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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.

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.

The SLC24A5 (Solute Carrier family 24, member 5) gene is implicated in skin pigmentation in zebrafish and humans as it regulates the morphogenesis of melanosomes, specialized lysosomes involved in melanin deposit. In humans, the ancestral allele predominates in African and East Asian populations, while the allelic variant is nearly fixed in European populations and correlates with lighter pigmentation. Considering the role of melanin in the protecting of DNA from ultraviolet radiation, the lack of information in cattle and the importance of polymorphisms associated with pigmentation phenotypes, we investigated the SLC24A5 gene in cattle with light and dark skin pigmentation. To identify SNPs (Single Nucleotide Polymorphisms) in this gene and their association to dark skin pigmentation in cattle, each of the nine SLC24A5 exons, three introns (1, 3 and 8) and a portion of intron 5, were sequenced in a set of sixteen animals belonging to four Italian cattle breeds, two African zebu breeds and two African sanga breeds. The region spanning exons 3 and 4 was sequenced in fifteen animals belonging to seven additional breeds. A total of sixteen SNPs were identified: eleven positioned in introns (six in intron 1, one in intron 5 and four in intron 8) and five in exons (one in exon 1, two in exon 6 and two in exon 7). Three SNPs (located in exons 1, 6 and 7) were non synonymous, determining Pro19Leu, Ala238Val, and Met341Ile amino acid changes, respectively. All the SNPs identified were polymorphic between Bos taurus, Bos indicus and Sanga, while none of them resulted associated with the studied phenotype and discriminated the three breeds (Chianina, Mucubal and Goudali) characterized by dark pigmented skin from the others.

We targeted quantitative trait loci (QTL) for milk protein percentage (P%) in two Italian Holstein granddaughter design families using selective genotyping in combination with high throughput amplified fragment length polymorphism (AFLP) markers. A total of 64 extreme high and low sires in respect to estimated breeding value (EBV) for P% (EBVP%) were genotyped with 25 AFLP primer combinations that revealed 305 and 291 polymorphisms in the two families. Association between markers and EBVP% was investigated by a linear model only on bands having paternal origin (105 and 96 AFLP bands in family D and S, respectively). Although no marker was significantly associated with the target trait after correction for multiple comparisons, 17 AFLP markers, significant without correction for multiple tests, were considered suggestive of the presence of a QTL. Eleven of these were successfully located on sixBos taurus(BTA) chromosomes by radiation hybrid or in-silico mapping. Ten of these mapped in the immediate neighbourhood (less than 10 cM) of already described QTL for P%. Suggestive association was verified in four regions by microsatellites analysis: one on BTA 10; one on BTA 28; and two on BTA 18. Microsatellites identified significant effects by single marker and interval mapping analyses on BTA 10 and BTA 28, while they were only suggestive of the presence of QTL on BTA 18. In summary, our results firstly indicate that AFLP markers may be used to seek QTL exploiting a selective genotyping approach in GDD, a wide used experimental design in cattle; secondly, propose two approaches for AFLP mapping, namely in-silico mapping exploiting most updated release from the bovine whole genome sequencing project, and physical mapping exploiting a panel of Bovine/Hamster Radiation Hybrids; and thirdly, provide new information on QTLs for an economic important trait in a never investigated Holstein cattle population. AFLP in combination with selective genotyping can be a useful strategy for QTL searching in minor livestock species, sometimes having large economic impact in marginal areas, where more informative markers are still poorly developed.

This report details a case of SRY-negative XX sex reversal in a mixed breed dog and surveys affected dogs of several breeds for mutations in RSPO1 coding regions. Genomic DNA from the mixed breed case was evaluated for mutations in candidate genes. Sequencing identified a homozygous G to A transition in RSPO1 exon 4 that changes a highly conserved amino acid codon in the thrombospondin domain. The possibility that this was a single nucleotide polymorphism (SNP) could not be excluded by genotyping family members. Therefore, the coding region of RSPO1 was sequenced in a survey of affected dogs, which identified a T to C transition (exon 3) in some, the above G to A transition (exon 4) in others, and no change in the remaining affected dogs. Genotypes at these base pair positions were not uniquely associated with the affected phenotype in any breed, indicating the identified transitions are most likely SNPs, not causative mutations for this canine disorder. However, the possibility that polymorphisms play a modifier role, such as changing threshold or severity of phenotypic expression in a mixed breed dog, cannot be excluded. This study emphasizes the importance of canine pedigree, breed, and population studies in evaluating candidate mutations.

