Identification of eQTLs and sQTLs associated with meat quality in beef.

BACKGROUND: Transcription has a substantial genetic control and genetic dissection of gene expression could help us understand the genetic architecture of complex phenotypes such as meat quality in cattle. The objectives of the present research were: 1) to perform eQTL and sQTL mapping analyses for meat quality traits in longissimus dorsi muscle; 2) to uncover genes whose expression is influenced by local or distant genetic variation; 3) to identify expression and splicing hot spots; and 4) to uncover genomic regions affecting the expression of multiple genes. RESULTS: Eighty steers were selected for phenotyping, genotyping and RNA-seq evaluation. A panel of traits related to meat quality was recorded in longissimus dorsi muscle. Information on 112,042 SNPs and expression data on 8588 autosomal genes and 87,770 exons from 8467 genes were included in an expression and splicing quantitative trait loci (QTL) mapping (eQTL and sQTL, respectively). A gene, exon and isoform differential expression analysis previously carried out in this population identified 1352 genes, referred to as DEG, as explaining part of the variability associated with meat quality traits. The eQTL and sQTL mapping was performed using a linear regression model in the R package Matrix eQTL. Genotype and year of birth were included as fixed effects, and population structure was accounted for by including as a covariate the first PC from a PCA analysis on genotypic data. The identified QTLs were classified as cis or trans using 1 Mb as the maximum distance between the associated SNP and the gene being analyzed. A total of 8377 eQTLs were identified, including 75.6% trans, 10.4% cis, 12.5% DEG trans and 1.5% DEG cis; while 11,929 sQTLs were uncovered: 66.1% trans, 16.9% DEG trans, 14% cis and 3% DEG cis. Twenty-seven expression master regulators and 13 splicing master regulators were identified and were classified as membrane-associated or cytoskeletal proteins, transcription factors or DNA methylases. These genes could control the expression of other genes through cell signaling or by a direct transcriptional activation/repression mechanism. CONCLUSION: In the present analysis, we show that eQTL and sQTL mapping makes possible positional identification of gene and isoform expression regulators.

Genome wide association and gene enrichment analysis reveal membrane anchoring and structural proteins associated with meat quality in beef.

BACKGROUND: Meat quality related phenotypes are difficult and expensive to measure and predict but are ideal candidates for genomic selection if genetic markers that account for a worthwhile proportion of the phenotypic variation can be identified. The objectives of this study were: 1) to perform genome wide association analyses for Warner-Bratzler Shear Force (WBSF), marbling, cooking loss, tenderness, juiciness, connective tissue and flavor; 2) to determine enriched pathways present in each genome wide association analysis; and 3) to identify potential candidate genes with multiple quantitative trait loci (QTL) associated with meat quality. RESULTS: The WBSF, marbling and cooking loss traits were measured in longissimus dorsi muscle from 672 steers. Out of these, 495 animals were used to measure tenderness, juiciness, connective tissue and flavor by a sensory panel. All animals were genotyped for 221,077 markers and included in a genome wide association analysis. A total number of 68 genomic regions covering 52 genes were identified using the whole genome association approach; 48% of these genes encode transmembrane proteins or membrane associated molecules. Two enrichment analysis were performed: a tissue restricted gene enrichment applying a correlation analysis between raw associated single nucleotide polymorphisms (SNPs) by trait, and a functional classification analysis performed using the DAVID Bioinformatic Resources 6.8 server. The tissue restricted gene enrichment approach identified eleven pathways including "Endoplasmic reticulum membrane" that influenced multiple traits simultaneously. The DAVID functional classification analysis uncovered eleven clusters related to transmembrane or structural proteins. A gene network was constructed where the number of raw associated uncorrelated SNPs for each gene across all traits was used as a weight. A multiple SNP association analysis was performed for the top five most connected genes in the gene-trait network. The gene network identified the EVC2, ANXA10 and PKHD1 genes as potentially harboring multiple QTLs. Polymorphisms identified in structural proteins can modulate two different processes with direct effect on meat quality: in vivo myocyte cytoskeletal organization and postmortem proteolysis. CONCLUSION: The main result from the present analysis is the uncovering of several candidate genes associated with meat quality that have structural function in the skeletal muscle.

Genomic Loci Influencing Cue-Reactivity in Heterogeneous Stock Rats.

