Analysis of 206 whole-genome resequencing reveals selection signatures associated with breed-specific traits in Hu sheep.

As an invaluable Chinese sheep germplasm resource, Hu sheep are renowned for their high fertility and beautiful wavy lambskins. Their distinctive characteristics have evolved over time through a combination of artificial and natural selection. Identifying selection signatures in Hu sheep can provide a straightforward insight into the mechanism of selection and further uncover the candidate genes associated with breed-specific traits subject to selection. Here, we conducted whole-genome resequencing on 206 Hu sheep individuals, each with an approximate 6-fold depth of coverage. And then we employed three complementary approaches, including composite likelihood ratio, integrated haplotype homozygosity score and the detection of runs of homozygosity, to detect selection signatures. In total, 10 candidate genomic regions displaying selection signatures were simultaneously identified by multiple methods, spanning 88.54 Mb. After annotating, these genomic regions harbored collectively 92 unique genes. Interestingly, 32 candidate genes associated with reproduction were distributed in nine genomic regions detected. Out of them, two stood out as star candidates: BMPR1B and GNRH2, both of which have documented associations with fertility, and a HOXA gene cluster (HOXA1-5, HOXA9, HOXA10, HOXA11 and HOXA13) had also been linked to fertility. Additionally, we identified other genes that are related to hair follicle development (LAMTOR3, EEF1A2), ear size (HOXA1, KCNQ2), fat tail formation (HOXA10, HOXA11), growth and development (FAF1, CCNDBP1, GJB2, GJA3), fat deposition (ACOXL, JAZF1, HOXA3, HOXA4, HOXA5, EBF4), immune (UBR1, FASTKD5) and feed intake (DAPP1, RNF17, NPBWR2). Our results offer novel insights into the genetic mechanisms underlying the selection of breed-specific traits in Hu sheep and provide a reference for sheep genetic improvement programs.

Allele-specific binding variants causing ChIP-seq peak height of histone modification are not enriched in expression QTL annotations.

BACKGROUND: Genome sequence variants affecting complex traits (quantitative trait loci, QTL) are enriched in functional regions of the genome, such as those marked by certain histone modifications. These variants are believed to influence gene expression. However, due to the linkage disequilibrium among nearby variants, pinpointing the precise location of QTL is challenging. We aimed to identify allele-specific binding (ASB) QTL (asbQTL) that cause variation in the level of histone modification, as measured by the height of peaks assayed by ChIP-seq (chromatin immunoprecipitation sequencing). We identified DNA sequences that predict the difference between alleles in ChIP-seq peak height in H3K4me3 and H3K27ac histone modifications in the mammary glands of cows. RESULTS: We used a gapped k-mer support vector machine, a novel best linear unbiased prediction model, and a multiple linear regression model that combines the other two approaches to predict variant impacts on peak height. For each method, a subset of 1000 sites with the highest magnitude of predicted ASB was considered as candidate asbQTL. The accuracy of this prediction was measured by the proportion where the predicted direction matched the observed direction. Prediction accuracy ranged between 0.59 and 0.74, suggesting that these 1000 sites are enriched for asbQTL. Using independent data, we investigated functional enrichment in the candidate asbQTL set and three control groups, including non-causal ASB sites, non-ASB variants under a peak, and SNPs (single nucleotide polymorphisms) not under a peak. For H3K4me3, a higher proportion of the candidate asbQTL were confirmed as ASB when compared to the non-causal ASB sites (P < 0.01). However, these candidate asbQTL did not enrich for the other annotations, including expression QTL (eQTL), allele-specific expression QTL (aseQTL) and sites conserved across mammals (P > 0.05). CONCLUSIONS: We identified putatively causal sites for asbQTL using the DNA sequence surrounding these sites. Our results suggest that many sites influencing histone modifications may not directly affect gene expression. However, it is important to acknowledge that distinguishing between putative causal ASB sites and other non-causal ASB sites in high linkage disequilibrium with the causal sites regarding their impact on gene expression may be challenging due to limitations in statistical power.

Using expression data to fine map QTL associated with fertility in dairy cattle.