Summary The verification of the breed origin of animal products is relevant for food safety andauthenticity. We assessed the suitability of AFLP molecular markers in the assignment ofcattle individuals to their breed of origin. Three hundred and ninety-six animals belongingto 16 cattle breeds genotyped with 141 AFLP markers were used as reference data set.Assignment was performed with likelihood ( AFLPOP ) and Bayesian ( STRUCTURE ) methods. TheBayesian approach was superior to the likelihood algorithm with respect to (i) the correctassignment of simulated individuals to their breed of origin (93% vs. 81% respectively), (ii)the correct assignment of 44 sampled Romagnola animals (91% vs. 45% respectively) and(iii) the correct classification of animals belonging to a breed that was not included withinthe reference dataset. Thus, AFLP profiling in combination with the Bayesian approachseems a useful tool for breed assignment.Keywords AFLP, Bayesian, cattle, likelihood, traceability. Introduction

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.

A total of 113 DNA polymorphisms in 32 candidate genes were pre-screened to assess their variability in 32 Italian Simmental bulls. A total of 64 variants successfully genotyped and having a minor allele frequency (MAF) higher than 0.03 were then analyzed in 477 Italian Simmental bulls. Among these 37 had a MAF ≥0.05 and were investigated for association with milk production and functional traits. Daughter yield deviations (DYD) were available for milk, protein and fat yield (MY, PY and FY, respectively), somatic cell count (SCC), udder score (US), feet and legs score (FLS), while for protein percent (PP), calving ease direct effect (CED), fertility (FE) and milkability (MA) associations were computed using (EBVs). Twelve SNPs were significantly associated with at least one trait ( P ≤0.05). The strongest association ( P =0.0009) was found between rs385640152 in the growth hormone receptor gene ( GHR ) and PP. Also marker rs42714483 in the thyroid hormone responsive gene ( THRSP ) was associated with PP ( P =0.004). Pleiotropic effects were detected only for β-lactoglobulin ( LGB or PAEP progestagen-associated endometrial protein) polymorphisms on. SNP rs29004488 in the leptin gene ( LEP ) and rs41974998 in milk fat globule-epidermal growth factor 8 protein gene ( MFGE8 ) were associated with MY ( P =0.043 and P =0.033, respectively). Suggestive associations with functional traits were also detected: rs43347906 in the leptin receptor gene ( LEPR ) with US ( P =0.029), rs29004485 in LEP with FLS ( P =0.049), rs41703837 in the v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog ( SRC ) with CED ( P =0.021) and rs109019599 in the oxidized low density lipoprotein receptor 1 gene ( OLR1 ) with MA ( P =0.024). Our results both provide loci newly associated with functional traits and confirm associations already detected in Italian Holstein and Italian Brown breeds.

With assignment methods are generally indicated several statistical approaches that use genetic information to identify or verify membership of individuals or groups of individuals. In the present research, the stepwise discriminant analysis (SDA), the canonical discriminant analysis (CDA) and the discriminant analysis (DA) were used to develop an assignment method based on genotypes obtained from the Illumina's OvineSNP50 BeadChip. The three statistical techniques were used to select the most informative markers for discriminating 21 different sheep populations (20 Italian and 1 Slovenian) and their 5 geographic areas of origin. The procedure selected two reduced pools of markers, 110 and 108, that were able to exactly discriminate the 5 geographic areas and the 21 sheep populations, respectively. Moreover, all animals were correctly allocated in the validation procedure. The obtained results indicated that the combined use of the SDA, CDA, and CD techniques represents an efficient strategy for selecting a reduced pool of highly discriminant markers. These SNPs could be further used to develop a low cost customized essay to trace the breed or the geographic area of animals or derived foodstuffs.

