Searching for homozygous haplotype deficiency in Manech Tête Rousse dairy sheep revealed a nonsense variant in the MMUT gene affecting newborn lamb viability.

BACKGROUND: Recessive deleterious variants are known to segregate in livestock populations, as in humans, and some may be lethal in the homozygous state. RESULTS: We used phased 50 k single nucleotide polymorphism (SNP) genotypes and pedigree data to scan the genome of 6845 Manech Tête Rousse dairy sheep to search for deficiency in homozygous haplotypes (DHH). Five Manech Tête Rousse deficient homozygous haplotypes (MTRDHH1 to 5) were identified, with a homozygous deficiency ranging from 84 to 100%. These haplotypes are located on Ovis aries chromosome (OAR)1 (MTRDHH2 and 3), OAR10 (MTRDHH4), OAR13 (MTRDHH5), and OAR20 (MTRDHH1), and have carrier frequencies ranging from 7.8 to 16.6%. When comparing at-risk matings between DHH carriers to safe matings between non-carriers, two DHH (MTRDHH1 and 2) were linked with decreased insemination success and/or increased stillbirth incidence. We investigated the MTRDHH1 haplotype, which substantially increased stillbirth rate, and identified a single nucleotide variant (SNV) inducing a premature stop codon (p.Gln409*) in the methylmalonyl-CoA mutase (MMUT) gene by using a whole-genome sequencing approach. We generated homozygous lambs for the MMUT mutation by at-risk mating between heterozygous carriers, and most of them died within the first 24 h after birth without any obvious clinical symptoms. Reverse transcriptase-qPCR and western blotting on post-mortem liver and kidney biological samples showed a decreased expression of MMUT mRNA in the liver and absence of a full-length MMUT protein in the mutant homozygous lambs. CONCLUSIONS: We identified five homozygous deficient haplotypes that are likely to harbor five independent deleterious recessive variants in sheep. One of these was detected in the MMUT gene, which is associated with lamb lethality in the homozygous state. A specific management of these haplotypes/variants in the MTR dairy sheep selection program would help enhance the overall fertility and lamb survival.

Variability in Global DNA Methylation Rate Across Tissues and Over Time in Sheep.

Recent studies highlighted the influence of epigenetic marks in the variability of many complex traits, both in plants and animals. These studied focused only on specific sites of the genome having differentially methylated profiles among individuals and/or tissues. In contrast, we recently used the methylation rate of the entire genome as a unique measure considered as a novel quantitative phenotype in sheep. This phenotype named global DNA methylation rate (GDMR), measured by luminometric assay, integrates the methylation level of each CpG dinucleotide within the 6 million of CCGG sites along the ovine genome. GDMR measured in blood previously showed moderate heritability of 0.20 and provided evidence for a genetic determinism. The main objective of the present study was to better characterize the GDMR phenotype in various tissues and investigate its variability in several breeds of sheep reared in the same environment. GDMR was measured on blood samples collected monthly from 59 growing male and female lambs (24 Romane, 23 Blackbelly and 12 Charollais), between birth and 4 months of age. Blood GDMR was on average around 80% and was influenced by the sampling date (p < 0.001), the breed (p = 0.002) and the sex (p = 0.002). In addition, GDMR was determined in 12 somatic (frontal lobe, pituitary gland, heart, lung, sub cutaneous and perirenal adipose tissue, skeletal muscle, liver, spleen, adrenal gland, medulla and cortical kidney) and 6 reproductive tissues (ovary, oviduct, uterus, testis, epididymis and seminal vesicle). GDMR was on average 70% in somatic tissues but marked variation was observed depending on the tissue. The GDMR measured in blood was higher than that measured in other somatic tissues, and is not a good proxy of less accessible tissues. Female reproductive tissues had a 10% higher GDMR than male reproductive tissues. We demonstrated a significant influence of the breed on blood GDMR, certainly reflecting the influence of different genetic backgrounds. The effect of the breed on GDMR may be related to their specific abilities to adapt to and live in different conditions.

A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure Associated with Increased Lamb Mortality in French Lacaune Dairy Sheep.

