Polymorphism analysis of AMY1, AMY2, SLC5A1, SLC2A2 and SLC2A5 genes in Italian horse breeds

The dietary demand of modern competition horse relies on high-cereal feeding daily interspersed by limited forage and it is frequently characterized by marked changes in carbohydrate fractions, above all starch, compared to natural grazing conditions. These dietary variations influence digestion, metabolism, and increase the risk for colic and laminitis, both main causes of equine mortality. Aim of this work has been to analyze the polymorphism of genes related to digestion and absorption of complex carbohydrates, particularly starch, to make a comparison between different cosmopolite and Italian native horse breeds, characterized by marked differences in feeding and management conditions. The analyzed genes were: salivary and pancreatic alpha-amylases (AMY1 and AMY2, respectively), solute carrier family 2, facilitated glucose transporter member 2 (SLC2A2) and member 5 (SLC2A5) and sodium/glucose cotransporter 1 (SLC5A1), code for GLUT2, GLUT5 and SGLT1 enzymes, respectively, main routes for the transport of dietary sugars from the lumen of the intestine into enterocytes. Horses belonging to three cosmopolite breeds, Throughbred, Purebred Arabian and Friesian, and three southern Italian native breeds, Sanfratellano, Indigeno Siciliano and Murgese, 2 DNA samples per breed, were pooled. SNP detection was performed by sequencing one DNA pool per breed. A total of 13 novel single nucleotide polymorphisms were found. Six SNPs are located in AMY1 gene and 3 are in exons, of which 1 is non-synonymous. A non-synonymous SNP was also identified in exon 1 of AMY2 gene. No SNPs were found in the SLC2A2 gene region analyzed, while 4 SNPs were characterized in 3\u2019UTR of SLC2A5 gene. Finally, 4 SNPs were found in SLC5A1 gene, all located in intron 3. Three out of the SNPs found in AMY1 and AMY2 genes were genotyped in 121 horses belonging to the analyzed breeds, with the addition of Haflinger and Sella Italiano breeds samples. The results of the SNP genotyping are presented

Use of the canonical discriminant analysis to select a reduced pool of single nucleotide polymorphism markers for sheep breed assignment and traceability purposes

Several market research studies have shown that consumers are concerned with the provenance of the food they eat. Then, the development of methodologies able to trace animal derived foodstuffs is become of primary interest. DNA-based technologies seem to be the most useful tools to completely prevent consumers from frauds. Moreover, to satisfy the increasing number of consumers demanding for edible products of certified origin, the development of a breed DNA traceability and authentication methods might represent an important marketing strategy. In this study, a new statistical method to discriminate among 21 different Italian sheep breeds and assign new individuals to groups was applied. For each breed, 22 animals were genotyped using the Illumina OvineSNP50 BeadChip: 20 animals were designed as training population (TP), 2 as validation population (VP). For each breed, monomorphic markers and SNP with MAF<0.15 were discharged and, at the end of the data editing, the retained markers were 26,450. The canonical discriminant analysis (CDA) was used to discriminate among breeds. CDA was first applied at chromosome level and markers whose canonical coefficients were higher than 0.27 were retained. A genome-wide CDA was then developed with only the selected markers to test the separation among breeds. Finally, the discriminant analysis (DA) technique was used to assign the VP to the proper breed. The by-chromosome CDA selected a total of 265 top discriminant SNPs and the genome-wide CDA gave a significant separation among all groups. The DA was able to assign 42 over 46 independent individuals to the proper breed. Only for four breeds, the assignment percentage was 50%. These results suggested that an assay with the selected 265 SNP could be used to routinely track monobreed products

Analysis of candidate genes for 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, seasonality of calving, long postpartum anoestrus and subsequent calving interval. Buffaloes living under Mediterranean latitudes can be considered to have a tendency to be seasonal breeding animals and their reproductive efficiency is usually negatively affected by increasing day-length. The main environmental factor affecting the regulation of reproductive seasonality is photoperiod, which regulates changes in the daily melatonin secretion by the pineal gland. The melatonin effect is carried out at hypothalamic level, by regulating of GnRH secretion. Aim of this work has been the polymorphism analysis of different candidate genes for fertility and seasonality traits (STAT5A, SERPINA14 and TNFA for fertility and MTNR1A for seasonality) in buffalo species. Two sets made up of 12 samples were used for SNP discovery analysis, one for fertility traits, containing animals belonging to the tails of distribution for calving interval, and the other for seasonality, composed by 6 animals with calving occurred from August to September and 6 with calving from March to May. Seven single nucleotide polymorphisms were found, of which a C>T substitution in MTNR1A gene, already found in literature and associated to seasonality in different species, and 6 novel SNPs, of which 2 in exon 4 of TNFA gene and 4 in STAT5A gene (1 in exon 8 and 3 in introns). The results of SNP genotyping analysis on a preliminary sample of 100 buffaloes are presented

