Beatrice Coizet

A method for single nucleotide polymorphism selection for parentage assessment in goats

Accurate pedigrees are essential to optimize genetic improvement and conservation of animal genetic resources. In goats, the use of mating groups and kidding management procedures hamper the identification of parentage. Small panels of single nucleotide polymorphisms (SNP) have been proposed in other species to substitute microsatellites for parentage assessment. Using data from the current GoatSNP50 chip, we developed a new 3-step procedure to identify a low-density SNP panel for highly accurate parentage assessment. Methodologies for SNP selection used in other species are less suitable in the goat because of uncertainties in the genome assembly. The procedure developed in this study is based on parent-offspring identification and on estimation of Mendelian errors, followed by canonical discriminant analysis identification and stepwise regression reduction. Starting from a reference sample of 109 Alpine goats with known pedigree relationships, we first identified a panel of 200 SNP that was further reduced to 2 final panels of 130 and 114 SNP with random coincidental match inclusion of 1.51×10(-57) and 2.94×10(-34), respectively. In our reference data set, all panels correctly identified all parent-offspring combinations, revealing a 40% pedigree error rate in the information provided by breeders. All reference trios were confirmed by official tests based on microsatellites. Panels were also tested on Saanen and Teramana breeds. Although the testing on a larger set of breeds in the reference population is still needed to validate these results, our findings suggest that our procedure could identify SNP panels for accurate parentage assessment in goats or in other species with unreliable marker positioning.

Associations of acetyl-coenzyme A carboxylase α, stearoyl-coenzyme A desaturase, and lipoprotein lipase genes with dairy traits in Alpine goats

Milk yield and composition are of great economic importance for the dairy goat industry. The identification of genes associated with phenotypic differences for these traits could allow for the implementation of gene-assisted selection programs in goats. Associations between polymorphisms at 3 candidate genes and milk production traits in Alpine goats farmed in Italy were investigated in the present research. Considered genes were acetyl-coenzyme A carboxylase α (ACACA), the major regulatory enzyme of fatty acid biosynthesis; stearoyl-coenzyme A desaturase (SCD), involved in the biosynthesis of monounsaturated fatty acids in the mammary gland; and lipoprotein lipase (LPL), which plays a central role in plasma triglyceride metabolism. An approach somewhat similar to the granddaughter design for detecting quantitative trait loci in dairy cattle was followed. Effects of genotypes of a sample of 59 Alpine bucks on phenotypes of their 946 daughters raised in 75 flocks were investigated. Data comprised 13,331 daily records for milk yields (L/d), fat and protein yields (kg/d), and fat and protein contents (%) of 2,200 lactations. Population genetics parameters were calculated and associations between milk production traits and 10 single nucleotide polymorphisms (SNP) at the 3 genes were tested. Two markers at the ACACA, 1 for the SCD and 1 at the LPL locus, deviated significantly from the Hardy-Weinberg equilibrium, with an observed heterozygosity lower than expected. Flock, age of the goat, kidding season, and stage of lactation affected all traits considered, except fat percentage. Three SNP were found to be significantly associated with milk production traits. The SNP located on the ACACA gene showed an effect on milk yield, with daughters of TT bucks having an average test-day milk yield of about 0.3 to 0.25 L/d lower than the other 2 genotypes. The marker on the LPL locus was highly associated with milk yield, with the largest values for CC daughters (about 0.50L more than GG). The TGT deletion located on the untranslated region of the SCD gene showed significant effects on average milk and protein yields. The homozygote-deleted genotype had values about 0.5 L/d and 16 g/d lower for milk and protein daily yield, respectively, compared with the TGT/TGT genotype. Differences between genotypes were quite constant across most of the lactation. Associations found in the present study, which should be tested in a larger sample, especially for those markers that show rare genotypes, may offer useful indications for the genetic improvement of dairy traits in goats.

Genomic analysis suggests KITLG is responsible for a roan pattern in two Pakistani goat breeds

The roan coat color pattern is described as the presence of white hairs intermixed with pigmented hairs. This kind of pigmentation pattern has been observed in many domestic species, including the goat. The molecular mechanisms and inheritance that underlie this pattern are known for some species and the KITLG gene has been shown associated with this phenotype. To date, no research effort has been carried out to find the gene(s) that control(s) roan coat color pattern in goats. In the present study, after genotyping with the GoatSNP50 BeadChip, 35 goats that showed a roan pattern and that belonged to two Pakistan breeds (Group A) were analyzed and then compared to 740 goats of 39 Italian and Pakistan goat breeds that did not have the same coat color pattern (Group B). Runs of homozygosity-based and XP-EHH analyses were used to identify unique genomic regions potentially associated with the roan pattern. A total of 3 regions on chromosomes 5, 6, and 12 were considered unique among the group A versus group B comparisons. The A region > 1.7 Mb on chromosome 5 was the most divergent between the two groups. This region contains six genes, including the KITLG gene. Our findings support the hypothesis that the KITLG gene may be associated with the roan phenotype in goats.

