- Email: [email protected]
Comparison of the myostatin (MSTN) gene in Russian Stavropol Merino sheep and New Zealand Merino sheep
Accepted Manuscript Title: Comparison of the Myostatin (MSTN) gene in Russian Stavropol Merino sheep and New Zealand Merino sheep Authors: V. Trukhachev, O. Yatsyk, E. Telegina, A. Krivoruchko, H. Zhou, J.G.H. Hickford PII: DOI: Reference:
S0921-4488(18)30008-7 https://doi.org/10.1016/j.smallrumres.2018.01.005 RUMIN 5614
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
20-7-2017 7-12-2017 7-1-2018
Please cite this article as: Trukhachev, V., Yatsyk, O., Telegina, E., Krivoruchko, A., Zhou, H., Hickford, J.G.H., Comparison of the Myostatin (MSTN) gene in Russian Stavropol Merino sheep and New Zealand Merino sheep.Small Ruminant Research https://doi.org/10.1016/j.smallrumres.2018.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Commutation
Comparison of the Myostatin (MSTN) gene in Russian Stavropol Merino sheep and New
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Zealand Merino sheep
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Faculty of Veterinary Medicine, Stavropol State Agrarian University, Stavropol, Russian
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a
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V. Trukhacheva, O. Yatsyka, E. Teleginaa, A. Krivoruchkoa, H. Zhoub, J. G. H. Hickfordb,*
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Federation. b
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Lincoln 7647, New Zealand.
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Gene-Marker Laboratory, Faculty of Agricultural and Life Sciences, Lincoln University,
*
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Address correspondence to: Jon GH Hickford, Department of Agricultural Sciences, Faculty
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of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, New Zealand
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E-mail: [email protected]
Highlights:
Stavropol Merino sheep and New Zealand Merino sheep were analysed at MSTN.
Targeted sequencing of ovine MSTN was performed using NimbleGen technology. 1
A total of 31 nucleotide variations are found, with eight of them being newly identified.
The location of nucleotide variations between the two types of Merino sheep is notably
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different.
Abstract
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The aim of this research was to study the myostatin gene (MSTN) in Russian Stavropol Merino sheep and compare them with Merino sheep from New Zealand (NZ). Thirty rams of the
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Stavropol Merino breed and ten NZ Merinos were investigated. In order to detect sequence
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variation in MSTN, enrichment and sequencing of targeted DNA fragments was undertaken
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using NimbleGen technology. Nucleotide sequence variations (n = 31) were found in MSTN,
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including 29 single nucleotide polymorphisms (SNPs), one deletion and one insertion. Of these, 21 of the SNPs, the insertion and the deletion, were found in the Stavropol Merino sheep and
c.373+396T>C,
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17 were found in the NZ Merinos. Eight variations in MSTN are described for the first time: c.374-362A>T,
c.374-16delT,
c.747+185C>A,
c.748-194C>A,
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c.782_783insT, c.940G>T and c.*310G>T, and these were unique to the Stavropol Merinos.
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The presence of SNP c.940G>T leads to the formation of a stop-codon and thus truncated protein at amino acid position 314 (GAA>TAA), while the insertion c.782_783insT changes the reading frame and would cause an amino acid sequence change from amino acid position
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263 onwards, with the formation of a stop-codon at position 265. The study confirms the presence of additional variability in both non-coding and coding regions of ovine myostatin, which might subsequently be analysed to ascertain whether it affects meat production Keywords: Myostatin gene (MSTN), variation, sheep, Stavropol Merino, New Zealand Merino. 2
1.
Introduction
Among the potential gene-markers for improved meat production, a leading candidate is the myostatin gene (MSTN). The myostatin protein (MSTN) inhibits the development of muscle tissue in higher vertebrates and mutations in MSTN have been revealed to result in an increased
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muscle mass in mice (McPherron et al., 1997), cattle (Grobet et al., 1997), dogs (Mosher et al., 2007), pigs (Stinckens et al., 2008) and sheep (Boman & Våge, 2009; Han et al., 2010; Hickford
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et al., 2010; Han et al., 2015).
Analysis of data in the NCBI GenBank database, reveals at least 62 single nucleotide variations in ovine MSTN, but only three of these are located in exons. This suggests that
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selective pressure results in conservation of the coding regions. Of the coding region changes,
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the exon 1 c.101G>A nucleotide variation leads to the substitution of a glutamic acid residue
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with glycine at codon 34 (Zhou et al., 2008), while Boman & Våge (2009) described a single
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nucleotide insertion (c.120_121insA) that produces a premature stop codon at amino acid
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position 49. Finally, a one base pair deletion has been described at position c.960 (c.960delG), and this results in a disrupted reading frame from amino acid position 320 onwards and the
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presence of a premature stop codon at position 359 (Boman et al. 2009). The MSTN stop codon is usually found at position c.1126_1128. These three single nucleotide changes have all been
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associated with variation in carcass muscle traits (Han et al. 2015; Boman & Våge 2009; Boman et al., 2009) and growth traits (Han et al., 2015).