BACKGROUND: Female fertility is an important trait in dairy cattle. Identifying putative causal variants associated with fertility may help to improve the accuracy of genomic prediction of fertility. Combining expression data (eQTL) of genes, exons, gene splicing and allele specific expression is a promising approach to fine map QTL to get closer to the causal mutations. Another approach is to identify genomic differences between cows selected for high and low fertility and a selection experiment in New Zealand has created exactly this resource. Our objective was to combine multiple types of expression data, fertility traits and allele frequency in high- (POS) and low-fertility (NEG) cows with a genome-wide association study (GWAS) on calving interval in Australian cows to fine-map QTL associated with fertility in both Australia and New Zealand dairy cattle populations. RESULTS: Variants that were significantly associated with calving interval (CI) were strongly enriched for variants associated with gene, exon, gene splicing and allele-specific expression, indicating that there is substantial overlap between QTL associated with CI and eQTL. We identified 671 genes with significant differential expression between POS and NEG cows, with the largest fold change detected for the CCDC196 gene on chromosome 10. Our results provide numerous candidate genes associated with female fertility in dairy cattle, including GYS2 and TIGAR on chromosome 5 and SYT3 and HSD17B14 on chromosome 18. Multiple QTL regions were located in regions with large numbers of copy number variants (CNV). To identify the causal mutations for these variants, long read sequencing may be useful. CONCLUSIONS: Variants that were significantly associated with CI were highly enriched for eQTL. We detected 671 genes that were differentially expressed between POS and NEG cows. Several QTL detected for CI overlapped with eQTL, providing candidate genes for fertility in dairy cattle.

Genome-wide analyses of variance in blood cell phenotypes provide new insights into complex trait biology and prediction.

Blood cell phenotypes are routinely tested in healthcare to inform clinical decisions. Genetic variants influencing mean blood cell phenotypes have been used to understand disease aetiology and improve prediction; however, additional information may be captured by genetic effects on observed variance. Here, we mapped variance quantitative trait loci (vQTL), i.e. genetic loci associated with trait variance, for 29 blood cell phenotypes from the UK Biobank (N~408,111). We discovered 176 independent blood cell vQTLs, of which 147 were not found by additive QTL mapping. vQTLs displayed on average 1.8-fold stronger negative selection than additive QTL, highlighting that selection acts to reduce extreme blood cell phenotypes. Variance polygenic scores (vPGSs) were constructed to stratify individuals in the INTERVAL cohort (N~40,466), where genetically less variable individuals (low vPGS) had increased conventional PGS accuracy (by ~19%) than genetically more variable individuals. Genetic prediction of blood cell traits improved by ~10% on average combining PGS with vPGS. Using Mendelian randomisation and vPGS association analyses, we found that alcohol consumption significantly increased blood cell trait variances highlighting the utility of blood cell vQTLs and vPGSs to provide novel insight into phenotype aetiology as well as improve prediction.

Recent advances in polygenic scores: translation, equitability, methods and FAIR tools.

Polygenic scores (PGS) can be used for risk stratification by quantifying individuals' genetic predisposition to disease, and many potentially clinically useful applications have been proposed. Here, we review the latest potential benefits of PGS in the clinic and challenges to implementation. PGS could augment risk stratification through combined use with traditional risk factors (demographics, disease-specific risk factors, family history, etc.), to support diagnostic pathways, to predict groups with therapeutic benefits, and to increase the efficiency of clinical trials. However, there exist challenges to maximizing the clinical utility of PGS, including FAIR (Findable, Accessible, Interoperable, and Reusable) use and standardized sharing of the genomic data needed to develop and recalculate PGS, the equitable performance of PGS across populations and ancestries, the generation of robust and reproducible PGS calculations, and the responsible communication and interpretation of results. We outline how these challenges may be overcome analytically and with more diverse data as well as highlight sustained community efforts to achieve equitable, impactful, and responsible use of PGS in healthcare.

Poor sleep and shift work associate with increased blood pressure and inflammation in UK Biobank participants.

