in the bull population. Nine SNP (located in BCAS, BSP3, CAST, FUT1, HSD17B7, OCLN, PCCB, PMM2, and TBC1D24) had a directional association with fertility in all 3 studies. Examination of the function of the genes with SNP associated with reproduction in more than one study indicates the importance of steroid hormones and immune function as determinants of reproductive function. All but 1 of the 68 evaluated SNP were variable in 11 breeds besides Holstein, indicating the potential effects of these SNP on reproductive function across breeds of cattle.

A mutation in the Warburg syndrome gene, RAB3GAP1, causes a similar syndrome with polyneuropathy and neuronal vacuolation in Black Russian Terrier dogs.

An autosomal recessive disease of Black Russian Terriers was previously described as a juvenile-onset, laryngeal paralysis and polyneuropathy similar to Charcot Marie Tooth disease in humans. We found that in addition to an axonal neuropathy, affected dogs exhibit microphthalmia, cataracts, and miotic pupils. On histopathology, affected dogs exhibit a spongiform encephalopathy characterized by accumulations of abnormal, membrane-bound vacuoles of various sizes in neuronal cell bodies, axons and adrenal cells. DNA from an individual dog with this polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV) was used to generate a whole genome sequence which contained a homozygous RAB3GAP1:c.743delC mutation that was absent from 73 control canine whole genome sequences. An additional 12 Black Russian Terriers with POANV were RAB3GAP1:c.743delC homozygotes. DNA samples from 249 Black Russian Terriers with no known signs of POANV were either heterozygotes or homozygous for the reference allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM), a severe developmental disorder characterized by abnormalities of the eye, genitals and nervous system including a predominantly axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, axonal transport, autophagy and synaptic transmission. The neuronal vacuolation and membranous inclusions and vacuoles in axons seen in this canine disorder likely reflect alterations of these processes. Thus, this canine disease could serve as a model for WARBM and provide insight into its pathogenesis and treatment.

RAPTR-SV: a hybrid method for the detection of structural variants.

MOTIVATION: Identification of structural variants (SVs) in sequence data results in a large number of false positive calls using existing software, which overburdens subsequent validation. RESULTS: Simulations using RAPTR-SV and other, similar algorithms for SV detection revealed that RAPTR-SV had superior sensitivity and precision, as it recovered 66.4% of simulated tandem duplications with a precision of 99.2%. When compared with calls made by Delly and LUMPY on available datasets from the 1000 genomes project, RAPTR-SV showed superior sensitivity for tandem duplications, as it identified 2-fold more duplications than Delly, while making ∼85% fewer duplication predictions. AVAILABILITY AND IMPLEMENTATION: RAPTR-SV is written in Java and uses new features in the collections framework in the latest release of the Java version 8 language specifications. A compiled version of the software, instructions for usage and test results files are available on the GitHub repository page: https://github.com/njdbickhart/RAPTR-SV. CONTACT: derek.bickhart@ars.usda.gov.

Lessons for livestock genomics from genome and transcriptome sequencing in cattle and other mammals.