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A well characterized single nucleotide polymorphism (SNP) or nucleotide substitution
(c.*1232G>A) located in the 3'-UTR of MSTN, has been identified as an important quantitative trait nucleotide (QTN) (Clop et al., 2006; Gan et al., 2008). This substitution has been found to affect muscle hypertrophy in Belgian Texel sheep (Clop et al., 2006), muscle depth in Charollais sheep (Hadjipavlou et al., 2008) and birth weight, loin yield and total muscle yield 3
in New Zealand (NZ) Romney sheep (Han et al., 2010). SNPs in non-coding regions are therefore of importance to gene activity, although the importance of SNPs in introns has tended to be over-looked. A splice-variant of ovine MSTN has been described and this suggests an intragenic
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regulatory mechanism that controls MSTN activity (Jeanplong et al., 2013). The splicing of a cryptic MSTN intron removes the receptor binding moiety from MSTN and results in a peptide
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with a 256 amino acid N-terminal region that is common to MSTN, and an additional 65 amino
acids at the C-terminal end. Over-expression of the splice variant resulted in an increased abundance of MyoD, MRF4 and Myogenin in a myoblast line (Jeanplong et al., 2013), and in
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vivo analysis revealed that the variant was more abundant during early post-natal muscle
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development, while normal MSTN levels remain unchanged. It follows therefore that gene-
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markers for improving meat production might not only be found in the coding region and
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3’UTR of ovine MSTN, but in other regions of the gene too.
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Traditionally in Russia, Merino sheep were used as a dual-purpose breed for meat and wool production. The Stavropol Merino sheep were created over the period 1921 to 1950 in
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Soviet breeding centres in the North Caucasus region. The base breed for the development was the Novokavkazskie fine-wool sheep. These sheep were adapted to the dry and cold climate of
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the Russian Steppe, but they are of small size with low live-weight and a low density wool coat. To increase meat production Novokavkazskie sheep were crossed with American
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Rambouillet sheep, and to improve wool quality; with Australian Merino sheep. The modern Stavropol Merino sheep is distinguished from other sheep breeds in Southern Russia by its larger size and increased meat production, the breed being one of the more prolific in the Russian Federation (Aboneev et al., 2011).
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The aim of this research was to study MSTN in Stavropol Merino sheep and compare these sheep with Merinos sourced from different studs in NZ so as to minimise the likelihood of these NZ sheep being related. 2.
Materials and methods
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2.1. Sheep investigated
All work was undertaken in the Genetics Laboratory of the Science-Diagnostic and
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Veterinary Care Centre (Stavropol State Agrarian University, Russian Federation). Thirty one-
year old Stavropol Merino rams were investigated. These were obtained from the breeding
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farm of the Stavropol Krai, or region, of the Russian Federation. They were healthy, kept in
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the same housed environment and fed with a same ration. Ten NZ Merinos were investigated.
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2.2. DNA collection
Venous blood was collected from the jugular vein into EDTA Vacutainers® (Becton
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Dickinson and Company, USA) and transported at 4 oC to the laboratory within 6 hours of collection. DNA was extracted from 0.2 mL of this blood, using a PureLink® Genomic DNA
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Mini Kit (Invitrogen, MA, USA). Extracted DNA from NZ Merinos obtained from the GeneMarker Laboratory of Lincoln University (Christchurch, NZ) was transported in aqueous
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solution to the Russian Federation for typing.
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2.3. Targeted enrichment and next generation sequencing of MSTN
In order to detect sequence variation, enrichment and sequencing of targeted DNA
fragments proximal to the MSTN coding sequence (Ensemble ENSOARG00000016285; http://www.ensembl.org/Ovis_aries/Gene/Summary?db=core;g=ENSOARG00000016285;r= 2:118144443-118149433;t=ENSOART00000017734) was undertaken using NimbleGen
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technology (NimbleGen, Roche, WI, USA). The probes for this MSTN typing were designed and made by NimbleGen (Roche). Briefly, high molecular weight double-stranded DNA, extracted using the PureLink® Genomic DNA Mini Kit (Invitrogen) from each sheep, was fragmented using the method described in the Rapid Library Preparation Method Manual (Standard Protocol GS Junior, Roche) with an enrichment step using SeqCap EZ Developer
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(Roche) and the protocol described in the SeqCap EZ Library LR User's Guide Version 2.0
(Roche). The enrichment of the target regions of DNA was carried out according to the protocol
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in the emPCR Amplification Method Manual, Lib-L (Roche). Sequencing was performed using a genomic sequencer (GS Junior genomic sequencer, Roche) and the sequences obtained
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mapped to the reference genome assembly Ovis aries oviAri3 [NCBI Genome. Ovis aries
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(sheep), 2015] using GS Reference Mapper v2.9 (Roche, USA).
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2.4. Sequence analyses
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SNPs were annotated using the Human Genome Variation Society (HGVS) nomenclature
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(http://varnomen.hgvs.org/) and putative changes in amino acid sequence were identified using
3.
Results
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UniPro UGENE 1.20 software (UniPro, Novosibirsk, Russia).
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Upon sequencing using the NimbleGen technology (Roche) the average depth of read per single nucleotide was 41-fold and the minimum reading depth was 32-fold. In total 31 nucleotide sequence variations were found in MSTN, including 29 SNPs, one deletion and one
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insertion. Of these variants, 21 of the SNPs, the insertion and the deletion were found in the Stavropol Merino sheep, and 17 of the SNPs were found in the NZ Merinos (Table 1). The frequencies of these variants in the Stravropol Merinos and the NZ Merinos are shown in Tables S1 and S2, respectively. Nine of the SNPs were found in both populations (Table 1). In the upstream regions of MSTN, there were seven SNPs identified in the NZ Merinos, but 6
only two in the Stavropol sheep. In contrast, intron 2 in the Stavropol sheep contained nine variations, while intron 2 in the NZ Merinos had only three. In the Stavropol Merinos c.374-645A>G was found predominantly (n = 28) in AG heterozygous individuals, with the rest (n = 2) being homozygous c.374-645GG. There were
found in one of the ten sheep studied, and in a heterozygous form.