We recently demonstrated that the Lacaune deficient homozygous haplotype 6 (LDHH6) potentially hosts a recessive perinatal lethal mutation in Lacaune dairy sheep mapped on OAR3. In the present study, we have analyzed the whole-genome sequences of two Lacaune ram heterozygous carriers of LDHH6. After variant calling and filtering against the variants of 86 non-carrier rams, we have identified a single nucleotide variant (SNV) in the two LDHH6 carriers whose variant allele induced a premature stop codon (p.Glu111*) in the Coiled-Coil Domain Containing 65 (CCDC65) gene. CCDC65 is involved in the assembly of the nexin-dynein regulatory complex for the formation of microtubules in ciliated cells. In order to identify the phenotype in homozygous sheep, we generated at-risk matings (n = 17) between rams and ewes heterozygous for the candidate variant in CCDC65. A total of 16 lambs were born alive with five genotyped as homozygous carriers. The homozygous lambs suffered from respiratory problems, and four of them died within the first month of life. At necropsy, we observed a broad hepatization of lung lobes possibly induced by infectious pneumonia. The management of this lethal recessive allele (frequency of 0.06) through reasoned mating in the Lacaune sheep selection schemes could reduce lamb mortality by 2%.

New Anti-Müllerian Hormone Target Genes Involved in Granulosa Cell Survival in Women With Polycystic Ovary Syndrome.

PURPOSE: A protective effect of anti-Müllerian hormone (AMH) on follicle atresia was recently demonstrated using long-term treatments, but this effect has never been supported by mechanistic studies. This work aimed to gain an insight into the mechanism of action of AMH on follicle atresia and on how this could account for the increased follicle pool observed in women with polycystic ovary syndrome (PCOS). METHODS: In vivo and in vitro experiments were performed to study the effects of AMH on follicle atresia and on the proliferation and apoptosis of granulosa cells (GCs). RNA-sequencing was carried out to identify new AMH target genes in GCs. The expression of some of these genes in GCs from control and PCOS women was compared using microfluidic real time quantitative RT-PCR. RESULTS: A short-term AMH treatment prevented follicle atresia in prepubertal mice. Consistent with this result, AMH inhibited apoptosis and promoted proliferation of different models of GCs. Moreover, integrative biology analyses of 965 AMH target genes identified in 1 of these GC models, confirmed that AMH had initiated a gene expression program favoring cell survival and proliferation. Finally, on 43 genes selected among the most up- and down-regulated AMH targets, 8 were up-regulated in GCs isolated from PCOS women, of which 5 are involved in cell survival. MAIN CONCLUSIONS: Our results provide for the first time cellular and molecular evidence that AMH protects follicles from atresia by controlling GC survival and suggest that AMH could participate in the increased follicle pool of PCOS patients.

Genome-Wide Identification of a Regulatory Mutation in BMP15 Controlling Prolificacy in Sheep.

The search for the genetic determinism of prolificacy variability in sheep has evidenced several major mutations in genes playing a crucial role in the control of ovulation rate. In the Noire du Velay (NV) sheep population, a recent genetic study has evidenced the segregation of such a mutation named FecL L . However, based on litter size (LS) records of FecL L non-carrier ewes, the segregation of a second prolificacy major mutation was suspected in this population. In order to identify this mutation, we have combined a case/control genome-wide association study with ovine 50k SNP chip genotyping, whole genome sequencing, and functional analyses. A new single nucleotide polymorphism (OARX:50977717T > A, NC_019484) located on the X chromosome upstream of the BMP15 gene was evidenced to be highly associated with the prolificacy variability (P = 1.93E-11). The variant allele was called FecX N and shown to segregate also in the Blanche du Massif Central (BMC) sheep population. In both NV and BMC, the FecX N allele frequency was estimated close to 0.10, and its effect on LS was estimated at +0.20 lamb per lambing at the heterozygous state. Homozygous FecX N carrier ewes were fertile with increased prolificacy in contrast to numerous mutations affecting BMP15. At the molecular level, FecX N was shown to decrease BMP15 promoter activity and supposed to impact BMP15 expression in the oocyte. This regulatory action was proposed as the causal mechanism for the FecX N mutation to control ovulation rate and prolificacy in sheep.

Genetic Determinism Exists for the Global DNA Methylation Rate in Sheep.