BACKGROUND: Decreasing sequencing costs and development of new protocols for characterizing global methylation, gene expression patterns and regulatory regions have stimulated the generation of large livestock datasets. Here, we discuss experiences in the analysis of whole-genome and transcriptome sequence data. METHODS: We analyzed whole-genome sequence (WGS) data from 132 individuals from five canid species (Canis familiaris, C. latrans, C. dingo, C. aureus and C. lupus) and 61 breeds, three bison (Bison bison), 64 water buffalo (Bubalus bubalis) and 297 bovines from 17 breeds. By individual, data vary in extent of reference genome depth of coverage from 4.9X to 64.0X. We have also analyzed RNA-seq data for 580 samples representing 159 Bos taurus and Rattus norvegicus animals and 98 tissues. By aligning reads to a reference assembly and calling variants, we assessed effects of average depth of coverage on the actual coverage and on the number of called variants. We examined the identity of unmapped reads by assembling them and querying produced contigs against the non-redundant nucleic acids database. By imputing high-density single nucleotide polymorphism data on 4010 US registered Angus animals to WGS using Run4 of the 1000 Bull Genomes Project and assessing the accuracy of imputation, we identified misassembled reference sequence regions. RESULTS: We estimate that a 24X depth of coverage is required to achieve 99.5 % coverage of the reference assembly and identify 95 % of the variants within an individual's genome. Genomes sequenced to low average coverage (e.g., <10X) may fail to cover 10 % of the reference genome and identify <75 % of variants. About 10 % of genomic DNA or transcriptome sequence reads fail to align to the reference assembly. These reads include loci missing from the reference assembly and misassembled genes and interesting symbionts, commensal and pathogenic organisms. CONCLUSIONS: Assembly errors and a lack of annotation of functional elements significantly limit the utility of the current draft livestock reference assemblies. The Functional Annotation of Animal Genomes initiative seeks to annotate functional elements, while a 70X Pac-Bio assembly for cow is underway and may result in a significantly improved reference assembly.

New World cattle show ancestry from multiple independent domestication events.

Previous archeological and genetic research has shown that modern cattle breeds are descended from multiple independent domestication events of the wild aurochs (Bos primigenius) ∼10,000 y ago. Two primary areas of domestication in the Middle East/Europe and the Indian subcontinent resulted in taurine and indicine lines of cattle, respectively. American descendants of cattle brought by European explorers to the New World beginning in 1493 generally have been considered to belong to the taurine lineage. Our analyses of 47,506 single nucleotide polymorphisms show that these New World cattle breeds, as well as many related breeds of cattle in southern Europe, actually exhibit ancestry from both the taurine and indicine lineages. In this study, we show that, although European cattle are largely descended from the taurine lineage, gene flow from African cattle (partially of indicine origin) contributed substantial genomic components to both southern European cattle breeds and their New World descendants. New World cattle breeds, such as Texas Longhorns, provide an opportunity to study global population structure and domestication in cattle. Following their introduction into the Americas in the late 1400s, semiferal herds of cattle underwent between 80 and 200 generations of predominantly natural selection, as opposed to the human-mediated artificial selection of Old World breeding programs. Our analyses of global cattle breed population history show that the hybrid ancestry of New World breeds contributed genetic variation that likely facilitated the adaptation of these breeds to a novel environment.

A simple procedure for directly obtaining haplotype sequences of diploid genomes.

BACKGROUND: Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism. RESULTS: The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed and divided into six aliquots (except for the sperm samples which were not divided into aliquots). Each aliquot was amplified with phi29 polymerase and sequenced. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 27-133 kb and a 88-98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97-201 kb but only ~27-68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data. CONCLUSIONS: We have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences.

What's in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual.

BACKGROUND: Next-generation sequencing projects commonly commence by aligning reads to a reference genome assembly. While improvements in alignment algorithms and computational hardware have greatly enhanced the efficiency and accuracy of alignments, a significant percentage of reads often remain unmapped. RESULTS: We generated de novo assemblies of unmapped reads from the DNA and RNA sequencing of the Bos taurus reference individual and identified the closest matching sequence to each contig by alignment to the NCBI non-redundant nucleotide database using BLAST. As expected, many of these contigs represent vertebrate sequence that is absent, incomplete, or misassembled in the UMD3.1 reference assembly. However, numerous additional contigs represent invertebrate species. Most prominent were several species of Spirurid nematodes and a blood-borne parasite, Babesia bigemina. These species are either not present in the US or are not known to infect taurine cattle and the reference animal appears to have been host to unsequenced sister species. CONCLUSIONS: We demonstrate the importance of exploring unmapped reads to ascertain sequences that are either absent or misassembled in the reference assembly and for detecting sequences indicative of parasitic or commensal organisms.