Disrupted circadian rhythms have been linked to an increased risk of hypertension and cardiovascular disease. However, many studies show inconsistent findings and are not sufficiently powered for targeted subgroup analyses. Using the UK Biobank cohort, we evaluate the association between circadian rhythm-disrupting behaviours, blood pressure (SBP, DBP) and inflammatory markers in >350,000 adults with European white British ancestry. The independent U-shaped relationship between sleep length and SBP/DBP is most prominent with a low inflammatory status. Poor sleep quality and permanent night shift work are also positively associated with SBP/DBP. Although fully adjusting for BMI in the linear regression model attenuated effect sizes, these associations remain significant. Two-sample Mendelian Randomisation (MR) analyses support a potential causal effect of long sleep, short sleep, chronotype, daytime napping and sleep duration on SBP/DBP. Thus, in the current study, we present a positive association between circadian rhythm-disrupting behaviours and SBP/DBP regulation in males and females that is largely independent of age.

Gene expression and RNA splicing explain large proportions of the heritability for complex traits in cattle.

Many quantitative trait loci (QTLs) are in non-coding regions. Therefore, QTLs are assumed to affect gene regulation. Gene expression and RNA splicing are primary steps of transcription, so DNA variants changing gene expression (eVariants) or RNA splicing (sVariants) are expected to significantly affect phenotypes. We quantify the contribution of eVariants and sVariants detected from 16 tissues (n = 4,725) to 37 traits of ∼120,000 cattle (average magnitude of genetic correlation between traits = 0.13). Analyzed in Bayesian mixture models, averaged across 37 traits, cis and trans eVariants and sVariants detected from 16 tissues jointly explain 69.2% (SE = 0.5%) of heritability, 44% more than expected from the same number of random variants. This 69.2% includes an average of 24% from trans e-/sVariants (14% more than expected). Averaged across 56 lipidomic traits, multi-tissue cis and trans e-/sVariants also explain 71.5% (SE = 0.3%) of heritability, demonstrating the essential role of proximal and distal regulatory variants in shaping mammalian phenotypes.

A sheep pangenome reveals the spectrum of structural variations and their effects on tail phenotypes.

Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here we generated high-quality genome assemblies for 15 individuals from genetically diverse sheep breeds using Pacific Biosciences (PacBio) high-fidelity sequencing, discovering 130.3 Mb nonreference sequences, from which 588 genes were annotated. A total of 149,158 biallelic insertions/deletions, 6531 divergent alleles, and 14,707 multiallelic variations with precise breakpoints were discovered. The SV spectrum is characterized by an excess of derived insertions compared to deletions (94,422 vs. 33,571), suggesting recent active LINE expansions in sheep. Nearly half of the SVs display low to moderate linkage disequilibrium with surrounding single-nucleotide polymorphisms (SNPs) and most SVs cannot be tagged by SNP probes from the widely used ovine 50K SNP chip. We identified 865 population-stratified SVs including 122 SVs possibly derived in the domestication process among 690 individuals from sheep breeds worldwide. A novel 168-bp insertion in the 5' untranslated region (5' UTR) of HOXB13 is found at high frequency in long-tailed sheep. Further genome-wide association study and gene expression analyses suggest that this mutation is causative for the long-tail trait. In summary, we have developed a panel of high-quality de novo assemblies and present a catalog of structural variations in sheep. Our data capture abundant candidate functional variations that were previously unexplored and provide a fundamental resource for understanding trait biology in sheep.

In it for the long run: perspectives on exploiting long-read sequencing in livestock for population scale studies of structural variants.

Studies have demonstrated that structural variants (SV) play a substantial role in the evolution of species and have an impact on Mendelian traits in the genome. However, unlike small variants (< 50 bp), it has been challenging to accurately identify and genotype SV at the population scale using short-read sequencing. Long-read sequencing technologies are becoming competitively priced and can address several of the disadvantages of short-read sequencing for the discovery and genotyping of SV. In livestock species, analysis of SV at the population scale still faces challenges due to the lack of resources, high costs, technological barriers, and computational limitations. In this review, we summarize recent progress in the characterization of SV in the major livestock species, the obstacles that still need to be overcome, as well as the future directions in this growing field. It seems timely that research communities pool resources to build global population-scale long-read sequencing consortiums for the major livestock species for which the application of genomic tools has become cost-effective.

Asymmetric growth-limiting development of the female conceptus.

