Deciphering the Wisent Demographic and Adaptive Histories from Individual Whole-Genome Sequences.

As the largest European herbivore, the wisent (Bison bonasus) is emblematic of the continent wildlife but has unclear origins. Here, we infer its demographic and adaptive histories from two individual whole-genome sequences via a detailed comparative analysis with bovine genomes. We estimate that the wisent and bovine species diverged from 1.7 × 106 to 850,000 years before present (YBP) through a speciation process involving an extended period of limited gene flow. Our data further support the occurrence of more recent secondary contacts, posterior to the Bos taurus and Bos indicus divergence (∼150,000 YBP), between the wisent and (European) taurine cattle lineages. Although the wisent and bovine population sizes experienced a similar sharp decline since the Last Glacial Maximum, we find that the wisent demography remained more fluctuating during the Pleistocene. This is in agreement with a scenario in which wisents responded to successive glaciations by habitat fragmentation rather than southward and eastward migration as for the bovine ancestors. We finally detect 423 genes under positive selection between the wisent and bovine lineages, which shed a new light on the genome response to different living conditions (temperature, available food resource, and pathogen exposure) and on the key gene functions altered by the domestication process.

Phenotypic and genotypic background underlying variations in fatty acid composition and sensory parameters in European bovine breeds.

BACKGROUND: Consuming moderate amounts of lean red meat as part of a balanced diet valuably contributes to intakes of essential nutrients. In this study, we merged phenotypic and genotypic information to characterize the variation in lipid profile and sensory parameters and to represent the diversity among 15 cattle populations. Correlations between fat content, organoleptic characteristics and lipid profiles were also investigated. METHODS: A sample of 436 largely unrelated purebred bulls belonging to 15 breeds and reared under comparable management conditions was analyzed. Phenotypic data -including fatness score, fat percentage, individual fatty acids (FA) profiles and sensory panel tests- and genotypic information from 11 polymorphisms was used. RESULTS: The correlation coefficients between muscle total lipid measurements and absolute vs. relative amounts of polyunsaturated FA (PUFA) were in opposite directions. Increasing carcass fat leads to an increasing amount of FAs in triglycerides, but at the same time the relative amount of PUFAs is decreasing, which is in concordance with the negative correlation obtained here between the percentage of PUFA and fat measurements, as well as the weaker correlation between total phospholipids and total lipid muscle content compared with neutral lipids. Concerning organoleptic characteristics, a negative correlation between flavour scores and the percentage of total PUFA, particularly to n-6 fraction, was found. The correlation between juiciness and texture is higher than with flavour scores. The distribution of SNPs plotted by principal components analysis (PCA) mainly reflects their known trait associations, although influenced by their specific breed allele frequencies. CONCLUSIONS: The results presented here help to understand the phenotypic and genotypic background underlying variations in FA composition and sensory parameters between breeds. The wide range of traits and breeds studied, along with the genotypic information on polymorphisms previously associated with different lipid traits, provide a broad characterization of beef meat, which allows giving a better response to the variety of consumers' preferences. Also, the development and implementation of low-density SNP panels with predictive value for economically important traits, such as those summarized here, may be used to improve production efficiency and meat quality in the beef industry.

The GENOTEND chip: a new tool to analyse gene expression in muscles of beef cattle for beef quality prediction.

BACKGROUND: Previous research programmes have described muscle biochemical traits and gene expression levels associated with beef tenderness. One of our results concerning the DNAJA1 gene (an Hsp40) was patented. This study aims to confirm the relationships previously identified between two gene families (heat shock proteins and energy metabolism) and beef quality. RESULTS: We developed an Agilent chip with specific probes for bovine muscular genes. More than 3000 genes involved in muscle biology or meat quality were selected from genetic, proteomic or transcriptomic studies, or from scientific publications. As far as possible, several probes were used for each gene (e.g. 17 probes for DNAJA1). RNA from Longissimus thoracis muscle samples was hybridised on the chips. Muscles samples were from four groups of Charolais cattle: two groups of young bulls and two groups of steers slaughtered in two different years. Principal component analysis, simple correlation of gene expression levels with tenderness scores, and then multiple regression analysis provided the means to detect the genes within two families (heat shock proteins and energy metabolism) which were the most associated with beef tenderness. For the 25 Charolais young bulls slaughtered in year 1, expression levels of DNAJA1 and other genes of the HSP family were related to the initial or overall beef tenderness. Similarly, expression levels of genes involved in fat or energy metabolism were related with the initial or overall beef tenderness but in the year 1 and year 2 groups of young bulls only. Generally, the genes individually correlated with tenderness are not consistent across genders and years indicating the strong influence of rearing conditions on muscle characteristics related to beef quality. However, a group of HSP genes, which explained about 40% of the variability in tenderness in the group of 25 young bulls slaughtered in year 1 (considered as the reference group), was validated in the groups of 30 Charolais young bulls slaughtered in year 2, and in the 21 Charolais steers slaughtered in year 1, but not in the group of 19 steers slaughtered in year 2 which differ from the reference group by two factors (gender and year). When the first three groups of animals were analysed together, this subset of genes explained a 4-fold higher proportion of the variability in tenderness than muscle biochemical traits. CONCLUSION: This study underlined the relevance of the GENOTEND chip to identify markers of beef quality, mainly by confirming previous results and by detecting other genes of the heat shock family as potential markers of beef quality. However, it was not always possible to extrapolate the relevance of these markers to all animal groups which differ by several factors (such as gender or environmental conditions of production) from the initial population of reference in which these markers were identified.

