Genetic variation in the first intron and exon of the myostatin gene in several Indonesian cattle populations

Background and Aim: Myostatin (MSTN), a member of the transforming growth factor-β family, is a negative regulator of muscle mass. This study aimed to detect the genetic variation of the 1160 bp fragment of exon 1 and part of intron 1 of the MSTN gene in several cattle populations raised in Indonesia. Materials and Methods: Polymerase chain reaction products of the MSTN gene amplified from 92 animals representing 10 cattle populations (Peranakan Ongole [PO], Belgian Blue x PO cross, Rambon, PO x Bali cross, Jabres, Galekan, Sragen, Donggala, Madura, and Bali) were sequenced, compared, and aligned with bovine MSTN of Bos taurus (GenBank Acc. No. AF320998.1) and Bos indicus (GenBank Acc. No. AY794986.1). Results: Four nucleotide substitutions (nt 1045 and 1066 in intron 1; nt 262 and 418 in exon 1) and two indels (nt 807 and 869 in intron 1) were synonymous mutations. Among these substitutions, only the nt 262G>C and nt 418A>G loci were polymorphic in all populations, except Bali cattle. The frequencies of the nt 262C (0.82) and nt 418A (0.65) alleles were highest. For the nt 262G>C locus, the CC genotype had the highest frequency (0.66) followed by GC (0.30) and CC (0.03). For the nt 418A>G locus, the AG genotype had the highest frequency (0.52) followed by AA (0.39) and GG (0.09). Conclusion: The results, showing genetic variations in exon 1 and intron 1 of the MSTN gene, might be helpful for future association studies.

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207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab207 ◽

2017 ◽

Vol 29 (1) ◽

pp. 212

Author(s):

C. A. Pinzon ◽

M. Snyder ◽

J. Pryor ◽

B. Thompson ◽

M. Golding ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Muscle Mass ◽

Transforming Growth Factor ◽

Fluorescent Protein ◽

Pcr Amplification ◽

Negative Regulator ◽

Bovine Embryos ◽

Myostatin Gene ◽

Fetal Fibroblasts ◽

Green Fluorescent

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL−1) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL−1 of total sgRNA, 10 ng µL−1 of Cas9 mRNA, and 30 ng µL−1 of Cas9 protein with 1 mg mL−1 of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.

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Molecular Characterization and Phylogeny Based Analysis of Intron I Sequence of Myostatin (MSTN) Gene in Iranian Makuei Sheep Breed

Annals of Animal Science ◽

10.1515/aoas-2016-0013 ◽

2016 ◽

Vol 16 (4) ◽

pp. 1007-1018

Author(s):

Mohammad Farhadian ◽

Ali Hashemi

Keyword(s):

Muscle Development ◽

Negative Regulator ◽

Nucleotide Sequencing ◽

Meat Production ◽

Potential Candidate ◽

Nucleotide Polymorphisms ◽

Single Strand Conformational Polymorphism ◽

Mstn Gene ◽

Potential Candidate Gene ◽

Intron 1

Abstract Myostatin (MSTN), a negative regulator of skeletal muscle development, acts as a potential candidate gene used to increase muscle mass. Likewise, sheep MSTN gene has an important role in meat production. MSTN is made up of 376 amino acids, and is synthesized as a precursor protein. To investigate the MSTN in Iranian native Makuei sheep, a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used. Genomic DNA was isolated from the blood samples. A 417-bp of MSTN intron I segment was amplified using locus-specific primers. Four SSCP patterns were identified and nucleotide sequencing of the Makuei sheep MSTN gene was done and registered in the NCBI GenBank with “KJ526625” number. Three novel single nucleotide polymorphisms (SNPs) were found in the samples. These SNPs are found in 224bp, 226bp and 242bp locations. Accordingly three substitutions (c.224C>T; c.226A>G; c.242G>T) were observed in the intron 1 region of MSTN gene. The effects of the observed SNPs on breeding values of some biometric traits were investigated and the substitution of c.226A>G was found to be associated with heart girth (HG) and leg circumference (LC). Phylogenetic analysis, based on the nucleotide sequences indicated similar evaluation with the GenBank reference sequences. It seems that the observed polymorphisms of the ovine MSTN gene are associated with HG and LC traits.

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Short Communication: Identification of the exon 1 myostatin gene polymorphism and its association with slaughtered weight in Indonesian Kampung and Broiler Chicken

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d210860 ◽

2020 ◽

Vol 21 (8) ◽

Author(s):

Linda Suhartati ◽

ISYANA KHAERUNNISA ◽

ASEP GUNAWAN ◽

RUKMIASIH RUKMIASIH ◽

SRI DARWATI ◽

...

Keyword(s):

Gene Polymorphism ◽

Broiler Chicken ◽

Muscle Development ◽

Short Communication ◽

Negative Regulator ◽

Silent Mutation ◽

Muscle Weight ◽

Myostatin Gene ◽

Exon 1 ◽

Meat Type

Abstract. Suhartati L, Khaerunnisa L, Gunawan A, Rukmiasih, Darwati S, Sumantri C, Rizqan. 2020. Short Communication: Identification of the exon 1 myostatin gene polymorphism and its association with slaughtered weight in Indonesian Kampung and Broiler Chicken. Biodiversitas 21: 3893-3897. Myostatin is widely known as a negative regulator of skeletal muscle development by controlling hypertrophy and hyperplasia. Silent mutation in exon 1 (c.234G>A) was then thought to be associated with chicken growth performances. This study was designed to analyze polymorphisms of c.234G>A mutation of the myostatin gene and its effect on slaughtered weight in Indonesian Kampung and meat-type chicken. The SatI restriction enzyme was used for genotype determination through the PCR-RFLP technique. The effect of genotype on the slaughtered weight was analyzed using the General Linear Model (GLM) procedure. Genotyping was performed on 138 chicken from Kampung (n=67) and meat type (cobb Broiler) (n=71) chicken. The amplification product contained 226 bp nucleotides. This myostatin gene was polymorphic in both chicken population, produced 2 alleles (G and A), and 3 genotypes (GG, GA, and AA). The A allele had a higher frequency than the G allele in all populations. In this study, genotype of AA and GA had higher live weight, carcass weight, breast weight, thigh weight, drumsticks weight, wings weight, breast muscle weight, thighs muscle weight, drumsticks muscle weight than GG genotype of 12 weeks-old Kampung chicken. The results suggested that the c.234G>A mutation in exon 1 could be potentially recommended as a genetic marker for chicken slaughtered weight traits.

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Myostatin: a modulator of skeletal-muscle stem cells

Biochemical Society Transactions ◽

10.1042/bst0331513 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1513-1517 ◽

Cited By ~ 26

Author(s):

F.S. Walsh ◽

A.J. Celeste

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Muscle Development ◽

Transforming Growth Factor ◽

Neutralizing Antibody ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Mdx Mouse ◽

Specific Expression ◽

Myostatin Gene

Myostatin, or GDF-8 (growth and differentiation factor-8), was first identified through sequence identity with members of the BMP (bone morphogenetic protein)/TGF-β (transforming growth factor-β) superfamily. The skeletal-muscle-specific expression pattern of myostatin suggested a role in muscle development. Mice with a targeted deletion of the myostatin gene exhibit a hypermuscular phenotype. In addition, inactivating mutations in the myostatin gene have been identified in ‘double muscled’ cattle breeds, such as the Belgian Blue and Piedmontese, as well as in a hypermuscular child. These findings define myostatin as a negative regulator of skeletal-muscle development. Myostatin binds with high affinity to the receptor serine threonine kinase ActRIIB (activin type IIB receptor), which initiates signalling through a smad2/3-dependent pathway. In an effort to validate myostatin as a therapeutic target in a post-embryonic setting, a neutralizing antibody was developed by screening for inhibition of myostatin binding to ActRIIB. Administration of this antimyostatin antibody to adult mice resulted in a significant increase in both muscle mass and functional strength. Importantly, similar results were obtained in a murine model of muscular dystrophy, the mdx mouse. Unlike the myostatin-deficient animals, which exhibit both muscle hypertrophy and hyperplasia, the antibody-treated mice demonstrate increased musculature through a hypertrophic mechanism. These results validate myostatin inhibition as a therapeutic approach to muscle wasting diseases such as muscular dystrophy, sarcopenic frailty of the elderly and amylotrophic lateral sclerosis.

