GENETIC ANALYSIS OF FERTILITY AND SEASONALITY TRAITS IN BUBALUS BUBALIS

Water buffalo is an important livestock resource, which occupies a critical niche in many ecologically disadvantaged agricultural systems, providing milk, meat, and work power. In Italy, the most important production related to buffalo breeding is milk, traditionally processed into mozzarella cheese. Reproductive efficiency is the main factor affecting productivity in female buffalo, but is greatly penalized by late achievement of puberty, long postpartum anoestrus and seasonality of calving. The main environmental factor affecting the reproductive seasonality is photoperiod, which regulates changes in the daily melatonin secretion by the pineal gland. Under Mediterranean latitudes, the reproductive efficiency of buffaloes is negatively affected by increasing day-length. In the present research, a polymorphism detection and an association study in candidate genes involved in fertility and seasonality of reproduction in Mediterranean Italian Buffalo were performed. The candidate genes analyzed were: signal transducer and activator of transcription 5A (STAT5A), serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 14 (SERPINA14) and tumor necrosis factor alpha (TNFA) for fertility, and melatonin receptor 1A (MTNR1A) for seasonality. The SNP discovery analysis allowed the identification of a total of 12 SNPs. Of these, 11 were detected for the first time in present work, while a C>T substitution in MTNR1A gene was already found in literature and associated to seasonality in buffalo species. Seven SNPs among those found resulted polymorphic in a sample of 36 buffaloes, including the one already present in literature and six SNPs identified in this work for the first time. Of these, 2 are located in exon 4 of TNFA gene and 4 in STAT5A gene (1 in exon 8 and 3 in introns). All the coding SNPs are synonymous. These polymorphisms were genotyped on a total of 491 female Mediterranean Italian Buffaloes, characterized by the periodic milking recording data and the dates of calving, provided by the Italian Buffalo Breeders\u2019 Association (ANASB). Population genetics parameters were calculated and associations with fertility, seasonality and milk production traits were tested. The marker at the MTNR1A gene and one at the STA5A gene deviated significantly from the Hardy-Weinberg equilibrium, with significantly different values for observed and expected heterozygosity. An interesting result emerged for the SNP located at the 3rd exon of TNFA gene, a candidate gene for fertility traits. A statistically significant association with calving interval was assessed in the fourth lactation subset, highlighting an improving effect for the A allele, which resulted associated to a reduction of the calving interval. Nevertheless, the frequency of A allele in the analyzed population is very low and decreases from the first lactation on. Concerning seasonality parameter, the C>T substitution in MTNR1A gene found in literature and associated to seasonality in buffalo species was confirmed in present work, but a relationship between this polymorphism and seasonal breeding was not completely established. The SNP at MTNR1A resulted also associated with milk, fat and protein yield (kg) and with protein percentage parameters. The associations with fat and protein yield were observed in the subset excluding data related to the first lactation. In all cases, animals homozygous for the minor allele T showed lower performances compared to the CC ones. For STAT5A gene, the TT genotype at the c.989+344C locus seems to be associated to a less seasonal reproductive pattern and to higher protein percentage performances. Concerning the latter, the worst mean value is shown by the heterozygous animals, for the phenomenon known as overdominance. Also the SNP STAT5Ac.1342+99A resulted associated with seasonality of reproduction, with GG animals showing a reduced sensitiveness to photoperiod. The G allele was the minor allele in the analyzed population and the homozygous genotype GG is very under-represented. No other statistically significant effects were detected for this polymorphism in the analyzed sample. Another polymorphism detected on the STAT5A gene, the C to G substitution at position c.128, showed a trend in respect to the protein yield (kg) in the subset including data related to classes of lactation number from 2 to 7. Also in this case, the lowest average protein yields were recorded for the heterozygous animals. The associations found, that could be tested in a larger sample, may offer useful indications for the genetic improvement of fertility, seasonality and production traits in buffalo species. Further researches on other genes and also including the SNP chip tool, which is now developing for buffalo species, should be performed to extend the knowledge about the molecular basis underlying the relationships between production and reproduction and the complex mechanisms behind seasonality and response to photoperiod. However, an accurate recording activity of phenotypic parameters, as well as genealogies registration, are critical elements in buffalo, and must be improved to enhance the genetic progress in this species. This could also allow obtaining further relevant results from the association analyses performed in present study

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

Genetic effects of αs1-casein locus on estimated cheese yields in Italian Alpine and Saanen goats

Cheese yield in dairy goat is influenced by milk fat and protein content and also by \u3b1s1-casein polymorphisms. The A allele at this locus is associated to better cheese yield due to smaller micellar casein, whereas the E allele seems to negatively affect milk technological properties. We evaluated the genetic effects of CSN1S1 alleles and genotypes on two estimated soft cheese yields, calculated from individual production data. Our results confirm the significant differences of CSN1S1 alleles on estimate cheese yield and on fat and protein content of goat milk. A allele showed the highest positive effect on all the parameters recorded and calculated. The lowest effect of the F allele on cheese yield and goat dairy traits was confirmed. The E allele showed a significant negative allele substitution effect for cheese yield and protein

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

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

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-SeasonMating technique is usually performed. We analysed three genes: (1) the signal transducer and activator of transcription 5A (STAT5A), (2) the tumour necrosis factor alpha (TNFa) 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 TNFa and four in the STAT5A. No associations were found for the SNPs in the MTNR1A and TNFa 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