Genetic variability and linkage disequilibrium patterns in the bovine DNAJA1 gene.

Correlation between expression level of the bovine DNAJA1 gene and meat tenderness was recently found in Charolais longissimus thoracis muscle samples, suggesting that this gene could play an important role in meat tenderness. Here, we report the validation of polymorphisms within the bovine DNAJA1 gene, and the haplotype variability and extent of linkage disequilibrium in the three main French beef breeds (Blonde d'Aquitaine, Charolais, Limousin). Genotyping 18 putative SNPs revealed that 16 SNPs were polymorphic within the breeds tested. Two SNPs were removed from further analyses as one SNP had a low genotyping call rate, while the other SNP was not in Hardy-Weinberg equilibrium. The degree of heterozygosity observed for the remaining 14 SNPs varied between breeds, with Charolais being the breed with the highest genetic variation and Blonde d'Aquitaine the lowest. Linkage disequilibrium and haplotype structure of DNAJA1 were different between breeds. Eighteen different haplotypes, including three shared by all breeds, were discovered, and two to three tag SNPs (depending on the breed) are sufficient to capture all the genetic variability seen in these haplotypes. The results of this study will facilitate the design of optimal future association studies evaluating the role of the DNAJA1 gene in meat tenderness.

An original SERPINA3 gene cluster: elucidation of genomic organization and gene expression in the Bos taurus 21q24 region.

BACKGROUND: The superfamily of serine proteinase inhibitors (serpins) is involved in numerous fundamental biological processes as inflammation, blood coagulation and apoptosis. Our interest is focused on the SERPINA3 sub-family. The major human plasma protease inhibitor, alpha1-antichymotrypsin, encoded by the SERPINA3 gene, is homologous to genes organized in clusters in several mammalian species. However, although there is a similar genic organization with a high degree of sequence conservation, the reactive-centre-loop domains, which are responsible for the protease specificity, show significant divergences. RESULTS: We provide additional information by analyzing the situation of SERPINA3 in the bovine genome. A cluster of eight genes and one pseudogene sharing a high degree of identity and the same structural organization was characterized. Bovine SERPINA3 genes were localized by radiation hybrid mapping on 21q24 and only spanned over 235 Kilobases. For all these genes, we propose a new nomenclature from SERPINA3-1 to SERPINA3-8. They share approximately 70% of identity with the human SERPINA3 homologue. In the cluster, we described an original sub-group of six members with an unexpected high degree of conservation for the reactive-centre-loop domain, suggesting a similar peptidase inhibitory pattern. Preliminary expression analyses of these bovSERPINA3s showed different tissue-specific patterns and diverse states of glycosylation and phosphorylation. Finally, in the context of phylogenetic analyses, we improved our knowledge on mammalian SERPINAs evolution. CONCLUSION: Our experimental results update data of the bovine genome sequencing, substantially increase the bovSERPINA3 sub-family and enrich the phylogenetic tree of serpins. We provide new opportunities for future investigations to approach the biological functions of this unusual subset of serine proteinase inhibitors.

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles.

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.

Purification of the skeletal muscle protein Endopin 1B and characterization of the genes encoding Endopin 1A and 1B isoforms.

In the present work, a new endopin-like serpin designed mEndopin 1B was purified from bovine muscle. Biochemical characterizations (amino acid sequencing and Maldi-Tof mass spectrometry peptide mapping) demonstrated that the purified protein is different from the previously described Endopin 1, renamed mEndopin 1A. The genes and cDNA of both endopins were characterized. The cDNA sequence of mEndopin 1B encodes a predicted protein of 411 amino-acids with a molecular mass of 43808Da. The mEndopin 1B gene comprised four coding exons and an additional 5' untranslated exon. The reactive site sequence of mEndopin 1B is somewhat different from that of mEndopin 1A. Nevertheless, both serpins have a similar peptidase inhibitory pattern against examined proteases (elastase, trypsin, plasmin and chymotrypsin). The high expression of both mEndopin 1A and 1B in bovine serum and tissues and their high efficiency to inhibit elastase (k(ass) approximately 10(6)-10(7) M(-1) s(-1)) suggested that these serpins might play a major role in inflammatory processes.

