Large-scale detection and characterization of interchromosomal rearrangements in normozoospermic bulls using massive genotype and phenotype data sets.

In this paper, we developed a highly sensitive approach to detect interchromosomal rearrangements in cattle by searching for abnormal linkage disequilibrium patterns between markers located on different chromosomes in large paternal half-sib families genotyped as part of routine genomic evaluations. We screened 5571 families of artificial insemination sires from 15 breeds and revealed 13 putative interchromosomal rearrangements, 12 of which were validated by cytogenetic analysis and long-read sequencing. These consisted of one Robertsonian fusion, 10 reciprocal translocations, and the first case of insertional translocation reported in cattle. Taking advantage of the wealth of data available in cattle, we performed a series of complementary analyses to define the exact nature of these rearrangements, investigate their origins, and search for factors that may have favored their occurrence. We also evaluated the risks to the livestock industry and showed significant negative effects on several traits in the sires and in their balanced or aneuploid progeny compared with wild-type controls. Thus, we present the most comprehensive and thorough screen for interchromosomal rearrangements compatible with normal spermatogenesis in livestock species. This approach is readily applicable to any population that benefits from large genotype data sets, and will have direct applications in animal breeding. Finally, it also offers interesting prospects for basic research by allowing the detection of smaller and rarer types of chromosomal rearrangements than GTG banding, which are interesting models for studying gene regulation and the organization of genome structure.

Targeted mutation and inactivation of the kinesin light chain 3 protein-encoding gene have no impact on mouse fertility†.

The kinesin light chain 3 protein (KLC3) is the only member of the kinesin light chain protein family that was identified in post-meiotic mouse male germ cells. It plays a role in the formation of the sperm midpiece through its association with both spermatid mitochondria and outer dense fibers (ODF). Previous studies showed a significant correlation between its expression level and sperm motility and quantitative semen parameters in humans, while the overexpression of a KLC3-mutant protein unable to bind ODF also affected the same traits in mice. To further assess the role of KLC3 in fertility, we used CRISPR/Cas9 genome editing in mice and investigated the phenotypes induced by the invalidation of the gene or of a functional domain of the protein. Both approaches gave similar results, i.e. no detectable change in male or female fertility. Testis histology, litter size and sperm count were not altered. Apart from the line-dependent alterations of Klc3 mRNA levels, testicular transcriptome analysis did not reveal any other changes in the genes tested. Western analysis supported the absence of KLC3 in the gonads of males homozygous for the inactivating mutation and a strong decrease in expression in males homozygous for the allele lacking one out of the five tetratricopeptide repeats. Overall, these observations raise questions about the supposedly critical role of this kinesin in reproduction, at least in mice where its gene mutation or inactivation did not translate into fertility impairment.

A recurrent de novo missense mutation in COL1A1 causes osteogenesis imperfecta type II and preterm delivery in Normande cattle.

BACKGROUND: Nine male and eight female calves born to a Normande artificial insemination bull named "Ly" were referred to the French National Observatory of Bovine Abnormalities for multiple fractures, shortened gestation, and stillbirth or perinatal mortality. RESULTS: Using Illumina BovineSNP50 array genotypes from affected calves and 84 half-sib controls, the associated locus was mapped to a 6.5-Mb interval on chromosome 19, assuming autosomal inheritance with germline mosaicism. Subsequent comparison of the whole-genome sequences of one case and 5116 control genomes, followed by genotyping in the affected pedigree, identified a de novo missense substitution within the NC1 domain of the COL1A1 gene (Chr19 g.36,473,965G > A; p.D1412N) as unique candidate variant. Interestingly, the affected residue was completely conserved among 243 vertebrate orthologs, and the same substitution in humans has been reported to cause type II osteogenesis imperfecta (OI), a connective tissue disorder that is characterized primarily by bone deformity and fragility. Moreover, three COL1A1 mutations have been described to cause the same syndrome in cattle. Necropsy, computed tomography, radiology, and histology confirmed the diagnosis of type II OI, further supporting the causality of this variant. In addition, a detailed analysis of gestation length and perinatal mortality in 1387 offspring of Ly and more than 160,000 progeny of 63 control bulls allowed us to statistically confirm in a large pedigree the association between type II OI and preterm delivery, which is probably due to premature rupture of fetal membranes and has been reported in several isolated cases of type II OI in humans and cattle. Finally, analysis of perinatal mortality rates and segregation distortion supported a low level of germ cell mosaicism in Ly, with an estimate of 4.5% to 7.7% of mutant sperm and thus 63 to 107 affected calves born. These numbers contrast with the 17 cases reported and raise concerns about the underreporting of congenital defects to heredo-surveillance platforms, even for textbook genetic syndromes. CONCLUSIONS: In conclusion, we describe a large animal model for a recurrent substitution in COL1A1 that is responsible for type II OI in humans. More generally, this study highlights the utility of such datasets and large half-sib families available in livestock species to characterize sporadic genetic defects.

