Truncation of IFT80 causes early embryonic loss in Holstein cattle associated with Holstein haplotype 2.

Recessive alleles represent genetic risk in populations that have undergone bottleneck events. We present a comprehensive framework for identification and validation of these genetic defects, including haplotype-based detection, variant selection from sequence data, and validation using knockout embryos. Holstein haplotype 2 (HH2), which causes embryonic death, was used to demonstrate the approach. Holstein haplotype 2 was identified using a deficiency-of-homozygotes approach and confirmed to negatively affect conception rate and stillbirths. Five carriers were present in a group of 183 sequenced Holstein bulls selected to maximize the coverage of unique haplotypes. Three variants concordant with haplotype calls were found in HH2: a high-priority frameshift mutation resulting, and 2 low-priority variants (1 synonymous variant, 1 premature stop codon). The frameshift in intraflagellar 80 (IFT80) was confirmed in a separate group of Holsteins from the 1000 Bull Genomes Project that shared no animals with the discovery set. IFT80-null embryos were generated by truncating the IFT80 transcript at exon 2 or 11 using a CRISPR-Cas9 system. Abattoir-derived oocytes were fertilized in vitro, and zygotes were injected at the one-cell stage either with a guide RNA and CAS9 mRNA complex (n = 100) or Cas9 mRNA (control, n = 100) before return to culture, and replicated 3 times. IFT80 is activated at the 8-cell stage, and IFT80-null embryos arrested at this stage of development, which is consistent with data from mouse hypomorphs and HH2 carrier-to-carrier matings. This frameshift in IFT80 on chromosome 1 at 107,172,615 bp (p.Leu381fs) disrupts WNT and hedgehog signaling, and is responsible for the death of homozygous embryos.

Cattle Sex-Specific Recombination and Genetic Control from a Large Pedigree Analysis.

Meiotic recombination is an essential biological process that generates genetic diversity and ensures proper segregation of chromosomes during meiosis. From a large USDA dairy cattle pedigree with over half a million genotyped animals, we extracted 186,927 three-generation families, identified over 8.5 million maternal and paternal recombination events, and constructed sex-specific recombination maps for 59,309 autosomal SNPs. The recombination map spans for 25.5 Morgans in males and 23.2 Morgans in females, for a total studied region of 2,516 Mb (986 kb/cM in males and 1,085 kb/cM in females). The male map is 10% longer than the female map and the sex difference is most pronounced in the subtelomeric regions. We identified 1,792 male and 1,885 female putative recombination hotspots, with 720 hotspots shared between sexes. These hotspots encompass 3% of the genome but account for 25% of the genome-wide recombination events in both sexes. During the past forty years, males showed a decreasing trend in recombination rate that coincided with the artificial selection for milk production. Sex-specific GWAS analyses identified PRDM9 and CPLX1 to have significant effects on genome-wide recombination rate in both sexes. Two novel loci, NEK9 and REC114, were associated with recombination rate in both sexes, whereas three loci, MSH4, SMC3 and CEP55, affected recombination rate in females only. Among the multiple PRDM9 paralogues on the bovine genome, our GWAS of recombination hotspot usage together with linkage analysis identified the PRDM9 paralogue on chromosome 1 to be associated in the U.S. Holstein data. Given the largest sample size ever reported for such studies, our results reveal new insights into the understanding of cattle and mammalian recombination.

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.

Identification of a nonsense mutation in APAF1 that is likely causal for a decrease in reproductive efficiency in Holstein dairy cattle.

The HH1 haplotype on chromosome 5 is associated with a reduced conception rate and a deficit of homozygotes at the population level in Holstein cattle. The source HH1 haplotype was traced to the bull Pawnee Farm Arlinda Chief (Chief), who was born in 1962 and has sired more than 16,000 daughters. We identified a nonsense mutation in APAF1 (apoptotic protease activating factor 1;APAF1 p.Q579X) within HH1 using whole-genome resequencing of Chief and 3 of his sons. This mutation is predicted to truncate 670 AA (53.7%) of the encoded APAF1 protein that contains a WD40 domain critical to protein-protein interactions. Initial screening revealed no homozygous individuals for the mutation in 758 animals previously genotyped, whereas all 497 HH1 carriers possessed 1 copy of the mutant allele. Subsequent commercial genotyping of 246,773 Holsteins revealed 5,299 APAF1 heterozygotes and zero homozygotes for the mutation. The causative role of this mutation is also supported by functional data in mice that have demonstrated Apaf1 to be an essential molecule in the cytochrome-c-mediated apoptotic cascade and directly implicated in developmental and neurodegenerative disorders. In addition, most Apaf1 homozygous knockouts die by day 16.5 of development. We thus propose that the APAF1 p.Q579X nonsense mutation is the functional equivalent of the Apaf1 knockout. This mutation has caused an estimated 525,000 spontaneous abortions worldwide over the past 35 years, accounting for approximately $420 million in losses. With the mutation identified, selection against the deleterious allele in breeding schemes has aided in eliminating this defect from the population, reducing carrier frequency from 8% in past decades to 2% in 2015.