Cotranscription and intergenic splicing of the PPARG and TSEN2 genes in cattle.

BACKGROUND: Intergenic splicing resulting in the combination of mRNAs sequences from distinct genes is a newly identified mechanism likely to contribute to protein diversity. Few cases have been described, most of them involving neighboring genes and thus suggesting a cotranscription event presumably due to transcriptional termination bypass. RESULTS: We identified bovine chimeric transcripts resulting from cotranscription and intergenic splicing of two neighboring genes, PPARG and TSEN2. These two genes encode the Peroxisome Proliferator Activated Receptors gamma1 and gamma2 and the tRNA Splicing Endonuclease 2 homolog and are situated in the same orientation about 50 kb apart on bovine chromosome 22q24. Their relative position is conserved in human and mouse. We identified two types of chimeric transcripts containing all but the last exon of the PPARG gene followed by all but the first exon of the TSEN2 gene. The two chimers differ by the presence/absence of an intermediate exon resulting from transcription of a LINE L2 sequence situated between the two genes. Both transcripts use canonical splice sites for all exons coming from both genes, as well as for the LINE L2 sequence. One of these transcripts harbors a premature STOP codon and the other encodes a putative chimeric protein combining most of the PPARgamma protein and the entire TSEN2 protein, but we could not establish the existence of this protein. CONCLUSION: By showing that both individual and chimeric transcripts are transcribed from PPARG and TSEN2, we demonstrated regulation of transcription termination. Further, the existence and functionality of a chimeric protein harboring active motifs that are a priori unrelated is hypothesized.

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts.

The bovine PRKAG3 gene encodes the AMPK gamma3 subunit, one isoform of the regulatory gamma subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the gamma subunits have been shown to influence AMPK activity. The gamma3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly influences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3' UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.

Correlation between bovine calpastatin mRNA transcripts and protein isoforms.

Calpastatin is a specific calpain protease inhibitor: calpains are a family of calcium-activated neutral proteases, which have been implicated in various processes. Despite all the available data concerning calpastatin, little is known about how this gene is regulated, particularly in bovine. The existence of four types of transcripts differing at their 5' ends (Type I, II, III, and IV) has been demonstrated. Here, we show that the Type I, II, and III transcripts are ubiquitous while Type IV is testis-specific. In addition, a Northern blot analysis revealed that the Type III transcript may have three different 3' termini. Using specific anti-peptide anti-sera, a correspondence between a 145 and a 125 kDa isoforms, and Type I and/or II and III transcripts, respectively, has been established. Finally, we discuss the origin of a 70 kDa isoform, recognized by anti-sera directed against the N-terminal region.

Four promoters direct expression of the calpastatin gene.

Calpastatin is a specific endogenous protein inhibitor of the ubiquitous calcium dependent proteinases mu- and m-calpain. The calpain-calpastatin system is involved in various physiological and pathological processes. In the present study, we determined the bovine calpastatin gene structure and demonstrated that four promoters direct its expression. The gene harbours 35 exons spanning at least 130kb on genomic DNA. Its structure is similar to that of mouse, pig, and human gene. Transient transfection assays in both C2C12 and COS7 cell lines demonstrated that the putative promoter regions situated 5' to exon 1xa, 1xb, 1u, and 14t were functional. We also established that the region situated upstream exon 14t is subjected to a tissue specific regulation. The implication of numerous high-scoring cis acting transcriptional motifs which are present in these regions will need to be determined. The existence of four promoters suggests differential expression patterns which must have a physiological significance.

Muscle endopin 1, a muscle intracellular serpin which strongly inhibits elastase: purification, characterization, cellular localization and tissue distribution.

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 microg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41x10(7) M(-1).s(-1)) and trypsin (kass=3.92x10(6) M(-1).s(-1)), whereas plasmin (kass=1.78x10(3) M(-1).s(-1)) and chymotrypsin (kass=1.0x10(2) M(-1).s(-1)) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.

Widespread expression of the bovine Agouti gene results from at least three alternative promoters.

