Study of some genes associated with meat productivity in Karnobat Merino sheep breed using PCR-RFLP

Abstract

The purpose of present study was the analysis of polymorphic variants of CAST, MSTN and CLPG genes associated with meat productivity in sheep. Karnobat Merino breed was established on the basis of local Karnobat sheep crossed with rams breed Kamvolmerino and Merinoflaysh and later Caucasus and Stavropol. Thirty five blood samples were collected from ewes of Karnobat Merino sheep breed. Genomic DNA was extracted and after PCR amplification with specific primers were obtained products with length 622 bp, 337 bp and 426 bp for CAST, MSTN and CLPG genes, respectively. The genotypes were determined using PCR-RFLP method with specific restriction endonucleases for each gene-MspI for CAST gene, HaeIII for MSTN gene and FaqI for CLPG gene. MSTN and CLPG genes were monomorphic. CAST gene was found to be polymorphic with allele frequencies: 0.94 for allele M and 0.06 for allele N and observed genotype frequencies: 0.89 for genotype MM and 0.11 for genotype MN. In this population the genotype NN was not established.

Full text

IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS)
e-ISSN: 2319-2380, p-ISSN: 2319-2372. Volume 10, Issue 8 Ver. III (August 2017), PP 61-65
www.iosrjournals.org
DOI: 10.9790/2380-1008036165 www.iosrjournals.org 61 | Page
Study of some genes associated with meat productivity in
Karnobat Merino sheep breed using PCR-RFLP
*Ivona Dimitrova1, Milena Bozhilova-Sakova1, Margarit Iliev2
1Faculty of Agronomy, University of Forestry, Sofia, Bulgaria
2 Institute of Agriculture, Karnobat, Bulgaria
Corresponding Author: Ivona Dimitrova
Abstract: The purpose of present study was the analysis of polymorphic variants of CAST, MSTN and CLPG
genes associated with meat productivity in sheep. Karnobat Merino breed was established on the basis of local
Karnobat sheep crossed with rams breed Kamvolmerino and Merinoflaysh and later Caucasus and Stavropol.
Thirty five blood samples were collected from ewes of Karnobat Merino sheep breed. Genomic DNA was
extracted and after PCR amplification with specific primers were obtained products with length 622 bp, 337 bp
and 426 bp for CAST, MSTN and CLPG genes, respectively. The genotypes were determined using PCR-RFLP
method with specific restriction endonucleases for each gene – MspI for CAST gene, HaeIII for MSTN gene and
FaqI for CLPG gene. MSTN and CLPG genes were monomorphic. CAST gene was found to be polymorphic
with allele frequencies: 0.94 for allele M and 0.06 for allele N and observed genotype frequencies: 0.89 for
genotype MM and 0.11 for genotype MN. In this population the genotype NN was not established.
Keywords: CAST gene, CLPG gene, Karnobat Merino sheep breed, MSTN gene, PCR-RFLP
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Date of Submission: 20-07-2017 Date of acceptance: 08-09-2017
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I. Introduction
In recent years the main role of sheep meat producers is to provide meat with good qualities such as
tenderness, nice colorization, low consistence of water and etc. Therefore the selection is based on fast-growing
animals with increased muscling.
There are three main genes affecting meat productivity in sheep and carcass quality which are mostly
used in marker-assisted selection – calpastatin, myostatin and callipyge [3, 4, 5]. Calpastatin is an endogenous
and specific inhibitor of calpains. Inhibiting the calpain activity in postmortem tissue CAST regulates the level
of postmortem meat tenderization [6]. Myostatin is part of the mammalian growth transforming family (TGF-
beta superfamily) [7]. Myostatin activity is related to inhibition of skeletal muscle growth. Mutations in MSTN
locus could lead to increased muscling [5, 8]. Callipyge mutation causes muscle hypertrophy in pelvic limbs
and loin when it’s inherited from the father. This effect is called “polar overdominance” [9, 10].
Bulgarian fine fleece breeds are traditionally created by combining the specific characteristics of
different breeds in order to increase productivity and genetic value and the resultant new breeds, populations and
lines are classified as composite [11]. Bulgarian fine fleece sheep breeds have excellent meat productivity and
adaptive ability, resistant to pests, insects and ticks and do not suffer from piroplasmosis. Karnobat Merino
breed was established on the basis of local Karnobat sheep crossed with rams breed Kamvolmerino and
Merinoflaysh and later Caucasus and Stavropol. In the Institute of Agriculture - Karnobat is kept the only herd
of 170 pure-bred animals. Animals have a strong constitution and good exterior. Ewes have average live weight
of about 55 kg, and rams - about 90 kg. Fertility is about 130% [12]. Nowadays because of the market needs it is
important to analyze the allelic variation of the genes associated with increasing of the productivity and quality
of sheep meat.
The aim of present study was to identify the allelic variants of three genes associated with meat
productivity in sheep of Bulgarian breed Karnobat Merino.
II. Materials And Methods
Animals and blood collection
In present study were tested 35 adult sheep of Bulgarian breed Karnobat Merino part of a larger
population kept in the Institute of Agriculture, Karnobat. Blood samples were collected from v.jugularis in
vacuum tubes containing EDTA.

