A combined genome-wide approach identifies a new potential candidate marker associated with the coat color sidedness in cattle

Coat color is one of the most important phenotypic features in livestock breeds. Cinisara is a local cattle breed generally of uniform black color which occasionally presents a particular phenotype, with animals typically display a white band along their spine, from the head to the tail, and on the ventral line (color sidedness). Therefore, this breed provides an ideal model to study the genetic components underlying phenotypic variation in coat color. A total of 63 animals, ten with sidedness phenotype and 53 with uniform black color were genotyped with Illumina Bovine 50 K. The comparison among genome-wide association study and FST analysis revealed a single nucleotide polymorphism (SNP), ARS-BFGL-NGS-55928, significantly associated with the trait. Only one gene (PLK2)was annotated near the associated SNP in a window of ±200 kb. The protein encoded by this gene is a member of the polo-like kinases, the same family of several known coat-color candidate genes. Based on the reported results, we draw the possible conclusion that the identified marker is potentially associated with the coat color sidedness in Cinisara. The local breeds with their genetic variability represent an important resource and model to study the genetic basis affecting peculiar traits. Future studies would be particularly relevant to refine these results and to better understand the genetic basis for this phenotype

Quantitative determination of casein genetic variants in goat milk: Application in Girgentana dairy goat breed

The study was conducted to develop a high-performance liquid chromatographic (HPLC) method to quantify casein genetic variants (s2-, β-, and κ-casein) in milk of homozygous individuals of Girgentana goat breed. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes. The described HPLC approach was precise, accurate and highly suitable for quantification of goat casein genetic variants of homozygous individuals. The amount of each casein per allele was: s2-casein A=2.9 ± 0.8 g/L and F=1.8 ± 0.4 g/L; β-casein C=3.0 ± 0.8 g/L and C1=2.0 ± 0.7 g/L and κ-casein A=1.6 ± 0.3 g/L and B=1.1 ± 0.2 g/L. A good correlation was found between the quantities of s2-casein genetic variants A and F, and β-casein C and C1 with other previously described method. The main important result was obtained for κ-casein because, till now, no data were available on quantification of single genetic variants for this protein

Genome-wide scan for Runs of Homozygosity in Valle del Belice sheep

The current availability of very large numbers of single nucleotide polymorphisms (SNPs) throughout the genome makes these markers particularly suitable for the detection of genomic regions where a reduction in heterozygosity occurred and offers new opportunities to improve the accuracy of inbreeding (F) estimates. Runs of homozygosity (ROH) are contiguous lengths of homozygous segments of the genome where the two haplotypes inherited from the parents are identical. Here, we investigated the occurrence and the distribution of ROH in medium-density SNP genotypes (~ 50 000) in order to characterize autozygosity in 512 individuals of Valle del Belice sheep and identify the regions of the genome with high ROH frequencies. A total of 11 629 ROH were identified. All individuals displayed at least one ROH > 1 Mb. The mean value of FROH>1Mb was 0.084\ub10.061. ROH that were shorter than 10 Mb predominated. The highest coverage of chromosome (OAR) by ROH was observed on OAR24, whereas the lowest one was observed on OAR1. A typical pattern was observed for the number of ROH per OAR with higher values in the first three chromosomes. There was a considerable difference among animals for the number of ROH segments and the length of the genome covered by ROH. The genomic regions most commonly associated with ROH were identified by selecting the top 1% of the SNPs most commonly observed in ROH within breed. A total of 239 SNPs were considered as candidate SNPs and we identified 107 potential candidate genes that may be under directional selection. Six genomic regions located on six chromosomes (OAR2, OAR3, OAR4, OAR10, OAR11 and OAR23), corresponding to ROH island, presented hotspot of autozygosity. According to KEGG database, a majority of the genes were involved in multiple signaling and signal transduction pathways in a wide variety of cellular and biochemical processes. The ROH islands spanned several candidate genes which influence traits that are associated with adaptability and with the regulation of immune responses (NPAS2, PDCL3, SERPINF1 and SERPINF2) and we did not identified candidate genes with important influence on milk production traits in sheep. The Valle del Belice breed is subjected to limited breeding selection programs for milk production traits, but shows excellent adaptability to the local environments. Therefore, these results suggest at least a partial role of natural selection in shaping the genome of Valle del Belice sheep breed