Development of a 200 single nucleotide polymorphism panel for parentage assessment for 14 Italian goat breeds

The recent availability of a medium density SNPs chip in goat offers the possibility to develop a useful and less expensive tool for parentage assessment. However, standard approaches of SNP selection for parentage assignment are still ineffective due to a lack of information about markers position. In this study, we describe the identification of a 200 SNPs panel for parentage testing in goat. Data on 350 goats of 14 different Italian breeds genotyped with the Illumina 50K SNP array were provided by the Italian Goat Consortium (IGC). The 200 SNPs panel was identified by a three-step procedure, as follows: 1) parentage assessment by mendelian errors and genomic parentage to identify true parent-offspring pairs; 2) identification of informative SNPs by canonical discriminant analysis and 3) reduction by mendelian errors and stepwise regression. The 200 SNPs panel was tested on pairwise comparison of all animals at each locus. Sensitivity, specificity and accuracy of the panel were assessed. The probability of exclusion (Pe) and the probability of a random coincidental match inclusion (Pi) for each breed were estimated. The panel showed good assessment power, with high sensitivity (0.9429), specificity (1.0) and accuracy (0.99997). Pe values ranged from a minimum of 0.99999981 for Maltese from Sardinia to a maximum of 0.999999999996 for Nicastrese. We further reduced panel size by stepwise regression to 174 SNPs showing the same performance of the 200 SNP panel. The development of tools for parentage assessment could improve breeding management also in species with low genetic information, as goat

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

BACKGROUND: DNA 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. RESULTS: Genome-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. CONCLUSIONS: The 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

Genomic retrospective evaluation of 20 years of selection in Italian Holstein bulls for feet and legs trait

Under strong directional selection,allelefrequencies rapidly change,allowing the identificationof genomic regions carrying genes and variantsthat control selected traits, as production, functional and morphologicaltraits. Here we searched selection sweeps by birth date regression on EBVs and the analysis of changes in allele frequencies. Genomic retrospective evaluation of recent selection wasperformed in 2918 Italian Holstein bulls born between 1979 and 2011. Genotypedata from SELMOL, PROZOO and INNOVAGEN projects were used.Estimated Breeding Value (EBVs) for 32 traitswereprovided by the Italian Holstein association (ANAFI). Bulls were genotyped with BovineSNP50 v.1 and BovineHD SNPchips. SNPs positions were updated to UMD3.1 using SNPchiMp v.3. Genotypes were imputed using BEAGLE (v.3.3.4) to obtain HD genotypes for all individuals. After quality control, a total of 2918 animals and 613,956 SNPs were included in the working dataset. Birth date regressed on Feet and LegsEBVshowsa strong positive trend in the birth date interval analyzed. To detect genomic regions involved, we first identifiedPLUS- and MINUS-variantanimalsfor the target EBV over the total year range (134 bulls, group OVERALL)and within each birth year (130 bulls, group BY_YEAR). Then,SNP allelic frequencies, within each group,wereobtainedfor PLUS and MINUS variantspools and the absolute allele frequency difference (delta)was calculated. Mean delta valueswere estimated in overlapping sliding windows of 50 SNPs.Only windows with the mean delta above the 75th percentile + 1.5*Interquartile rangewere retained. Only overlapping regions between OVERALL and BY_YEAR group were retained. These regions cover the 0.84% of the total windows analyzed.Among these, two regions seem particularly interesting. The ~686Kb region on BTA10 (from position 62,578 to 63,264 Kb) had the highest mean delta on BY_YEAR. The~417Kb region onBTA20(from position 40,738 to 41,155 Kb)had thehighest mean delta on OVERALL.Bioinformatic analysis is underway to identify candidate genes, QTLsand metabolic pathways under selection for this trait

The DNA Methylation Pattern of Prepubertal and Pubertal Alpine Goats

Puberty timing is controlled by many genes and the elements coordinating this process have not completely been identified. Hypothalamus is a pivotal organ in the control of sexual maturation. There is evidence that epigenetic modifications, such as DNA methylation, play a key role in the process. The methylome of the hypothalamus of 10 Alpine goats, 5 at a prepubertal stage (93\ub18 days old) and 5 at their pubertal stage (230\ub117 days old) was analysed to investigate the differences at the DNA methylation level behind these physiological changes. In order to evaluate differentially methylated regions, Methylated DNA Binding Domain sequencing (MBD-seq) with enrichment of methylated DNA fragments and next generation sequencing (Hiseq2000 Illumina) were performed. An average of 24,13 million of reads (range 18.00 and 30.11 million of reads) were produced per sample and peaks corresponding to hyper-methylated regions were estimated using the software ChIPseeqer. The analysis showed that there was an increase in methylation before puberty.The extent of methylation had a median value (\ub1IQR) of 12.32\ub110.21 Mbp of the genome for prepubertal goats, compared with 8.18\ub19.71 Mbp for pubertal goats. Significantly increased methylation was seen on 11 chromosomes in prepubertal goats. Among these, chromosomes 4 and 7 were the most highly significant differentially methylated. In showing that female puberty in goats is associated with amodification of the DNA methylation pattern in the hypothalamus, these results add information on the complex mechanisms that control puberty in mammals

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