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Sequencing the exon 1 of MSTN in Mongolian horse (Equus caballus)

Mongolian Journal of Agricultural Sciences ◽

10.5564/mjas.v27i02.1278 ◽

2019 ◽

Vol 27 (02) ◽

pp. 3-7

Author(s):

Sergelen B ◽

Khaliunaa T ◽

Myagmarsuren P

Keyword(s):

Muscle Development ◽

Equus Caballus ◽

Reference Sequence ◽

Amino Acid Difference ◽

Muscular Tissue ◽

Long Distance ◽

Myostatin Gene ◽

Mstn Gene ◽

Exon 1 ◽

Mongolian Horse

Myostatin (MSTN) is a protein responsible for muscular tissue differentiation, development, and growth in mammals. Studies on cattle, mice, pig, and dog have provided sample demonstrations of mutations on myostatin, which strongly affected animal Ankole93200phenotype, particularly muscle development. In horse, myostatin gene is usually studied with respect to racing performance. Most Mongolian native horses do race for long distance and such racing is a very popular sport in Mongolia. On the other hand, the MSTN gene also affects meat yield and quality. We partially sequenced of MSTN gene to explore its polymorphic features in Mongolian horse. A 238 bp long segment of exon 1 was sequenced, using 3130 Genetic Analyser and MEGAX. The sequence was registered with accession number LC216412 in the Gene bank database of NCBI and aligned to four reference sequences (GQ183900.1, KC708233.1, AY840554.2, NM 001081817) from this database. The nucleotide diversity was 0.0084, Tajima test statistic was -1.12. so that there were 233 invariable (monomorphic) sites and 5 variable (polymorphic) sites, in the group of animals compared. The Mongolian horse differed from the reference sequence at 3 sites. There was an amino acid difference in the partial sequence, in which isoleucine in the reference sequence was converted into valine. The results of the study indicate that the Mongolian horse seems to differ substantially from the reference genome of equus caballus. Sequencing of the full length of exon 1 or even the whole MSTN gene, including introns, is strongly advised, to get more insights with regard to the identification and characterization of variants at the myostatin locus in Mongolian horses.

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Myostatin Gene as a Genetic Marker for Growth and Carcass Traits in Beef Cattle

Indonesian Bulletin of Animal and Veterinary Sciences ◽

10.14334/wartazoa.v31i1.2530 ◽

2021 ◽

Vol 31 (1) ◽

pp. 37

Author(s):

Peni Wahyu Prihandini ◽

D N H Hariyono ◽

Y A Tribudi

Keyword(s):

Beef Cattle ◽

Muscle Mass ◽

Gene Polymorphisms ◽

Carcass Traits ◽

Phenotypic Expression ◽

Negative Regulator ◽

Myostatin Gene ◽

Mstn Gene ◽

Growth Performances ◽

Superior Genotype

<p>Growth and carcass traits are of economic importances in livestock breeding, because they affect the profitability of animal production. The phenotypic expression of these traits is controlled by multiple genes (polygenes), such as myostatin (MSTN) gene. This paper aims to discuss the expression, polymorphism and potential application of MSTN gene as a marker-assisted selection (MAS) for growth and carcass traits in beef cattle based on data from published studies. MSTN gene or known as growth and differentiation factor 8 (GDF8) is a member of the transforming growth factor-β (TGF-β) superfamily, which acts as a negative regulator of skeletal muscle mass deposition. Several published studies showed that mutations in the MSTN gene can inhibit the activation of myostatin, which leads to an increased muscle mass (hypertrophy). Several <em>MSTN</em> gene polymorphisms were reported to be associated with growth and carcass traits in local cattle in several countries, including Indonesia, namely Bali cattle. Based on several assumptions: 1) there is MTSN gene polymorphisms in a population, 2) there is a significant association between MSTN gene polymorphisms and growth and carcass traits, as reported in several beef cattle populations and 3) those cattle with superior genotype have better growth performances, we expect that there will be improvement in growth performances in the future if those cattle are selected. Understanding MSTN gene polymorphisms would be useful to make strategies for the genetic improvement for growth and carcass traits of local cattle.</p>

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Variants in the Myostatin Gene and Physical Performance Phenotype of Elite Athletes

Genes ◽

10.3390/genes12050757 ◽

2021 ◽

Vol 12 (5) ◽

pp. 757

Author(s):

Valentina Ginevičienė ◽

Audronė Jakaitienė ◽

Erinija Pranckevičienė ◽

Kazys Milašius ◽

Algirdas Utkus

Keyword(s):

