Structural variation and introgression from wild populations in East Asian cattle genomes confer adaptation to local environment.

BACKGROUND: Structural variations (SVs) in individual genomes are major determinants of complex traits, including adaptability to environmental variables. The Mongolian and Hainan cattle breeds in East Asia are of taurine and indicine origins that have evolved to adapt to cold and hot environments, respectively. However, few studies have investigated SVs in East Asian cattle genomes and their roles in environmental adaptation, and little is known about adaptively introgressed SVs in East Asian cattle. RESULTS: In this study, we examine the roles of SVs in the climate adaptation of these two cattle lineages by generating highly contiguous chromosome-scale genome assemblies. Comparison of the two assemblies along with 18 Mongolian and Hainan cattle genomes obtained by long-read sequencing data provides a catalog of 123,898 nonredundant SVs. Several SVs detected from long reads are in exons of genes associated with epidermal differentiation, skin barrier, and bovine tuberculosis resistance. Functional investigations show that a 108-bp exonic insertion in SPN may affect the uptake of Mycobacterium tuberculosis by macrophages, which might contribute to the low susceptibility of Hainan cattle to bovine tuberculosis. Genotyping of 373 whole genomes from 39 breeds identifies 2610 SVs that are differentiated along a "north-south" gradient in China and overlap with 862 related genes that are enriched in pathways related to environmental adaptation. We identify 1457 Chinese indicine-stratified SVs that possibly originate from banteng and are frequent in Chinese indicine cattle. CONCLUSIONS: Our findings highlight the unique contribution of SVs in East Asian cattle to environmental adaptation and disease resistance.

Graph construction method impacts variation representation and analyses in a bovine super-pangenome.

BACKGROUND: Several models and algorithms have been proposed to build pangenomes from multiple input assemblies, but their impact on variant representation, and consequently downstream analyses, is largely unknown. RESULTS: We create multi-species super-pangenomes using pggb, cactus, and minigraph with the Bos taurus taurus reference sequence and eleven haplotype-resolved assemblies from taurine and indicine cattle, bison, yak, and gaur. We recover 221 k nonredundant structural variations (SVs) from the pangenomes, of which 135 k (61%) are common to all three. SVs derived from assembly-based calling show high agreement with the consensus calls from the pangenomes (96%), but validate only a small proportion of variations private to each graph. Pggb and cactus, which also incorporate base-level variation, have approximately 95% exact matches with assembly-derived small variant calls, which significantly improves the edit rate when realigning assemblies compared to minigraph. We use the three pangenomes to investigate 9566 variable number tandem repeats (VNTRs), finding 63% have identical predicted repeat counts in the three graphs, while minigraph can over or underestimate the count given its approximate coordinate system. We examine a highly variable VNTR locus and show that repeat unit copy number impacts the expression of proximal genes and non-coding RNA. CONCLUSIONS: Our findings indicate good consensus between the three pangenome methods but also show their individual strengths and weaknesses that need to be considered when analysing different types of variants from multiple input assemblies.

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.

Novel functional sequences uncovered through a bovine multiassembly graph.

Many genomic analyses start by aligning sequencing reads to a linear reference genome. However, linear reference genomes are imperfect, lacking millions of bases of unknown relevance and are unable to reflect the genetic diversity of populations. This makes reference-guided methods susceptible to reference-allele bias. To overcome such limitations, we build a pangenome from six reference-quality assemblies from taurine and indicine cattle as well as yak. The pangenome contains an additional 70,329,827 bases compared to the Bos taurus reference genome. Our multiassembly approach reveals 30 and 10.1 million bases private to yak and indicine cattle, respectively, and between 3.3 and 4.4 million bases unique to each taurine assembly. Utilizing transcriptomes from 56 cattle, we show that these nonreference sequences encode transcripts that hitherto remained undetected from the B. taurus reference genome. We uncover genes, primarily encoding proteins contributing to immune response and pathogen-mediated immunomodulation, differentially expressed between Mycobacterium bovis-infected and noninfected cattle that are also undetectable in the B. taurus reference genome. Using whole-genome sequencing data of cattle from five breeds, we show that reads which were previously misaligned against the Bos taurus reference genome now align accurately to the pangenome sequences. This enables us to discover 83,250 polymorphic sites that segregate within and between breeds of cattle and capture genetic differentiation across breeds. Our work makes a so-far unused source of variation amenable to genetic investigations and provides methods and a framework for establishing and exploiting a more diverse reference genome.

