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Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle

Abstract

The 1000 bull genomes project supports the goal of accelerating the rates of genetic gain in domestic cattle while at the same time considering animal health and welfare by providing the annotated sequence variants and genotypes of key ancestor bulls. In the first phase of the 1000 bull genomes project, we sequenced the whole genomes of 234 cattle to an average of 8.3-fold coverage. This sequencing includes data for 129 individuals from the global Holstein-Friesian population, 43 individuals from the Fleckvieh breed and 15 individuals from the Jersey breed. We identified a total of 28.3 million variants, with an average of 1.44 heterozygous sites per kilobase for each individual. We demonstrate the use of this database in identifying a recessive mutation underlying embryonic death and a dominant mutation underlying lethal chrondrodysplasia. We also performed genome-wide association studies for milk production and curly coat, using imputed sequence variants, and identified variants associated with these traits in cattle.

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Figure 1: Accuracy of imputing sequence variants.
Figure 2: Sequence-based fine-mapping of a locus underlying curly hair in 2,253 Fleckvieh bulls.
Figure 3: Sequence-based association study for early-lactation milk fat percentage.

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Sequence Read Archive

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Acknowledgements

H.P. and R.F. acknowledge funding from the German Federal Ministry of Education and Research (BMBF) within the AgroClustEr 'Synbreed—Synergistic Plant and Animal Breeding' (grant 0315527B). We acknowledge the Bavarian State Research Center and Vereinigte Informationssysteme Tierhaltung (VIT) Verden for providing phenotype data. Swissgenetics is acknowledged for providing the DNA material for a Swiss Simmental bull. P.S. and X.L. acknowledge funding from the Genome Canada project entitled 'Whole-Genome Sequence Selection Through Genome-Wide Imputation in Beef Cattle' and acknowledge WestGrid and Compute/Calcul Canada for providing computing resources. D.A.B., A.C., S.F. and D.R. acknowledge the Agence Nationale de la Recherche and Apisgene for funding the Cartoseq project (ANR-10-GENM-0018). T. Simon (EVOLUTION), S. Legrain and P. Laurent (INRA) are acknowledged for providing samples from bulldog calves and information. J.-M. Vacelet (GEN'IAtest), G. Fayolle (UMOTEST) and D. Peinturier (Jura Bétail) are acknowledged for providing samples and phenotypic information for curly coat in Montbeliarde cattle. A. Baur (UNCEIA) and A. Barbat (INRA) are acknowledged for providing haplotype and fertility information for the HH3 study. We would like to thank E. Mullaart (CRV) for providing sequences for ten of the bulls used in this project. B.G., R.F.B. and M.S.L. acknowledge support from grant 3405-10-0137 from the Green Development and Demonstration Programme of the Danish Ministry of Food, Agriculture and Fisheries, the Milk Levy Fund, grant 12-132452 (GenSAP) from the Strategic Research Council and Viking Genetics. C.P.V. acknowledges funding from USDA-ARS project 1245-31000-104-00D and USDA-National Institute of Food and Agriculture (NIFA) competitive grant 2009-65205-05635 from the Animal Genome Program. B.J.H., H.D.D., A.J.C., P.J.B. and M.E.G. acknowledge funding from the Dairy Futures Cooperative Research Centre, Dairy Australia and the Cooperative Research Centre for Beef Genetic Technologies.

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Authors and Affiliations

Authors

Contributions

B.J.H., D.A.B., R.F.V., M.S.L., B.G., M.E.G., R.F., D.R. and C.P.V. designed the study. A.C., H.P., H.D.D., R.v.B., R.F.B. and S.F. performed statistical analyses. P.S. and X.L. performed variant annotation. A.J.C., P.S., X.L., C.A., P.J.B., D.C., I.H. and H.D.D. performed quality control analyses and prepared data. H.D.D., D.C. and P.J.B. designed and developed software to process sequences. A.D., A.E., S.C.R., C.G., D.E., M.-N.R., C.K. and O.B. contributed genotype and phenotype data. H.D.D., R.F., H.P., A.C., D.A.B., R.F.V., B.G., M.S.L., C.P.V., P.S. and B.J.H. contributed to writing the manuscript.