Polymorphism of the STAT5A, MTNR1A and TNFα genes and their effect on dairy production in Bubalus bubalis

Abstract The water buffalo is a fundamental resource, especially in developing countries, however, differently from other species, its genetic potential is still poorly investigated. In this work, we performed a candidate gene association study for milk composition in 491 female buffaloes. Animals were from four farms located in Southern Italy, where the Out-of-Breeding-Season-Mating technique is usually performed. We analysed three genes: (1) the signal transducer and activator of transcription 5A (STAT5A), (2) the tumour necrosis factor alpha (TNFα) and (3) the melatonin receptor 1A (MTNR1A). We confirmed the mutation at the MTNR1A gene and we found five novel single nucleotide polymorphisms (SNPs): one in the TNFα and four in the STAT5A. No associations were found for the SNPs in the MTNR1A and TNFα genes, while we identified a marked association with milk protein % for a C > T substitution at the STAT5A gene. At this locus, the TT buffaloes showed significantly higher protein percentage in milk. Conversely, this genotype class was the less frequent in the population. Moreover, an A > G substitution at the STAT5A showed an influence on reproductive seasonality, with the advantageous allele most frequent in the population, suggesting a possible effect of selection for this trait. The C > T substitution on STAT5A detected in present study could be used in marker assisted selection of Mediterranean Italian buffalo, and should be monitored to understand the reasons behind the low frequency of the favourable genotype at this locus and to stop this unfavourable trend in the population.

Genome-wide analysis of DNA methylation in hypothalamus and ovary of Capra hircus

BackgroundDNA methylation is a frequently studied epigenetic modification due to its role in regulating gene expression and hence in biological processes and in determining phenotypic plasticity in organisms. Rudimentary DNA methylation patterns for some livestock species are publically available: among these, goat methylome deserves to be further explored.ResultsGenome-wide DNA methylation maps of the hypothalamus and ovary from Saanen goats were generated using Methyl-CpG binding domain protein sequencing (MBD-seq). Analysis of DNA methylation patterns indicate that the majority of methylation peaks found within genes are located gene body regions, for both organs. Analysis of the distribution of methylated sites per chromosome showed that chromosome X had the lowest number of methylation peaks. The X chromosome has one of the highest percentages of methylated CpG islands in both organs, and approximately 50% of the CpG islands in the goat epigenome are methylated in hypothalamus and ovary. Organ-specific Differentially Methylated Genes (DMGs) were correlated with the expression levels.ConclusionsThe comparison between transcriptome and methylome in hypothalamus and ovary showed that a higher level of methylation is not accompanied by a higher gene suppression. The genome-wide DNA methylation map for two goat organs produced here is a valuable starting point for studying the involvement of epigenetic modifications in regulating goat reproduction performance.

Initial genomic characterization of Italian, Egyptian and Pakistani goat breeds.

Selection and breeding practices in goats have differed greatly among countries and populations. These processes, together with natural selection and regional drift, have shaped the phenotypic variability of goat breeds (Kim et al., 2015). The availability of improved genomic analysis tools for this species may provide useful information on the history of selection, adaptation and differentiation of goats from different areas of the world, that can be evaluated by the study of gene frequencies and length of the Runs of Homozigosity (contiguous length of homozygous genotypes, ROH; Purfield et al., 2012). In current study, we examined using a goat medium density SNP chip animals from three different countries: Egypt (with lack of selection scheme), Italy (with several standardized breeds; Nicoloso et al., 2015) and Pakistan (with several breeds showing peculiar phenotypes) to produce a genomic landscape of goats breeds in these countries. A total of 1,123 animals of 39 different populations, and 48,895 SNP markers were analyzed. Genotypes were imputed on a country-based approach, and markers without known position in the genome were excluded from the analysis. MDS and ADMIXTURE plots confirmed the good differentiation among populations from the three countries. Runs of Homozygosity (ROH) were performed for each country and population allowed the detection of genomic regions with high homozygosity levels, common in at least two out of three sampling areas. These results provide new insights into goat genome structure within and among breeds and countries. The detection of conserved regions with different lengths may explain recent selection strategies or adaptation to different, extreme environmental conditions. The research was funded by INNOVAGEN project. Support by Iowa State University and the Ensminger funds for AE and AT as well as support by the Fulbright Foundation for AE are gratefully acknowledged. Sampling from Pakistan was funded by PAK-USAID project.