Addiction vulnerability is associated with the tendency to attribute incentive salience to reward predictive cues; both addiction and the attribution of incentive salience are influenced by environmental and genetic factors. To characterize the genetic contributions to incentive salience attribution, we performed a genome-wide association study (GWAS) in a cohort of 1,645 genetically diverse heterogeneous stock (HS) rats. We tested HS rats in a Pavlovian conditioned approach task, in which we characterized the individual responses to food-associated stimuli ("cues"). Rats exhibited either cue-directed "sign-tracking" behavior or food-cup directed "goal-tracking" behavior. We then used the conditioned reinforcement procedure to determine whether rats would perform a novel operant response for unrewarded presentations of the cue. We found that these measures were moderately heritable (SNP heritability, h2 = .189-.215). GWAS identified 14 quantitative trait loci (QTLs) for 11 of the 12 traits we examined. Interval sizes of these QTLs varied widely. 7 traits shared a QTL on chromosome 1 that contained a few genes (e.g. Tenm4, Mir708) that have been associated with substance use disorders and other mental health traits in humans. Other candidate genes (e.g. Wnt11, Pak1) in this region had coding variants and expression-QTLs in mesocorticolimbic regions of the brain. We also conducted a Phenome-Wide Association Study (PheWAS) on other behavioral measures in HS rats and found that regions containing QTLs on chromosome 1 were also associated with nicotine self-administration in a separate cohort of HS rats. These results provide a starting point for the molecular genetic dissection of incentive salience and provide further support for a relationship between attribution of incentive salience and drug abuse-related traits.

Genomic Approaches Reveal Pleiotropic Effects in Crossbred Beef Cattle.

Carcass and meat quality are two important attributes for the beef industry because they drive profitability and consumer demand. These traits are of even greater importance in crossbred cattle used in subtropical and tropical regions for their superior adaptability because they tend to underperform compared to their purebred counterparts. Many of these traits are challenging and expensive to measure and unavailable until late in life or after the animal is harvested, hence unrealistic to improve through traditional phenotypic selection, but perfect candidates for genomic selection. Before genomic selection can be implemented in crossbred populations, it is important to explore if pleiotropic effects exist between carcass and meat quality traits. Therefore, the objective of this study was to identify genomic regions with pleiotropic effects on carcass and meat quality traits in a multibreed Angus-Brahman population that included purebred and crossbred animals. Data included phenotypes for 10 carcass and meat quality traits from 2,384 steers, of which 1,038 were genotyped with the GGP Bovine F-250. Single-trait genome-wide association studies were first used to investigate the relevance of direct additive genetic effects on each carcass, sensory and visual meat quality traits. A second analysis for each trait included all other phenotypes as covariates to correct for direct causal effects from identified genomic regions with pure direct effects on the trait under analysis. Five genomic windows on chromosomes BTA5, BTA7, BTA18, and BTA29 explained more than 1% of additive genetic variance of two or more traits. Moreover, three suggestive pleiotropic regions were identified on BTA10 and BTA19. The 317 genes uncovered in pleiotropic regions included anchoring and cytoskeletal proteins, key players in cell growth, muscle development, lipid metabolism and fat deposition, and important factors in muscle proteolysis. A functional analysis of these genes revealed GO terms directly related to carcass quality, meat quality, and tenderness in beef cattle, including calcium-related processes, cell signaling, and modulation of cell-cell adhesion. These results contribute with novel information about the complex genetic architecture and pleiotropic effects of carcass and meat quality traits in crossbred beef cattle.

RNA-seq analysis identifies cytoskeletal structural genes and pathways for meat quality in beef.

RNA sequencing (RNA-seq) has allowed for transcriptional profiling of biological systems through the identification of differentially expressed (DE) genes and pathways. A total of 80 steers with extreme phenotypes were selected from the University of Florida multibreed Angus-Brahman herd. The average slaughter age was 12.91±8.69 months. Tenderness, juiciness and connective tissue assessed by sensory panel, along with marbling, Warner-Bratzler Shear Force (WBSF) and cooking loss, were measured in longissimus dorsi muscle. Total RNA was extracted from muscle and one RNA-seq library per sample was constructed, multiplexed, and sequenced based on protocols by Illumina HiSeq-3000 platform to generate 2×101 bp paired-end reads. The overall read mapping rate using the Btau_4.6.1 reference genome was 63%. A total of 8,799 genes were analyzed using two different methodologies, an expression association and a DE analysis. A gene and exon expression association analysis was carried out using a meat quality index on all 80 samples as a continuous response variable. The expression of 208 genes and 3,280 exons from 1,565 genes was associated with the meat quality index (p-value ≤ 0.05). A gene and isoform DE evaluation was performed analyzing two groups with extreme WBSF, tenderness and marbling. A total of 676 (adjusted p-value≤0.05), 70 (adjusted p-value≤0.1) and 198 (adjusted p-value≤0.1) genes were DE for WBSF, tenderness and marbling, respectively. A total of 106 isoforms from 98 genes for WBSF, 13 isoforms from 13 genes for tenderness and 43 isoforms from 42 genes for marbling (FDR≤0.1) were DE. Cytoskeletal and transmembrane anchoring genes and pathways were identified in the expression association, DE and the gene enrichment analyses; these proteins can have a direct effect on meat quality. Cytoskeletal proteins and transmembrane anchoring molecules can influence meat quality by allowing cytoskeletal interaction with myocyte and organelle membranes, contributing to cytoskeletal structure and architecture maintenance postmortem.