Corresponding author

Correspondence to Ben J Hayes.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Improvement in accuracy of imputation as a result of BEAGLE correction in Fleckvieh bulls, at increasing fold coverage.

Supplementary Figure 2 The rate of opposing homozygotes from comparison of genotypes for 54 parent-offspring pairs.

Rates for chromosome 1 (a) and chromosome 12 (b). The rate of opposing homozygotes is calculated as the proportion of genotypes within a sliding window of 500 variants that are opposing homozygotes for parent offspring pairs. Genomic regions potentially with problematic assembly are revealed by their high rate of opposing homozygotes, as on chromosome 12. The 1000 bull genomes data has identified a number of such regions.

Supplementary Figure 3 Variants segregating within and across breeds.

Absolute counts of variants segregating within and across breeds (a). Polymorphic sites (SNPs, indels) in randomly selected subsets of 60 animals (15 Angus, 15 Fleckvieh, 15 Holstein, 15 Jersey) were evaluated to assess the number of variants segregating within and across breeds. Across ten replicates, an average number of 8.74 million variants were identified in all 4 breeds (b). Among those, 116,489 variants were homozygous for the alternate allele to the reference sequence in all animals. Holstein and Fleckvieh shared the most variants that were segregating across breeds, most likely reflecting a recent introgression of Holstein into Fleckvieh3. Angus and Jersey shared the least, consistent with FST estimates from array data (The Bovine Hap Consortium, 2009). Compared to Angus, the number of variants segregating within the breed was considerably higher in Fleckvieh, Holstein and Jersey, possibly reflecting that the resequenced animals are key ancestors of these breeds. Fleckvieh had the highest genetic diversity, corroborating its large effective population size4. Principal-component analysis on the sequence variants clearly separated animals by breed (c).

Supplementary Figure 4 Distribution of length of insertion deletions (bp).

(a) All indels. (b) Indels in coding regions.

Supplementary Figure 5 Allele frequency spectrum of variant classes.

(a) Intergenic and intragenic and, for the intragenic variants, synonymous versus nonsynonymous, (b) for nonsynonymous variants, missense and premature stop codons, (c) also for nonsynonymous variants, SIFT predicted tolerated and SIFT predicted deleterious variants. The allele frequency spectrum is reflected around 0.5.

Supplementary Figure 6 Alignment of the SMC2 protein sequences from different eukaryotic organisms around amino acid position F1135 in bovine.

Species names and sequence accession numbers in Ensembl are: Bta, Bos taurus (ENSBTAP00000011562); Has, Homo sapiens (ENSP00000286398); Gga, Gallus gallus (ENSGALP00000036817); Aca, Anolis carolinensis (ENSACAP00000012737); Xtr, Xenopus tropicalis (ENSXETP00000022515); Gmo, Gadus morhua (ENSGMOP00000005092); Csa, Ciona savignyi (ENSCSAVP00000005455); Dme, Drosophila melanogaster (FBpp0086591); Cel, Caenorhabditis elegans (M106.1); Tgo, Toxoplasma gondii (TGME49_097800); Ptr, Phaeodactylum tricornutum (Phatr30352); Ppa, Physcomitrella patens (PP1S52_57V6.1); Ath, Arabidopsis thaliana (AT5G62410.1); Afl, Aspergillus flavus (CADAFLAP00007264); Sce, Saccharomyces cerevisiae (YFR031C). Animals are shown in normal character, protists are underlined, plants are shown in bold type, and fungi are italicized.

Supplementary Figure 7 Identification and characterization of the COL2A1 g.32475732G>A mutation on BTA5, associated with bulldog calf syndrome.

(a) IGV snapshot and (b) DNA sequencing chromatogram showing the g.32475732G>A mutation of a bulldog calf. (b) Genotyping of the same mutation using a PCR-RFLP system (see material and methods). BD, bulldog calves; Mo, wild-type mother of a bulldog calf; IG, IGALE MASC. The 341-bp undigested fragment corresponds to the mutant allele, whereas the 166- and 175-bp fragments result from the digestion of the wild-type allele with BpmI endonuclease. Note incomplete restriction of the IGALE PCR fragment, consistent with mosaicism. (c) DNA sequencing chromatogram showing the g.32475732G>A mutation of a bulldog calf. Confirmation of mosaicism by Sanger sequencing of IGALE PCR fragments prior to RFLP (d) and of a nested PCR on the unrestricted fragment after RFLP (e). (f) Multispecies alignment of the COL2A1 protein sequence around the p.G960R substitution using CLUSTALW. Cattle (Bos taurus), human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), anolis (Anolis carolinensis), Xenopus (Xenopus tropicalis) and zebrafish (Danio rerio) sequence accession numbers are, respectively, ENSBTAP00000017505, ENSP00000369889, ENSMUSP00000023123, ENSGALP00000035064, ENSACAP00000006225, ENSXETP00000043834 and ENSDARP00000047843 in Ensembl . Note the succession of GXY amino acids triplets, typical of the triple-helical domain of collagen proteins, and the perfect across-species conservation of the first G residues.

Supplementary Figure 8 Sequence-based association study for curly hair.

Manhattan plot showing the association of 17,640,970 imputed variants with the proportion of daughters with curly hair in 3222 Fleckvieh bulls (a). Red dots represent variants with P < 10−9. Detailed overview of the associated regions on chromosomes 5 (b) and 19 (c). Variants in the promoter (defined to encompass 1,000 bp upstream of the transcription start), in the untranslated regions (UTR) and in the amino acid coding region are highlighted with different color. The associated region on BTA5 encompasses Krt71, which underlies curly hair in various species. Variant calling yielded four missense mutations in Krt71 (p.R133W, p.F143I, p.N177I, p.P452H); however, none of them was polymorphic in the 43 resequenced Fleckvieh animals. Functional annotation of the variants within the QTL region on BTA5 revealed that 12 closely linked missense mutations in Krt73, Krt2 and Krt76 are highly significantly associated with curly hair in Fleckvieh cattle. Among those, only the p.R201Q mutation in Krt73 (c.G602A, chr. 5: 27,445,800 bp, ss682156288) was predicted to be damaging by PolyPhen-2 and SIFT analysis.

Supplementary Figure 9 Analysis of the sequence conservation of the KRT27 and KRT73 proteins.

(a) Multispecies alignment of the KRT27 proteins from Bos taurus (ENSBTAP00000040718), Procavia capensis (ENSPCAP00000010617), Equus caballus (ENSECAP00000009042), Pteropus vampyrus (ENSPVAP00000010717), Mus musculus (ENSMUSP00000017732), Canis familiaris (ENSCAFP00000023584), Homo sapiens (ENSP00000301656), Loxodonta africana (ENSLAFP00000005744), Erinaceus europaeus (ENSEEUP00000003333), Choloepus hoffmanni (ENSCHOP00000004382), Echinops telfairi (ENSETEP00000010909), Ornithorhynchus anatinus (ENSOANP00000004600), Sarcophilus harrisii (ENSSHAP00000009933) and Monodelphis domestica (ENSMODP00000038545). (b) Multiple-sequence alignment of mammalian-specific paralogs of the KRT27 protein type I keratins within the helix initiation motif in bovine KRT9 (ENSBTAP00000054159), KRT10 (ENSBTAP00000017140), KRT12 (ENSBTAP00000020622), KRT14 (ENSBTAP00000036252), KRT15 (ENSBTAP00000006445), KRT16 (ENSBTAP00000045066), KRT17 (ENSBTAP00000008948), KRT18 (ENSBTAP00000001988), KRT19 (ENSBTAP00000006450), KRT20 (ENSBTAP00000010255), KRT23 (ENSBTAP00000051816), KRT24 (ENSBTAP00000027749), KRT25 (ENSBTAP00000040707), KRT26 (ENSBTAP00000040717), KRT27 (ENSBTAP00000040718), KRT28 (ENSBTAP00000040707), KRT31 (ENSBTAP00000018185), KRT32 (ENSBTAP00000013107), KRT33A (ENSBTAP00000023926), KRT34 (ENSBTAP00000012807), KRT35 (ENSBTAP00000040659), KRT36 (ENSBTAP00000006440), KRT37 (ENSBTAP00000000746), KRT38 (ENSBTAP00000000746) KRT39 (ENSBTAP00000026752) and KRT40 (ENSBTAP00000023156). (c) Multiple-sequence alignment of type I keratins within the helix initiation motif in human. The protein sequence and domain information of human type I keratins was obtained from the Human Intermediate Filament Database (http://www.interfil.org/). KRT24 (ENSBTAP00000027749), KRT25 (ENSBTAP00000040707), KRT26 (ENSBTAP00000040717) and KRT28 (ENSBTAP00000034743). (d) Multispecies alignment of the KRT73 proteins from Bos taurus (ENSBTAP00000010393), Homo sapiens (ENSP00000307014), Pteropus vampyrus (ENSPVAP00000006226), Loxodonta africana (ENSLAFP00000006274), Procavia capensis (ENSPCAP00000005278), Equus caballus (ENSECAP00000012147), Erinaceus europaeus (ENSEEUP00000013894), Mus musculus (ENSMUSP00000065349) and Felis catus (ENSFCAP00000000268).