To individuate candidate genes (CGs) for a set of barley developmental mutants, a synteny approach comparing the genomes of barley and rice has been introduced. Based on map positions of mutants, sequenced RFLP markers linked to the target loci were selected. The markers were mapped in silico by BLAST searches against the rice genome sequence and chromosomal regions syntenous to barley target intervals were identified. Rice syntenous regions were defined for 15 barley chromosomal intervals hosting 23 mutant loci affecting plant height (brh1; brh2; sld4), shoot and inflorescence branching (als; brc1; cul-2, -3, -5, -15, -16; dub1; mnd6; vrs1), development of leaves (lig) and leaf-like organs (cal-b19, -C15, -d4; lks5; suKD-25; suKE-74; suKF-76; trd; trp). Annotation of 110 Mb of rice genomic sequence made it possible to screen for putative CGs which are listed together with the reasons supporting mutant-gene associations. For two loci, CGs were identified with a clear probability to represent the locus considered. These include FRIZZY PANICLE, a candidate for the brc1 barley mutant, and the rice ortholog of maize Liguleless1 (Lg1), a candidate for the barley lig locus on chromosome 2H. For this locus, the validity of the approach was supported by the PCR-amplification of a genomic fragment of the orthologous barley sequence. SNP mapping located this fragment on chromosome 2H in the region hosting the lig genetic locus.

Summary Italy counts several sheep breeds, arisen over centuries as a consequence of ancient and recent genetic and demographic events. To finely reconstruct genetic structure and relationships between Italian sheep, 496 subjects from 19 breeds were typed at 50K single nucleotide polymorphism loci. A subset of foreign breeds from the Sheep HapMap dataset was also included in the analyses. Genetic distances (as visualized either in a network or in a multidimensional scaling analysis of identical by state distances) closely reflected geographic proximity between breeds, with a clear north–south gradient, likely because of high levels of past gene flow and admixture all along the peninsula. Sardinian breeds diverged more from other breeds, a probable consequence of the combined effect of ancient sporadic introgression of feral mouflon and long-lasting genetic isolation from continental sheep populations. The study allowed the detection of previously undocumented episodes of recent introgression (Delle Langhe into the endangered Altamurana breed) as well as signatures of known, or claimed, historical introgression (Merino into Sopravissana and Gentile di Puglia; Bergamasca into Fabrianese, Appenninica and, to a lesser extent, Leccese). Arguments that would question, from a genomic point of view, the current breed classification of Bergamasca and Biellese into two separate breeds are presented. Finally, a role for traditional transhumance practices in shaping the genetic makeup of Alpine sheep breeds is proposed. The study represents the first exhaustive analysis of Italian sheep diversity in an European context, and it bridges the gap in the previous HapMap panel between Western Mediterranean and Swiss 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.

Genetic variation at the ?S1-casein locus (CSN1S1) is recognized as being crucial in the selection of dairy goats for cheese yield. At this locus, the existence of alleles that have strong, intermediate, weak, and null favorable effects on cheese yield and curd firmness is well known. Selection for alleles that have a strong favorable effect has been deliberately carried out, espe- cially in France. In fact, the importance of ?S1-casein in selection was recently confirmed in the selling policies of semen, where bucks are marketed according to their genotypes. We evaluated genotypes and alleles frequen- cies at the ?S1-casein locus in 491 Italian Saanen and Alpine goats and compared them with previous data to investigate their evolution over the past decade. We also estimated soft cheese yield in a subset of the most represented genotypes to quantify the economic im- portance of considering the genetic trend of ?S1-casein genotype frequencies. We found a significant increase in frequency of the allele with the strongest favorable ef- fect, A (+12 and +13%), and of the intermediate allele E (+17 and +7%) in Saanen and Alpine goats, respec- tively. Surprisingly, the frequency of the strong allele B decreased strikingly over time (-12% in Saanen, -6% in Alpine from 2004 to 2012). This is consistent with the current marketing of semen, in that bucks that are homozygous for strong (AA and BB) and intermediate alleles (EE) and even heterozygous for these alleles (BE and AE) are considered equal. It is worth noting that this practice strongly penalizes the best breeders that have flocks composed almost entirely of goats that are homozygous for strong alleles. For heterozygous goats, we estimated an economic loss of EUR85 and EUR215 per goat per lactation, respectively, for AE and BE, compare with AA and BB genotypes. The marketing of buck se- men should clearly differentiate these 2 alleles to ensure the best economic genetic progress at this locus.

The identification of outlier loci in multilocus datasets is a critical step for reliably inferring population demographic histories and for detecting signatures of adaptive selection, since the presence of just a few of them can lead to a biased estimation of genetic parameters. Here, we show the effect of four outliers out of 30 microsatellites on phylogeography and relationship between eight native Italian goat breeds. The presence of the outliers influenced the relationships between individuals as estimated by factorial correspondence analysis, and between breeds as estimated by Reynolds’ distances. The outliers also affected the neighbour-joining (NJ) tree topology and the computation of the putative genetic barriers separating the populations. The complete dataset supported the existence of two significant barriers: the first isolates Orobica from the surrounding northern Alpine populations and the second separates Girgentana from the southern and Sarda breeds. After removing the outliers, a single and different barrier was significant. It separated the Sarda breed from the continental populations. Our results give new insights into the genetic structure of several native goat breeds across Italy and provide a clear example of the importance of testing for the occurrence of non-neutral loci in population genetics studies. Four outlier loci demonstrated the ability to blur the effect of geography, hiding the natural genetic barrier that separates the Sarda breed from the continental populations, while highlighting the peculiar demographic and selection history of Orobica and Girgentana.

DNA analysis from ancient and old remains offers new tools to answer archaeozoological questions and investigate the origin of the genetic variability in domestic animal species. Molecular genetics techniques contribute to identify the species supporting classical osteological studies and to establish the relationship to modern species and breeds.Mitochondrial DNA (mtDNA) sequences are useful to reconstruct the history of maternal lineages comparing haplotype variations of present and old DNA samples. Mitochondrial data from modern cattle populations show a high diversity in Anatolia and in the Middle East supporting a near-Eastern matrilineal centre of origin. On the contrary in Europe a single family of mitochondrial haplotypes strongly dominates.A number of recent studies reported the successful recovery of ancient and old nuclear DNA (nuDNA) sequences. Such studies represent an important breakthrough, as nuDNA can be used for the characterisation of genetic loci directly involved in phenotypic...

Chantier qualité GA; The effectiveness of single nucleotide polymorphisms (SNPs) for the assignment of cattle to their source breeds was investigated by analysing a panel of 90 SNPs assayed on 24 European breeds. Breed assignment was performed by comparing the Bayesian and frequentist methods implemented in the structure 2.2 and geneclass 2 software programs. The use of SNPs for the reallocation of known individuals to their breeds of origin and the assignment of unknown individuals was tested. In the reallocation tests, the methods implemented in structure 2.2 performed better than those in geneclass 2, with 96% vs. 85% correct assignments respectively. In contrast, the methods implemented in geneclass 2 showed a greater correct assignment rate in allocating animals treated as unknowns to a reference dataset (62% vs. 51% and 80% vs. 65% in field tests 1 and 2 respectively). These results demonstrate that SNPs are suitable for the assignment of individuals to reference breeds. The results also indicate that structure 2.2 and geneclass 2 can be complementary tools to assess breed integrity and assignment. Our findings also stress the importance of a high-quality reference dataset in allocation studies.

Stearoyl CoA desaturase (SCD) is the key enzyme involved in the endogenous synthesis of conjugated linoleic acid (CLA) in ruminants. Changes in the enzymatic activity as a result of SCD gene polymorphism and regulation have been hypothesized to cause diet-independent variations of CLA content in milk. Evidences for the direct influence of SCD polymorphism on fatty acid composition of milk and beef have also been reported. To evaluate genetic differences because of breed and/or selection goal, we investigated the polymorphism of three previously reported single nucleotide polymorphisms located in exon 5 of the SCD gene in 11 cattle breeds raised in Italy and selected for different production goals. Results obtained: (i) evidenced a high variability in the allele frequencies across breeds; (ii) detected three novel haplotypes, one of which is private to indigenous beef breeds, and (iii) showed a significant association between haplotypes and selective goal.

Livestock skin is largely employed in the manufacturing of clothing and shoes, sector in which Italy is a world leader. To sustain Italian products against foreign competition in the globalization era particular attention is to be focus on product quality. Here we investigate the association of SNP mutations in genes coding for collagen proteins present in animal skin with a number of phisico-chemical parameters influencing skin quality for the tanning industry.Skin and blood were sampled from 73 Italian Friesian and Italian Brown bovines and from 43 Bergamasca and Sarda ovines, classified by sex and age. Skins were characterised for a set of chemico-physical parameters (thickness, density, humidity, protein content, ashes, lipid content, hydrossi-proline and DNA content).Regions of the collagen type I, III and IV were screened for SNP discovery in the two species by sequencing a set of reference animals. In bovine 15 polymorphisms were identified: (2 in collagen type I, 9 in collagen type III alp...