Candidate genes for barley mutants involved in plant architecture : an in silico approach

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

Evolution of alphaS1-casein polymorphism in Italian Alpine and Saanen breeds

AlphaS1-casein, coded by CSN1S1 gene, is one of the most important goat milk protein, although, in this species, it represents on average only the 5.6% of the total casein content. On the basis of the milk content of alphaS1-casein, the CSN1S1 variants can be grouped into 4 classes: strong alleles (A, B1, B2, B3, B4, C, H, L, and M), producing almost 3.5 g/L of \uf061S1-casein each; intermediate alleles (E and I; 1.1 g/L); weak alleles (F and G; 0.45 g/L); and null alleles (01, 02, and N) apparently producing no alphaS1-casein. Previous studies have reported that the CSN1S1 can affect casein, protein and fat levels, total solids, milk rheology, as well as cheese yield and quality. The aim of the study was to evaluate how the frequencies of the major CSN1S1 allelic variant have changed during the last 10 years in the two main dairy goat breeds raised in the North of Italy: Italian Alpine and Saanen. Two hundred and twenty three Alpine and 129 Saanen were sampled from 8 farms spread all over Lombardy. AS-PCR and RFLP-PCR were performed to genotype the major allelic variants at the alphaS1-casein locus. Only the Saanen breed proved to be in Hardy-Weinberg equilibrium at this locus. The allele frequencies observed in the present work showed statistically significant differences (\uf0632 test, P<0.005) compared to those reported in two studies published in 2004. This difference was due to an increase of the strong allele A (12 and 18%) and the intermediate allele E (15 and 8%) nearby a reduction of the weak allele F (17 and 12%) and the strong allele B (11 and 13%) respectively in Saanen and Alpine breeds. The A allele is well known for being associated to smaller micellar casein, with a consequent better cheese yield. Recent indications of selection concerning the Alpine and Saanen bucks favours both strong and intermediate alleles, consistent with the described strong frequency increase of CSN1S1*E. For the top farms analysed, with over 85% genotypes with strong alleles, these directive could lead to an increaseof the allele E and consequently to a reduction of protein content and cheese yield

Use of different statistical approaches to study selection signatures in sheep breeds farmed in Italy

Natural and artificial selection affect genome structure causing genetic variation between breeds. Dense marker maps of thousand SNP disseminated across the whole genome allow for the investigation of chromosomal regions that differ between breeds. Several statistical approaches have been proposed to study selection signatures in livestock species. In this work, four approaches were used to study selection signatures in a sample of 496 sheep belonging to 20 Italian breeds, different for geographical origin and production aptitude. The four approaches were: I) Fst Outlier Detection (FOD), implemented in the LOSITAN software. II) comparison of Breed LS means of the sum of differences in SNP allele frequencies along sliding windows (SNP_DIFF).. III) Correspondence analysis (CA). VI) Canonical Discriminant Analysis (CDA). Animal were genotyped with the Illumina OvineSNP50 BeadChip. The first five chromosomes were considered. After data editing, a total of 20,194 SNP were retained for the analysis. The different approaches were able to identify the same regions expressing variation between breeds. On OAR6, for example, all methods highlighted a region located between 35 and 41 Mb, where BMPR1b and ABCG2 loci map. Moreover, SNP able to differentiate between breeds were also detected at 76, 96 and 107 Mb, near to KIT, IL8 and SCD5 loci, respectively. All methods were able to discriminate breeds and, in general, a geographical pattern of variation has been detected. However each approach may supply different kind of information. FOD detected a relatively low number of markers in divergent selection but it was able to identify loci under balanced selection. CA and CDA decomposed the total variability of SNP markers among breeds in different and uncorrelated variables that could be useful for the identification of genes influencing complex traits

Mutations in the RSPO1 coding region are not the main cause of canine SRY-negative XX sex reversal in several breeds

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