A. Crisà

Double muscling in Marchigiana beef breed is caused by a stop codon in the third exon of myostatin gene

Double muscling is a partially recessive trait present in some beef breeds. It shows a high frequency in some breeds, while in others the frequency is low, and double-muscled individuals are rare. The double muscling is caused by an allelic series of mutations that cause a loss of function of the myostatin gene (GDF8). We describe here a new mutation in the myostatin gene in Marchigiana breed, a typical beef breed of Central Italy, in which rare double-muscling individuals have been described. A PCR product of the third exon was sequenced in subjects phenotypically showing double muscling, and a G > T transversion was discovered that introduces a premature stop codon. The variant found adds to the large series of mutations present in cattle, and particularly to the only two causative of double muscling in the third exon. A PCR-RFLP test is described for the rapid and effective identification of both heterozygous and homozygous subjects. It was applied to a larger survey carried on the same and also in two other beef breeds, Chianina and Romagnola. Further individuals carrying the new variant were found in Marchigiana, but none in the other breeds. The results may be important for a better comprehension of the role of myostatin in muscular development, for commercial use and for the inference of phylogeny of this gene.

Discovery, characterization and validation of single nucleotide polymorphisms within 206 bovine genes that may be considered as candidate genes for beef production and quality

A large number of putative single nucleotide polymorphisms (SNPs) have been identified from the bovine genome-sequencing project. However, few of these have been validated and many will turn out to be sequencing artefacts or have low minor allele frequencies. In addition, there is little information available on SNPs within coding regions, which are likely to be responsible for phenotypic variation. Therefore, additional SNP discovery is necessary to identify and validate polymorphisms both in specific genes and genome-wide. Sequence-tagged sites within 286 genes were resequenced from a panel of animals representing a wide range of European cattle breeds. For 80 genes, no polymorphisms were identified, and 672 putative SNPs were identified within 206 genes. Fifteen European cattle breeds (436 individuals plus available parents) were genotyped with these putative SNPs, and 389 SNPs were confirmed to have minor allele frequencies above 10%. The genes containing SNPs were localized on chromosomes by radiation hybrid mapping and on the bovine genome sequence by Blast. Flanking microsatellite loci were identified, to facilitate the alignment of the genes containing the SNPs in relation to mapped quantitative trait loci. Of the 672 putative SNPs discovered in this work, only 11 were found among the validated SNPs and 100 were found among the approximately 2.3 million putative SNPs currently in dbSNP. The genes studied in this work could be considered as candidates for traits associated with beef production and the SNPs reported will help to assess the role of the genes in the genetic control of muscle development and meat quality. The allele frequency data presented allows the general utility of the SNPs to be assessed.

Exploring polymorphisms and effects of candidate genes on milk fat quality in dairy sheep

The aim of the present study was to investigate the genetic control of the fatty acid (FA) composition in milk from 3 breeds of sheep: Altamurana, Gentile di Puglia, and Sarda. Single nucleotide polymorphisms within genes, encoding enzymes putatively involved in the synthesis and metabolism of milk fat, were selected for analysis, and the allele substitution effects were determined for 16 genes, which were polymorphic in the 3 sheep breeds, upon the milk fat composition. Four genes (alpha-1-antichymotrypsin-2; diacylglycerol O-acyltransferase homolog-2; propionyl Coenzyme A carboxylase, beta polypeptide; and insulin-like growth factor-I) play a role in the desaturation of stearic FA into polyunsaturated fatty acids. Furthermore, 2 genes (growth hormone receptor and zona pellucida glycoprotein-2) affect the variability of the total fat content in addition to the butyric and stearic FA profile, and the fatty acid synthetase gene has an influence on the medium-chain FA. Milk FA profiles play an important role in dairy sheep farming because they have a large effect on cheese characteristics and also because sheep milk may be marketed as a source of nutraceuticals because it contains higher levels of conjugated linoleic acid than milk from other ruminants. The current study evaluated the global effects of a large number of single nucleotide polymorphisms and haplotypes on traits that are not commonly investigated in sheep but that are potentially very useful for improving milk quality.

Milk fatty acid variability: effect of some candidate genes involved in lipid synthesis.

