Skeletal muscle transcriptional profiles in two Italian beef breeds, Chianina and Maremmana, reveal breed specific variation.

Chianina and Maremmana breeds play an important role in the Italian cattle meat market. The Chianina breed is an ancient breed principally raised for draught. Now this breed is the worldwide recognized producer of top quality beef, tasteful and tender, specifically the famous "Florentine steak". The Maremmana characterized by a massive skeletal structure, is a rustic cattle breed selected for adaptability to the marshy land of the Maremma region. We used a high throughput mRNA sequencing to analyze gene expression in muscle tissues of two Italian cattle breeds, Maremmana (MM) and Chianina (CN) with different selection history. We aim to examine the specific genetic contribution of each breed to meat production and quality, comparing the skeletal muscle tissue from Maremmana and Chianina. Most of the differentially expressed genes were grouped in the Glycolysis/Gluconeogenesis pathways. The rate and the extent of post-mortem energy metabolism have a critical effect on the conversion of muscle to meat. Furthermore, we aim at discovering the differences in nucleotide variation between the two breeds which might be attributable to the different history of selection/divergence. In this work we could emphasize the involvement of pathways of post-mortem energy metabolism. Moreover, we detected a collection of coding SNPs which could offer new genomic resources to improve phenotypic selection in livestock breeding program.

Transcriptomic investigation of meat tenderness in two Italian cattle breeds.

Our objectives for this study were to understand the biological basis of meat tenderness and to provide an overview of the gene expression profiles related to meat quality as a tool for selection. Through deep mRNA sequencing, we analyzed gene expression in muscle tissues of two Italian cattle breeds: Maremmana and Chianina. We uncovered several differentially expressed genes that encode for proteins belonging to a family of tripartite motif proteins, which are involved in growth, cell differentiation and apoptosis, such as TRIM45, or play an essential role in regulating skeletal muscle differentiation and the regeneration of adult skeletal muscle, such as TRIM32. Other differentially expressed genes (SCN2B, SLC9A7 and KCNK3) emphasize the involvement of potassium-sodium pumps in tender meat. By mapping splice junctions in RNA-Seq reads, we found significant differences in gene isoform expression levels. The PRKAG3 gene, which is involved in the regulation of energy metabolism, showed four isoforms that were differentially expressed. This distinct pattern of PRKAG3 gene expression could indicate impaired glycogen storage in skeletal muscle, and consequently, this gene very likely has a role in the tenderization process. Furthermore, with this deep RNA-sequencing, we captured a high number of expressed SNPs, for example, we found 1462 homozygous SNPs showing the alternative allele with a 100% frequency when comparing tender and tough meat. SNPs were then classified into categories by their position and also by their effect on gene coding (174 non-synonymous polymorphisms) based on the available UMD_3.1 annotations.

Genome-wide analysis of Italian sheep diversity reveals a strong geographic pattern and cryptic relationships between breeds.

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 ACACA gene is a potential candidate gene for fat content in sheep milk.

No major gene has yet been reported in sheep that explains the variation of milk fat content. The coding region of the acetyl-CoA carboxylase alpha (ACACA) gene, which plays an important role in de novo fatty acid synthesis, had been investigated, but no non-synonymous mutations have been reported. In this study, the genomic regions encoding the three promoters of the ACACA gene were directly sequenced in 264 sheep of three different breeds, and 10 SNPs were identified. Allele frequencies of most SNPs significantly differed (P = 0.05-0.0001) between breeds. The SNPs that potentially altered either gene regulatory elements or putative binding sites of transcription factors were made evident through in silico analysis. The association analysis with milk traits, performed for one SNP of PIII (GenBank AJ292286, g.1330G>T), showed a significant allelic substitution effect (+0.33%, P < 0.0001 and +0.35%, P < 0.01) in the Altamurana and Gentile breeds respectively. Because this SNP was located in the binding site of the paired box protein transcription factors, which was shown to function as an efficient promoter element, and because PIII transcripts are expressed in the mammary gland, the SNP in PIII of the ACACA gene might affect the variation of fat content in sheep milk.

Analysis of SLC11A1 gene expression in healthy water buffalo (Bubalus bubalis) blood cells using qPCR.

SLC11A1 (solute carrier family 11 member 1 protein) gene influences the initial phase of bacterial cellular infections through macrophage activation. Recent literature on buffalo has attempted to associate the genotype of the polymorphic microsatellite located in the 3'untranslated region (3'UTR) of the gene, with either susceptibility to brucellosis or with improved macrophage function. Carriers of the (GT)16 allele have been reported to be resistant to brucellosis. In this study we analyzed the steady-state level of SLC11A1 expression in a serologically negative herd of 26 animals differing by the number of (GT)n microsatellite repeats by using a reverse transcriptase quantitative real-time polymerase chain reaction approach. We evaluated five different reference genes, which had not been reported previously, for use in gene expression experiments in buffalo blood. However, we did not find any significant difference between buffalo carriers of the different microsatellite alleles, with respect to SLC11A1 expression in whole blood or in blood fractions [peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes/granulocytes (PMN/G)]. Conversely, there was a difference between the blood fractions in their SLC11A1 expression levels, with the PMN/G fraction having a higher expression level than the PBMC fraction (P < 0.015).

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.

Ovine acyl CoA:diacylglycerol acyltransferase 1- molecular characterization, polymorphisms and association with milk traits.

The objective of this work was to characterize the complete coding region of the ovine acylCoA:diacylglycerol acyltransferase 1 (DGAT1) gene of three Italian sheep breeds: Sarda, Altamurana and Gentile di Puglia. Characterization was accomplished by direct sequencing of 8676 bp of the relevant DNA, including introns and partial 5' and 3' untranslated regions (UTRs). We detected five novel SNPs; one SNP (g.5553C>T) is located in intron 2, has similar frequencies in the three breeds and showed a negative association with milk fat content. More interesting is an SNP in the 5' UTR (g.127C>A), the occurrence of which is rare in the higher milk-fat breeds (Altamurana and Gentile di Puglia); it is located in the core sequence of Sp1, a putative binding site of transcription factors. This SNP showed a significant negative association with milk fat content in the Sarda sheep. Because DGAT1 plays a fundamental role in triacylglycerol synthesis, the novel detected SNP in the 5' UTR of the DGAT1 gene might explain, at least partially, the variation of fat content in the milk of Sarda sheep.

Fatty acid composition and sensory properties of Italian Simmental beef as affected by gene frequency of Montbéliarde origin.

The aim of this study was to investigate the effect of Montbéliarde (Mb) gene frequency on fatty acid composition and sensory properties of Italian Simmental (IS) steaks (longissimus thoracis m.). Twenty-seven bulls belonging to three strains with different percentages of Mb genes: traditional (ISt), without Mb ascendants (ISt=0% Mb genes), cross-strain (ISmt=37.5-50% Mb genes), Montbéliarde strain (ISm=87.5-100% Mb genes) and balanced for stearoyl Co-A desaturase genotype were considered. ISt has the highest C20:4 n-6 (P<0.01), C22:4 n-6 (P<0.05) and total PUFA n-3 level (P<0.01), while ISt and ISmt have higher C18:3 n-3 (P<0.05) and slightly lower MUFA (P=0.08) than ISm. Sensory tests indicated that the three experimental groups can be differentiated; moreover, ISmt meat is perceived as less hard (P<0.01), less chewable (P<0.01) and less fibrous (P<0.05) than ISt meat.

Genome-wide association study for longevity in the Holstein cattle population.

Longevity is one of the most important traits determining dairy cow profitability. In the last decades dairy cows suffered a lowering in the age at culling. With the aim to identify the genes involved in longevity, dates of birth, yields, dates of calving during lifespan and culling dates were collected for 946 culled cows which had been genotyped with the Bovine High Density panel. Using the GenABEL package in R, genome-wide association analysis was performed on three potential traits of longevity: (1) 'days in production,' (2) 'days in herd,' (3) number of calvings over lifespan.' Five genome-wide significant single nucleotide polymorphisms (SNPs) associated with all three longevity traits were detected. Several consecutive SNPs identified on chromosomes 16 and 30 indicated the presence of two suggestive quantitative trait loci (QTL). The genes comprised in the QTL regions had biological functions related to fertility, reproductive disorders, heat stress and welfare of cows. These findings might contribute to improving breeding strategies to improve longevity.