Distribution of ncRNAs expression across hypothalamic-pituitary-gonadal axis in Capra hircus

BackgroundMolecular regulation of the hypothalamic-pituitary-gonadal (HPG) axis plays an essential role in the fine tuning of seasonal estrus in Capra hircus. Noncoding RNAs (ncRNAs) are emerging as key regulators in sexual development and mammalian reproduction. In order to identify ncRNAs and to assess their expression patterns, along the HPG axis, we sequenced ncRNA libraries from hypothalamus, pituitary and ovary of three goats.ResultsAmong the medium length noncoding RNAs (mncRNAs) identified, small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs) were found to be more abundant in ovary and hypothalamus, respectively. The observed GC content was representative for different classes of ncRNAs, allowing the identification of a tRNA-derived RNA fragments (tRFs) subclass, which had a peak distribution around 32–38% GC content in the hypothalamus. Differences observed among organs confirmed the specificity of microRNA (miRNA) profiles for each organ system.ConclusionsData on ncRNAs in organs constituting the HPG axis will contribute to understanding their role in the physiological regulation of reproduction in goats.

The methylome of the hypothalamus of prepubertal and pubertal goats

Puberty is the fulfillment of fertility, a process involving physiological and morphological development. It is well known that the increased hypothalamic secretion of the gonadotropin-releasing hormone (GnRH) is essential for the activation of this process, even if the elements coordinating the timing of puberty have not been fully identified1,2. Recent studies provide proof that there is an epigenetic regulation of female puberty, and DNA methylation, the most studied epigenetic modification, plays a major role in it3. We analyzed DNA methylation patterns of 5 Alpine goats at their prepubertal stage and 5 that reached puberty in order to highlight differences in their methylome. Detection of methylated regions across the goat genome involved a Methyl Binding Domain (MBD) enrichment followed by deep sequencing (Hiseq2000 Illumina). The software ChIPseeqer4 permitted the identification of peaks corresponding to hyper-methylated regions. We have observed a higher methylation level in prepubertal goats. The distribution of the methylation peaks across the genome and within CpG islands per chromosome per group of animals has been analyzed. Furthermore, we have investigated differential methylation in genes associated with puberty. Specifically, Cbx7, coding for a core component of the Polycomb group silencing complex, and GnRHR, the gene coding for GnRH receptor, showed a higher number of peaks into two intragenic fragments within prepubertal goats. These results, accompanied by transcriptome analysis, provide a foundation for elucidating the role of DNA methylation in the complex mechanisms that drive puberty in goat species.

A first glance on the epigenome of Capra hircus

DNA methylation and microRNAs (miRNA) are two important forms of epigenetic modifications that play an important role in gene regulation in animals. Methylation at the carbon 5 position of cytosine residues is a fundamental layer of cellular differentiation through the control of transcriptional potential. MiRNA are small noncoding RNA molecules that regulate gene expression. Complete DNA methylomes for several organisms are now available; at the present, methylome of the domestic goat is unexplored. There is also still limited knowledge about miRNAs expression profiles in small ruminant species. Therefore, to contribute information on epigenetic modification in Capra hircus, we analysed the methylome and the miRNA population of three tissues (hypothalamus, pituitary and ovary) from 3 adult Saanen goats. We used Methylated DNA binding domain sequencing with enrichment of methylated DNA fragments and next generation sequencing. We produced least 23 million reads per sample, which were aligned to the goat reference genome. Further analyses were performed to identify peaks corresponding to hyper-methylated regions. We sequenced miRNAs expressed in the three tissues with Illumina high-throughput sequencing. Reads were mapped on the Capra hircus reference genome and both known and novel miRNAs, and miRNA target sites were identified using information collected in miRBase and using specific bioinformatic tools. This study produced a comprehensive miRNA profile related to the biology of goat. Furthermore, this is the first work dealing with methylome in Capra hircus: our preliminary results could provide new information for a deeper comprehension of epigenetic mechanisms of this species.

Variation in Salivary and Pancreatic Alpha-Amylase Genes in Italian Horse Breeds

The dietary demand of the modern horse relies on high-cereal feeding and limited forage compared with natural grazing conditions, predisposing the horse to several important diseases. Salivary and pancreatic alpha-amylases (coded by AMY1 and AMY2 genes, respectively) play a crucial role in carbohydrate digestion in nonruminants, but little is known about these 2 genes in the horse. Aim of this work has been to distinguish genomic sequences of horse AMY1 and AMY2 genes and to analyze any polymorphisms in breeds historically characterized by marked differences in nutritional management. A single nucleotide polymorphism detection was performed and 7 novel single nucleotide polymorphisms were found. Three single nucleotide polymorphisms are in exons and were genotyped in 112 horses belonging to 6 breeds. One single nucleotide polymorphism in AMY1 gene distinguished Haflinger and the Italian native Murgese from the other breeds, whereas both the single nucleotide polymorphisms in AMY2 gene showed different allelic frequencies in Friesian compared with the other breeds. These differences are confirmed by quite high fixation index (Fst) values for these 2 nonsynonymous single nucleotide polymorphisms. These preliminary results highlight marked divergences in allele frequencies of AMY1 and AMY2 genes, involved in starch digestion, between horse breeds characterized by different histories of selection, thus providing first indications of possible relations between genetics and nutritional management.