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no sheep homozygous AA for this SNP. In the NZ Merinos, the G variant of the SNP was only
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In the NZ Merinos, two SNPs in the 5' flanking region, c.-1128T>C and c.-40C>A were
found in heterozygous and both homozygous forms, but these were not found in the Stavropol
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Merinos.
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Eight variations in MSTN are described for the first time: c.373+396T>C, c.374-
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362A>T, c.374-16delT, c.747+185C>A, c.748-194C>A, c.782_783insT, c.940G >T and
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c.*310G>T, and these were unique to the Stavropol Merinos.
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Most of the substitutions detected were in introns. However, a single nucleotide insertion (c.782_783insT) was detected in exon 3 and a new SNP c.940G>T was found. The
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presence of SNP c.940G>T would lead to the formation of a stop-codon in the place of glutamic acid at position 314 (GAA>TAA), and would therefore result in a truncated peptide, with that
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truncation being of the receptor binding moiety of the mature peptide. The insertion c.782_783insT changes the reading frame and thus the amino acid sequence from amino acid
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position 263 onwards and would introduce a pre-mature stop codon at codon 265 (Figure 1). 4.
Discussion
The aim of this work was to identify polymorphism in the MSTN gene in Stavropol Merinos and compare it to variation in NZ Merinos. The Stavropol breed is used for meat production in Russia, while in NZ the Merino is considered to be a wool breed, with meat being 7
a secondary product. NZ Merinos are typically slower growing, especially relative to the dualpurpose Romney-cross type sheep that are used for lamb production and export in NZ (Beef+LambNZ, 2016). The difference between the two types of Merino sheep in respect to the location of the
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SNPs (Table 1) is notable. Some of the SNPs present in the Stavropol Merinos are not found in the NZ Merinos, and vice versa. Given the relatively small number of sheep studied, this
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cannot be taken to be a breed difference, but it does suggest that either the Rambouillet Merinos or the Australian Merinos (or both populations), have introduced this novel genetic variation and larger size into the Stavropol sheep, especially given the historic observation that the
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Novokavkazskie sheep were of small size and low live-weight. However, modern NZ Merino
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genetics are also derived from Australian flocks and the Rambouillet historically influenced
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Australian Merino genetics (Hone, 1974), so the source of the differences is contentious.
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Some insight into the two types of Merinos can also be gained by analysis of the
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frequency of the two nucleotides for any given SNP. This does assume that the Stavropol and NZ Merinos are representative of their breed. For example, with SNP: c.-1213C>T, which is
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in a region upstream of the gene, there appears to be a breed difference. According to Ensembl (http://www.ensembl.org/Ovis_aries/Variation/Population?db=core;r=2:118142730-
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118143730;v=rs398560354;vdb=variation;vf=3475990), this substitution also occurs in Moroccan and Iranian sheep (EVA_Livestock, European Molecular Biology Laboratory,
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European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom). In 20 Iranian sheep, the frequency of the T variant was 18%, which is higher than for the NZ Merino sheep studied (10%) and lower than in the Stavropol Merino (27%). None of the Iranian sheep were homozygous TT, and neither were the NZ Merino sheep, but 27% of
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the Stavropol sheep were TT homozygous. In the 159 Moroccan sheep described, the frequency of the T variant was 16%, with three homozygous TT sheep. SNP c.373+323C>T has previously been described in NZ Romney sheep (Hickford et al., 2010; Ibrahim & Hickford, 2016), NZ cross-bred sheep (Han et al., 2013) and some Chinese
4%
recorded
in
Ensembl
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breeds (Gan et al., 2008). The frequency of the T variant in NZ Merino (5%) is similar to the for
Moroccan
sheep
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(http://www.ensembl.org/Ovis_aries/Variation/Population?db=core;r=2:118144638-
118145638;v=rs407388367;vdb=variation;vf = 12303786), but the T variant is found with an eight-fold higher frequency (40%) in the Stavropol Merinos.
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Substitution c.747+164A>G has been described in Dorset Down, Poll Dorset, Suffolk
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and NZ Merino sheep (Han et al., 2013), and some Chinese breeds (Gan et al., 2008). The
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frequency of the G nucleotide in Stavropol Merinos is more than four times higher than in the
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NZ Merinos. In 180 Moroccan the G nucleotide has a frequency of 34%
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(http://www.ensembl.org/Ovis_aries/Variation/Population?db=core;r=2:118146686118147686;v=rs426500486;vdb=variation;vf=11353799).
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Substitution c.373+18G>T is located in intron 1 near the donor-site splice. It has been
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described in Latvian dark-sheep (Sjakste et al., 2011), a number of NZ sheep breeds (Han et al., 2013; Ibrahim & Hickford, 2016), Chinese breeds (Gan et al., 2008), and Australian Suffolk and Texel sheep (Kijas et al., 2007). Changes near splice sites in introns may affect mRNA
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splicing, with Sjakste et al. (2011) using an in-silico analysis to predict that the substitution would influence pre-mRNA secondary structure. In NZ Romney sheep, Hickford et al. (2010) have described an association between c.373+18G>T and carcass lean meat yield. The presence of their allele A, which contains the c.373+18G sequence, was associated with decreased leg, loin and total yield of lean meat. 9
Conversely, the presence of allele B, which contains the c.373+18T sequence, was associated with increased loin yield and proportion loin yield (loin yield divided by total yield). Equally, Schuelke et al. (2004) described the presence of a g.IVS1+5 G>A mutation in a human child, near to c.373+18G>T in sheep. In a homozygous form g.IVS1+5A was associated with extraordinary muscling in the thighs and upper arms. In the context of the Stavropol and
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NZ Merinos described in this study, the T variant of c.373+18G>T was found with a 40% frequency in the Stavropol Merinos, but was not present in the NZ Merino sheep. Given, that
NZ Merino sheep are not typically bred for meat production, then this would again suggest that
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the c.373+18G sequence in Stavropol Merinos is connected to their breeding and use for meat
production. However, in a separate study of Russian sheep, SNP c.373+18G>T was not shown to be associated with most of the meat productivity indicators in the Dzhalginsky Merino
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(Trukhachev et al., 2015).