Genome wide Copy Number Variation (CNV) detection in Cinisara cattle breed

Copy Number Variations (CNVs) are classes of polymorphic genomic regions including deletions, duplications and insertions of DNA fragments from at least 0.5 kb up to several Mb. CNV represents an important source of genetic variability that provides genomics structural information complementary to the single nucleotide polymorphism (SNP) data. Some CNVs have been shown to be important in both normal phenotypic variability and disease susceptibility in livestock. Several approaches to identify CNVs including FISH, aCGH, SNP array or NGS, were proposed and among these SNP genotyping is relatively low cost, high-throughput and high coverage method. The aim of this study was to identify the CNVs in 71 animals of Cinisara breed using Illumina BovineSNP50 BeadChip v2. PennCNV software, which incorporates Log R ratio and B allele frequency at each SNP marker, was used to identify CNVs. Seven animals showed not shared CNVs, as well as autosomes 19, 21, 22. Chromosome 25 presented no CNVs at all. A final number of 322 CNVs were detected. The average number of CNVs was 4.5 per individual, with an average length and median size of 143.04 kb and 122.14 kb, respectively. All CNVs were grouped in CNV regions (CNVRs) and a total of 107 CNVRs, ranged from 50 to ~500 kb, were detected, which covered 4.90 Mb of polymorphic sequence and corresponded to 0.18% of the total genome length. In particular, we found 81 CNVRs with only gain (duplication), 22 with only loss (deletion), and four CNVRs with both. Furthermore, 8 CNVRs with >1%, 77 with >2.5%, and 22 with >5% frequency, were found. CNVRs having the highest frequency were located on Chr3:120501439-120647330 and Chr23:34673581-35007295, whereas the greatest number of genes was mapped in only one CNVR located on Chr 17:74123863-74393620. A total of 241 genes were included in the identified CNVRs. According to KEGG and DAVID database, most of the genes were involved in multiple signaling and signal transduction pathways in a wide variety of cellular and biochemical processes, such as immune response, adaptability, and olfactory receptors pathway. Further studies, using different algorithms and validating the CNVs discovered, will be conducted to corroborate these preliminary results on the CNVRs detected. These results will be used for the investigation of genomic changes and features of interest in the Cinisara breed, such as for association with functional or production traits and for biodiversity studies

Maedi Visna virus infection and TMEM154 genotypes in Valle del Belìce sheep breed

Maedi Visna (MV) is a viral infection in sheep caused by Lentivirus and characterised by a long incubation period, slow progression, weight loss and eventually death. TMEM154 was reported in the ovine as major candidate gene associated with host susceptibility/resistance. The aim of this study was to verify the prevalence of MV infection within Sicilian herds of Valle del Belìce breed using an ELISA serological test and to estimate the frequencies of the resistant/susceptible genotypes to the TMEM154 gene. Finally, we investigated the association between TMEM154 E/K genotype and MV infectious status. A total of 1,083 animals from different flocks were tested. The ELISA method showed 15.33% (n = 166) of infected individuals. Analyses of the sequences showed the presence of both K and E alleles with frequencies of 0.151 and 0.849, respectively. On the total individuals, only 23 carried KK genotype while 280 were heterozigous EK, and 780 homozygous EE. The locus was in Hardy-Weinberg equilibrium in the breed (p-value <0.05) and observed and expected heterozygosity values were 0.258 and 0.256, respectively. Moreover, a significant association (p-value < 0.001) between TMEM154 genotypes and seroprevalence status (positive vs. negative) was found. This study allowed us to know the prevalence of MV infection in Sicilian flocks of Valle del Belìce breed and could be helpful in establishing selection programs aimed at controlling and eradicating this virus

Distribution of functional variants within Runs of Homozygosity in four Italian cattle breeds

Several methods have been used to identify genomic regions subjected to selective sweeps. An alternative method can make use of runs of homozygosity (ROH), defined as stretches of homozygous regions in a genome. The aim of the present study was to detect selective sweeps using ROH in four cattle breeds. Individuals of Cinisara (71), Modicana (72),Reggiana (168) and Italian Holstein (96) were genotyped with the Illumina Bovine50SNP v2 BeadChip. To identify genomic regions that were most commonly associated with ROH within each breed, the percentage of occurrences of a single nucleotide polymorphism (SNP) in ROH was calculated across animals. The genomic regions most commonly associated with ROH were identified by selecting the top 1% of the SNPs most commonly observed in ROH in each breed. This approach resulted in the identification of 11 genomic regions in the Cinisara and Italian Holstein, and 8 in Modicana and Reggiana showing increased frequency of ROH. Generally, ROH patterns differed between breeds. There were two commons genomic regions between breed pairs, and in particular one in BTA6 between Modicana and Reggiana and one in BTA10 between Cinisara and Italian Holstein. A highly homozygous region (> 45% of individuals with ROH) was found only in Modicana breed in BTA6 (6:37,019,972- 39,069,719) within a QTL affecting milk fat and protein concentration. In these genomic regions we identified from 126 to 347 genes for each breed. According to Panther and KEGG database, a majority of the genes was involved in multiple signaling and signal transduction pathways in a wide variety of cellular and biochemical processes. Several of these genes were also comprised in a list of genes related to phenotypes for which cattle breeds have been subjected to strong positive selection. For most genes associated with ROH islands, a biological link to traits such as milk yield and composition, reproduction, immune response, coat colour, genetic disorders and resistance/susceptibility to infectious and diseases, which are known to be under selection, can be hypothesized. These results showed that selective sweeps detected with ROH approach are shared among breeds and that scanning the genome for ROH might be an alternative or complementary strategy to detect selective sweep related with important economically traits