In wild-type mice, it is well known that Agouti is only expressed in skin where it controls the banded-hair phenotype. As a first step to investigate the physiological role of Agouti in cattle, we isolated the corresponding gene and studied its expression pattern. We found no evidence of coding-region sequence variation within and between eight breeds representing a large panel of coat colour phenotypes. We detected by northern hybridization two Agouti mRNA isoforms in brain, heart, lung, liver, kidney, spleen and a third in skin. We characterized the full-length Agouti transcript in skin and isolated the 5'UTR of two mRNAs expressed in the other tissues. The three mRNAs have the same coding region but differ by their 5' untranslated regions. Upstream regulatory sequences display two alternative promoters involved with the broad expression in tissues other than skin. Interestingly, these sequences are highly homologous to upstream sequences of the orthologous human (76-85% identity) and pig (82-86% identity) ASIP genes. In addition to its potential role in pigmentation (as seen in mice), we suggest that bovine Agouti could be involved in various physiological functions. Furthermore, the significant homology between cattle, pig and human regulatory sequences indicate that these orthologous genes are regulated alike. Lastly, since the 5'UTR of many eukaryotic mRNAs are physiologically relevant, their impact on bovine Agouti mRNA performance is discussed.

Pheomelanin coat colour dilution in French cattle breeds is not correlated with the TYR, TYRP1 and DCT transcription levels.

In this study we report the isolation of full-length cDNAs and the expression patterns of TYR, TYRP1 and DCT in four e/e cattle breeds exhibiting different pheomelanic coat colours ranging from reddish brown to creamy white phenotypes. Predicted proteins encoded by bovine TYR, TYRP1 and DCT display high levels of homology and contain all characteristic domains shared between their mouse and human counterparts. The full expression of these three genes is observed in melanocytes of black areas of E(D)/E(D) Prim'Holstein's animals. On the other hand, e/e melanocytes of animals belonging to the Blonde d'Aquitaine (blond), Limousine (red) and Salers (reddish brown) breeds present different levels of down-regulated TYR and DCT expression and a complete repression of TYRP1. Surprisingly, e/e melanocytes of animals belonging to the Charolais breed (creamy white) present an inverse relationship between TYR, TYRP1 and DCT expression and its lower melanogenic activity. The sum of these results shows that the dilution of the coat colour in French cattle breeds is not correlated with a transcription level of TYR family genes. Other possible modifier loci are suggested.

Combined analysis of data from two granddaughter designs: A simple strategy for QTL confirmation and increasing experimental power in dairy cattle.

A joint analysis of five paternal half-sib Holstein families that were part of two different granddaughter designs (ADR- or Inra-design) was carried out for five milk production traits and somatic cell score in order to conduct a QTL confirmation study and to increase the experimental power. Data were exchanged in a coded and standardised form. The combined data set (JOINT-design) consisted of on average 231 sires per grandsire. Genetic maps were calculated for 133 markers distributed over nine chromosomes. QTL analyses were performed separately for each design and each trait. The results revealed QTL for milk production on chromosome 14, for milk yield on chromosome 5, and for fat content on chromosome 19 in both the ADR- and the Inra-design (confirmed within this study). Some QTL could only be mapped in either the ADR- or in the Inra-design (not confirmed within this study). Additional QTL previously undetected in the single designs were mapped in the JOINT-design for fat yield (chromosome 19 and 26), protein yield (chromosome 26), protein content (chromosome 5), and somatic cell score (chromosome 2 and 19) with genomewide significance. This study demonstrated the potential benefits of a combined analysis of data from different granddaughter designs.

Detection of genes influencing economic traits in three French dairy cattle breeds.

A project of QTL detection was carried out in the French Holstein, Normande, and Montbéliarde dairy cattle breeds. This granddaughter design included 1 548 artificial insemination bulls distributed in 14 sire families and evaluated after a progeny-test for 24 traits (production, milk composition, persistency, type, fertility, mastitis resistance, and milking ease). These bulls were also genotyped for 169 genetic markers, mostly microsatellites. The QTL were analysed by within-sire linear regression of daughter yield deviations or deregressed proofs on the probability that the son receives one or the other paternal QTL allele, given the marker information. QTL were detected for all traits, including those with a low heritability. One hundred and twenty QTL with a chromosome-wise significance lower than 3% were tabulated. This threshold corresponded to a 15% false discovery rate. Amongst them, 32 were genome-wise significant. Estimates of their contribution to genetic variance ranged from 6 to 40%. Most substitution effects ranged from 0.6 to 1.0 genetic standard deviation. For a given QTL, only 1 to 5 families out of 14 were informative. The confidence intervals of the QTL locations were large and always greater than 20 cM. This experiment confirmed several already published QTL but most of them were original, particularly for non-production traits.