In this work, the genetic variation of milk FA was investigated in three different bovine breeds, the Jersey, the Piedmontese and the Valdostana, and at different lactation stages. All animals were genotyped for 21 Single Nucleotide Polymorphisms located within nine candidate genes involved in lipid synthesis: diacylglycerol acyltransferase 1 and 2 (DGAT1, 2); stearoyl-CoA desaturase (SCD); growth hormone receptor (GHR); fatty acid synthase (FASN); acyl-CoA dehydrogenase (ACAD); fatty acid binding protein (FABP4); lipoprotein lipase (LPL); and leptin gene (LEP). The highest milk-fat Jersey breed also showed the highest content of saturated FA. Throughout lactation, the breeds showed a similar variation in the FA, with a decrease in the short-chain, this was accompanied by a general increase in the long chain FA at the end of lactation. The increase in long chain saturated FA was particularly evident in the case of the Jersey. The effect of SCD gene on the C14 desaturation index was confirmed; the DGAT1 gene was polymorphic only in the Jersey breed, but its effect was confirmed only on milk fat content; three further potential candidate genes were identified: first, the FABP4 gene, which was found to influence medium and long chain FA in all the breeds, but not the desaturation indices; second, the FASN gene, which was found to influence the amount of PUFA in the Piedmontese and the Valdostana, and third, the LPL gene, which was found to affect fat content in the Piedmontese.

Leptin gene haplotypes are associated with change in immunological and hematological variables in dairy cow during the peripartum period.

In this study, the effect of polymorphisms in the leptin gene on the hematological variables in periparturient dairy cows was investigated. The hematological profile of 67 Holstein cows was assessed for 6 wk around calving. The DNA of the cows was genotyped at 6 polymorphic loci within the leptin gene, and 7 haplotypes were reconstructed. Significant haplotype substitution effects were found, for haplotype 1, on total white blood cell count for 2 wk around calving (+0.70 10(3)/μL, P = 0.05; +1.38 10(3)/μL, P = 0.0001); on neutrophil cell count in the first week after calving (+0.94 10(3)/μL, P = 0.001); on lymphocyte count during the 3 wk before and the first week after calving (+0.32 10(3)/μL, P = 0.05; +0.27 10(3)/μL, P = 0.03; +0.26 10(3)/μL, P = 0.04; +0.34 10(3)/μL, P = 0.01); on red blood cell count during the last week before calving and wk 1 and 2 after calving (+0.21 10(6)/μL, P = 0.02; +0.23 10(6)/μL, P = 0.01; +0.20 10(6)/μL, P = 0.03); on mean corpuscular volume (-1.35 fL, P = 0.01; -1.29 fL, P = 0.002; -1.18 fL, P = 0.004; -1.09 fL, P = 0.008; -1.23 fL, P = 0.003; -1.31 fL, P = 0.003); and on mean corpuscular hemoglobin (-0.37 pg, P = 0.05; -0.38 pg, P = 0.02; -0.39 pg, P = 0.01; -0.34 pg, P = 0.03; -0.40 pg, P = 0.01; -0.40 pg, P = 0.01) during all 6 wk analyzed. Significant haplotype substitution effects, but opposite those of haplotype-1, were found for haplotype-2 on white blood cell count (-1.10 10(3)/μL, P = 0.01; -1.30 10(3)/μL, P = 0.002; -1.09 10(3)/μL, P = 0.01) and neutrophil count (-0.82 10(3)/μL, P = 0.02; -0.95 10(3)/μL, P = 0.005; -0.92 10(3)/μL, P = 0.01). Haplotype-3 influenced red blood cell count (-0.23 10(6)/μL, P = 0.03; -0.28 10(6)/μL, P = 0.01; -0.34 10(6)/μL, P = 0.002) during the last 2 wk before and the first week after calving, and also, with effects evident only in wk 3 and 2 before calving, mean corpuscular volume (+1.38 fL, P = 0.03; +0.97 fL, P = 0.05; +1.08 fL, P = 0.05), mean corpuscular hemoglobin (+0.58 pg, P = 0.02; +0.38 pg, P = 0.04; 0.51 pg, P = 0.01), and red blood cell distribution width (-0.56% P = 0.02; -0.47%, P = 0.05). The current study provided evidence that several polymorphisms in the leptin gene play a role in the variability of hematological variables during the peripartum period, and might be used as genetic markers for improving the immunological conditions of dairy cows in critical productive periods.