Whole Genome Sequence Data Provides Novel Insights Into the Genetic Architecture of Meat Quality Traits in Beef.

Tenderness is a major quality attribute for fresh beef steaks in the United States, and meat quality traits in general are suitable candidates for genomic research. The objectives of the present analysis were to (1) perform genome-wide association (GWA) analysis for marbling, Warner-Bratzler shear force (WBSF), tenderness, and connective tissue using whole-genome data in an Angus population, (2) identify enriched pathways in each GWA analysis; (3) construct a protein-protein interaction network using the associated genes and (4) perform a µ-calpain proteolysis assessment for associated structural proteins. An Angus-sired population of 2,285 individuals was assessed. Animals were transported to a commercial packing plant and harvested at an average age of 457 ± 46 days. After 48 h postmortem, marbling was recorded by graders' visual appraisal. Two 2.54-cm steaks were sampled from each muscle for recording of WBSF, and tenderness, and connective tissue by a sensory panel. The relevance of additive effects on marbling, WBSF, tenderness, and connective tissue was evaluated on a genome-wide scale using a two-step mixed model-based approach in single-trait analysis. A tissue-restricted gene enrichment was performed for each GWA where all polymorphisms with an association p-value lower than 1 × 10-3 were included. The genes identified as associated were included in a protein-protein interaction network and a candidate structural protein assessment of proteolysis analyses. A total of 1,867, 3,181, 3,926, and 3,678 polymorphisms were significantly associated with marbling, WBSF, tenderness, and connective tissue, respectively. The associate region on BTA29 (36,432,655-44,313,046 bp) harbors 13 highly significant markers for meat quality traits. Enrichment for the GO term GO:0005634 (Nucleus), which includes transcription factors, was evident. The final protein-protein network included 431 interations between 349 genes. The 42 most important genes based on significance that encode structural proteins were included in a proteolysis analysis, and 81% of these proteins were potential µ-Calpain substrates. Overall, this comprehensive study unraveled genetic variants, genes and mechanisms of action responsible for the variation in meat quality traits. Our findings can provide opportunities for improving meat quality in beef cattle via marker-assisted selection.

Structural Equation Modeling and Whole-Genome Scans Uncover Chromosome Regions and Enriched Pathways for Carcass and Meat Quality in Beef.

Structural equation models involving latent variables are useful tools for formulating hypothesized models defined by theoretical variables and causal links between these variables. The objectives of this study were: (1) to identify latent variables underlying carcass and meat quality traits and (2) to perform whole-genome scans for these latent variables in order to identify genomic regions and individual genes with both direct and indirect effects. A total of 726 steers from an Angus-Brahman multibreed population with records for 22 phenotypes were used. A total of 480 animals were genotyped with the GGP Bovine F-250. The single-step genomic best linear unbiased prediction method was used to estimate the amount of genetic variance explained for each latent variable by chromosome regions of 20 adjacent SNP-windows across the genome. Three types of genetic effects were considered: (1) direct effects on a single latent phenotype; (2) direct effects on two latent phenotypes simultaneously; and (3) indirect effects. The final structural model included carcass quality as an independent latent variable and meat quality as a dependent latent variable. Carcass quality was defined by quality grade, fat over the ribeye and marbling, while the meat quality was described by juiciness, tenderness and connective tissue, all of them measured through a taste panel. From 571 associated genomic regions (643 genes), each one explaining at least 0.05% of the additive variance, 159 regions (179 genes) were associated with carcass quality, 106 regions (114 genes) were associated with both carcass and meat quality, 242 regions (266 genes) were associated with meat quality, and 64 regions (84 genes) were associated with carcass quality, having an indirect effect on meat quality. Three biological mechanisms emerged from these findings: postmortem proteolysis of structural proteins and cellular compartmentalization, cellular proliferation and differentiation of adipocytes, and fat deposition.

Association of µ-Calpain and Calpastatin Polymorphisms with Meat Tenderness in a Brahman-Angus Population.

Autogenous proteolytic enzymes of the calpain family are implicated in myofibrillar protein degradation. As a result, the µ-calpain gene and its specific inhibitor, calpastatin, have been repeatedly investigated for their association with meat quality traits in cattle; however, no functional mutation has been identified for these two genes. The objectives of this study were: (1) to assess breed composition effect on tenderness; (2) to perform a linkage disequilibrium (LD) analysis in µ-calpain and calpastatin genes as well as an association analyses with tenderness; and (3) to analyze putative functional SNPs inside the significant LD block for an effect on tenderness. Tenderness measurements and genotypes for 16 SNPs in µ-calpain gene and 28 SNPs in calpastatin gene from 673 steers were analyzed. A bioinformatic analysis identified "putative functional SNPs" inside the associated LD block - polymorphisms able to produce a physical and/or chemical change in the DNA, mRNA, or translated protein in silico. Breed composition had a significant (P < 0.0001) effect on tenderness where animals with more than 80% Angus composition had the most tender meat. One 11-kb LD-block and three LD-blocks of 37, 17, and 14 kb in length were identified in the µ-calpain and calpastatin genes, respectively. Out of these, the LD-block 3 in calpastatin, tagged by SNPs located at 7-98566391 and 7-98581038, had a significant effect on tenderness with the TG-CG diplotype being approximately 1 kg more tender than the toughest diplotype, TG-CG. A total of 768 SNPs in the LD-block 3 of calpastatin were included in the bioinformatic analysis, and 28 markers were selected as putative functional SNPs inside the LD-block 3 of calpastatin; however, none of them were polymorphic in this population. Out of 15 initial polymorphisms segregating inside the LD-block 3 of calpastatin in this population, markers ARSUSMARC116, Cast5, rs730723459, and rs210861835 were found to be significantly associated with tenderness.

Fatty acid profile, mineral content, and palatability of beef from a multibreed Angus-Brahman population.

Consumers demand meat that is both healthy and palatable, 2 attributes of meat that are affected by lipid content. Many cattle in the southern United States are Bos indicus influenced, as this improves the ability to survive and thrive in these subtropical regions. However, these animals tend to have leaner carcasses and less marbled meat products. Thus, the objective of this study was to examine the effect of percent Brahman genetics on carcass characteristics, palatability, fatty acids profile, and minerals content in LM of steers from a multibreed population ranging from 100% Angus to 100% Brahman. Breed effect was significant for birth weight (P = 0.0003), weaning weight (P < 0.0001), HCW (P < 0.0001), dressing percentage (P = 0.0008), ribeye area (P = 0.002), quality grade (P < 0.0001), and marbling score (P < 0.0001), and all these traits except dressing percentage decreased as the percentage of Brahman increased. Among palatability traits, breed group had a significant effect only on tenderness (TEND) and connective tissue (CT) scores (P < 0.0001). Least squares means decreased from Angus (5.75 ± 0.13 TEND score and 6.29 ± 0.14 CT score, respectively) to Brahman (4.84 ± 0.10 TEND score and 5.49 ± 0.11 CT score, respectively) as indicated by a significant linear effect. Breed group significantly affected the percentage of several individual fatty acids, saturated fatty acids (SFA), and polyunsaturated fatty acids (PUFA), but not monounsaturated fatty acids (MUFA). The 100% Angus group had the highest percentage of SFA at 49.92%, which was significantly higher (P < 0.05) than the SFA percentage in the 50%, 75%, and 100% Brahman breed groups. Brangus animals also had an increased SFA percentage compared with the 100% Brahman animals (P < 0.05). No significant effect was identified for the concentration of PUFA across the 6 breed groups (P = 0.14). Least squares means decreased from 100% Angus to 100% Brahman for concentration of total fat, SFA, and MUFA (g/mg meat). The concentration of magnesium (P < 0.0001), phosphorus (P = 0.06), and potassium (P = 0.06) increased as the percentage of Brahman increased. Our study shows that breed has a significant effect on the fatty acid profile of beef. Cattle with high Brahman percentage, which are characterized by lower marbled meat, will present a more favorable healthfulness profile with reduced content of SFA and MUFA but the same content of PUFA as purebred Angus animals.