Gelsolin expression in sheep milk somatic cells during lactation.

The identification of genes involved in phenotypes related to milk quality is important for both economic and health aspects in livestock production. The aim of this study was to assess the level of gelsolin gene expression in two breeds of dairy sheep - Sarda and Gentile - with pronounced differences in quantitative and qualitative milk traits. Gelsolin, a type of actin-modulating proteins is involved in the processes of actin remodeling during cell growth and apoptosis; therefore a role of this protein in mammary changes during lactation was here hypothesized. Individual milk samples were collected three times during lactation from 26 ewes of the two breeds. The differential gene expression of gelsolin in the two breeds and the three lactation times was estimated by quantitative PCR on RNA extracted from milk somatic cells. Correlations of gelsolin gene expression with milk yield and quality and days of lactation were also estimated. The results showed that gelsolin gene expression was significantly higher in the Sarda compared to the Gentile at each lactation stage, in agreement with the longer lactation duration and the higher daily milk yield of the first breed. Significant correlations of gelsolin gene expression were found with milk fat content in Sarda breed (-0.46, P<0.05). Gelsolin expression analysis confirmed the link between gelsolin gene function and milk fat content of sheep.

Short communication: Effect of stearoyl-coenzyme A desaturase polymorphism on fatty acid composition of milk.

The effect of the stearoyl-CoA desaturase (SCD) gene on milk fatty acid composition was tested. Cows of 3 breeds of northern Italy, Piedmontese, Valdostana, and Jersey, were genotyped at exon 5 of the SCD gene. This has been suggested as a primary candidate gene to change the proportion of saturated vs. unsaturated fatty acids in milk, wherein a single nucleotide polymorphism (C/T) gives rise to a different AA codon. It was possible to ascribe a reduced desaturase activity to the T allele only in the case of caproleic and myristoleic fatty acids. In contrast with the findings of SCD effects on carcass fat, it was not possible to confirm the higher desaturation activity of this single nucleotide polymorphism on long-chain fatty acids, due to the different pathways that originate milk fatty acids of different carbon length; long-chain fatty acids are highly influenced by the complex metabolic events that affect the ingested nutrients during their transfer to milk fat.

Genetic parameters and genome-wide associations of twinning rate in a local breed, the Maremmana cattle.

This study seeks to verify the feasibility of increasing twinning in a herd of the Italian autochtonous Maremmana breed. The data set included 1260 individuals born from 1963 to 2014, 527 males and 733 females, 402 of them calving at least once from 1983 through 2015. Breeding values for twinning were estimated by a single-trait linear animal model. However, since twinning is a dichotomous trait and the frequency of twins is far smaller than the frequency of single births, breeding values were also estimated by a single-trait animal threshold model. Heritability of twinning was 0.014±0.018 and 0.062±0.093 for the linear and the threshold models, respectively. Repeatability was 0.071±0.004 and 0.286± 0.012, respectively, for the two models. Genotyping with the Illumina BovineSNP54 BeadChip was performed for cows living on farm in 2012 (119 cows) and a genome-wide association analysis was performed on the corrected phenotype of all calving during the lifespan of each cow, using the GenABEL package in R and a three step GRAMMAR-GC approach. Genomic heritability, calculated from the genomic kinship matrix estimated through genomic marker data, was 0.29±0.021. The most significant detected single nucleotide polymorphisms (Hapmap22923-BTA-129564) was located in proximity of two genes, ARHGAP8 and TMEM200C, which might be potential functional candidates for twinning rate in cattle.

Meat molecular traceability: How to choose the best set of microsatellites?

To set up routine assays for molecular meat traceability along the food chain, the availability of a simple and low cost test for the identification of cattle carcasses is required. For this purpose, we evaluated 13 microsatellites for their ability in the identification of animals belonging to four Italian cattle breeds. Here we propose a criterion for a microsatellite-based test with the best reliability when reducing the number of loci to be analysed. The method is based on the observation that in the same loci breeds can show differences in frequencies and number of fixed alleles. This non-uniform distribution of alleles between breeds results in differences in the informative content of the same loci in different breeds. Taking into account these differences, it is possible to perform tests for the allocation of samples to specific animals utilizing a small number of microsatellites. The proposed approach allows cost reduction and ease in performing the analyses.

Signatures of selection identify loci associated with fat tail in sheep.

Identification of genomic regions that have been targets of selection for phenotypic traits is one of the most challenging applications of dense marker panels in animal genetics. In this study, a genomewide scan using approximately 50,000 SNP was performed in an attempt to identify genomic regions associated with fat deposition in sheep, the importance of this not only being limited to livestock facing future climate changes but also for elucidating the physiology of lipid metabolism. The genotyping results obtained with the Ovine SNP50K BeadChip in 2 fat tail breeds were compared with those obtained in 13 thin tail breeds. Direct sequencing of the annotated genes located in proximity to the markers with opposite allele frequency in thin tail vs. fat tail sheep gave additional SNP of interest. To further confirm the results of the genomewide scan, we genotyped the SNP within these genes in the 2 groups of sheep. A missense mutation in the gene, with different allele frequency in the 2 groups, was detected. The results indicated and as the most probable genes involved in the fat tail phenotype.

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.

Association analyses of single nucleotide polymorphisms in the LEP and SCD1 genes on the fatty acid profile of muscle fat in Simmental bulls.

The aim was to investigate the effect of the genetic polymorphisms of leptin (LEP) and stearoyl-CoA desaturase (SCD1) genes on the fatty acid (FA) composition of the muscle of 103 Simmental bulls. Ten single nucleotide polymorphisms (SNP) were detected in exons 2 and 3 of the LEP gene, two of them encoding non-synonymous mutations. Allelic substitution effects of all the SNP on 28 single fatty acids, monounsaturated (MUFA) and polyunsaturated (PUFA) and desaturation indexes were estimated. Both the SCD1 SNP, as well as three SNP of the leptin gene, affected, to different extents, the desaturation of FA into MUFA. Because it was previously proposed that leptin's metabolic action involves down-regulation of SCD1, it is possible that, beyond the mere additive effect of SCD1 gene on FA desaturation, the non-synonymous mutations in the leptin gene also contribute to the variability of FA composition in muscle fat.

Single nucleotide polymorphism detection in promoter III of the acetyl-CoA carboxylase-alpha gene in sheep.

Three novel SNPs were identified in the locus OAR292286, encoding the DNA sequence of promoter III of the ovine acetyl-CoA carboxylase-alpha gene, in Italian sheep of four breeds: Gentile di Puglia (25 individuals) and Sopravissana (31) which are triple-purpose local endangered breeds, Comisana (25) which is a local non-selected, non-endangered dairy breed and Sarda (15) which is a popular selected high yielding dairy breed. Variant alleles are: G/T at 1330 bp, C/G at 1338 bp and C/T at 1430 bp. Frequencies of the variant alleles were calculated and chi-squared analysis of the differences in allele frequency between breed pairs was performed. Allele frequencies of the Sarda breed differ significantly from the other considered breeds.

Genetic evaluation of dairy sheep with an animal model for annual or partial lactation production.

Two traits were compared as breeding objectives for the selection of dairy sheep: the total milk produced during 1 yr and the production over the first 70 d in milk. Both traits are proposed for sheep breeds that have more than one lambing per year and, consequently, highly variable lactation length. Breeding values for both traits were obtained with an animal model including flock-year-season of lambing and age class as fixed effects and additive genetic and permanent environmental as random effects. A total of 12,759 ewes and 547 rams of the Masses breed of sheep were evaluated for both traits, which were built up from a total of 31,170 lactations of 11,106 ewes. Average annual production was 161.8 kg; average length of lactation was 146.9 d/yr, and average partial lactation production from d 31 to 100 after lambing was 90.9 kg. Estimated breeding values ranged from -54.8 to 74.5 kg of milk for annual production and from -26.2 to 33.1 kg of milk for partial lactation production. Correlation of the estimated breeding values for the two traits was 67.4%. Annual genetic trend from 1980 to 1988 was .48 kg of milk for annual production and .07 kg of milk for 70 d lactation production.