Introduction: Sex differences in prenatal growth may contribute to sex-dependent programming effects on postnatal phenotype. Methods: We integrated for the first time phenotypic, histomorphological, clinico-chemical, endocrine and gene expression analyses in a single species, the bovine conceptus at mid-gestation. Results: We demonstrate that by mid-gestation, before the onset of accelerated growth, the female conceptus displays asymmetric lower growth compared to males. Female fetuses were smaller with lower ponderal index and organ weights than males. However, their brain:body weight, brain:liver weight and heart:body weight ratios were higher than in males, indicating brain and heart 'sparing'. The female placenta weighed less and had lower volumes of trophoblast and fetal connective tissue than the male placenta. Female umbilical cord vessel diameters were smaller, and female-specific relationships of body weight and brain:liver weight ratios with cord vessel diameters indicated that the umbilico-placental vascular system creates a growth-limiting environment where blood flow is redistributed to protect brain and heart growth. Clinico-chemical indicators of liver perfusion support this female-specific growth-limiting phenotype, while lower insulin-like growth factor 2 (IGF2) gene expression in brain and heart, and lower circulating IGF2, implicate female-specific modulation of key endocrine mediators by nutrient supply. Conclusion: This mode of female development may increase resilience to environmental perturbations in utero and contribute to sex-bias in programming outcomes including susceptibility to non-communicable diseases.

Genetic variation in histone modifications and gene expression identifies regulatory variants in the mammary gland of cattle.

BACKGROUND: Causal variants for complex traits, such as eQTL are often found in non-coding regions of the genome, where they are hypothesised to influence phenotypes by regulating gene expression. Many regulatory regions are marked by histone modifications, which can be assayed by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Sequence reads from ChIP-seq form peaks at putative regulatory regions, which may reflect the amount of regulatory activity at this region. Therefore, eQTL which are also associated with differences in histone modifications are excellent candidate causal variants. RESULTS: We assayed the histone modifications H3K4Me3, H3K4Me1 and H3K27ac and mRNA in the mammary gland of up to 400 animals. We identified QTL for peak height (histone QTL), exon expression (eeQTL), allele specific expression (aseQTL) and allele specific binding (asbQTL). By intersecting these results, we identify variants which may influence gene expression by altering regulatory regions of the genome, and may be causal variants for other traits. Lastly, we find that these variants are found in putative transcription factor binding sites, identifying a mechanism for the effect of many eQTL. CONCLUSIONS: We find that allele specific and traditional QTL analysis often identify the same genetic variants and provide evidence that many eQTL are regulatory variants which alter activity at regulatory regions of the bovine genome. Our work provides methodological and biological updates on how regulatory mechanisms interplay at multi-omics levels.

A multi-tissue atlas of regulatory variants in cattle.

Characterization of genetic regulatory variants acting on livestock gene expression is essential for interpreting the molecular mechanisms underlying traits of economic value and for increasing the rate of genetic gain through artificial selection. Here we build a Cattle Genotype-Tissue Expression atlas (CattleGTEx) as part of the pilot phase of the Farm animal GTEx (FarmGTEx) project for the research community based on 7,180 publicly available RNA-sequencing (RNA-seq) samples. We describe the transcriptomic landscape of more than 100 tissues/cell types and report hundreds of thousands of genetic associations with gene expression and alternative splicing for 23 distinct tissues. We evaluate the tissue-sharing patterns of these genetic regulatory effects, and functionally annotate them using multiomics data. Finally, we link gene expression in different tissues to 43 economically important traits using both transcriptome-wide association and colocalization analyses to decipher the molecular regulatory mechanisms underpinning such agronomic traits in cattle.

Mutant alleles differentially shape fitness and other complex traits in cattle.

Mutant alleles (MAs) that have been classically recognised have large effects on phenotype and tend to be deleterious to traits and fitness. Is this the case for mutations with small effects? We infer MAs for 8 million sequence variants in 113k cattle and quantify the effects of MA on 37 complex traits. Heterozygosity for variants at genomic sites conserved across 100 vertebrate species increase fertility, stature, and milk production, positively associating these traits with fitness. MAs decrease stature and fat and protein concentration in milk, but increase gestation length and somatic cell count in milk (the latter indicative of mastitis). However, the frequency of MAs decreasing stature and fat and protein concentration, increasing gestation length and somatic cell count were lower than the frequency of MAs with the opposite effect. These results suggest bias in the mutations direction of effect (e.g. towards reduced protein in milk), but selection operating to reduce the frequency of these MAs. Taken together, our results imply two classes of genomic sites subject to long-term selection: sites conserved across vertebrates show hybrid vigour while sites subject to less long-term selection show a bias in mutation towards undesirable alleles.