Genetic parameters, genome-wide association study, and selection perspective on gestation length in 16 French cattle breeds.

In this paper, we present a comprehensive study of gestation length (GL) in 16 cattle breeds by using large genotype and animal record databases. Data included over 20 million gestations since 2000 and genotypes from one million calves. The study addressed the GL variability within and between breeds, estimation of its direct and maternal heritability coefficients, association with fitness and several economic traits, and QTL detection. The breed average GL varied from 279.7 to 294.4 d in Holstein and Blonde d'Aquitaine breeds, respectively. Standard deviations per breed were similar and ranged from 5.2 to 5.8 d. Direct heritability (i.e., for GL defined as a trait of the calf) was moderate to high (h2 = 0.40-0.67), whereas the maternal heritability was low (0.04-0.06). Extreme breeding values for GL were strongly associated with a higher mortality during the first 2 d of life and were associated with milk production of dams for dairy breeds and precocity of females. Finally, several QTL were detected affecting GL with cumulated effects up to a few days, and at least 2 QTL were found to be shared between different breeds. Our study highlights the risks that would be associated with selection toward a reduced GL. Further genomic studies are needed to identify the causal variants and their association with juvenile mortality and other economic traits.

Genetic parameters, GWAS and selection perspective on gestation length in 16 French cattle breeds.

In this paper, we present a comprehensive study of gestation length (GL) in 16 cattle breeds by using large genotype and animal record databases. Data included over 20 million gestations since 2000 and genotypes from one million calves. The study addressed the GL variability within and between breeds, estimation of its direct and maternal heritability coefficients, association with fitness and several economic traits, and QTL detection. The breed average GL varied from 279.7 to 294.4 d, in Holstein and Blonde d'Aquitaine breeds, respectively. Standard deviations per breed were similar and ranged from 5.2 to 5.8 d. Direct heritability (i.e., for GL defined as a trait of the calf) was moderate to high (h2 = 0.40 to 0.67), whereas the maternal heritability was low (0.04 to 0.06). Extreme breeding values for GL were strongly associated with a higher mortality during the first 2 d of life and were associated with milk production of dams for dairy breeds and precocity of females. Finally, several QTL were detected affecting GL with cumulated effects up to a few days, and at least 2 QTL were found to be shared between different breeds. Our study highlights the risks that would be associated with selection toward a reduced gestation length. Further genomic studies are needed to identify the causal variants, and their association with juvenile mortality and other economic traits.

X-linked genes influence various complex traits in dairy cattle.

BACKGROUND: The search for quantitative trait loci (QTL) affecting traits of interest in mammals is frequently limited to autosomes, with the X chromosome excluded because of its hemizygosity in males. This study aimed to assess the importance of the X chromosome in the genetic determinism of 11 complex traits related to milk production, milk composition, mastitis resistance, fertility, and stature in 236,496 cows from three major French dairy breeds (Holstein, Montbéliarde, and Normande) and three breeds of regional importance (Abondance, Tarentaise, and Vosgienne). RESULTS: Estimates of the proportions of heritability due to autosomes and X chromosome (h²X) were consistent among breeds. On average over the 11 traits, h²X=0.008 and the X chromosome explained ~ 3.5% of total genetic variance. GWAS was performed within-breed at the sequence level (~ 200,000 genetic variants) and then combined in a meta-analysis. QTL were identified for most breeds and traits analyzed, with the exception of Tarentaise and Vosgienne and two fertility traits. Overall, 3, 74, 59, and 71 QTL were identified in Abondance, Montbéliarde, Normande, and Holstein, respectively, and most were associated with the most-heritable traits (milk traits and stature). The meta-analyses, which assessed a total of 157 QTL for the different traits, highlighted new QTL and refined the positions of some QTL found in the within-breed analyses. Altogether, our analyses identified a number of functional candidate genes, with the most notable being GPC3, MBNL3, HS6ST2, and DMD for dairy traits; TMEM164, ACSL4, ENOX2, HTR2C, AMOT, and IRAK1 for udder health; MAMLD1 and COL4A6 for fertility; and NRK, ESX1, GPR50, GPC3, and GPC4 for stature. CONCLUSIONS: This study demonstrates the importance of the X chromosome in the genetic determinism of complex traits in dairy cattle and highlights new functional candidate genes and variants for these traits. These results could potentially be extended to other species as many X-linked genes are shared among mammals.

