Growth performance and meat quality are economically important traits with the highest impact on the production of small ruminant. Recently, the development of molecular genetics approaches help for improvement of these traits. Generally, the detection of genetic markers for quantitative traits is the principal step to establish best selection programs1. Many studies were done in this area to detect the potential genes associated with these economic traits in different livestock, like cattle, sheep and chicken2. Myostatin (MSTN) and callipyge (CLPG) are the most well known major genes which are related with growth and meat quality traits in domestic animals.
The mammalian growth transforming family has some members, one of them is myostatin which has an essential role in the development of embryo and regulation of tissue homeostasis3. Due to the impact role of myostatin gene in muscle growth, it is considered as one of the important candidate genes in meat quality and growth traits in domestic animals4,5. Sequence variations in the MSTN gene can alter its expression and produce a non-functional protein, which leads to double-muscling phenomenon in many species6. This effect of a single gene on processing yields can open a best way to improve the processing yields of animals using knockout technology7.
The muscular hypertrophy phenomena, callipyge phenotype are proclaimed in the pelvic limb muscles8,9. In lambs expressing callipyge, the muscles are enlarged by different degrees in some muscles while others are not affected. The weights of callipyge expressed muscles range from 14% in thoracic muscles to 50% in torso muscles. This muscle hypertrophy develops after 21 days from birth10. The muscle hypertrophy phenomena occur in the proportion as well as the gycolytic myofibers which lead to the increase of muscle size11,12.
Due to the positive effect of MSTN and CLPG variations on the growth performance and quality of meat in different livestock, the aim of this study was to identify the genetic polymorphism and SNPs for these two economically important genes in major Egyptian small ruminant breeds.
MATERIALS AND METHODS
Blood samples and genomic DNA extraction: The whole blood samples were collected from 171 animals belonging to six native major small ruminant breeds, in sheep animals: 47 Barki, 30 Rahmani and 30 Ossimi, while for goat animals: 24 Baladi, 17 Barki and 23 Zaraibi.
Genomic DNA was extracted from the whole blood according to the method described by Miller et al.13 with minor modifications. Briefly, blood samples were mixed with cold 2x sucrose-triton and centrifuged at 5000 rpm for 15 min at 4°C. The nuclear pellet was suspended in lysis buffer, sodium dodecyl sulfate and proteinase K and incubated overnight in a shaking water bath at 37°C. Nucleic acids were extracted with saturated NaCl solution. The DNA was picked up and washed in 70% ethanol. The DNA was dissolved in 1x TE buffer. The DNA concentration was determined, using Nano Drop1000 Thermo Scientific spectrophotometer and then diluted to the working concentration of 50 ng μL1.
Polymerase Chain Reaction (PCR): A PCR cocktail consisted of 1.0 mM upper and lower primers specific for each tested gene (Table 1), 0.2 mM dNTPs and 1.25 units of Taq polymerase. The cocktail was aliquoted into PCR tubes with 100 ng of sheep or goat DNA. The reaction was cycled for 1 min at 94°C, 1 min at an optimized annealing temperature that was determined for each primer (Table 1) and 1-2 min at 72°C for 35 cycles. The PCR products were electrophoresed on 2% agarose gel stained with ethidium bromide to test the amplification success.
Restriction Fragment Length Polymorphism (RFLP): The PCR products for the two tested genes were digested with specific restriction enzyme for each gene (Table 1). Ten microliters of PCR product were digested with 1 μL of FastDigest restriction enzymes specific for each tested gene for 15 min at the optimum temperature for maximum activity of each restriction enzyme. For CLPG gene, the restriction fragments were subjected to electrophoresis in 2% agarose/ethidium bromide gel in 1x TBE buffer (0.09 M tris-boric acid and 0.002 M EDTA).
|Table 1:|| Sequences and information of primers used in this study
Gels were visualized under UV light and documented in FX Molecular Imager apparatus (BIO-RAD). Whereas the digestion products of MSTN gene were separated by electrophoresis on 12% non-denaturing polyacrylamide gels then stained by silver nitrate staining method14.
Sequence analysis: The PCR products of each tested gene were purified and sequenced by Macrogen Incorporation (Seoul, Korea). Sequence analysis and alignment were carried out using ClustalW2. Results of endouclease restriction were carried out using FastPCR. The nucleotide sequences of the two tested genes in Egyptian sheep and goat were submitted to GenBank (NCBI, BankIt).
RESULTS AND DISCUSSION
Despite of the fast progress in breeding programs of farm animals in recent years, little attentions was focused on the meat quality performance15,16. Growth performance and meat quality are economically important traits in domestic animals and there are some factors; like breed and genotype that affect on them17. Recently many studies were carried out to test some genes, markers and chromosome regions which are related with these economic traits in many farm animals.
Myostatin (MSTN) and callipyge (CLPG) are the most well known major genes which are related to growth performance as well as meat quality traits. Due to the positive effect of these two genes on farm animal breeding, this study aimed to genetically characterize MSTN and CLPG genes in 6 main Egyptian sheep and goat breeds as a step towards the improvement of their productivity traits.
Myostatin (MSTN) gene: Myostatin (also known as GDF8) is a member of the mammalian growth transforming family (TGF-beta superfamily). This super-family includes differentiation and growth factors which have major roles in embryonic development regulating and maintainability of tissue homeostasis in adult animals. The major regulation of myogenesis is controlled by myostatin gene which negatively regulates muscle growth in mammals18.
Three exons and two introns are the major components of myostatin gene19 which maps to sheep chromosome 218. Muscular hypertrophy allele (mh allele) in the double muscle breeds was attributed to mutations within myostatin gene20. Knockout technology was used to improve the processing yields of animals after notifying such a major effect of a single gene7. Therefore, myostatin gene characterization in farm animals is essential for future direction of selection programs, especially marker-assisted selection for economic traits.
Ethidium bromide-stained gel of PCR products representing amplification of MSTN gene in Egyptian sheep and goat animals, Lane M: 100 bp ladder marker, Lane1-7: 337 bp PCR product amplified from sheep and goat DNA
Table 1 presents the primers investigated in this study, which were found to flank a 337 bp segment of sheep and goat MSTN gene exon 3. The segments amplified in all tested sheep and goat DNA gave the expected fragment of 337 bp (Fig. 1).
The alignment of nucleotide sequences of the amplified fragments from Egyptian goat and sheep MSTN showed 100% identity in both species (Fig. 2) and were given the accession No. KP120861 and KP120862, respectively through the submission to nucleotide sequences database NCBI/ Bankit/GenBank.
Restriction endonuclease HaeIII was used to digest the amplified PCR fragments (337 bp). The differentiation between the different genotypes could be performed depending on the presence or absence of the restriction site (GG^CC), at positions 125^126 and 219^220, where 3 different genotypes: "MM" with one undigested fragment at 337 bp, "mm" with three digested fragments at 125, 118 and 94 bp and "Mm" with four fragments at 337, 125, 118 and 94 bp. The results showed that all 171 tested sheep and goat animals, investigated for this gene, were genotyped as "mm" (Fig. 3 and 4) due to the presence of the restriction site (GG^CC) at positions 125^126 and 219^220 (Fig. 5).
Four goat populations were used to investigate MSTN polymorphism using DNA sequence analysis and PCR-SSCP21 which revealed two SNPs (368A>C) and (4911C>T). Female Boer goats with AA genotype showed increased body weight and greater withers height compared to those with AC genotype. The biochemical and physiological functions of MSTN along with the obtained results suggested that the gene might play crucial role in affecting goat growth traits.
Dehnavi et al.2 used PCR-RFLP and PCR-SSCP methods to investigate the association between Zel sheep yearling weight records and Myostatin gene polymorphism. Three fragments: 337, 222 and 311 bp were amplified using polymerase chain reaction, comprising a part of exon 3, intron 1 and intron 2 of myostatin gene, respectively.
|Fig. 2:|| Nucleotide sequences and alignment between Egyptian sheep and goat MSTN gene
Electrophoretic pattern (polyacrylamide gel) obtained after digestion of PCR amplified fragment of MSTN gene from sheep and goat DNA with HaeIII restriction enzyme. Lane M: 100 bp ladder marker, Lanes 1-6: "mm" homozygous genotype with three digested fragment at 125, 118 and 94 bp
|Fig. 4:||Endonuclease restriction of amplified fragment from sheep and goat MSTN using FastPCR GG^CC shows the restriction site in red
The RFLP method was conducted to digest exon 3 by HaeIII enzyme, while SSCP was used to study introns 1 and 2.
||Restriction site GG^CC at position (a) 125^126 and (b) 219^220
All samples displayed "mm" genotype with RFLP method, while intron 1 was found to be monomorphic using SSCP method and intron 2 was polymorphic (AA, AB and BB). The A and B showed allelic frequencies of 75.5 and 24.5%, respectively. Yearling weights were found not to be significantly affected by myostatin gene.
Farhadian et al.22 identified four SSCP patterns, representing four different genotypes in intron I of MSTN gene in Iranian Makoei sheep. The genotypes showed frequencies of 0.413, 0.293, 0.130 and 0.163 for AD, AC, AE and BC, respectively. These genotypes were investigated to test their effect on some traits and birth weight was found to be associated with the AD genotype. Other genotypes showed no phenotypic associations.
Two novel single nucleotide polymorphisms: 197G>A and 345A>T were detected in MSTN gene of two goat breeds by Zhang et al.23, where the 5-untranslated region showed three potential genotypes (AA, AB and BB) with the substitution 197G>A. Exon I segregated the polymorphism (CC and CD) of substitution 345A>T. Boer goat and Anhui white goat showed significant associations between the genotypes and body length, weight and height (p<0.05).
Han et al.24 identified 28 nucleotide substitutions through MSTN gene in New Zealand sheep breeds, where the promoter region showed 3 substitutions, while the 5UTR showed 3; 11 in intron 1, 5 in intron 2 and 5 substitutions were detected in the 3UTR. One substitution in exon 1 (101G>A) potentially resulted in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. This study revealed genetic variation that suggests that MSTN gene is more variable and can be considered as a foundation for future investigation of the effect of this variation on muscle and growth traits.
The results of the study showed that all tested Egyptian sheep and goat animals are genotyping as "mm" genotype for MSTN gene. This result agrees with the findings obtained by Dehnavi et al.2 where there is no RFL polymorphism in exon 3 of MSTN gene in Iranian Zel sheep breed. Also, there is no report about the presence of nucleotide variation in exon 3 of MSTN gene in small ruminant breeds with the exception of the findings of An et al. 21 who reported one SNP (4911C>T) in Chinese goat breeds with frequency of allele C ranged from 0.76-0.82 against allele T with frequencies ranged from 0.18-0.24.
Callipyge (CLPG) gene: Selecting animals with superior reproduction capacities is considered to be the main focus of breeding schemes for increased meat production. Reproductive traits have been primarily the main concern of many studies directed at the genetic improvement of sheep and goat. Another trait that could be important in sheep and goat breeding is the improvement of growth efficiency and meat quality25. Callipyge gene is the most well known major gene concerned with these traits. The locus of CLPG gene was mapped to the telomeric region of ovine chromosome 1826.
Several desirable production qualifications and meat quality traits are exhibited by callipyge lambs. Documentations for callipyge carcasses showed superior lean composition, larger longissimus (loin eye) areas, higher dressing percentages and leg scores8,9. These superior carcass traits reflect improved yields of wholesale leg, loin, rack and shoulder from callipyge animals by 11.8, 4.7, 2.5 and 2.3%, respectively, over normally muscled lambs27. It was also documented that callipyge lambs show superior feed efficiencies and lower daily feed intakes10, which leads to lower production expenses.
The primers tested in this study (Table 1) covered a fragment of 214 bp of sheep and goat CLPG gene. The amplified fragments resulting from all tested sheep and goat DNA gave the expected fragment at 214 bp (Fig. 6).
Three nucleotide substitutions were detected between Egyptian sheep and goat CLPG at positions 60 (C/A), 175 (T/C) and 193 (A/G), after sequencing the amplified fragments (Fig. 7). Accession No. KM597158 and KM569669, respectively were given to the nucleotide sequences of Egyptian goat and sheep submitted to NCBI/Bankit/GenBank.
Restriction endonuclease AvaII was used to digest the PCR amplified fragments (214 bp). The presence or absence of the restriction site (G^GWCC) (W = A or T) at position 77^78 allowed the differentiation between 3 different genotypes: "MM" with one 214 bp undigested fragment, "NN" with two 137 and 77 bp fragments and "MN" with three 214, 137 and 77 bp digested fragments.
The obtained results indicated that all 171 sheep and goat animals tested for this gene showed the presence of the restriction site G^GACC at position 77^78 and therefore were genotyped as "NN" (Fig. 8-10).
Ethidium bromide-stained gel of PCR products representing amplification of CLPG gene in Egyptian sheep and goat animals. Lane 1: 100 bp ladder marker, Lanes 2-6: 214 bp PCR products amplified from sheep and goat DNA
|Fig. 7:||Nucleotide sequences and alignment between Egyptian goat and sheep CLPG gene nucleotide substitutions are shown in red
|Fig. 8:||Endonuclease restriction of amplified fragment from sheep and goat CLPG using FastPCR. G^GACC restriction site is shown in red
Electrophoretic pattern obtained after digestion of PCR amplified fragment of CLPG gene from sheep and goat DNA with AvaII restriction enzyme. Lane 1: 100 bp ladder marker, Lanes 2-8: "NN" homozygous genotype with two digested fragments at 137 and 77 bp
|Fig. 10:|| Restriction site G^GACC at position 77^78
Sheep expressing CLPG mutation exhibited marked enlargement or hypertrophy of certain muscles, notably those of the hind legs and loin. The CLPG lambs showed greater percentages of total weight of excised muscles from the pelvic, torso and thoracic limbs by 42, 50 and 14%, respectively than in normally muscled lambs9. Interestingly there is no increased risk of dystocia for CLPG lambs since this muscle hypertrophy develops after about a few weeks of age10. Therefore, there would be increased profitability of the sheep industry and lowering of lamb costs for consumers through widespread production of CLPG lambs.
The polymorphism of CLPG gene was analyzed in Dorset, Suffolk and Xinjiang sheep breeds by Liu et al.28. The results showed that there was no PCR-RFLP polymorphism which suggested that hindquarters over-development was not controlled by the CLPG in Xinjiang meat sheep group. On the other hand, SNPs were detected by PCR-SSCP which resulted in the presence of three genotypes AA, AB and AC with AA being the major one. Results indicated that the AC genotype had a significant impact on the hindquarters hypertrophy, while the other two were not related to the muscling trait.
Qanbari et al.15 surveyed the presence of responsible mutations in Afshari sheep breeding flock. Direct test for CLPG alleles were conducted on 58 DNA samples by PCR-RFLP assay. The banding patterns resulted from AvaII digestion of CLPG amplicons approved the absence of the mutations in this flock. On contrary, Li et al.29 recognized one SNP (184C→T) of goat CLPG gene using ForkI. Boer goat was found to have the characteristics of double muscle, having higher T allele frequency (0.2465) and lower C allele frequency (0.7535) compared to other breeds. Therefore, it could be inferred that the double muscle characteristics of the Boer goat might be related to the 184 C→T mutation.
On the other hand, Cao et al.30 identified the polymorphism in goat CLPG gene and its association with production traits. A partial DNA fragment of 250 bp was obtained from the goat callipyge gene which shared 96.04% with the corresponding regions of ovine. The results of sequencing showed no A→C mutation corresponding to the ovine CLPG gene, although one A→C transversion was located 147 bp downstream from the CLPG site. Allele A was found to be dominant in four of the goat populations, with the exception of Mongolia Alashan White cashmere goats. In last population where the allele C is dominant, least-square means of birth weight, production of cashmere and body weight gain from birth to weaning did not differ significantly between the AA and AC phenotypes.
The results of the study showed that all tested Egyptian sheep and goat animals are genotyping as "NN" genotype for CLPG gene. This finding agrees with the previous results obtained by Qanbari et al.15 which showed no mutation or polymorphism in this site of CLPG gene and Gui-Ling et al.30 which showed the presence of dominant nucleotide A (allele N) in highest frequency than that of nucleotide C (allele M). Also the present results declared that Egyptian sheep is similar to Iranian Afshari sheep breeds where the polymorphism in this site of CLPG gene is absent15.
It can be concluded that although there were no polymorphism or variation detected through the amplified fragments of MSTN and CLPG genes, further analysis will be needed to find out other molecular loci associated with muscular performance and meat quality in Egyptian sheep and goat breed, in order to use these markers in breeding programs as a step for improvement of carcass trait and meat quality in these native breeds.
The authors acknowledge the National Research Centre, Egypt for funding this study. Funding source code: 10120506A11.
This study contributes as a preliminary step for meat and growth trait improvement in native small ruminant breeds. It is the first study to focus on the genetic characterization, polymorphism and nucleotide sequencing of two important genes associated with meat and growth traits in Egyptian sheep and goat breeds. This molecular information for such economically important trait genes allows their improvement through marker-assisted selection based on favorable genotypes correlated with superior reproduction traits.