Study of some genes associated with meat productivity in Karnobat Merino sheep breed using PCR-
DOI: 10.9790/2380-1008036165 www.iosrjournals.org 62 | Page
Figure 1: Genomic DNA tested on 1% agarose gel by agarose gel electrophoresis. All samples were with
concentration approximately 10 ng/µl.
DNA extraction
Genomic DNA was extracted from whole blood with QIAamp DNA Blood Mini Kit (Qiagen)
according to the manufacture’s instruction. DNA concentration and purity were determined using Biodrop and
agarose electrophoresis on 1% agarose gel and 1x TBE buffer (Thermo) (Figure 1).
Polymerase chain reaction (PCR)
PCR amplification reactions were carried out in total volume 10 µl containing 4 µl genomic DNA, 5 µl
Red Taq DNA polymerase Master Mix (VWR), 0.4 µl of each primer (Bioneer) and 0.2 µl ddH2O. For CAST
gene was used primer sequence suggested by Palmer et al., (1997) [13], for MSTN locus by Dehnavi et al.,
(2012) [14] and for CLPG locus by Gabor et al., (2009) [15]. For each gene were used specific primer sequences
showed in Table 1 and specific
Table 1: Locus, primer sequences and length of PCR fragments of the investigated genes
Locus
Primer sequence (5’→3’)
PCR fragment
CAST
F:5’-TGG GGC CCA ATG ACG CCA TCG ATG-3’
R:5’-GGT GGA GCA GCA CTT CTG ATC ACC-3”
622 bp
MSTN
F: 5’-CCG GAG AGA CTT TGG GCT TGA-3’
R: 5’- TCA TGA GCA CCC ACA GCG GTC-3’
337 bp
CLPG
F: 5’- TGA AAA CGT GAA CCC AGA AGC-3’
R: 5’- GTC CTA AAT AGG TCC TCT CG-3’
426 bp
PCR conditions showed in Table 2. All PCR reactions were accomplished by QB-96 Quanta Biotech
thermocycler. Table 2: PCR conditions
Stages/Gene
CAST
MSTN
CLPG
To
Time
To
Time
To
Time
Primary denaturation
94 oC
5 min
94 oC
5 min
95 oC
4 min
Cycles
30
30
35
Denaturation
94 oC
30 s
94 oC
30 s
94 oC
20 s
Annealing
62 oC
45 s
58 oC
45 s
58 oC
30 s
Elongation
72 oC
1 min
72 oC
1 min
72 oC
1 min
Final elongation
72 oC
10 min
72 oC
10 min
72 oC
10 min
Store
10 Co
Restriction fragment length polymorphism analysis
Table 3: Restriction conditions and restrictions enzymes of the investigated genes
Locus
Endonuclease
Duration
Incubation ot
CAST
MspI (Bioneer)
15 h
37 oC
MSTN
HaeIII (Bioneer)
15 h
37 oC
CLPG
FaqI (Thermo)
16 h
37 oC
The genotypes of investigated animals were established using RFLP method for the three genes. The
restriction reactions were carried out in 10 µl final volume containing 6 µl PCR product, 0,5 µl specific
endonuclease for each gene, buffer and ddH2O. The incubation process was performed at 37 oC in thermostat
for all reactions. The restriction enzymes and conditions are shown in Table 3. The fragment sizes were
determined by agarose gel electrophoresis using 50 bp DNA Ladder (Thermo) on 2% agarose gel stained by
10000x RedGelTM NucleicAcid Stain (Biotuim) and 1x TBE buffer. The results were visualized under UV
light. III. Results And Discussion
CAST locus
The amplified region of CAST gene produced a 622 bp PCR fragment. After digestion with
endonuclease MspI two alleles were observed allele M and allele N with frequencies 0.94 and 0.06 respectively.
Enzyme MspI digests the allele M and as a result produces two fragments of 336 bp and 286 bp, but not the
allele N. Only two genotypes were detected in this population – genotype MM (two fragments were observed –

Study of some genes associated with meat productivity in Karnobat Merino sheep breed using PCR-
DOI: 10.9790/2380-1008036165 www.iosrjournals.org 63 | Page
286 bp and 336 bp) and genotype MN (three fragments were observed – 286 bp, 336 bp and 622 bp) (Figure 2)
with frequencies 0.89 and 0.11, respectively (Table 4). Genotype NN was not found. Observed and the expected
heterozygosity were calculated on base of chi-square test using Hardy-Weinberg equilibrium. This population
was found to be in HWE for CAST locus.
These results are in agreement with results reported by other authors in different sheep breeds where
the allele M was the most frequent [2, 13, 15, 17, 18, 19]. Hristova et al. [20] found similar results in another
Bulgarian sheep breed. They investigated 96 animals from Local Karnobat and Stara Zagora sheep breeds and
reported the presence of only two genotypes in Stara Zagora sheep breed with frequencies 0.97 and 0.03 for MM
and MN, respectively. In previous our study in Synthetic population Bulgarian Milk sheep breed we detected the
presence of all three possible genotypes of CAST gene with frequencies 0,84, 0,15 and 0,01 for MM, MN and
NN, respectively.
Gorlov et al. [21] studied two breeds - Salsk sheep which have only 2 genotypes, MM and MN, with
frequencies 0.78 and 0.22, respectively, and Soviet Merino sheep which shown 3 genotypes, MM, MN, and NN,
with frequencies of 0.82, 0.12, and 0.06, respectively. Asadi et al. [22] also detected all three possible
genotypes. Genotype frequencies in Dalagh sheep were 0.36, 0.38, 0.26 for the MM, MN and NN genotypes,
respectively [23]. Santos et al. [24] investigating four Brazilian breeds Pantaneira, Ile de France, Suffolk and
Brazilian Bergamacia have established the presence of all three genotypes in each of them. In Iran Tohidi et al.
[25] found the presence of genotype NN with frequency more than 0,50 in Mehraban and Arcmerino sheep
breeds. Sumantri et al. [26] studied animals from eight Indonesian sheep breeds and found extremely high level
of genotype NN reach up to 1.00 in one of the breeds.
The obtained polymorphism in the CAST locus in Karnobat Merino population allows it to be used for
improvement of meat quality in this breed through appropriate breeding programs.
Figure 2: Restriction analysis of PCR product of CAST gene with MspI restriction enzyme on 2% agarose gel
electrophoresis in Karnobat Merino sheep breed. 1 – PCR product 622 bp; 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14 –
homozygous genotype MM; 12 – heterozygous genotype MN; 7 – DNA Ladder 50 bp.
MSTN locus
After amplification of exon 3 of ovine MSTN gene a PCR fragment with size 337 bp was obtained.
After digestion with specific enzyme HaeIII it was detected only allele m and only one possible genotype mm
with frequency 1,00 (Table 4). HaeIII digests allele m and produces three fragments of 83 bp, 123 bp and 131 bp
(Figure 3). Allele M and genotypes Mm and MM were not detected in this population. In the present study exon
3 of MSTN locus was found to be monomorphic.
Results in this paper are in agreement with results reported from different authors. In previous our
studies in three Bulgarian sheep breeds we tasted 25 adult animals (22 ewes and 3 rams) of Karakachan sheep
breed, 32 rams of Northeast Bulgarian Merino and 121 sheep of Synthetic Population Bulgarian Milk and we
found similar results - all animals were carried only the genotype mm [5, 27, 28]. Ahani Azari et al. [23]
reported that exon 3 of MSTN gene was also monomorphic in 110 native Dalagh sheep. Elkorshi et al. [29]
studied the MSTN gene in 140 animals belonging to four Egyptian and two Saudi sheep breeds. All samples
were analyzed by PCR-RFLP and only allele m and genotype mm were found. Khederzadeh et al. [30] obtained
same results in 100 Zandi fat-tailed sheep in Iran.
Fig 3: Restriction analysis of PCR products of MSTN gene with HaeIII restriction enzyme on 2% agarose gel
electrophoresis in Karnobat Merino sheep breed. 1 – PCR product 337 bp; 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14 –
homozygous genotype mm; 7 – DNA Ladder 50 bp.

Study of some genes associated with meat productivity in Karnobat Merino sheep breed using PCR-
DOI: 10.9790/2380-1008036165 www.iosrjournals.org 64 | Page
Jamshidi et al. [31] randomly collected 95 DNA samples of Mehraban’s sheep and announced for
presence of the two alleles of MSTN gene with frequencies 0.97 and 0.03 for m and M, respectively. In Iran in
Sanjabi sheep was found diversity in exon 3 of MSTN [32]. They also detected the two alleles m and M with
frequencies 0.97 and 0.03, respectively and all three possible genotypes – mm, Mm and MM with frequencies
0.97, 0.01 and 0.02, respectively. In the contrast in 105 Teleorman Black Head lambs in Romania was identified
a high diversity in exon 3 of MSTN gene - two genotypes - Mm and mm with frequencies 0.83 and 0.17,
respectively [33]. In this case M allele frequency was 0.42 and for allele m was 0.58.
CLPG locus
After PCR amplification reaction of ovine CLPG locus it was produced a fragment with length 426 bp.
It was performed RFLP analysis with FaqI restriction enzyme which digests the PCR products and it was
detected only the wild allele A - with three fragments of 278 bp, 117 bp and 31 bp. The mutant allele G (with
two fragments 395 bp, 31 bp) was not observed in this study. As a result only one possible genotype was
detected– genotype AA with frequency 1,00 (Table 4) (three fragments – 278 bp, 117 bp and 31 bp). The other
two genotypes - genotype AG (four fragments – 395 bp, 278 bp, 117 bp, 31 bp) and genotype GG (two
fragments – 395 bp, 31 bp) were not presented in this population. In this study CLPG locus was found to be
monomorphic in Karnobat Merino sheep breed (Figure 4).
Fig 4: Restriction analysis of PCR product of CLPG gene with FaqI restriction enzyme on 2% agarose gel
electrophoresis in Karnobat Merino sheep breed. 1 – PCR product of CLPG gene 426 bp; 2, 3, 4, 5, 6, 8, 9, 10,
11, 12, 13, 14 – homozygous genotype AA ;7 – DNA Ladder 50 bp
In previous our study in Bulgarian indigenous sheep breed Karakachan it was also observed only
genotype AA with frequency 1,00 [34]. Same results were announced by Alakilli et al. [35] in two Saudi sheep
breeds – Najdi and Harri. They were monomorphic for CLPG locus and only genotype AA was found. Gabor et
al. [15] collected 96 samples from five different sheep breeds, kept in Slovakia. They observed only wild allele
A in all tested animals. Same results were reported by Nanekerani et al. [4] in 124 Lori sheep. They all were
monomorphic for CLPG locus.
In the contrast Jackson et al. [36] reported the presence of mutant allele G and the heterozygous
genotype AG in crossbred of 15/16 Rambouillet and 1/16 Dorset rams.
Table 4: Frequencies of alleles and genotypes, observed (Ho) and expected (He) heterozygosity.
IV. Conclusion
In present study it may be concluded that there is polymorphism in CAST locus of investigated animals
from Karnobat Merino breed which could be used in future investigations referring to candidate genes for meat
productivity in sheep. MSTN and CLPG loci were monomorphic in this population.
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Ivona Dimitrova. “Study of some genes associated with meat productivity in Karnobat Merino sheep
breed using PCR-RFLP .” IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS), vol. 10,
no. 8, 2017, pp. 61–65.