Physical Performance ◽

Elite Athletes ◽

Performance Status ◽

Muscle Growth ◽

Endurance Performance ◽

Negative Regulator ◽

Myostatin Gene ◽

Mstn Gene ◽

Homozygous Genotype ◽

The Status

The MSTN gene is a negative regulator of muscle growth that is attracting attention as a candidate gene for physical performance traits. We hypothesised that variants of MSTN might be associated with the status of elite athlete. We therefore sought to study the potential role of MSTN in the physical performance of athletes by analysing the whole coding sequence of the MSTN gene in a cohort of Lithuanian elite athletes (n = 103) and non-athletes (n = 127). Consequently, two genetic variants were identified: the deletion of one of three adenines in the first intron (c.373+90delA, rs11333758) and a non-synonymous variant in the second exon (c.458A>G, p.Lys(K)153Arg(R), rs1805086). Among all samples, the MSTN rs1805086 Lys(K) allele was the most common form in both groups. Homozygous genotype for the less common Arg(R) allele was identified in only one elite canoe rower, and we could find no direct association between rs1805086 and successful results in elite athletes. Surprisingly, the intronic variant (rs11333758) was abundant among all samples. The main finding was that endurance-oriented athletes had 2.1 greater odds of being MSTN deletion genotype than non-athletes (13.6% vs. 0.8%). The present study confirms the association of the polymorphism rs11333758 with endurance performance status in Lithuanian elite athletes.

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Effects of Intronic SNPs in the Myostatin Gene on Growth and Carcass Traits in Colored Polish Merino Sheep

Genes ◽

10.3390/genes11010002 ◽

2019 ◽

Vol 11 (1) ◽

pp. 2 ◽

Cited By ~ 1

Author(s):

Ewa Grochowska ◽

Bronisław Borys ◽

Sławomir Mroczkowski

Keyword(s):

Carcass Traits ◽

Muscle Growth ◽

Single Strand Conformation Polymorphism ◽

Negative Regulator ◽

Meat Production ◽

Nucleotide Polymorphisms ◽

Myostatin Gene ◽

Merino Sheep ◽

Mstn Gene ◽

First Intron

Myostatin acts as a negative regulator of muscle growth; therefore, its role is important with regard to animal growth and meat production. This study was undertaken with the objective to detect polymorphisms in the first intron and c.*1232 position of the MSTN gene and to analyze effects of the detected alleles/genotypes on growth and carcass traits in Colored Polish Merino sheep. In total, 23 traits were analyzed, i.e., seven describing lamb growth and 16 carcass traits. Single nucleotide polymorphisms (SNPs) in the first intron and the c.*1232 position were identified using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) methods, respectively. The MIXED procedure of the SAS software package was used to analyze allelic and genotypic effects of the MSTN gene on growth and carcass traits. Polymorphisms were only detected in the first intron of the MSTN gene. All investigated sheep were monomorphic G in the c.*1232 position. The MSTN genotype was found to have significant effect on body weight at 2nd day of life (BW2) and loin and fore shank weights. Significant allelic effects were detected with respect to BW2, scrag, leg, fore, and hind shank weights. These results suggest that polymorphisms in the first intron of the MSTN gene are relevant with respect to several carcass traits and BW2 in Colored Polish Merino sheep.

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Polymorphism of Myostatin Gene and its Association with Body Weight of Iraqi Local Ducks

Basrah Journal of Agricultural Sciences ◽

10.37077/25200860.2019.260 ◽

2019 ◽

Vol 32 ◽

pp. 97-104

Author(s):

Ali. J. Ghali ◽

Sajida A. Al-shaheen

Keyword(s):

Body Weight ◽

Dna Sequence ◽

Transforming Growth Factor ◽

Base Change ◽

The Body ◽

Average Heterozygosity ◽

Nitrogen Base ◽

Myostatin Gene ◽

Mstn Gene ◽

Expected Heterozygosity

Myostatin, or growth and differentiation factor-8(GDF-8) is a member of the transforming growth factor (TGF)-ß superfamily. The objective of this study was to investigate the association between genetic polymorphism of the myostatin gene with the body-weight of Iraqi local ducks by DNA sequence analysis. The results of DNA sequence showed nitrogen base change (G?A) at 129 base pair sites of the exon1 of MSTN gene. Therefore, two alleles, G and A and two patterns of genotypes, GG, AG were observed. The observed numbers of GG and AG genotypes were 25 and 6, respectively. The G allele was of the highest frequency (0.9) while A allele showed the lowest frequency (0.1). The GG genotype showed the highest frequency (80.65%) and AG genotype showed the lowest frequency (19.35%). Additionally, the percentages of observed and expected heterozygosity were 0.19 and 0.17 respectively. Moreover, the percentages of observed and expected homozygosity were 0.80 and 0.82, respectively. The average heterozygosity was 0.17. The results revealed no significant (P?0.05) effect of the MSTN gene on body weight.

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