Recent studies showed that epigenetic marks, including DNA methylation, influence production and adaptive traits in plants and animals. So far, most studies dealing with genetics and epigenetics considered DNA methylation sites independently. However, the genetic basis of the global DNA methylation rate (GDMR) remains unknown. The main objective of the present study was to investigate genetic determinism of GDMR in sheep. The experiment was conducted on 1,047 Romane sheep allocated into 10 half-sib families. After weaning, all the lambs were phenotyped for global GDMR in blood as well as for production and adaptive traits. GDMR was measured by LUminometric Methylation Analysis (LUMA) using a pyrosequencing approach. Association analyses were conducted on some of the lambs (n = 775) genotyped by using the Illumina OvineSNP50 BeadChip. Blood GDMR varied among the animals (average 70.7 ± 6.0%). Female lambs had significantly higher GDMR than male lambs. Inter-individual variability of blood GDMR had an additive genetic component and heritability was moderate (h 2 = 0.20 ± 0.05). No significant genetic correlation was found between GDMR and growth or carcass traits, birthcoat, or social behaviors. Association analyses revealed 28 QTLs associated with blood GDMR. Seven genomic regions on chromosomes 1, 5, 11, 17, 24, and 26 were of most interest due to either high significant associations with GDMR or to the relevance of genes located close to the QTLs. QTL effects were moderate. Genomic regions associated with GDMR harbored several genes not yet described as being involved in DNA methylation, but some are already known to play an active role in gene expression. In addition, some candidate genes, CHD1, NCO3A, KDM8, KAT7, and KAT6A have previously been described to be involved in epigenetic modifications. In conclusion, the results of the present study indicate that blood GDMR in domestic sheep is under polygenic influence and provide new insights into DNA methylation genetic determinism.

Prenatal programming by testosterone of follicular theca cell functions in ovary.

In mammalian ovaries, the theca layers of growing follicles are critical for maintaining their structural integrity and supporting androgen synthesis. Through combining the postnatal monitoring of ovaries by abdominal magnetic resonance imaging, endocrine profiling, hormonal analysis of the follicular fluid of growing follicles, and transcriptomic analysis of follicular theca cells, we provide evidence that the exposure of ovine fetuses to testosterone excess activates postnatal follicular growth and strongly affects the functions of follicular theca in adulthood. Prenatal exposure to testosterone impaired androgen synthesis in the small antral follicles of adults and affected the expression in their theca cells of a wide array of genes encoding extracellular matrix components, their membrane receptors, and signaling pathways. Most expression changes were uncorrelated with the concentrations of gonadotropins, steroids, and anti-Müllerian hormone in the recent hormonal environment of theca cells, suggesting that these changes rather result from the long-term developmental effects of testosterone on theca cell precursors in fetal ovaries. Disruptions of the extracellular matrix structure and signaling in the follicular theca and ovarian cortex can explain the acceleration of follicle growth through altering the stiffness of ovarian tissue. We propose that these mechanisms participate in the etiology of the polycystic ovarian syndrome, a major reproductive pathology in woman.

Corrigendum: Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

This corrects the article DOI: 10.1038/srep27059.

Revealing the selection history of adaptive loci using genome-wide scans for selection: an example from domestic sheep.

BACKGROUND: One of the approaches to detect genetics variants affecting fitness traits is to identify their surrounding genomic signatures of past selection. With established methods for detecting selection signatures and the current and future availability of large datasets, such studies should have the power to not only detect these signatures but also to infer their selective histories. Domesticated animals offer a powerful model for these approaches as they adapted rapidly to environmental and human-mediated constraints in a relatively short time. We investigated this question by studying a large dataset of 542 individuals from 27 domestic sheep populations raised in France, genotyped for more than 500,000 SNPs. RESULTS: Population structure analysis revealed that this set of populations harbour a large part of European sheep diversity in a small geographical area, offering a powerful model for the study of adaptation. Identification of extreme SNP and haplotype frequency differences between populations listed 126 genomic regions likely affected by selection. These signatures revealed selection at loci commonly identified as selection targets in many species ("selection hotspots") including ABCG2, LCORL/NCAPG, MSTN, and coat colour genes such as ASIP, MC1R, MITF, and TYRP1. For one of these regions (ABCG2, LCORL/NCAPG), we could propose a historical scenario leading to the introgression of an adaptive allele into a new genetic background. Among selection signatures, we found clear evidence for parallel selection events in different genetic backgrounds, most likely for different mutations. We confirmed this allelic heterogeneity in one case by resequencing the MC1R gene in three black-faced breeds. CONCLUSIONS: Our study illustrates how dense genetic data in multiple populations allows the deciphering of evolutionary history of populations and of their adaptive mutations.

Transcriptome analysis of ovine granulosa cells reveals differences between small antral follicles collected during the follicular and luteal phases.