Single nucleotide polymorphisms in candidate genes associated with fertilizing ability of sperm and subsequent embryonic development in cattle.

Fertilization and development of the preimplantation embryo is under genetic control. The present goal was to test 434 single nucleotide polymorphisms (SNPs) for association with genetic variation in fertilization and early embryonic development. The approach was to produce embryos from 93 bulls using in vitro procedures (n = 3-6 replicates per bull) and relate cleavage rate (CR) and development of cleaved embryos to the blastocyst stage (BDRC) to the genotype for each SNP. Bulls were selected to have either high or low estimates for predicted transmitted ability for daughter pregnancy rate (DPR), an estimate of female fertility. The repeatability was 0.84 for CR and 0.55 for BDRC. Semen extender affected CR, with lower results for milk extender than yolk extender. There was no significant correlation between DPR and either CR or BDRC. A total of 100 SNPs had a minor allele frequency sufficiently high (>5%) to allow association analysis. There were nine genes with SNPs associated with CR (AVP, DEPP, EPAS1, HSD17B6, NT5E, SERPINE2, SLC18A2, TBC1D24, and a noncharacterized gene) and 12 genes with SNPs associated with BDRC (C1QB, FAM5C, HSPA1A, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3, and WBP1). Results demonstrate that in vitro fertilization and blastocyst development are under genetic control and point out the potential importance of some previously unknown genes in these processes. Selection of cattle based on the genotype at one or more of these 19 loci may prove useful in conjunction with other genetic markers for improving genetic ability for fertility.

Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle.

BACKGROUND: Identification of single nucleotide polymorphisms (SNPs) for specific genes involved in reproduction might improve reliability of genomic estimates for these low-heritability traits. Semen from 550 Holstein bulls of high (≥ 1.7; n = 288) or low (≤ -2; n = 262) daughter pregnancy rate (DPR) was genotyped for 434 candidate SNPs using the Sequenom MassARRAY® system. Three types of SNPs were evaluated: SNPs previously reported to be associated with reproductive traits or physically close to genetic markers for reproduction, SNPs in genes that are well known to be involved in reproductive processes, and SNPs in genes that are differentially expressed between physiological conditions in a variety of tissues associated in reproductive function. Eleven reproduction and production traits were analyzed. RESULTS: A total of 40 SNPs were associated (P < 0.05) with DPR. Among these were genes involved in the endocrine system, cell signaling, immune function and inhibition of apoptosis. A total of 10 genes were regulated by estradiol. In addition, 22 SNPs were associated with heifer conception rate, 33 with cow conception rate, 36 with productive life, 34 with net merit, 23 with milk yield, 19 with fat yield, 13 with fat percent, 19 with protein yield, 22 with protein percent, and 13 with somatic cell score. The allele substitution effect for SNPs associated with heifer conception rate, cow conception rate, productive life and net merit were in the same direction as for DPR. Allele substitution effects for several SNPs associated with production traits were in the opposite direction as DPR. Nonetheless, there were 29 SNPs associated with DPR that were not negatively associated with production traits. CONCLUSION: SNPs in a total of 40 genes associated with DPR were identified as well as SNPs for other traits. It might be feasible to include these SNPs into genomic tests of reproduction and other traits. The genes associated with DPR are likely to be important for understanding the physiology of reproduction. Given the large number of SNPs associated with DPR that were not negatively associated with production traits, it should be possible to select for DPR without compromising production.

Integrating Signals from Sperm Methylome Analysis and Genome-Wide Association Study for a Better Understanding of Male Fertility in Cattle.

Decreased male fertility is a big concern in both human society and the livestock industry. Sperm DNA methylation is commonly believed to be associated with male fertility. However, due to the lack of accurate male fertility records (i.e., limited mating times), few studies have investigated the comprehensive impacts of sperm DNA methylation on male fertility in mammals. In this study, we generated 10 sperm DNA methylomes and performed a preliminary correlation analysis between signals from sperm DNA methylation and signals from large-scale (n = 27,214) genome-wide association studies (GWAS) of 35 complex traits (including 12 male fertility-related traits). We detected genomic regions, which experienced DNA methylation alterations in sperm and were associated with aging and extreme fertility phenotypes (e.g., sire-conception rate or SCR). In dynamic hypomethylated regions (HMRs) and partially methylated domains (PMDs), we found genes (e.g., HOX gene clusters and microRNAs) that were involved in the embryonic development. We demonstrated that genomic regions, which gained rather than lost methylations during aging, and in animals with low SCR were significantly and selectively enriched for GWAS signals of male fertility traits. Our study discovered 16 genes as the potential candidate markers for male fertility, including SAMD5 and PDE5A. Collectively, this initial effort supported a hypothesis that sperm DNA methylation may contribute to male fertility in cattle and revealed the usefulness of functional annotations in enhancing biological interpretation and genomic prediction for complex traits and diseases.

Comparative analyses of sperm DNA methylomes among human, mouse and cattle provide insights into epigenomic evolution and complex traits.

Sperm DNA methylation is crucial for fertility and viability of offspring but epigenome evolution in mammals is largely understudied. By comparing sperm DNA methylomes and large-scale genome-wide association study (GWAS) signals between human and cattle, we aimed to examine the DNA methylome evolution and its associations with complex phenotypes in mammals. Our analysis revealed that genes with conserved non-methylated promoters (e.g., ANKS1A and WNT7A) among human and cattle were involved in common system and embryo development, and enriched for GWAS signals of body conformation traits in both species, while genes with conserved hypermethylated promoters (e.g., TCAP and CD80) were engaged in immune responses and highlighted by immune-related traits. On the other hand, genes with human-specific hypomethylated promoters (e.g., FOXP2 and HYDIN) were engaged in neuron system development and enriched for GWAS signals of brain-related traits, while genes with cattle-specific hypomethylated promoters (e.g., LDHB and DGAT2) mainly participated in lipid storage and metabolism. We validated our findings using sperm-retained nucleosome, preimplantation transcriptome, and adult tissue transcriptome data, as well as sequence evolutionary features, including motif binding sites, mutation rates, recombination rates and evolution signatures. In conclusion, our results demonstrate important roles of epigenome evolution in shaping the genetic architecture underlying complex phenotypes, hence enhance signal prioritization in GWAS and provide valuable information for human neurological disorders and livestock genetic improvement.

Convergent Evolution of Slick Coat in Cattle through Truncation Mutations in the Prolactin Receptor.

Evolutionary adaptations are occasionally convergent solutions to the same problem. A mutation contributing to a heat tolerance adaptation in Senepol cattle, a New World breed of mostly European descent, results in the distinct phenotype known as slick, where an animal has shorter hair and lower follicle density across its coat than wild type animals. The causal variant, located in the 11th exon of prolactin receptor, produces a frameshift that results in a truncated protein. However, this mutation does not explain all cases of slick coats found in criollo breeds. Here, we obtained genome sequences from slick cattle of a geographically distinct criollo breed, namely Limonero, whose ancestors were originally brought to the Americas by the Spanish. These data were used to identify new causal alleles in the 11th exon of the prolactin receptor, two of which also encode shortened proteins that remove a highly conserved tyrosine residue. These new mutations explained almost 90% of investigated cases of animals that had slick coats, but which also did not carry the Senepol slick allele. These results demonstrate convergent evolution at the molecular level in a trait important to the adaptation of an animal to its environment.

Diversity and population-genetic properties of copy number variations and multicopy genes in cattle.

The diversity and population genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analysed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, and Romagnola), sequenced to 11-fold coverage to identify 1,853 non-redundant CNV regions. Supported by high validation rates in array comparative genomic hybridization (CGH) and qPCR experiments, these CNV regions accounted for 3.1% (87.5 Mb) of the cattle reference genome, representing a significant increase over previous estimates of the area of the genome that is copy number variable (∼2%). Further population genetics and evolutionary genomics analyses based on these CNVs revealed the population structures of the cattle taurine and indicine breeds and uncovered potential diversely selected CNVs near important functional genes, including AOX1, ASZ1, GAT, GLYAT, and KRTAP9-1 Additionally, 121 CNV gene regions were found to be either breed specific or differentially variable across breeds, such as RICTOR in dairy breeds and PNPLA3 in beef breeds. In contrast, clusters of the PRP and PAG genes were found to be duplicated in all sequenced animals, suggesting that subfunctionalization, neofunctionalization, or overdominance play roles in diversifying those fertility-related genes. These CNV results provide a new glimpse into the diverse selection histories of cattle breeds and a basis for correlating structural variation with complex traits in the future.

Genome-wide association mapping for identification of quantitative trait loci for rectal temperature during heat stress in Holstein cattle.

Heat stress compromises production, fertility, and health of dairy cattle. One mitigation strategy is to select individuals that are genetically resistant to heat stress. Most of the negative effects of heat stress on animal performance are a consequence of either physiological adaptations to regulate body temperature or adverse consequences of failure to regulate body temperature. Thus, selection for regulation of body temperature during heat stress could increase thermotolerance. The objective was to perform a genome-wide association study (GWAS) for rectal temperature (RT) during heat stress in lactating Holstein cows and identify SNPs associated with genes that have large effects on RT. Records on afternoon RT where the temperature-humidity index was ≥78.2 were obtained from 4,447 cows sired by 220 bulls, resulting in 1,440 useable genotypes from the Illumina BovineSNP50 BeadChip with 39,759 SNP. For GWAS, 2, 3, 4, 5, and 10 adjacent SNP were averaged to identify consensus genomic regions associated with RT. The largest proportion of SNP variance (0.07 to 0.44%) was explained by markers flanking the region between 28,877,547 and 28,907,154 bp on Bos taurus autosome (BTA) 24. That region is flanked by U1 (28,822,883 to 28,823,043) and NCAD (28,992,666 to 29,241,119). In addition, the SNP at 58,500,249 bp on BTA 16 explained 0.08% and 0.11% of the SNP variance for 2- and 3-SNP analyses, respectively. That contig includes SNORA19, RFWD2 and SCARNA3. Other SNPs associated with RT were located on BTA 16 (close to CEP170 and PLD5), BTA 5 (near SLCO1C1 and PDE3A), BTA 4 (near KBTBD2 and LSM5), and BTA 26 (located in GOT1, a gene implicated in protection from cellular stress). In conclusion, there are QTL for RT in heat-stressed dairy cattle. These SNPs could prove useful in genetic selection and for identification of genes involved in physiological responses to heat stress.

Identification of a nonsense mutation in CWC15 associated with decreased reproductive efficiency in Jersey cattle.

With the recent advent of genomic tools for cattle, several recessive conditions affecting fertility have been identified and selected against, such as deficiency of uridine monophosphate synthase, complex vertebral malformation, and brachyspina. The current report refines the location of a recessive haplotype affecting fertility in Jersey cattle using crossover haplotypes, discovers the causative mutation using whole genome sequencing, and examines the gene's role in embryo loss. In an attempt to identify unknown recessive lethal alleles in the current dairy population, a search using deep Mendelian sampling of 5,288 Jersey cattle was conducted for high-frequency haplotypes that have a deficit of homozygotes at the population level. This search led to the discovery of a putative recessive lethal in Jersey cattle on Bos taurus autosome 15. The haplotype, denoted JH1, was associated with reduced fertility, and further investigation identified one highly-influential Jersey bull as the putative source ancestor. By combining SNP analysis of whole-genome sequences aligned to the JH1 interval and subsequent SNP validation a nonsense mutation in CWC15 was identified as the likely causative mutation underlying the fertility phenotype. No homozygous recessive individuals were found in 749 genotyped animals, whereas all known carriers and carrier haplotypes possessed one copy of the mutant allele. This newly identified lethal has been responsible for a substantial number of spontaneous abortions in Jersey dairy cattle throughout the past half-century. With the mutation identified, selection against the deleterious allele in breeding schemes will aid in reducing the incidence of this defect in the population. These results also show that carrier status can be imputed with high accuracy. Whole-genome resequencing proved to be a powerful strategy to rapidly identify a previously mapped deleterious mutation in a known carrier of a recessive lethal allele.