Integrin alpha 6 homozygous splice-site mutation causes a new form of junctional epidermolysis bullosa in Charolais cattle.

BACKGROUND: Inherited epidermolysis bullosa (EB) is a group of painful and life-threatening genetic disorders that are characterized by mechanically induced blistering of the skin and mucous membranes. Congenital skin fragility resembling EB was recently reported in three Charolais calves born in two distinct herds from unaffected parents. Phenotypic and genetic analyses were carried out to describe this condition and its molecular etiology. RESULTS: Genealogical, pathological and histological investigations confirmed the diagnosis of recessive EB. However, the affected calves showed milder clinical signs compared to another form of EB, which was previously reported in the same breed and is caused by a homozygous deletion of the ITGB4 gene. Homozygosity mapping followed by analysis of the whole-genome sequences of two cases and 5031 control individuals enabled us to prioritize a splice donor site of ITGA6 (c.2160 + 1G > T; Chr2 g.24112740C > A) as the most compelling candidate variant. This substitution showed a perfect genotype-phenotype correlation in the two affected pedigrees and was found to segregate only in Charolais, and at a very low frequency (f = 1.6 × 10-4) after genotyping 186,154 animals from 15 breeds. Finally, RT-PCR analyses revealed increased retention of introns 14 and 15 of the ITGA6 gene in a heterozygous mutant cow compared with a matched control. The mutant mRNA is predicted to cause a frameshift (ITGA6 p.I657Mfs1) that affects the assembly of the integrin α6ß4 dimer and its correct anchoring to the cell membrane. This dimer is a key component of the hemidesmosome anchoring complex, which ensures the attachment of basal epithelial cells to the basal membrane. Based on these elements, we arrived at a diagnosis of junctional EB. CONCLUSIONS: We report a rare example of partial phenocopies observed in the same breed and due to mutations that affect two members of the same protein dimer, and provide the first evidence of an ITGA6 mutation that causes EB in livestock species.

Genomic regions underlying positive selection in local, Alpine cattle breeds.

We used genome-wide SNP data from 18 local cattle breeds from six countries of the Alpine region to characterize population structure and identify genomic regions underlying positive selection. The geographically close breeds Evolèner, Eringer, Valdostana Pezzata Nera, and Valdostana Castana were found to differ from all other Alpine breeds. In addition, three breeds, Simmental, and Original Braunvieh from Switzerland and Pinzgauer from Austria built three separate clusters. Of the 18 breeds studied, the intra-alpine Swiss breed Evolèner had the highest average inbreeding based on runs of homozygosity (FROH ) and the highest average genomic relationship within the breed. In contrast, Slovenian Cika cattle had the lowest average genomic inbreeding and the lowest average genomic relationship within the breed. We found selection signatures on chromosome 6 near known genes such as KIT and LCORL explaining variation in coat color and body size in cattle. The most prominent selection signatures were similar regardless of marker density and the breeds in the data set. In addition, using available high-density SNP data from 14 of the breeds we identified 47 genome regions as ROH islands. The proportion of homozygous animals was higher in all studied animals of local breeds than in Holstein and Brown Swiss cattle, the two most important commercial breeds in the Alpine region. We report ROH islands near genes related to thermoregulation, coat color, production, and stature. The results of this study serve as a basis for the search for causal variants underlying adaptation to the alpine environment and other specific characteristics selected during the evolution of local Alpine cattle breeds.

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Identification of two new recessive MC1R alleles in red-coloured Evolèner cattle and other breeds.

