Eduard Murani

QTL for microstructural and biophysical muscle properties and body composition in pigs

BackgroundThe proportion of muscle fibre types and their size affect muscularity as well as functional properties of the musculature and meat quality. We aimed to identify QTL for microstructural muscle properties including muscle fibre size, their numbers and fibre type proportions as well as biophysical parameters of meat quality and traits related to body composition, i.e. pH, conductivity, area of M. longissimus dorsi and lean meat content. A QTL scan was conducted in a porcine experimental population that is based on Duroc and Berlin Miniature Pig.ResultsLeast square regression interval mapping revealed five significant and 42 suggestive QTL for traits related to muscle fibre composition under the line-cross model as well as eight significant and 40 suggestive QTL under the half-sib model. For traits related to body composition and biophysical parameters of meat quality five and twelve significant plus nine and 22 suggestive QTL were found under the line-cross and half-sib model, respectively. Regions with either significant QTL for muscle fibre traits or significant QTL for meat quality and muscularity or both were detected on SSC1, 2, 3, 4, 5, 13, 14, 15, and 16. QTL for microstructural properties explained a larger proportion of variance than did QTL for meat quality and body composition.ConclusionMicrostructural properties of pig muscle and meat quality are governed by genetic variation at many loci distributed throughout the genome. QTL analysis under both, the line-cross and half-sib model, allows detecting QTL in case of fixation or segregation of the QTL alleles among the founder populations and thus provide comprehensive insight into the genetic variation of the traits under investigation. Genomic regions affecting complex traits of muscularity and meat quality as well as microstructural properties might point to QTL that in first instance affect muscle fibre traits and by this in second instance meat quality. Disentangling complex traits in their constituent phenotypes might facilitate the identification of QTL and the elucidation of the pleiotropic nature of QTL effects.

Trait correlated expression combined with expression QTL analysis reveals biological pathways and candidate genes affecting water holding capacity of muscle

BackgroundLeakage of water and ions and soluble proteins from muscle cells occurs during prolonged exercise due to ischemia causing muscle damage. Also post mortem anoxia during conversion of muscle to meat is marked by loss of water and soluble components from the muscle cell. There is considerable variation in the water holding capacity of meat affecting economy of meat production. Water holding capacity depends on numerous genetic and environmental factors relevant to structural and biochemical muscle fibre properties a well as ante and post slaughter metabolic processes.ResultsExpression microarray analysis of M. longissimus dorsi RNAs of 74 F2 animals of a resource population showed 1,279 transcripts with trait correlated expression to water holding capacity. Negatively correlated transcripts were enriched in functional categories and pathways like extracellular matrix receptor interaction and calcium signalling. Transcripts with positive correlation dominantly represented biochemical processes including oxidative phosphorylation, mitochondrial pathways, as well as transporter activity. A linkage analysis of abundance of trait correlated transcripts revealed 897 expression QTL (eQTL) with 104 eQTL coinciding with QTL regions for water holding capacity; 96 transcripts had trans acting and 8 had cis acting regulation.ConclusionThe complex relationships between biological processes taking place in live skeletal muscle and meat quality are driven on the one hand by the energy reserves and their utilisation in the muscle and on the other hand by the muscle structure itself and calcium signalling. Holistic expression profiling was integrated with QTL analysis for the trait of interest and for gene expression levels for creation of a priority list of genes out of the orchestra of genes of biological networks relevant to the liability to develop elevated drip loss.

Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity

BackgroundPostnatal muscle growth is largely depending on the number and size of muscle fibers. The number of myofibers and to a large extent their metabolic and contractile properties, which also influence their size, are determined prenatally during the process of myogenesis. Hence identification of genes and their networks governing prenatal development of skeletal muscles will provide insight into the control of muscle growth and facilitate finding the source of its variation. So far most of the genes involved in myogenesis were identified by in vitro studies using gene targeting and transgenesis. Profiling of transcriptome changes during the myogenesis in vivo promises to obtain a more complete picture. In order to address this, we performed transcriptome profiling of prenatal skeletal muscle using differential display RT-PCR as on open system with the potential to detect novel transcripts. Seven key stages of myogenesis (days 14, 21, 35, 49, 63, 77 and 91 post conception) were studied in two breeds, Pietrain and Duroc, differing markedly in muscularity and muscle structure.ResultsEighty prominent cDNA fragments were sequenced, 43 showing stage-associated and 37 showing breed-associated differences in the expression, respectively. Out of the resulting 85 unique expressed sequence tags, EST, 52 could be assigned to known genes. The most frequent functional categories represented genes encoding myofibrillar proteins (8), genes involved in cell adhesion, cell-cell signaling and extracellular matrix synthesis/remodeling (8), genes regulating gene expression (8), and metabolism genes (8). Some of the EST that showed no identity to any known transcripts in the databases are located in introns of known genes and most likely represent novel exons (e.g. HMGA2). Expression of thirteen transcripts along with five reference genes was further analyzed by means of real-time quantitative PCR. Nine of the target transcripts showed higher than twofold differences in the expression between the two breeds (GATA3, HMGA2, NRAP, SMC6L1, SPP1, RAB6IP2, TJP1 and two EST).ConclusionThe present study revealed several genes and novel transcripts not previously associated with myogenesis and expands our knowledge of genetic factors operating during myogenesis. Genes that exhibited differences between the divergent breeds represent candidate genes for muscle growth and structure.