Corrigendum: Putative Causal Variants Are Enriched in Annotated Functional Regions From Six Bovine Tissues.

[This corrects the article DOI: 10.3389/fgene.2021.664379.].

New loci and neuronal pathways for resilience to heat stress in cattle.

While understanding the genetic basis of heat tolerance is crucial in the context of global warming's effect on humans, livestock, and wildlife, the specific genetic variants and biological features that confer thermotolerance in animals are still not well characterized. We used dairy cows as a model to study heat tolerance because they are lactating, and therefore often prone to thermal stress. The data comprised almost 0.5 million milk records (milk, fat, and proteins) of 29,107 Australian Holsteins, each having around 15 million imputed sequence variants. Dairy animals often reduce their milk production when temperature and humidity rise; thus, the phenotypes used to measure an individual's heat tolerance were defined as the rate of milk production decline (slope traits) with a rising temperature-humidity index. With these slope traits, we performed a genome-wide association study (GWAS) using different approaches, including conditional analyses, to correct for the relationship between heat tolerance and level of milk production. The results revealed multiple novel loci for heat tolerance, including 61 potential functional variants at sites highly conserved across 100 vertebrate species. Moreover, it was interesting that specific candidate variants and genes are related to the neuronal system (ITPR1, ITPR2, and GRIA4) and neuroactive ligand-receptor interaction functions for heat tolerance (NPFFR2, CALCR, and GHR), providing a novel insight that can help to develop genetic and management approaches to combat heat stress.

Putative Causal Variants Are Enriched in Annotated Functional Regions From Six Bovine Tissues.

Genetic variants which affect complex traits (causal variants) are thought to be found in functional regions of the genome. Identifying causal variants would be useful for predicting complex trait phenotypes in dairy cows, however, functional regions are poorly annotated in the bovine genome. Functional regions can be identified on a genome-wide scale by assaying for post-translational modifications to histone proteins (histone modifications) and proteins interacting with the genome (e.g., transcription factors) using a method called Chromatin immunoprecipitation followed by sequencing (ChIP-seq). In this study ChIP-seq was performed to find functional regions in the bovine genome by assaying for four histone modifications (H3K4Me1, H3K4Me3, H3K27ac, and H3K27Me3) and one transcription factor (CTCF) in 6 tissues (heart, kidney, liver, lung, mammary and spleen) from 2 to 3 lactating dairy cows. Eighty-six ChIP-seq samples were generated in this study, identifying millions of functional regions in the bovine genome. Combinations of histone modifications and CTCF were found using ChromHMM and annotated by comparing with active and inactive genes across the genome. Functional marks differed between tissues highlighting areas which might be particularly important to tissue-specific regulation. Supporting the cis-regulatory role of functional regions, the read counts in some ChIP peaks correlated with nearby gene expression. The functional regions identified in this study were enriched for putative causal variants as seen in other species. Interestingly, regions which correlated with gene expression were particularly enriched for potential causal variants. This supports the hypothesis that complex traits are regulated by variants that alter gene expression. This study provides one of the largest ChIP-seq annotation resources in cattle including, for the first time, in the mammary gland of lactating cows. By linking regulatory regions to expression QTL and trait QTL we demonstrate a new strategy for identifying causal variants in cattle.

Evolution of tissue and developmental specificity of transcription start sites in Bos taurus indicus.

To further the understanding of the evolution of transcriptional regulation, we profiled genome-wide transcriptional start sites (TSSs) in two sub-species, Bos taurus taurus and Bos taurus indicus, that diverged approximately 500,000 years ago. Evolutionary and developmental-stage differences in TSSs were detected across the sub-species, including translocation of dominant TSS and changes in TSS distribution. The 16% of all SNPs located in significant differentially used TSS clusters across sub-species had significant shifts in allele frequency (472 SNPs), indicating they may have been subject to selection. In spleen and muscle, a higher relative TSS expression was observed in Bos indicus than Bos taurus for all heat shock protein genes, which may be responsible for the tropical adaptation of Bos indicus.