Supplementary Figure 10 Association of 425,607 SNPs located on BTA19 conditional on the ss699911276 polymorphism.

Black dots represent the P values of the initial screen, and orange dots represent the P values obtained from association analysis conditional on the ss699911276 polymorphism. The red vertical line corresponds to the Bonferroni-corrected threshold of significance (P = 2.8 × 10−9).

Supplementary Figure 11 Sequence-based association study of early-lactation milk fat content on bovine chromosome 14.

The sequence-based association study identified the causal p.A232K polymorphism in Dgat1 among the top association signals in the Holstein breed (a). In the Fleckvieh breed, the association of the causal p.A232K polymorphism in Dgat1 with early-lactation milk fat content is considerably lower compared to the top marker (P = 2.6 × 10−57 versus P = 3.2 × 10−106) (b).

Supplementary Figure 12 Sequence-based association study of early-lactation milk fat content on bovine chromosome 14 in the Fleckvieh breed with an increased reference population.

During the review process of this manuscript, the number of resequenced Fleckvieh animals increased to 78. We reimputed the sequence information for the entire Fleckvieh population on the basis of 78 reference animals and reperformed the association analysis. Now, with an increased reference population, the association of the causal p.A232K polymorphism in Dgat1 with early-lactation milk fat content is only marginally lower compared to the top marker (P = 6.1 × 10−111 versus P = 4.5 × 10−111).

Supplementary Figure 13 Transcription factor binding site (TFBS) prediction for associated promoter variants of AGPAT6 (transcript ID ENSBTAT00000007532).

TFBS were predicted with Alibaba2.1 (red; ref. 5) and Contra (green)6. Only TFBS that differ between the reference and alternative sequence are displayed. The blue box highlights the polymorphic sites.

Supplementary Figure 14 Association studies on chromosome 27 for early-lactation milk fat content with sequence variants imputed from the 1000 bull genomes

In Fleckvieh (a) and Holstein (b) populations. Black dots represent the P values of the initial screen, and orange dots represent the P values after conditioning on the indel polymorphism (36,211,252 bp). The red vertical line represents the Bonferroni-corrected threshold of genome-wide significance (P = 2.8 × 10−9). Conditioning on the indel polymorphism eliminated all association signals in the immediate vicinity of AGPAT6. However, there might be an additional QTL for early-lactation milk fat content segregating in both breeds near the Thrb gene encoding the thyroid hormone receptor beta (green triangle).

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Daetwyler, H., Capitan, A., Pausch, H. et al. Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle. Nat Genet 46, 858–865 (2014). https://doi.org/10.1038/ng.3034

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