Combined line-cross and half-sib QTL analysis in Duroc–Pietrain population

A Duroc–Pietrain resource population was built to detect quantitative trait loci (QTL) that affect growth, carcass composition, and pork quality. The data were analyzed by applying three least-squares Mendelian models: a line-cross (LC) model, a half-sib (HS) model, and a combined LC and HS model (CB), which enabled the detection of QTL that had fixed, equal, and different allele frequencies for alternate breed alleles, respectively. Permutation tests were performed to determine 5% chromosome-wide and 5% genome-wide threshold values. A total of 40 (137) QTL were detected at the 5% genome-wide (chromosome-wide) level for the 35 traits analyzed. Of the 137 QTL detected, 62 were classified as the LC type (LC-QTL), 47 as the HS type (HS-QTL), and 28 as the CB type (CB-QTL). The results indicate that implementation of a series of model-based framework is not only beneficial to detect QTL, but also provides us with a new and more robust interpretation from which further methodology could be developed.

Association of HPA axis-related genetic variation with stress reactivity and aggressive behaviour in pigs

BackgroundStress, elicited for example by aggressive interactions, has negative effects on various biological functions including immune defence, reproduction, growth, and, in livestock, on product quality. Stress response and aggressiveness are mutually interrelated and show large interindividual variation, partly attributable to genetic factors. In the pig little is known about the molecular-genetic background of the variation in stress responsiveness and aggressiveness. To identify candidate genes we analyzed association of DNA markers in each of ten genes (CRH g.233C>T, CRHR1 c.*866_867insA, CRHBP c.51G>A, POMC c.293_298del, MC2R c.306T>G, NR3C1 c.*2122A>G, AVP c.207A>G, AVPR1B c.1084A>G, UCN g.1329T>C, CRHR2 c.*13T>C) related to the hypothalamic-pituitary-adrenocortical (HPA) axis, one of the main stress-response systems, with various stress- and aggression-related parameters at slaughter. These parameters were: physiological measures of the stress response (plasma concentrations of cortisol, creatine kinase, glucose, and lactate), adrenal weight (which is a parameter reflecting activity of the central branch of the HPA axis over time) and aggressive behaviour (measured by means of lesion scoring) in the context of psychosocial stress of mixing individuals with different aggressive temperament.ResultsThe SNP NR3C1 c.*2122A>G showed association with cortisol concentration (p = 0.024), adrenal weight (p = 0.003) and aggressive behaviour (front lesion score, p = 0.012; total lesion score p = 0.045). The SNP AVPR1B c.1084A>G showed a highly significant association with aggressive behaviour (middle lesion score, p = 0.007; total lesion score p = 0.003). The SNP UCN g.1329T>C showed association with adrenal weight (p = 0.019) and aggressive behaviour (front lesion score, p = 0.029). The SNP CRH g.233C>T showed a significant association with glucose concentration (p = 0.002), and the polymorphisms POMC c.293_298del and MC2R c.306T>G with adrenal weight (p = 0.027 and p < 0.0001 respectively).ConclusionsThe multiple and consistent associations shown by SNP in NR3C1 and AVPR1B provide convincing evidence for genuine effects of their DNA sequence variation on stress responsiveness and aggressive behaviour. Identification of the causal functional molecular polymorphisms would not only provide markers useful for pig breeding but also insight into the molecular bases of the stress response and aggressive behaviour in general.

Expression Profiling of Muscle Reveals Transcripts Differentially Expressed in Muscle That Affect Water-Holding Capacity of Pork

To identify biological processes as well as molecular markers for drip loss, a parameter for water holding capacity of meat, the M. longissimus dorsi transcriptomes of six divergent sib pairs were analyzed using Affymetrix Porcine Genome Array. Functional categories of differentially regulated transcripts were determined by single-gene analysis and gene set analysis. The transcripts being up-regulated at high drip loss belong to groups of genes functionally categorized as genes of membrane proteins, signal transduction, cell communication, response to stimulus, and cytoskeleton. Among genes down-regulated with high drip loss, functional groups of oxidoreductase activity, lipid metabolism, and electron transport were identified. Differential regulation of the abundance of transcripts of these biological networks in live muscle affect mortem biochemical processes of meat maturation. Knowledge of this functional link is indicative for the identification of candidate genes for improvement of meat quality.

Maternal dietary protein restriction and excess affects offspring gene expression and methylation of non-SMC subunits of condensin I in liver and skeletal muscle

Recent evidence indicates that maternal nutrition during pregnancy influences gene expression in offspring through epigenetic alterations. In the present study we evaluated the effect of protein excess and deficiency during porcine pregnancy on offspring hepatic and skeletal muscular expression patterns of key genes of methionine metabolism (DNMT1, DNMT3a, DNMT3b, BHMT, MAT2B and AHCYL1), condensin I subunit genes (NCAPD2, NCAPG and NCAPH), important for chromosome condensation and segregation, global DNA methylation and gene-specific DNA methylation. German Landrace sows were randomly assigned to control (CO), high protein (HP) and low protein (LP) diet groups. Tissue samples of offspring were collected from fetal (dpc95), newborn (dpn1), weanling (dpn28) and finisher pigs (dpn188). Gene expression of DNMT1, DNMT3a and DNMT3b was influenced by both HP and LP diets, indicating an involvement of DNA methylation in fetal programming by maternal protein supply. Moreover, hepatic global methylation was significantly affected by protein restriction at dpc95 (p = 0.004) and by protein excess at dpn188 (p = 0.034). Gene expression in fetal liver was significantly different between CO and LP for NCAPD2 (p = 0.0005), NCAPG (p = 0.0009) and NCAPH (p < 0.0001). In skeletal muscle, LP fetuses had significantly altered gene expression of NCAPD2 (p = 0.020) and NCAPH (p = 0.001), compared with CO. Furthermore, NCAPG was differentially methylated among LP, HP and CO; indeed, a significant positive correlation was detected with transcript amount in fetal pigs (r = 0.47, p = 0.002). These data demonstrate that both restriction and excess dietary protein during pregnancy alters the offspring’s epigenetic marks and influences gene expression.

Porcine muscle sensory attributes associate with major changes in gene networks involving CAPZB, ANKRD1, and CTBP2

Principal component analysis of traits related to carcass and meat properties were combined with microarray expression data for the identification of functional networks of genes and biological processes taking place during the conversion of muscle to meat. Principal components (PCs) with high loadings of meat quality traits were derived from phenotypic data of 572 animals of a porcine crossbreed population. Microarray data of 74 M. longissimus dorsi samples were correlated with PC datasets. Lists of significantly correlated genes were analyzed for enrichment of functional annotation groups as defined in the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases as well as the Ingenuity Pathways Analysis library. Ubiquitination, phosphorylation, mitochondrion dysfunction, actin, integrin, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor, and Ca signaling pathways are correlated with meat quality. Among the significantly trait-associated genes, CAPZB, ANKRD1, and CTBP2 are promoted as candidate genes for meat quality that provide a link between the highlighted pathways. Knowledge of the relevant biological processes and the differential expression of members of the pathway will provide tools that are predictive for traits related to meat quality and that may also be diagnostic for many muscle defects or damages including muscle atrophy, dystrophy, and hypoxia.

Detection of quantitative trait loci for carcass traits in the pig by using AFLP

For evaluation of the suitability of Amplified Fragment Length Polymorphism (AFLP) for detection of quantitative trait loci in farm animals, a combination of AFLP and selective genotyping has been applied as a rapid screening method for marker–QTL associations. Focusing on loci affecting eye muscle area, six extreme discordant sib pairs were selected from a Duroc × Berlin Miniature Pig F2 experimental cross and examined by using 48 AFLP primer combinations. Two prominent AFLP markers were converted into simple codominant PCR markers (STS-Bo1 and STS-Bo3) and assigned to Sscr4 by physical and linkage mapping. Single marker analysis indicated association of the STS markers with a putative QTL influencing eye muscle area. Interval mapping confirmed the presence of a significant QTL for eye muscle area (Pgenomewide < 0.01) on the Sscr4, with STS-Bo1 being the closer marker. At the same location, significant effects (Pgenomewide < 0.01) on carcass length and backfat thickness were also detected. Our results demonstrate the capability of the combination of AFLP analysis and selective genotyping as a method for detection of genome regions containing QTL in livestock.

Molecular genetic analysis of porcine mannose-binding lectin genes, MBL1 and MBL2, and their association with complement activity.

Mannose‐binding lectin (MBL) mediates activation of the complement system via the lectin pathway. Two forms of MBL, MBL‐A and MBL‐C, were characterized in rodents, rabbits, bovine and rhesus monkeys, whereas only one form was identified in humans, chimpanzees and chickens. The two forms are encoded by two distinct genes named MBL1 and MBL2, which have been identified in many species including the pig. In this report, we studied the two porcine genes MBL1 and MBL2. The porcine MBL genes had higher identities to bovine rather than primate and rodent sequences. Both genes were assigned to chromosome 14 by radiation hybrid panel and linkage mapping. Both MBL genes were highly expressed in liver. MBL1 was also found to be expressed in the lung, testis and brain, whereas low expression of MBL2 was detected in the testis and kidney. New single nucleotide polymorphisms of porcine MBL2 gene were found and genotyped in an experimental F2 pig population, together with a previously reported SNP of MBL1. MBL1 genotypes differed in C3c serum concentration, i.e. in vivo complement activity, at P < 0.1. Correspondingly, linkage analysis revealed a quantitative trait locus for C3c serum level close to the position of the MBL genes. The study thus promotes the porcine MBL genes as functional and positional candidate gene for complement activity.