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In summary, the study confirms the conserved nature of MSTN exons, but reveals further variability in non-coding regions, including eight novel SNPs, a deletion and an insertion. The
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study suggests once again that MSTN SNP c.373+18G>T affects meat production traits in
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sheep.
Conflict of Interest Statement
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The authors have declared that no conflict of interest exists.
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Acknowledgements
This project was funded by Ministry of Agriculture of the Russian Federation
(agreement on the procedure and conditions for granting subsidies to financial security the state
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order for the provision of public services (works) by December 30, 2013 № 3119/13).
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250
292
Reference c.940G>T c.782_783insT
NPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEA -------------------------------------------------------KI*
Reference c.940G>T
FGWDWIIAPKRYKANYCSGECEFLFLQKYPHTHLVHQANPKGS ---------------------*
Reference
AGPCCTPTKMSPINMLYFNGKEQIIYGKIPGMVVDRCGCS*
335
IP T
293
375
SC R
336
A
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ED
M
A
N
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Figure 1. Predicted amino acid changes of the two newly identified SNPs in exon 3 of ovine MSTN. Amino acid sequences encoded by exon 3 are shown in one letter code, and the positions refer to the whole protein. The stop codons are indicated with *.
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Table 1. Comparison of polymorphisms found in Stavropol Merino sheep and New Zealand Merino sheep.
Gene region
Polymorphism
Identifier in the
Position in
Presence of
NCBI database
chromosome
polymorphism
c.-1499G>A
rs401553933
118142944
x
region
с.-1404A>T
rs412722044
118143039
x
с.-1401G>A
rs424217443
118143042
x
с.-1213C>T
rs398560354
118143230
x
с.-1128T>C
rs414042681
118143315
x
с.-958T>C
rs425338021
118143485
x
с.-40C>A
rs411139795
118144403
x
c.373+18G>T
rs119102825
118144833
x
c.373+241T>C
rs119102826
118145056
x
c.373+243G>A
rs427811339
118145058
x
c.373+249T>C
rs417602601
118145064
x
c.373+259G>T
rs119102828
c.373+323C>T
rs407388367
c.373+396T>C
Not in database
c.373+563G>A
U
N
118145074
x
118145138
x
A
Intron 1
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Upstream
St
IP T
NZ
x x
x
x
x x
rs408710650
118145378
x
c.373+913A>G
rs413881846
118145728
x
c.374-645A>G
rs420853334
118146004
c.374-362A>T
Not in database
118146287
x
c.374-16delT
Not in database
118146633
x
с.747+164A>G
rs426500486
118147186
с.747+185C>A
Not in database
118147207
x
с.747+309T>A
rs404916326
118147331
x
с.748-810C>T
rs423466211
118148243
x
с.748-475A>C
rs406265773
118148578
с.748-468C>T
rs417558185
118148585
x
с.748-229G>A
rs596160146
118148824
x
с.748-194C>A
Not in database
118148859
x
с.748-54C>T
rs428638621
118148999
Exon 3 coding
с.782_783insT
Not in database
118149087
x
region
c.940G>T
Not in database
118149245
x
3’-UTR
c.*310G>T
Not in database
118149743
x
A
ED
PT
CC E
Intron 2
M
118145211
16
x
x
x
x
x
x
x
x
S0921-4488(18)30008-7 https://doi.org/10.1016/j.smallrumres.2018.01.005 RUMIN 5614
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
20-7-2017 7-12-2017 7-1-2018
Please cite this article as: Trukhachev, V., Yatsyk, O., Telegina, E., Krivoruchko, A., Zhou, H., Hickford, J.G.H., Comparison of the Myostatin (MSTN) gene in Russian Stavropol Merino sheep and New Zealand Merino sheep.Small Ruminant Research https://doi.org/10.1016/j.smallrumres.2018.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Commutation
Comparison of the Myostatin (MSTN) gene in Russian Stavropol Merino sheep and New
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Zealand Merino sheep
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Faculty of Veterinary Medicine, Stavropol State Agrarian University, Stavropol, Russian
N
a
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V. Trukhacheva, O. Yatsyka, E. Teleginaa, A. Krivoruchkoa, H. Zhoub, J. G. H. Hickfordb,*
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Federation. b
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Lincoln 7647, New Zealand.
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Gene-Marker Laboratory, Faculty of Agricultural and Life Sciences, Lincoln University,
*
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Address correspondence to: Jon GH Hickford, Department of Agricultural Sciences, Faculty
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of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, New Zealand
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E-mail: [email protected]
Highlights:
Stavropol Merino sheep and New Zealand Merino sheep were analysed at MSTN.
Targeted sequencing of ovine MSTN was performed using NimbleGen technology. 1
A total of 31 nucleotide variations are found, with eight of them being newly identified.
The location of nucleotide variations between the two types of Merino sheep is notably
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different.
Abstract
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The aim of this research was to study the myostatin gene (MSTN) in Russian Stavropol Merino sheep and compare them with Merino sheep from New Zealand (NZ). Thirty rams of the
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Stavropol Merino breed and ten NZ Merinos were investigated. In order to detect sequence
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variation in MSTN, enrichment and sequencing of targeted DNA fragments was undertaken
A
using NimbleGen technology. Nucleotide sequence variations (n = 31) were found in MSTN,
M
including 29 single nucleotide polymorphisms (SNPs), one deletion and one insertion. Of these, 21 of the SNPs, the insertion and the deletion, were found in the Stavropol Merino sheep and
c.373+396T>C,
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17 were found in the NZ Merinos. Eight variations in MSTN are described for the first time: c.374-362A>T,
c.374-16delT,
c.747+185C>A,
c.748-194C>A,
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c.782_783insT, c.940G>T and c.*310G>T, and these were unique to the Stavropol Merinos.
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The presence of SNP c.940G>T leads to the formation of a stop-codon and thus truncated protein at amino acid position 314 (GAA>TAA), while the insertion c.782_783insT changes the reading frame and would cause an amino acid sequence change from amino acid position
A
263 onwards, with the formation of a stop-codon at position 265. The study confirms the presence of additional variability in both non-coding and coding regions of ovine myostatin, which might subsequently be analysed to ascertain whether it affects meat production Keywords: Myostatin gene (MSTN), variation, sheep, Stavropol Merino, New Zealand Merino. 2
1.
Introduction
Among the potential gene-markers for improved meat production, a leading candidate is the myostatin gene (MSTN). The myostatin protein (MSTN) inhibits the development of muscle tissue in higher vertebrates and mutations in MSTN have been revealed to result in an increased
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muscle mass in mice (McPherron et al., 1997), cattle (Grobet et al., 1997), dogs (Mosher et al., 2007), pigs (Stinckens et al., 2008) and sheep (Boman & Våge, 2009; Han et al., 2010; Hickford
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et al., 2010; Han et al., 2015).
Analysis of data in the NCBI GenBank database, reveals at least 62 single nucleotide variations in ovine MSTN, but only three of these are located in exons. This suggests that
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selective pressure results in conservation of the coding regions. Of the coding region changes,
N
the exon 1 c.101G>A nucleotide variation leads to the substitution of a glutamic acid residue
A
with glycine at codon 34 (Zhou et al., 2008), while Boman & Våge (2009) described a single
M
nucleotide insertion (c.120_121insA) that produces a premature stop codon at amino acid
ED
position 49. Finally, a one base pair deletion has been described at position c.960 (c.960delG), and this results in a disrupted reading frame from amino acid position 320 onwards and the
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presence of a premature stop codon at position 359 (Boman et al. 2009). The MSTN stop codon is usually found at position c.1126_1128. These three single nucleotide changes have all been
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associated with variation in carcass muscle traits (Han et al. 2015; Boman & Våge 2009; Boman et al., 2009) and growth traits (Han et al., 2015).
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A well characterized single nucleotide polymorphism (SNP) or nucleotide substitution
(c.*1232G>A) located in the 3'-UTR of MSTN, has been identified as an important quantitative trait nucleotide (QTN) (Clop et al., 2006; Gan et al., 2008). This substitution has been found to affect muscle hypertrophy in Belgian Texel sheep (Clop et al., 2006), muscle depth in Charollais sheep (Hadjipavlou et al., 2008) and birth weight, loin yield and total muscle yield 3
in New Zealand (NZ) Romney sheep (Han et al., 2010). SNPs in non-coding regions are therefore of importance to gene activity, although the importance of SNPs in introns has tended to be over-looked. A splice-variant of ovine MSTN has been described and this suggests an intragenic
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regulatory mechanism that controls MSTN activity (Jeanplong et al., 2013). The splicing of a cryptic MSTN intron removes the receptor binding moiety from MSTN and results in a peptide
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with a 256 amino acid N-terminal region that is common to MSTN, and an additional 65 amino
acids at the C-terminal end. Over-expression of the splice variant resulted in an increased abundance of MyoD, MRF4 and Myogenin in a myoblast line (Jeanplong et al., 2013), and in
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vivo analysis revealed that the variant was more abundant during early post-natal muscle
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development, while normal MSTN levels remain unchanged. It follows therefore that gene-
A
markers for improving meat production might not only be found in the coding region and
M
3’UTR of ovine MSTN, but in other regions of the gene too.
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Traditionally in Russia, Merino sheep were used as a dual-purpose breed for meat and wool production. The Stavropol Merino sheep were created over the period 1921 to 1950 in
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Soviet breeding centres in the North Caucasus region. The base breed for the development was the Novokavkazskie fine-wool sheep. These sheep were adapted to the dry and cold climate of
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the Russian Steppe, but they are of small size with low live-weight and a low density wool coat. To increase meat production Novokavkazskie sheep were crossed with American
A
Rambouillet sheep, and to improve wool quality; with Australian Merino sheep. The modern Stavropol Merino sheep is distinguished from other sheep breeds in Southern Russia by its larger size and increased meat production, the breed being one of the more prolific in the Russian Federation (Aboneev et al., 2011).
4
The aim of this research was to study MSTN in Stavropol Merino sheep and compare these sheep with Merinos sourced from different studs in NZ so as to minimise the likelihood of these NZ sheep being related. 2.
Materials and methods
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2.1. Sheep investigated
All work was undertaken in the Genetics Laboratory of the Science-Diagnostic and
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Veterinary Care Centre (Stavropol State Agrarian University, Russian Federation). Thirty one-
year old Stavropol Merino rams were investigated. These were obtained from the breeding
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farm of the Stavropol Krai, or region, of the Russian Federation. They were healthy, kept in
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the same housed environment and fed with a same ration. Ten NZ Merinos were investigated.
M
A
2.2. DNA collection
Venous blood was collected from the jugular vein into EDTA Vacutainers® (Becton
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Dickinson and Company, USA) and transported at 4 oC to the laboratory within 6 hours of collection. DNA was extracted from 0.2 mL of this blood, using a PureLink® Genomic DNA
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Mini Kit (Invitrogen, MA, USA). Extracted DNA from NZ Merinos obtained from the GeneMarker Laboratory of Lincoln University (Christchurch, NZ) was transported in aqueous
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solution to the Russian Federation for typing.
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2.3. Targeted enrichment and next generation sequencing of MSTN
In order to detect sequence variation, enrichment and sequencing of targeted DNA
fragments proximal to the MSTN coding sequence (Ensemble ENSOARG00000016285; http://www.ensembl.org/Ovis_aries/Gene/Summary?db=core;g=ENSOARG00000016285;r= 2:118144443-118149433;t=ENSOART00000017734) was undertaken using NimbleGen
5
technology (NimbleGen, Roche, WI, USA). The probes for this MSTN typing were designed and made by NimbleGen (Roche). Briefly, high molecular weight double-stranded DNA, extracted using the PureLink® Genomic DNA Mini Kit (Invitrogen) from each sheep, was fragmented using the method described in the Rapid Library Preparation Method Manual (Standard Protocol GS Junior, Roche) with an enrichment step using SeqCap EZ Developer
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(Roche) and the protocol described in the SeqCap EZ Library LR User's Guide Version 2.0
(Roche). The enrichment of the target regions of DNA was carried out according to the protocol
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in the emPCR Amplification Method Manual, Lib-L (Roche). Sequencing was performed using a genomic sequencer (GS Junior genomic sequencer, Roche) and the sequences obtained
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mapped to the reference genome assembly Ovis aries oviAri3 [NCBI Genome. Ovis aries
N
(sheep), 2015] using GS Reference Mapper v2.9 (Roche, USA).
A
2.4. Sequence analyses
M
SNPs were annotated using the Human Genome Variation Society (HGVS) nomenclature
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(http://varnomen.hgvs.org/) and putative changes in amino acid sequence were identified using
3.
Results
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UniPro UGENE 1.20 software (UniPro, Novosibirsk, Russia).
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Upon sequencing using the NimbleGen technology (Roche) the average depth of read per single nucleotide was 41-fold and the minimum reading depth was 32-fold. In total 31 nucleotide sequence variations were found in MSTN, including 29 SNPs, one deletion and one
A
insertion. Of these variants, 21 of the SNPs, the insertion and the deletion were found in the Stavropol Merino sheep, and 17 of the SNPs were found in the NZ Merinos (Table 1). The frequencies of these variants in the Stravropol Merinos and the NZ Merinos are shown in Tables S1 and S2, respectively. Nine of the SNPs were found in both populations (Table 1). In the upstream regions of MSTN, there were seven SNPs identified in the NZ Merinos, but 6
only two in the Stavropol sheep. In contrast, intron 2 in the Stavropol sheep contained nine variations, while intron 2 in the NZ Merinos had only three. In the Stavropol Merinos c.374-645A>G was found predominantly (n = 28) in AG heterozygous individuals, with the rest (n = 2) being homozygous c.374-645GG. There were
found in one of the ten sheep studied, and in a heterozygous form.
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no sheep homozygous AA for this SNP. In the NZ Merinos, the G variant of the SNP was only
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In the NZ Merinos, two SNPs in the 5' flanking region, c.-1128T>C and c.-40C>A were
found in heterozygous and both homozygous forms, but these were not found in the Stavropol
U
Merinos.
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Eight variations in MSTN are described for the first time: c.373+396T>C, c.374-
A
362A>T, c.374-16delT, c.747+185C>A, c.748-194C>A, c.782_783insT, c.940G >T and
M
c.*310G>T, and these were unique to the Stavropol Merinos.
ED
Most of the substitutions detected were in introns. However, a single nucleotide insertion (c.782_783insT) was detected in exon 3 and a new SNP c.940G>T was found. The
PT
presence of SNP c.940G>T would lead to the formation of a stop-codon in the place of glutamic acid at position 314 (GAA>TAA), and would therefore result in a truncated peptide, with that
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truncation being of the receptor binding moiety of the mature peptide. The insertion c.782_783insT changes the reading frame and thus the amino acid sequence from amino acid
A
position 263 onwards and would introduce a pre-mature stop codon at codon 265 (Figure 1). 4.
Discussion
The aim of this work was to identify polymorphism in the MSTN gene in Stavropol Merinos and compare it to variation in NZ Merinos. The Stavropol breed is used for meat production in Russia, while in NZ the Merino is considered to be a wool breed, with meat being 7
a secondary product. NZ Merinos are typically slower growing, especially relative to the dualpurpose Romney-cross type sheep that are used for lamb production and export in NZ (Beef+LambNZ, 2016). The difference between the two types of Merino sheep in respect to the location of the
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SNPs (Table 1) is notable. Some of the SNPs present in the Stavropol Merinos are not found in the NZ Merinos, and vice versa. Given the relatively small number of sheep studied, this
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cannot be taken to be a breed difference, but it does suggest that either the Rambouillet Merinos or the Australian Merinos (or both populations), have introduced this novel genetic variation and larger size into the Stavropol sheep, especially given the historic observation that the
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Novokavkazskie sheep were of small size and low live-weight. However, modern NZ Merino
N
genetics are also derived from Australian flocks and the Rambouillet historically influenced
A
Australian Merino genetics (Hone, 1974), so the source of the differences is contentious.
M
Some insight into the two types of Merinos can also be gained by analysis of the
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frequency of the two nucleotides for any given SNP. This does assume that the Stavropol and NZ Merinos are representative of their breed. For example, with SNP: c.-1213C>T, which is
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in a region upstream of the gene, there appears to be a breed difference. According to Ensembl (http://www.ensembl.org/Ovis_aries/Variation/Population?db=core;r=2:118142730-
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118143730;v=rs398560354;vdb=variation;vf=3475990), this substitution also occurs in Moroccan and Iranian sheep (EVA_Livestock, European Molecular Biology Laboratory,
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European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom). In 20 Iranian sheep, the frequency of the T variant was 18%, which is higher than for the NZ Merino sheep studied (10%) and lower than in the Stavropol Merino (27%). None of the Iranian sheep were homozygous TT, and neither were the NZ Merino sheep, but 27% of
8
the Stavropol sheep were TT homozygous. In the 159 Moroccan sheep described, the frequency of the T variant was 16%, with three homozygous TT sheep. SNP c.373+323C>T has previously been described in NZ Romney sheep (Hickford et al., 2010; Ibrahim & Hickford, 2016), NZ cross-bred sheep (Han et al., 2013) and some Chinese
4%
recorded
in
Ensembl
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breeds (Gan et al., 2008). The frequency of the T variant in NZ Merino (5%) is similar to the for
Moroccan
sheep
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(http://www.ensembl.org/Ovis_aries/Variation/Population?db=core;r=2:118144638-
118145638;v=rs407388367;vdb=variation;vf = 12303786), but the T variant is found with an eight-fold higher frequency (40%) in the Stavropol Merinos.
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Substitution c.747+164A>G has been described in Dorset Down, Poll Dorset, Suffolk
N
and NZ Merino sheep (Han et al., 2013), and some Chinese breeds (Gan et al., 2008). The
A
frequency of the G nucleotide in Stavropol Merinos is more than four times higher than in the
M
NZ Merinos. In 180 Moroccan the G nucleotide has a frequency of 34%
ED
(http://www.ensembl.org/Ovis_aries/Variation/Population?db=core;r=2:118146686118147686;v=rs426500486;vdb=variation;vf=11353799).
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Substitution c.373+18G>T is located in intron 1 near the donor-site splice. It has been
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described in Latvian dark-sheep (Sjakste et al., 2011), a number of NZ sheep breeds (Han et al., 2013; Ibrahim & Hickford, 2016), Chinese breeds (Gan et al., 2008), and Australian Suffolk and Texel sheep (Kijas et al., 2007). Changes near splice sites in introns may affect mRNA
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splicing, with Sjakste et al. (2011) using an in-silico analysis to predict that the substitution would influence pre-mRNA secondary structure. In NZ Romney sheep, Hickford et al. (2010) have described an association between c.373+18G>T and carcass lean meat yield. The presence of their allele A, which contains the c.373+18G sequence, was associated with decreased leg, loin and total yield of lean meat. 9
Conversely, the presence of allele B, which contains the c.373+18T sequence, was associated with increased loin yield and proportion loin yield (loin yield divided by total yield). Equally, Schuelke et al. (2004) described the presence of a g.IVS1+5 G>A mutation in a human child, near to c.373+18G>T in sheep. In a homozygous form g.IVS1+5A was associated with extraordinary muscling in the thighs and upper arms. In the context of the Stavropol and
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NZ Merinos described in this study, the T variant of c.373+18G>T was found with a 40% frequency in the Stavropol Merinos, but was not present in the NZ Merino sheep. Given, that
NZ Merino sheep are not typically bred for meat production, then this would again suggest that
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the c.373+18G sequence in Stavropol Merinos is connected to their breeding and use for meat
production. However, in a separate study of Russian sheep, SNP c.373+18G>T was not shown to be associated with most of the meat productivity indicators in the Dzhalginsky Merino
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(Trukhachev et al., 2015).
N
In summary, the study confirms the conserved nature of MSTN exons, but reveals further variability in non-coding regions, including eight novel SNPs, a deletion and an insertion. The
A
study suggests once again that MSTN SNP c.373+18G>T affects meat production traits in
ED
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sheep.
Conflict of Interest Statement
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The authors have declared that no conflict of interest exists.
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Acknowledgements
This project was funded by Ministry of Agriculture of the Russian Federation
(agreement on the procedure and conditions for granting subsidies to financial security the state
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order for the provision of public services (works) by December 30, 2013 № 3119/13).
10
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fatness in Norwegian White Sheep (Ovis aries). Anim. Genet. 40, 418-422. Clop A., Marcq F., Takeda H., Pirottin D., Tordoir X.B., Bibé B., Bouix J., Caiment F., Elsen
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J.M., Eychenne F., Larzul C., Laville E., Meish F., Milenkovic D., Tobin J., Charlier C.,
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Georges M., 2006. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat. Genet. 38, 813-818.
Felsenstein J., 1985. Confidence limits on phylogenies: An approach using the bootstrap.
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928-935. Grobet L., Martin L.J., Poncelet D., Pirottin D., Brouwers B., Riquet J., Schoeberlein A., Dunner S., Menissier F., Massabanda J., Fries R., Hanset R., Georges M., 1997. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat. Genet.
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17, 71-74. Hadjipavlou G., Matika O., Clop A., Bishop S.C., 2008. Two single nucleotide polymorphisms
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Han J., Zhou H., Forrest R.H., Sedcole J.R., Frampton C.M., Hickford J.G.H., 2010. Effect of
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myostatin (MSTN) g+6223G>A on production and carcass traits in New Zealand Romney
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Sheep. Asian-Australas. J. Anim. Sci. 23, 863-866.
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Han J., Forrest R.H., Hickford J.G.H., 2013. Genetic Variations in the myostatin gene (MSTN)
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in New Zealand sheep breeds. Mol. Biol. Rep. 40, 6379-6384. Han J., Forrest R.H., Sedcole J.R., Hickford J.G.H., 2015. Myostatin (MSTN) gene haplotypes
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and their association with growth and carcass traits in New Zealand Romney lambs. Small
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Hickford J.G.H., Forrest R.H., Zhou H., Fang Q., Han J., Frampton C.M., Horrell A.L., 2010. Polymorphisms in the ovine myostatin gene (MSTN) and their association with growth
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Ibrahim A.H.M., Zhou H., Hickford J.G.H., 2015. Variation in intron 1 of the ovine GDF8 gene and its association with growth and carcass characteristics of dual purpose sheep. Egyptian J. Anim. Prod. 52, 39-46. Ibrahim A.H.M., Hickford J.G.H., 2016. Correlation analysis between myostatin gene
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polymorphisms and carcass traits in New Zealand Romney sheep. Egyptian J. Genet. Cytology 44, 189-204.
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Sjakste T., Paramonova N., Grislis Z., Trapina I., Kairisa D., 2011. Analysis of the SingleNucleotide Polymorphism in the 5’UTR and Part of Intron I of the Sheep MSTN Gene. DNA Cell Biol. 30, 433-444. Stinckens A., Luyten T., Bijttebier J., van den Maagdenberg K., Dieltiens D., Janssens S., de
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Smet S., Georges M., Buys N., 2008. Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity. Anim. Genet. 39, 586-
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Tamura K., Nei M., 1993. Estimation of the number of nucleotide substitutions in the control
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region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10, 512-526.
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Trukhachev V., Belyaev V., Kvochko A., Kulichenko A., Kovalev D., Pisarenko S., Volynkina
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A., Selionova M., Aybazov M., Shumaenko S., Omarov A., Mamontova T., Golovanova
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N., Yatsyk O., Krivoruchko A., 2015. Myostatin gene (MSTN) polymorphism with a negative effect on meat productivity in Dzhalginsky Merino sheep breed. J. BioSci.
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Biotechnol. 4, 191-199.
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Zhou H., Hickford J.G.H., Fang Q., 2008. Variation in the coding region of the myostatin
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(GDF8) gene in sheep. Mol. Cell. Probes 22, 67-68.
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250
292
Reference c.940G>T c.782_783insT
NPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEA -------------------------------------------------------KI*
Reference c.940G>T
FGWDWIIAPKRYKANYCSGECEFLFLQKYPHTHLVHQANPKGS ---------------------*
Reference
AGPCCTPTKMSPINMLYFNGKEQIIYGKIPGMVVDRCGCS*
335
IP T
293
375
SC R
336
A
CC E
PT
ED
M
A
N
U
Figure 1. Predicted amino acid changes of the two newly identified SNPs in exon 3 of ovine MSTN. Amino acid sequences encoded by exon 3 are shown in one letter code, and the positions refer to the whole protein. The stop codons are indicated with *.
15
Table 1. Comparison of polymorphisms found in Stavropol Merino sheep and New Zealand Merino sheep.
Gene region
Polymorphism
Identifier in the
Position in
Presence of
NCBI database
chromosome
polymorphism
c.-1499G>A
rs401553933
118142944
x
region
с.-1404A>T
rs412722044
118143039
x
с.-1401G>A
rs424217443
118143042
x
с.-1213C>T
rs398560354
118143230
x
с.-1128T>C
rs414042681
118143315
x
с.-958T>C
rs425338021
118143485
x
с.-40C>A
rs411139795
118144403
x
c.373+18G>T
rs119102825
118144833
x
c.373+241T>C
rs119102826
118145056
x
c.373+243G>A
rs427811339
118145058
x
c.373+249T>C
rs417602601
118145064
x
c.373+259G>T
rs119102828
c.373+323C>T
rs407388367
c.373+396T>C
Not in database
c.373+563G>A
U
N
118145074
x
118145138
x
A
Intron 1
SC R
Upstream
St
IP T
NZ
x x
x
x
x x
rs408710650
118145378
x
c.373+913A>G
rs413881846
118145728
x
c.374-645A>G
rs420853334
118146004
c.374-362A>T
Not in database
118146287
x
c.374-16delT
Not in database
118146633
x
с.747+164A>G
rs426500486
118147186
с.747+185C>A
Not in database
118147207
x
с.747+309T>A
rs404916326
118147331
x
с.748-810C>T
rs423466211
118148243
x
с.748-475A>C
rs406265773
118148578
с.748-468C>T
rs417558185
118148585
x
с.748-229G>A
rs596160146
118148824
x
с.748-194C>A
Not in database
118148859
x
с.748-54C>T
rs428638621
118148999
Exon 3 coding
с.782_783insT
Not in database
118149087
x
region
c.940G>T
Not in database
118149245
x
3’-UTR
c.*310G>T
Not in database
118149743
x
A
ED
PT
CC E
Intron 2
M
118145211
16
x
x
x
x
x
x
x
x