Characterization of a haplotype-reference panel for genotyping by low-pass sequencing in Swiss Large White pigs.

BACKGROUND: The key-ancestor approach has been frequently applied to prioritize individuals for whole-genome sequencing based on their marginal genetic contribution to current populations. Using this approach, we selected 70 key ancestors from two lines of the Swiss Large White breed that have been selected divergently for fertility and fattening traits and sequenced their genomes with short paired-end reads. RESULTS: Using pedigree records, we estimated the effective population size of the dam and sire line to 72 and 44, respectively. In order to assess sequence variation in both lines, we sequenced the genomes of 70 boars at an average coverage of 16.69-fold. The boars explained 87.95 and 95.35% of the genetic diversity of the breeding populations of the dam and sire line, respectively. Reference-guided variant discovery using the GATK revealed 26,862,369 polymorphic sites. Principal component, admixture and fixation index (FST) analyses indicated considerable genetic differentiation between the lines. Genomic inbreeding quantified using runs of homozygosity was higher in the sire than dam line (0.28 vs 0.26). Using two complementary approaches, we detected 51 signatures of selection. However, only six signatures of selection overlapped between both lines. We used the sequenced haplotypes of the 70 key ancestors as a reference panel to call 22,618,811 genotypes in 175 pigs that had been sequenced at very low coverage (1.11-fold) using the GLIMPSE software. The genotype concordance, non-reference sensitivity and non-reference discrepancy between thus inferred and Illumina PorcineSNP60 BeadChip-called genotypes was 97.60, 98.73 and 3.24%, respectively. The low-pass sequencing-derived genomic relationship coefficients were highly correlated (r > 0.99) with those obtained from microarray genotyping. CONCLUSIONS: We assessed genetic diversity within and between two lines of the Swiss Large White pig breed. Our analyses revealed considerable differentiation, even though the split into two populations occurred only few generations ago. The sequenced haplotypes of the key ancestor animals enabled us to implement genotyping by low-pass sequencing which offers an intriguing cost-effective approach to increase the variant density over current array-based genotyping by more than 350-fold.

Bovine breed-specific augmented reference graphs facilitate accurate sequence read mapping and unbiased variant discovery.

BACKGROUND: The current bovine genomic reference sequence was assembled from a Hereford cow. The resulting linear assembly lacks diversity because it does not contain allelic variation, a drawback of linear references that causes reference allele bias. High nucleotide diversity and the separation of individuals by hundreds of breeds make cattle ideally suited to investigate the optimal composition of variation-aware references. RESULTS: We augment the bovine linear reference sequence (ARS-UCD1.2) with variants filtered for allele frequency in dairy (Brown Swiss, Holstein) and dual-purpose (Fleckvieh, Original Braunvieh) cattle breeds to construct either breed-specific or pan-genome reference graphs using the vg toolkit. We find that read mapping is more accurate to variation-aware than linear references if pre-selected variants are used to construct the genome graphs. Graphs that contain random variants do not improve read mapping over the linear reference sequence. Breed-specific augmented and pan-genome graphs enable almost similar mapping accuracy improvements over the linear reference. We construct a whole-genome graph that contains the Hereford-based reference sequence and 14 million alleles that have alternate allele frequency greater than 0.03 in the Brown Swiss cattle breed. Our novel variation-aware reference facilitates accurate read mapping and unbiased sequence variant genotyping for SNPs and Indels. CONCLUSIONS: We develop the first variation-aware reference graph for an agricultural animal ( https://doi.org/10.5281/zenodo.3759712 ). Our novel reference structure improves sequence read mapping and variant genotyping over the linear reference. Our work is a first step towards the transition from linear to variation-aware reference structures in species with high genetic diversity and many sub-populations.

Activation of cryptic splicing in bovine WDR19 is associated with reduced semen quality and male fertility.

Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10-27), head (P = 2.0 x 10-44) and tail anomalies (P = 7.2 x 10-49) and insemination success (P = 9.9 x 10-13). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10-32) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24% in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle.

Assessing genomic diversity and signatures of selection in Original Braunvieh cattle using whole-genome sequencing data.

BACKGROUND: Autochthonous cattle breeds are an important source of genetic variation because they might carry alleles that enable them to adapt to local environment and food conditions. Original Braunvieh (OB) is a local cattle breed of Switzerland used for beef and milk production in alpine areas. Using whole-genome sequencing (WGS) data of 49 key ancestors, we characterize genomic diversity, genomic inbreeding, and signatures of selection in Swiss OB cattle at nucleotide resolution. RESULTS: We annotated 15,722,811 SNPs and 1,580,878 Indels including 10,738 and 2763 missense deleterious and high impact variants, respectively, that were discovered in 49 OB key ancestors. Six Mendelian trait-associated variants that were previously detected in breeds other than OB, segregated in the sequenced key ancestors including variants causal for recessive xanthinuria and albinism. The average nucleotide diversity (1.6  × 10- 3) was higher in OB than many mainstream European cattle breeds. Accordingly, the average genomic inbreeding derived from runs of homozygosity (ROH) was relatively low (FROH = 0.14) in the 49 OB key ancestor animals. However, genomic inbreeding was higher in OB cattle of more recent generations (FROH = 0.16) due to a higher number of long (> 1 Mb) runs of homozygosity. Using two complementary approaches, composite likelihood ratio test and integrated haplotype score, we identified 95 and 162 genomic regions encompassing 136 and 157 protein-coding genes, respectively, that showed evidence (P < 0.005) of past and ongoing selection. These selection signals were enriched for quantitative trait loci related to beef traits including meat quality, feed efficiency and body weight and pathways related to blood coagulation, nervous and sensory stimulus. CONCLUSIONS: We provide a comprehensive overview of sequence variation in Swiss OB cattle genomes. With WGS data, we observe higher genomic diversity and less inbreeding in OB than many European mainstream cattle breeds. Footprints of selection were detected in genomic regions that are possibly relevant for meat quality and adaptation to local environmental conditions. Considering that the population size is low and genomic inbreeding increased in the past generations, the implementation of optimal mating strategies seems warranted to maintain genetic diversity in the Swiss OB cattle population.

Accurate sequence variant genotyping in cattle using variation-aware genome graphs.

BACKGROUND: Genotyping of sequence variants typically involves, as a first step, the alignment of sequencing reads to a linear reference genome. Because a linear reference genome represents only a small fraction of all the DNA sequence variation within a species, reference allele bias may occur at highly polymorphic or divergent regions of the genome. Graph-based methods facilitate the comparison of sequencing reads to a variation-aware genome graph, which incorporates a collection of non-redundant DNA sequences that segregate within a species. We compared the accuracy and sensitivity of graph-based sequence variant genotyping using the Graphtyper software to two widely-used methods, i.e., GATK and SAMtools, which rely on linear reference genomes using whole-genome sequencing data from 49 Original Braunvieh cattle. RESULTS: We discovered 21,140,196, 20,262,913, and 20,668,459 polymorphic sites using GATK, Graphtyper, and SAMtools, respectively. Comparisons between sequence variant genotypes and microarray-derived genotypes showed that Graphtyper outperformed both GATK and SAMtools in terms of genotype concordance, non-reference sensitivity, and non-reference discrepancy. The sequence variant genotypes that were obtained using Graphtyper had the smallest number of Mendelian inconsistencies between sequence-derived single nucleotide polymorphisms and indels in nine sire-son pairs. Genotype phasing and imputation using the Beagle software improved the quality of the sequence variant genotypes for all the tools evaluated, particularly for animals that were sequenced at low coverage. Following imputation, the concordance between sequence- and microarray-derived genotypes was almost identical for the three methods evaluated, i.e., 99.32, 99.46, and 99.24% for GATK, Graphtyper, and SAMtools, respectively. Variant filtration based on commonly used criteria improved genotype concordance slightly but it also decreased sensitivity. Graphtyper required considerably more computing resources than SAMtools but less than GATK. CONCLUSIONS: Sequence variant genotyping using Graphtyper is accurate, sensitive and computationally feasible in cattle. Graph-based methods enable sequence variant genotyping from variation-aware reference genomes that may incorporate cohort-specific sequence variants, which is not possible with the current implementation of state-of-the-art methods that rely on linear reference genomes.

Widespread gene duplication and adaptive evolution in the RNA interference pathways of the Drosophila obscura group.

BACKGROUND: RNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show gene duplication and loss-most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the obscura species-group of Drosophila. RESULTS: Here we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression. CONCLUSIONS: These results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, have undergone multiple independent duplications and that their history has been particularly labile within the obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible.