Associations between the 11-bp deletion in the myostatin gene and carcass quality in Angus-sired cattle

An 11-bp deletion in the bovine myostatin ( MSTN ) gene was identified as the causative mutation for the double-muscling phenotype in Belgian Blue and Asturiana cattle. More recently, this mutation was also identified in the South Devon breed of cattle, in which it has been found to be associated with a general increase in muscle mass. The present study found that the mutant allele was also segregating in a commercial population of Scottish Aberdeen Angus beef cattle. The mutation was found at a low frequency (0.04) with no animals homozygous for the mutation in the sample population (536 animals). The effects of this mutation on various carcass traits of economic interest were then tested. We found that the mutation significantly increased carcass weight, sirloin weight, hindquarter weight, muscle conformation score and eye muscle area, but had no effect on the fat traits.     

Myostatin and its implications on animal breeding: a review

Myostatin, or growth and differentiation factor 8 (GDF8), has been identified as the factor causing a phenotype known as double muscling, in which a series of mutations render the gene inactive, and therefore, unable to regulate muscle fibre deposition. This phenotype occurs at a high frequency in some breeds of cattle such as Belgian Blue and Peidmontese. Phylogenetic analysis has shown that there has been positive selection pressure for non-synonymous mutations within the myostatin gene family, around the time of the divergence of cattle, sheep and goats, and these positive selective pressures on non-ancestral myostatin are relatively recent. To date, there have been reports of nine mutations in coding regions of myostatin that cause non-synonymous changes, of which three cause missense mutations, including two in exon 1 and one in exon 2. The remaining six mutations, located in exons 2 and 3, result in premature stop codons, which are the mutations responsible for the double-muscling phenotype. Unfortunately, breed management problems exist for double-muscled cattle, such as birthing difficulties, which can be overcome through genetically controlled breeding programmes, as shown in this review.     

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.     

Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle

We have determined the entire myostatin coding sequence for 32 double-muscled cattle sampled from ten European cattle breeds. Seven DNA sequence polymorphisms were identified, of which five would be predicted to disrupt the function of the protein, one is a conservative amino acid substitution, and one a silent DNA sequence variant. Four additional DNA sequence polymorphisms were identified in myostatin intronic sequences. In all but two breeds, all double-muscled animals were either homozygous or compound heterozygotes for one of the five loss-of-function mutations. The absence of obvious loss-of-function mutations in the coding sequence of the two remaining breeds points either towards additional mutations in unexplored segments of the gene, or towards locus heterogeneity of double-muscling. Received: 20 September 1997 / Accepted: 11 October 1997     

Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity

Myostatin (MSTN), a transforming growth factor beta superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5' and 3' UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks.     

Muscle-related traits in cattle: The role of the myostatin gene in the South Devon breed

In this paper, we examined the effects of an 11-bp mutation within the GDF-8 gene, originally identified in Belgian Blue cattle, in the South Devon breed. The mutation was found at moderate frequency (0.37) in the South Devon population. We quantified the effects of this mutation on growth, body composition and calving traits in South Devon cattle. We found that the mutation significantly increased muscle score and calving difficulty and decreased fat depth. The mutation did not significantly affect weight at 200 and 400 days or muscle depth. Its effect on muscle score and fat depth was additive while its effect on calving difficulty was recessive. The mutation accounted for a significant proportion of the phenotypic variance in muscle score and calving difficulty. There was an economic benefit of the mutation for this data set, however, calculations were sensitive to changes in the parameter values. Additional data would be required to refine these calculations.     

Assessment of selection mapping near the myostatin gene (GDF-8) in cattle

Domestic species provide a unique opportunity to examine the effects of selection on the genome. The myostatin gene ( GDF-8 ) has been under strong selection in a number of cattle breeds because of its influence on muscle conformation and association with the 'double-muscling' phenotype. This study examined genetic diversity near this gene in a set of breeds including some nearly fixed for the allele associated with double-muscling (MH), some where the allele is segregating at intermediate frequency and some where the allele is absent. A set of microsatellites and SNPs were used to examine patterns of diversity at the centromeric end of bovine chromosome 2, the region where GDF-8 is located, using various statistical methods. The putative position of a selected gene was moved across the genomic region to determine, by regression, a best position of reduced heterozygosity. Additional analyses examined extended homozygous regions and linkage disequilibrium patterns. While the SNP data was not found to be very informative for selection mapping in this dataset, analyses of the microsatellite data provided evidence of selection on GDF-8 in several breeds. These results suggested that, of the breeds examined, the allele was most recently introduced into the South Devon. Limitations to the selection-mapping approach were highlighted from the analysis of the SNP data and the situation where the MH allele was at intermediate frequency.     

Real-time PCR genotyping and frequency of the myostatin F94L mutation in beef cattle breeds

This research developed two real-time PCR assays, employing high-resolution melt and allele-specific analysis to accurately genotype the F94L mutation in cattle. This mutation (g.433C > A) in the growth differentiation factor 8 or myostatin gene has recently been shown to be functionally associated with increased muscle mass and carcass yield in cattle. The F94L mutation is not, like other myostatin mutations, associated with reduced fertility and dystocia. It is therefore a candidate for introgression into other breeds to improve retail beef yield and the development of a simple and accurate test to genotype this specific mutation is warranted. Variations in the efficiency of enzyme cleavage compromised the accuracy of genotyping by published methods, potentially resulting in an overestimation of the frequency of the mutant allele. The frequency of the F94L mutation was determined by real-time PCR in 1140 animals from 15 breeds of cattle in Australia. The mutation was present in Simmental (0.8%), Piedmontese (2%), Droughtmaster (4%) and Limousin (94.2%) but not found in Salers, Angus, Poll Hereford, Hereford, Gelbvieh, Charolais, Jersey, Brahman, Holstein, Shorthorn or Maine Anjou. The low prevalence of F94L in all beef breeds except Limousin indicates the significant potential for this mutation to improve retail yield in Australian beef cattle.     

Towards interbreed IBD fine mapping of the mh locus: Double-muscling in the Asturiana de los Valles breed involves the same locus as in the Belgian Blue cattle breed

The Spanish ``Asturiana' cattle breed is characterized by the segregation of a genetically determined muscular hypertrophy referred to as double-muscling or ``culones'. We demonstrate by linkage analysis that this muscular hypertrophy involves the mh locus previously shown to cause double-muscling in the Belgian Blue cattle breed, pointing towards locus homogeneity of this trait across both breeds. Moreover, using a twopoint and multipoint maximum likelihood approach, we show that flanking microsatellite markers are in linkage disequilibrium with the mh locus in both breeds albeit with different alleles. Finally, we discuss how allelic homogeneity across breeds might be exploited to achieve efficient genetic fine-mapping of the mh locus. Received: 13 September 1996 / Accepted: 20 January 1997     

The mh gene causing double-muscling in cattle maps to bovine Chromosome 2

While the hereditary nature of the double-muscling phenotype (a generalized muscular hypertrophy documented in several cattle breeds) is well established, its precise segregation mode has remained controversial. Both monogenic models (autosomal dominant or recessive) and oligogenic models have been proposed. Using a panel of 213 bovine microsatellite markers, and an experimental pedigree obtained by backcrossing double-muscled (Belgian Blue)xconventional (Friesian) F₁ dams to double-muscled sire, we have mapped a locus on bovine Chromosome (Chr) 2 that accounts for all the phenotypic variance in the backcross generation. This locus, referred to as mh (muscular hypertrophy), has been positioned with respect to a map composed of seven Chr 2-specific microsatellites, at 2 cM from the closest marker. This result confirms the validity in the Belgian Blue population of the monogenic model involving an autosomal mh locus, characterized by a wild-type + and a recessive mh allele, causing the double-muscling phenotype in the homozygous condition. The linkage relationship between the mh locus and the Chr 2 markers was confirmed in three informative pedigrees collected from the general Belgian Blue Cattle population, reinforcing the notion of genetic homogeneity of the double-muscling trait in this breed. This work paves the way towards marker-assisted selection for or against the double-muscling trait, and towards positional cloning of the corresponding gene.     

CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs

CRISPR/Cas9 has emerged as one of the most popular genome editing tools due to its simple design and high efficiency in multiple species. Myostatin (MSTN) negatively regulates skeletal muscle growth and mutations in myostatin cause double-muscled phenotype in various animals. Here, we generated myostatin mutation in Erhualian pigs using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. The protein level of myostatin precursor decreased dramatically in mutant cloned piglets. Unlike myostatin knockout Landrace, which often encountered health issues and died shortly after birth, Erhualian pigs harboring homozygous mutations were viable. Moreover, myostatin knockout Erhualian pigs exhibited partial double-muscled phenotype such as prominent muscular protrusion, wider back and hip compared with wild-type piglets. Genome editing in Chinese indigenous pig breeds thus holds great promise not only for improving growth performance, but also for protecting endangered genetic resources.     

Characterization of gene expression in double-muscled and normal-muscled bovine embryos

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth. Cattle with mutations that inactivate myostatin exhibit a remarkable increase in mass of skeletal muscle called double muscling that is accompanied by an equally remarkable decrease in carcass fat. Although a mouse knockout model has been created which results in mice with a 200% increase in skeletal muscle mass, molecular mechanisms whereby myostatin regulates skeletal muscle and fat mass are not fully understood. Using suppressive subtractive hybridization, genes that were differentially expressed in double-muscled vs. normal-muscled cattle embryos were identified. Genetic variation at other loci was minimized by using embryonic samples collected from related Piedmontese x Angus dams or Belgian Blue x Hereford dams bred to a single sire of the same breed composition. Embryos were collected at 31-33 days of gestation, which is 2-4 days after high-level expression of myostatin in the developing bovine embryo. The suppressive subtraction resulted in 30 clones that were potentially differentially expressed, 19 of which were confirmed by macroarray analysis. Several of these genes have biological functions that suggest that they are directly involved in myostatin's regulation of skeletal muscle development. Furthermore, several of these genes map to quantitative trait loci known to interact with variation in the myostatin gene.     

Signatures of selection? Patterns of microsatellite diversity on a chromosome containing a selected locus

This paper explores patterns of genetic diversity near a locus known to have been under selection. The myostatin gene (GDF-8) has been shown to be associated with double muscling, a phenotype selected for in a number of cattle breeds. We examined population genetic parameters for microsatellite loci at varying distances from GDF-8 in double-muscled (DM) and non-double-muscled (non-DM) cattle breeds in order to assess patterns of diversity. A theoretical analysis was also performed to predict the patterns of diversity expected under different scenarios. We found differences in the patterns of heterozygosity, allele diversity and linkage disequilibrium between DM and non-DM breeds. However, there were some exceptions to the predicted patterns. These are discussed in light of the histories of the breeds and the potential for using microsatellite diversity for mapping trait genes in livestock populations.     

Convenient genotyping of six myostatin mutations causing double-muscling in cattle using a multiplex oligonucleotide ligation assay

We herein describe a procedure that allows for simultaneous genotyping of six loss-of-function mutations in the bovine myostatin gene associated with the double-muscling phenotype. The proposed method relies on a multiplex oligonucleotide ligation assay and detection of the fluorescently labelled products using automatic sequencers.     

myostatin基因打靶的成肌细胞制备

Myostatin(MSTN), β-转化生长因子超家族的一员,是肌肉生长的负调控因子,该基因自然突变的比利时蓝牛和皮尔蒙特牛出现双肌性状。基因敲除该基因,是实现家畜产肉量的提高的有效方法。通过建立无启动子打靶载体MSTN-GFP, MSTN-neo,转染新生和胎儿绵羊骨骼肌细胞,经过表达绿色荧光蛋白报告基因和G418筛选获得骨骼肌细胞克隆,PCR,Southern blot,DNA序列检测获得阳性细胞克隆,为制备myostatin基因敲除的体细胞克隆绵羊提供核移植供体。     

Breed variation and genetic parameters for growth and body development in diverse beef cattle genotypes

Conformation scores can account for more than 20% of cattle price variation at Australian livestock sales. However, there are limited available references which define genetic factors relating objective live developmental traits to carcass composition. Weaning and post-weaning weight, height, length, girth, muscle (ratio of stifle to hip width) and fat depth of 1202 progeny from mature Hereford cows (637) mated to seven sire breeds (Jersey, Wagyu, Angus, Hereford, South Devon, Limousin and Belgian Blue) were examined for growth and development across ages. Crossbred Wagyu and Jersey were both lighter in weight and smaller in size (height, length and girth) than purebred Hereford and crossbred Angus, South Devon, Limousin and Belgian Blue. Within the five larger crossbreds, there were significant changes in relative weight from weaning to 600 days. Sire breeds differed in fat depth, with Angus being the fattest (9% on average fatter than Hereford and Wagyu), and Jersey 5% less fat than Hereford, followed by South Devon and Limousin (19% lower than Hereford) and Belgian Blue (39% lower than Hereford). Direct heritability ranged from 19 to 42% and was higher than the proportion of total phenotypic variance accounted for by maternal effects (which ranged from 0 to 17%) for most body measurement traits except for weight (38 v. 18%) and girth (36 v. 9%) traits at weaning, an indication of maternal effect on some body conformation traits at early ages. Muscularity (19 to 44%) and fat depth (26 to 43%) were moderately to highly heritable across ages. There were large differences for growth and the objective measures of body development between crossbreds with a degree of overlap among the progeny of the seven sire breeds. The variation between genetic (positive) and environmental (negative) correlations for dry versus wet season average daily gains in weight and fat, suggested the potential use of live-animal conformation traits for within breed selection of genetically superior animal in these traits across seasons.     

Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects

Since its identification in 1997, myostatin has been considered as a novel and unique negative regulator of muscle growth, as mstn-/- mice display a dramatic and widespread increase in skeletal muscle mass. Myostatin also appears to be involved in muscle homeostasis in adults as its expression is regulated during muscle atrophy. Moreover, deletion of the myostatin gene seems to affect adipose tissue mass in addition to skeletal muscle mass. Natural myostatin gene mutations occur in cattle breeds such as Belgian Blue, exhibiting an obviously increased muscle mass, but also in humans, as has recently been demonstrated. Here we review these natural mutations and their associated phenotypes as well as the physiological influence of the alterations in myostatin expression and the physiopathological consequences of changes in myostatin expression, especially with regard to satellite cells. Interestingly, studies have demonstrated some rescue effects of myostatin in muscular pathologies such as myopathies, providing a novel pharmacological strategy for treatment. Furthermore, the myostatin pathway is now better understood thanks to in vitro studies and it consists of inhibition of myoblast progression in the cell cycle, inhibition of myoblast terminal differentiation, in both cases associated to protection from apoptosis. The molecular pathway driving the myogenic myostatin influence is currently under extensive study and many molecular partners of myostatin have been identified, suggesting novel potent muscle growth enhancers for both human and agricultural applications.