Sequence variations in the melanocortin-1 receptor (MC1R) gene are associated with melanism in different animal species. Six functionally relevant alleles have been described in cattle to date. In a hypothesis-free approach we performed a genome-wide allelic association study with black, red and wild-coloured cattle of three Alpine cattle breeds (Eringer, Evolèner and Valdostana), revealing a single significant association signal close to the MC1R gene. We searched for candidate causative variants by sequencing the entire coding sequence and identified two novel protein-changing variants. We propose designating the mutant alleles at MC1R:c.424C>T as ev1 and at MC1R:c.263G>A as ev2 . Both affect conserved amino acid residues in functionally important transmembrane domains (p.Arg142Cys and p.Ser88Asn). Both alleles segregate predominantly in the Swiss Evolèner breed. They occur in other European cattle breeds such as Abondance and Rotes Höhenvieh as well. We observed almost perfect association between the MC1R genotypes and the coat colour phenotype in a cohort of 513 black, red and wild-coloured cattle. Animals carrying two copies of MC1R loss-of-function alleles or that were compound heterozygous for e, ev1 , or ev2 have a red to dark red (chestnut-like red) coat colour. These findings expand the spectrum of causal MC1R variants causing recessive red in cattle.

Genome-wide association study with imputed whole-genome sequence variants including large deletions for female fertility in 3 Nordic dairy cattle breeds.

Fertility is an economically important trait in livestock. Poor fertility in dairy cattle can be due to loss-of-function variants affecting any essential gene that causes early embryonic mortality in homozygotes. To identify fertility-associated quantitative trait loci, we performed single-marker association analyses for 8 fertility traits in Holstein, Jersey, and Nordic Red Dairy cattle using imputed whole-genome sequence variants including SNPs, indels, and large deletion. We then performed stepwise selection of independent markers from GWAS loci using conditional and joint association analyses. From single-marker analyses for fertility traits, we reported genome-wide significant associations of 30,384 SNPs, 178 indels, and 3 deletions in Holstein; 23,481 SNPs, 189 indels, and 13 deletions in Nordic Red; and 17 SNPs in Jersey cattle. Conditional and joint association analyses identified 37 and 23 independent associations in Holstein and Nordic Red Dairy cattle, respectively. Fertility-associated GWAS loci were enriched for developmental and cellular processes (Gene Ontology enrichment, false discovery rate < 0.05). For these quantitative trait loci regions (top marker and 500 kb of surrounding regions), we proposed several candidate genes with functional annotations corresponding to embryonic lethality and various fertility-related phenotypes in mouse and cattle. The inclusion of these top markers in future releases of the custom SNP chip used for genomic evaluations will enable their validation in independent populations and improve the accuracy of genomic predictions.

Are scurs in heterozygous polled (Pp) cattle a complex quantitative trait?

BACKGROUND: Breeding genetically hornless, i.e. polled, cattle provides an animal welfare-friendly and non-invasive alternative to the dehorning of calves. However, the molecular regulation of the development of horns in cattle is still poorly understood. Studying genetic characters such as polledness and scurs, can provide valuable insights into this process. Scurs are hornlike formations that occur occasionally in a wide variety of sizes and forms as an unexpected phenotype when breeding polled cattle. METHODS: We present a unique dataset of 885 Holstein-Friesian cattle with polled parentage. The horn phenotype was carefully examined, and the phenotypic heterogeneity of the trait is described. Using a direct gene test for polledness, the polled genotype of the animals was determined. Subsequently, the existence of a putative scurs locus was investigated using high-density genotype data of a selected subset of 232 animals and two mapping approaches: mixed linear model-based association analyses and combined linkage disequilibrium and linkage analysis. RESULTS: The results of an exploratory data analysis indicated that the expression of scurs depends on age at phenotyping, sex and polled genotype. Scurs were more prevalent in males than in females. Moreover, homozygous polled animals did not express any pronounced scurs and we found that the Friesian polled allele suppresses the development of scurs more efficiently than the Celtic polled allele. Combined linkage and linkage disequilibrium mapping revealed four genome-wide significant loci that affect the development of scurs, one on BTA5 and three on BTA12. Moreover, suggestive associations were detected on BTA16, 18 and 23. The mixed linear model-based association analysis supports the results of the combined linkage and linkage disequilibrium analysis. None of the mapping approaches provided convincing evidence for a monogenic inheritance of scurs. CONCLUSIONS: Our results contradict the initial and still broadly accepted model for the inheritance of horns and scurs. We hypothesise an oligogenetic model to explain the development of scurs and polledness.

Α de novo 3.8-Mb inversion affecting the EDA and XIST genes in a heterozygous female calf with generalized hypohidrotic ectodermal dysplasia.

BACKGROUND: In mammals, hypohidrotic ectodermal dysplasia (HED) is a genetic disorder that is characterized by sparse hair, tooth abnormalities, and defects in cutaneous glands. Only four genes, EDA, EDAR, EDARADD and WNT10A account for more than 90% of HED cases, and EDA, on chromosome X, is involved in 50% of the cases. In this study, we explored an isolated case of a female Holstein calf with symptoms similar to HED. RESULTS: Clinical examination confirmed the diagnosis. The affected female showed homogeneous hypotrichosis and oligodontia as previously observed in bovine EDAR homozygous and EDA hemizygous mutants. Under light microscopy, the hair follicles were thinner and located higher in the dermis of the frontal skin in the affected animal than in the control. Moreover, the affected animal showed a five-fold increase in the number of hair follicles and a four-fold decrease in the diameter of the pilary canals. Pedigree analysis revealed that the coefficient of inbreeding of the affected calf (4.58%) was not higher than the average population inbreeding coefficient (4.59%). This animal had ten ancestors in its paternal and maternal lineages. By estimating the number of affected cases that would be expected if any of these common ancestors carried a recessive mutation, we concluded that, if they existed, other cases of HED should have been reported in France, which is not the case. Therefore, we assumed that the causal mutation was dominant and de novo. By analyzing whole-genome sequencing data, we identified a large chromosomal inversion with breakpoints located in the first introns of the EDA and XIST genes. Genotyping by PCR-electrophoresis the case and its parents allowed us to demonstrate the de novo origin of this inversion. Finally, using various sources of information we present a body of evidence that supports the hypothesis that this mutation is responsible for a skewed inactivation of X, and that only the normal X can be inactivated. CONCLUSIONS: In this article, we report a unique case of X-linked HED affected Holstein female calf with an assumed full inactivation of the normal X-chromosome, thus leading to a severe phenotype similar to that of hemizygous males.

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle.

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation.

A missense variant in the coil1A domain of the keratin 25 gene is associated with the dominant curly hair coat trait (Crd) in horse.

BACKGROUND: Curly horses present a variety of curl phenotypes that are associated with various degrees of curliness of coat, mane, tail and ear hairs. Their origin is still a matter of debate and several genetic hypotheses have been formulated to explain the diversity in phenotype, including the combination of autosomal dominant and recessive alleles. Our purpose was to map the autosomal dominant curly hair locus and identify the causal variant using genome-wide association study (GWAS) and whole-genome sequencing approaches. RESULTS: A GWAS was performed using a Bayesian sparse linear mixed model, based on 51 curly and 19 straight-haired French and North American horses from 13 paternal families genotyped on the Illumina EquineSNP50 BeadChip. A single strong signal was observed on equine chromosome 11, in a region that encompasses the type I keratin gene cluster. This region was refined by haplotype analysis to a segment including 36 genes, among which are 10 keratin genes (KRT-10, -12, -20, -23, -24, -25, -26, -27, -28, -222). To comprehensively identify candidate causal variants within all these genes, whole-genome sequences were obtained for one heterozygous curly stallion and its straight-haired son. Among the four non-synonymous candidate variants identified and validated in the curly region, only variant g.21891160G>A in the KRT25 gene (KRT25:p.R89H) was in perfect agreement with haplotype status in the whole pedigree. Genetic association was then confirmed by genotyping a larger population consisting of 353 horses. However, five discordant curly horses were observed, which carried neither the variant nor the main haplotype associated with curliness. Sequencing of KRT25 for two discordant horses did not identify any other deleterious variant, which suggests locus rather than allelic heterogeneity for the curly phenotype. CONCLUSIONS: We identified the KRT25:p.R89H variant as responsible for the dominant curly trait, but a second dominant locus may also be involved in the shape of hairs within North American Curly horses.

A missense mutation in PFAS (phosphoribosylformylglycinamidine synthase) is likely causal for embryonic lethality associated with the MH1 haplotype in Montbéliarde dairy cattle.

A candidate mutation in the sex hormone binding globulin gene was proposed in 2013 to be responsible for the MH1 recessive embryonic lethal locus segregating in the Montbéliarde breed. In this follow-up study, we excluded this candidate variant because healthy homozygous carriers were observed in large-scale genotyping data generated in the framework of the genomic selection program. We fine mapped the MH1 locus in a 702-kb interval and analyzed genome sequence data from the 1,000 bull genomes project and 54 Montbéliarde bulls (including 14 carriers and 40 noncarriers). We report the identification of a strong candidate mutation in the gene encoding phosphoribosylformylglycinamidine synthase (PFAS), a protein involved in de novo purine synthesis. This mutation, located in a class I glutamine amidotransferase-like domain, results in the substitution of an arginine residue that is entirely conserved among eukaryotes by a cysteine (p.R1205C). No homozygote for the cysteine-encoding allele was observed in a large population of more than 25,000 individuals despite a 6.7% allelic frequency and 122 expected homozygotes under neutrality assumption. Genotyping of 18 embryos collected from heterozygous parents as well as analysis on nonreturn rates suggested that most homozygous carriers died between 7 and 35 d postinsemination. The identification of this strong candidate mutation will enable the accurate testing of the reproducers and the efficient selection against this lethal recessive embryonic defect in the Montbéliarde breed.

Construction of a large collection of small genome variations in French dairy and beef breeds using whole-genome sequences.

BACKGROUND: In recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability. RESULTS: In this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data. CONCLUSIONS: To our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection.

A reverse genetic approach identifies an ancestral frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds.

BACKGROUND: Domestication and artificial selection have resulted in strong genetic drift, relaxation of purifying selection and accumulation of deleterious mutations. As a consequence, bovine breeds experience regular outbreaks of recessive genetic defects which might represent only the tip of the iceberg since their detection depends on the observation of affected animals with distinctive symptoms. Thus, recessive mutations resulting in embryonic mortality or in non-specific symptoms are likely to be missed. The increasing availability of whole-genome sequences has opened new research avenues such as reverse genetics for their investigation. Our aim was to characterize the genetic load of 15 European breeds using data from the 1000 bull genomes consortium and prove that widespread harmful mutations remain to be detected. RESULTS: We listed 2489 putative deleterious variants (in 1923 genes) segregating at a minimal frequency of 5 % in at least one of the breeds studied. Gene enrichment analysis showed major enrichment for genes related to nervous, visual and auditory systems, and moderate enrichment for genes related to cardiovascular and musculoskeletal systems. For verification purposes, we investigated the phenotypic consequences of a frameshift variant in the retinitis pigmentosa-1 gene segregating in several breeds and at a high frequency (27 %) in Normande cattle. As described in certain human patients, clinical and histological examination revealed that this mutation causes progressive degeneration of photoreceptors leading to complete blindness in homozygotes. We established that the deleterious allele was even more frequent in the Normande breed before 1975 (>40 %) and has been progressively counter-selected likely because of its associated negative effect on udder morphology. Finally, using identity-by-descent analysis we demonstrated that this mutation resulted from a unique ancestral event that dates back to ~2800 to 4000 years. CONCLUSIONS: We provide a list of mutations that likely represent a substantial part of the genetic load of domestication in European cattle. We demonstrate that they accumulated non-randomly and that genes related to cognition and sensory functions are particularly affected. Finally, we describe an ancestral deleterious variant segregating in different breeds causing progressive retinal degeneration and irreversible blindness in adult animals.

Whole-genome sequencing identifies a homozygous deletion encompassing exons 17 to 23 of the integrin beta 4 gene in a Charolais calf with junctional epidermolysis bullosa.

BACKGROUND: Since 2010, four Charolais calves with a congenital mechanobullous skin disorder that were born in the same herd from consanguineous matings were reported to us. Clinical and histopathological examination revealed lesions that are compatible with junctional epidermolysis bullosa (JEB). RESULTS: Fifty-four extended regions of homozygosity (>1 Mb) were identified after analysing the whole-genome sequencing (WGS) data from the only case available for DNA sampling at the beginning of the study. Filtering of variants located in these regions for (i) homozygous polymorphisms observed in the WGS data from eight healthy Charolais animals and (ii) homozygous or heterozygous polymorphisms found in the genomes of 234 animals from different breeds did not reveal any deleterious candidate SNPs (single nucleotide polymorphisms) or small indels. Subsequent screening for structural variants in candidate genes located in the same regions identified a homozygous deletion that includes exons 17 to 23 of the integrin beta 4 (ITGB4), a gene that was previously associated with the same defect in humans. Genotyping of a second case and of six parents of affected calves (two sires and four dams) revealed a perfect association between this mutation and the assumed genotypes of the individuals. Mining of Illumina BovineSNP50 Beadchip genotyping data from 6870 Charolais cattle detected only 44 heterozygous animals for a 5.6-Mb haplotype around ITGB4 that was shared with the carriers of the mutation. Interestingly, none of the 16 animals genotyped for the deletion carried the mutation, which suggests a rather recent origin for the mutation. CONCLUSIONS: In conclusion, we successfully identified the causative mutation for a very rare autosomal recessive mutation with only one case by exploiting the most recent DNA sequencing technologies.