Full-length sequencing and identification of novel polymorphisms in the ACACA gene of Valle del Belice sheep breed

The essential role of the acetyl-CoA carboxylase (ACACA) enzyme in milk fatty acid (FA) synthesis suggests that it may be responsible for the phenotypic variability observed in milk. Before attempting association analyses between this gene and/or enzyme and phenotypic traits, a study on the genetic variability within this locus is required. The aim of this work was to sequence the entire coding region of ACACA gene in Valle del Belice sheep breed to identify polymorphic sites. A total of 51 coding exons of ACACA gene were sequenced in 32 individuals of Valle del Belice sheep breed. Sequencing analysis and alignment of obtained sequences showed the presence of 23 polymorphic sites. The most polymorphic was exon 53 which showed presence of 12 single-nucleotide polymorphisms (SNPs), of which eight were missense mutations, caused amino acid changes and therefore may affect protein function or stability causing variation in phenotype. The identified polymorphisms showed high variability of the ACACA gene. Sequences analysis allowed to find six new SNPs in exon 53 (6832C>T; 6835C>A; 6840G>A; 6847G>T; 6852C>T and 6860G>C). A total of 31 haplotypes were inferred. Although this study could not provide association study with production traits, it shows finding of novel SNPs that might be important in future studies and laid the basis for further association analyses needed to evaluate the potential use of these SNPs as genetic markers for fat content and FAs composition in milk of Valle del Belice sheep breed

Population genetic structure and milk production traits in Girgentana goat breed

The aim of this work was to evaluate the genetic status of the Girgentana goat, an endangered breed from Sicily (Italy), using microsatellite markers. Furthermore, as the main purpose of the Girgentana breed is milk production, quantitative milk traits were investigated, including fatty acid profile. Molecular data from CSN1S1, CSN2, CSN1S2, and CSN3 casein genes were also used to infer haplotypes. A total of 264 individuals were collected. Samples of Maltese (n 64 41) and Derivata di Siria (n 64 33) goat breeds were also used to understand the genetic relationship among breeds. Test-day records for milk production were collected to determine daily milk yield, fat, protein, casein, lactose, and somatic cell count. Individual milk samples were also collected for fatty acid extraction. Wright's statistics, gene flow, Nei genetic distance, factorial correspondence analysis, and Bayesian assignment test showed the existence of genetic variability and differentiation among breeds. The AMOVA results indicated that 89.96% of the total variance was partitioned within populations. The Girgentana breed appears to have a subdivided population, and has not experienced a recent bottleneck. A high variability in milk yield was observed. Mean morning milk yield was 1448 \ub1 404 g, with 4.30 \ub1 0.87% and 3.72 \ub1 0.44% of fat and protein percentages, respectively. The average somatic cell count found in Girgentana goat milk was higher than the threshold of 1 500000 cells/mL advised in Europe for fresh milk. Gross milk and fatty acid composition were similar to that reported in the literature for other local goat breeds

Genome-wide scan for runs of homozygosity identifies potential candidate genes associated with local adaptation in Valle del Belice sheep

Background: Because very large numbers of single nucleotide polymorphisms (SNPs) are now available throughout the genome, they are particularly suitable for the detection of genomic regions where a reduction in heterozygosity has occurred and they offer new opportunities to improve the accuracy of inbreeding (F F) estimates. Runs of homozygosity (ROH) are contiguous lengths of homozygous segments of the genome where the two haplotypes inherited from the parents are identical. Here, we investigated the occurrence and distribution of ROH using a medium-dense SNP panel to characterize autozygosity in 516 Valle del Belice sheep and to identify the genomic regions with high ROH frequencies. Results: We identified 11,629 ROH and all individuals displayed at least one ROH longer than 1 Mb. The mean value of F F estimated from ROH longer than1 Mb was 0.084 \uc2\ub1 0.061. ROH that were shorter than 10 Mb predominated. The highest and lowest coverages of Ovis aries chromosomes (OAR) by ROH were on OAR24 and OAR1, respectively. The number of ROH per chromosome length displayed a specific pattern, with higher values for the first three chromosomes. Both number of ROH and length of the genome covered by ROH varied considerably between animals. Two hundred and thirty-nine SNPs were considered as candidate markers that may be under directional selection and we identified 107 potential candidate genes. Six genomic regions located on six chromosomes, corresponding to ROH islands, are presented as hotspots of autozygosity, which frequently coincided with regions of medium recombination rate. According to the KEGG database, most of these genes were involved in multiple signaling and signal transduction pathways in a wide variety of cellular and biochemical processes. A genome scan revealed the presence of ROH islands in genomic regions that harbor candidate genes for selection in response to environmental stress and which underlie local adaptation. Conclusions: These results suggest that natural selection has, at least partially, a role in shaping the genome of Valle del Belice sheep and that ROH in the ovine genome may help to detect genomic regions involved in the determinism of traits under selection