Polymorphisms within the Toll-like receptor (TLR)-2, -4, and -6 genes in cattle

Abstract Toll-like receptors (TLR) have an important role in the defence against a variety of infectious diseases. TLR recognize various microbial components and are important in the innate immune response to pathogens and in crosstalk between innate immunity and adaptive immunity. Polymorphisms in TLRs influence their abilities of recognition of pathogen-derived molecules and therefore could be of great relevance in livestock selection once genetic variation in these loci has been associated with resistance. Several single nucleotide polymorphisms (SNPs) within TLR genes have been described in humans and some of them seem to be associated with susceptibility to infection diseases. Recently, all the 10 TLR genes have been mapped in cattle by radiation hybrid panel. In this study, we screened the nucleotide sequences of bovine TLR2, TLR4, and TLR6 genes (located on BTA17, BTA8 and BTA6, respectively) searching for SNPs potentially linked with disease resistance. We could identify 11 SNPs that were used for screening 900 individuals belonging to 16 different bovine European breeds. Eight of the analysed breeds resulted polymorphic at all the analysed loci, and six of the loci were polymorphic in all the breeds. Allelic frequencies, Gene Diversity, Heterozygosity and PIC were calculated. Heterozygosity and PIC ranged respectively from 0.05 to 0.47 and from 0.06 and 0.37. Heterozygosity and Fst were calculated using the Fdist software (http://www.rubic.rdg.ac.uk/~mab/software.html). Population phylogeny datasets were built by bootstrapping 200,000 replications on real data using a coalescent model. None of the SNPs were found to lie outside the 95% confidence limits assumed for conditional joint distribution of Fst vs mean heterozygosity, and therefore the genes do not appear to be under selection in the analysed samples according to the employed model. However, several SNPs were not in HW equilibrium. We hypothesize that some of the polymorphisms were fixated since many generations within breed and the coalescent model could not be powerful enough to reveal selection event so far in the past.

Structure of cattle, sheep, goat and buffalo populations using single nucleotide polymorphisms in genes affecting lipid metabolism

Abstract Single nucleotide polymorphism (SNPs) have the potential to become the genetic marker of choice in studies on the ecology and the conservation of natural populations because of their high frequency across the genome. Being biallelic markers, SNPs are intrinsically less variable than microsatellites, so individual locus information content is low. However, in population studies, this can be compensated for by the use of many more SNPs to obtain a suitable statistical power. SNPs in candidate genes could reveal chromosomal regions subject to natural or artificial selection directly or by linkage disequilibrium. In this study we used 69 SNPs, identified in 20 candidates genes involved in milk fat composition, to assess the genetic diversity within and across four ruminants species (cattle, sheep, goat and buffalo). SNPs were characterised and genotyped on 459 individuals belonging to 15 breeds and the allelic frequencies of the 2 alleles were calculated both at species and breed level. Individuals were clustered applying a parametric genetic mixture analysis implemented in the Structure 2.0 software. The number of genetic clusters (K) was tested using the no admixture model, a burning period of 100,000 followed by 200,000 MCMC repeats and considering SNPs frequencies correlated. The likelihood values and the variance of the bootstrap samples so obtained were plotted against K (ranging from 2 to 20, a number higher of the true number of breeds) in order to estimate the optimal K value. The method resolved 10 populations, being able to correctly identify breeds of cows, sheep and buffalo. Goat breeds remained unresolved, demonstrating the lower diversity found in this species in comparison to the other species. These findings are in agreement with other studies that used neutral markers like microsatellites.

Effect of some candidate genes on meat characteristics of three cattle breeds.

Abstract With the aim to assess if some molecular markers can help to select animals for meat characteristics, we studied 84 individuals equally representing the Marchigiana, Maremmana, and Holstein Friesian cattle breeds genotyped at 288 SNPs located within candidate genes. Several SNPs were found associated with meat quality parameters but with P which was higher than the Bonferroni threshold. However, several SNPs had a low P at different times during meat maturation, suggesting their involvement in the meat quality variation. Of particular interest for the biological role and potential for selection were: cathepsin G affecting MFI, IGF1R affecting pH and collagen XVIII affecting colour.