Ovarian follicular growth occurs in both the follicular and luteal phases of the estrous cycle but in very different endocrine contexts. In both phases, many small antral follicles with similar morphologic and histologic characteristics are present within the ovaries as a reserve for the terminal folliculogenesis. However, there are several gaps in our molecular knowledge of the gene expression profiles of small antral follicles in the follicular and luteal phases. The aim of the present study was to use RNA sequencing to compare and analyze the global transcriptional profile of ovine granulosa cells collected from small antral follicles (1-3 mm) either during the follicular or the luteal phase of the estrous cycle, with the hypothesis that they should be differential. We identified 663 genes whose mRNA was differentially expressed or accumulated in the granulosa cell layer of small antral follicles in the two phases. A comprehensive interpretation of these data was performed through integrative analyses (Gene Ontology, Ingenuity Pathway Analysis) and the exploitation of already available transcriptomic data on follicular growth and atresia. In particular, we observed that the contrasted endocrine context between follicular and luteal phases may have an impact on estradiol, follicle-stimulating hormone (FSH), and on the activin/inhibin signaling pathways. Furthermore, we reveal the possible initiation of early follicular atresia in small antral follicles during the follicular phase in interaction with the presence of immune cells. This study provides new insights into the gene expression profile in ovine granulosa cells, and we suggest that these molecular changes may have an implication at the time of follicle selection.

Genome-Wide Identification of the Mutation Underlying Fleece Variation and Discriminating Ancestral Hairy Species from Modern Woolly Sheep.

The composition and structure of fleece variation observed in mammals is a consequence of a strong selective pressure for fiber production after domestication. In sheep, fleece variation discriminates ancestral species carrying a long and hairy fleece from modern domestic sheep (Ovis aries) owning a short and woolly fleece. Here, we report that the "woolly" allele results from the insertion of an antisense EIF2S2 retrogene (called asEIF2S2) into the 3' UTR of the IRF2BP2 gene leading to an abnormal IRF2BP2 transcript. We provide evidence that this chimeric IRF2BP2/asEIF2S2 messenger 1) targets the genuine sense EIF2S2 RNA and 2) creates a long endogenous double-stranded RNA which alters the expression of both EIF2S2 and IRF2BP2 mRNA. This represents a unique example of a phenotype arising via a RNA-RNA hybrid, itself generated through a retroposition mechanism. Our results bring new insights on the sheep population history thanks to the identification of the molecular origin of an evolutionary phenotypic variation.

Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians.

A genome scan for quantitative trait loci affecting the Salmonella carrier-state in the chicken.

Selection for increased resistance to Salmonella colonisation and excretion could reduce the risk of foodborne Salmonella infection. In order to identify potential loci affecting resistance, differences in resistance were identified between the N and 6(1) inbred lines and two QTL research performed. In an F2 cross, the animals were inoculated at one week of age with Salmonella enteritidis and cloacal swabs were carried out 4 and 5 wk post inoculation (thereafter called CSW4F2 and CSW4F2) and caecal contamination (CAECF2) was assessed 1 week later. The animals from the (N x 6(1)) x N backcross were inoculated at six weeks of age with Salmonella typhimurium and cloacal swabs were studied from wk 1 to 4 (thereafter called CSW1BC to CSW4BC). A total of 33 F2 and 46 backcross progeny were selectively genotyped for 103 and 135 microsatellite markers respectively. The analysis used least-squares-based and non-parametric interval mapping. Two genome-wise significant QTL were observed on Chromosome 1 for CSW2BC and on Chromosome 2 for CSW4F2, and four suggestive QTL for CSW5F2 on Chromosome 2, for CSW5F2 and CSW2BC on chromosome 5 and for CAECF2 on chromosome 16. These results suggest new regions of interest and the putative role of SAL1.

AFLP linkage map of the Japanese quail Coturnix japonica.

The quail is a valuable farm and laboratory animal. Yet molecular information about this species remains scarce. We present here the first genetic linkage map of the Japanese quail. This comprehensive map is based solely on amplified fragment length polymorphism (AFLP) markers. These markers were developed and genotyped in an F2 progeny from a cross between two lines of quail differing in stress reactivity. A total of 432 polymorphic AFLP markers were detected with 24 TaqI/EcoRI primer combinations. On average, 18 markers were produced per primer combination. Two hundred and fifty eight of the polymorphic markers were assigned to 39 autosomal linkage groups plus the ZW sex chromosome linkage groups. The linkage groups range from 2 to 28 markers and from 0.0 to 195.5 cM. The AFLP map covers a total length of 1516 cM, with an average genetic distance between two consecutive markers of 7.6 cM. This AFLP map can be enriched with other marker types, especially mapped chicken genes that will enable to link the maps of both species and make use of the powerful comparative mapping approach. This AFLP map of the Japanese quail already provides an efficient tool for quantitative trait loci (QTL) mapping.

ChickRH6: a chicken whole-genome radiation hybrid panel.

As a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6,000 rads with HPRT-deficient hamster Wg3hCl2 cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs.