Genetic variation of CAST gene in Local Karnobat and Karnobat merino sheep breeds

Abstract

Karnobat sheep plays an important role in the development of sheep breeding in Southeastern region of Bulgaria. They are valuable source of genetic material. The aim of present experiment was to determine the allele variation of CAST gene in Local Karnobat and Karnobat Merino sheep breeds. A total of 60 blood samples were collected-30 per breed. DNA was extracted and genotypes of all animals were identified by means of PCR-RFLP technique. The restriction reactions were accomplished by specific enzyme MspI. As expected both breeds were characterized with low level of genetic diversity due to the fact that mostly maintaining selection has been implemented. In Local Karnobat sheep breed was identified only one heterozygous individual from all 30. In Karnobat merino were identified allele M with frequency 0,97 and allele N with frequency 0,03. Genotypes MM and MN were revealed with frequencies 0,93 and 0,07, respectively. According to the statistical analysis both breeds were in HWE equilibrium.

Full text

Genetic variation of CAST gene in Local
Karnobat and Karnobat merino sheep breeds
Ivona Dimitrova1,
*
, Milena Bozhilova-Sakova2, Margarit Iliev3, and Atanaska Teneva1
1Agronomy Faculty, University of Forestry, Sofia, Bulgaria
2Institute of Animal Science, Kostinbrod, Bulgaria
3Institute of Agriculture, Karnobat, Bulgaria
Abstract. Karnobat sheep plays an important role in the development of
sheep breeding in Southeastern region of Bulgaria. They are valuable
source of genetic material. The aim of present experiment was to determine
the allele variation of CAST gene in Local Karnobat and Karnobat Merino
sheep breeds. A total of 60 blood samples were collected – 30 per breed.
DNA was extracted and genotypes of all animals were identified by means
of PCR-RFLP technique. The restriction reactions were accomplished by
specific enzyme MspI. As expected both breeds were characterized with
low level of genetic diversity due to the fact that mostly maintaining
selection has been implemented. In Local Karnobat sheep breed was
identified only one heterozygous individual from all 30. In Karnobat
merino were identified allele M with frequency 0,97 and allele N with
frequency 0,03. Genotypes MM and MN were revealed with frequencies
0,93 and 0,07, respectively. According to the statistical analysis both
breeds were in HWE equilibrium.
1 Introduction
Sheep farming is an economically important industry for agriculture. Sheep breeding
efficiency is directly affected by the quality and quantity of produced meat. Providing of
lamb meat with higher quality is a key priority for this industry, because the main part of
the income in the sheep farms, in particular in Bulgaria, is formed from the sale of lambs
[1]. The quantity and quality of the obtained meat are formed under the influence of a
complex of genes and environmental conditions. Improving the meat quality is the main
objective of livestock production whereby the meat tenderness is one of the most important
factors for measuring the quality of meat [2]. However, the quality of meat can hardly be
improved by traditional selection, as the inheritance of meat quality is low [3] and the
measuring of quality characteristics is difficult, expensive and possible only after slaughter.
The application of new methods based on DNA technologies allows to make sheep
breeding a modern, competitive and profitable industry [4]. In this regard, DNA methods
based on the latest advances in molecular genetics are increasingly used to assess the
genotype of breeding animals [5].
*
Corresponding author: ivonna_dimitrova@yahoo.co.uk
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 42, 01029 (2022)
SDGE 2021
https://doi.org/10.1051/bioconf/20224201029

The genes CAST (calpastatin), MSTN (myostatin), FABP3 (fatty acid binding protein
3), CLPG (calypig), DGAT1 (diacylglycerol acyltransferase 1) and LEP (leptin) were
studied as candidate genes for meat productivity and meat quality in sheep [7]. One of the
promising markers for growth intensity and meat quality in sheep is the calpastatin gene
(CAST) [6, 7]. The CAST gene is located at locus 5q15 on chromosome 5 of the sheep
genome (Ovis aries L.), it is composed of 29 exons separated by introns and has a total size
of 89553 bp [8]. Genetic polymorphism has been identified in the following regions: exon
1, exon 6, exon 12, intron 12, and domains II to IV of the CAST gene [9, 10]. CAST
inhibits calpain activity and this effects regulation of meat post mortem tenderness, birth
weight, and growth rate to weaning. The calpastatin gene shows large and significant
effects on birth weight and fat thickness [11]. A significant association between CAST
genotypes and growth rate and final body weight was reported, with lambs of the MN
genotype having a higher mean daily gain and final body weight than lambs of the MM
genotype. The genotypes of the CAST gene have a significant effect on some components
of the carcass and the quality parameters of the meat - the MN genotype shows a lower total
bone mass and a higher meat to bone ratio compared to the MM genotype [12]. The
presence of polymorphism in the locus of the calpastatin gene indicates that the quality of
meat in the studied breeds of animals can be improved by applying an appropriate breeding
program. Genetic polymorphisms, which are significantly associated with certain
interesting traits, are very useful [13].
Different breeds of thin-tailed sheep are bred in Bulgaria - including local breeds and
others intended for specific purposes such as milk, meat and wool. Many of the commercial
Bulgarian sheep breeds are created based on local breeds. Such is the case with the
composite Karnobat merino breed, in the creation of which were also used local Karnobat
sheep characterized by high quality meat [14]. The establishment of genetic polymorphism
in existing breeds of sheep is a major task, on the one hand in local breeds in order to
preserve genetic resources, and on the other in cultivated animal breeds in order to their
effective management and exploitation
The present study was aimed to investigate the genetic polymorphism of the CAST gene
in 60 ewes of one local and one merino sheep breed – Local Karnobat and Karnobat
merino.
2 Materials and Methods
The present experiment was conducted with a total of 60 ewes belonging to two sheep
breeds from the Institute of Agriculture, Karnobat – 30 animals from Local Karnobat breed
and 30 animals from composite Karnobat Merino breed. Blood samples were collected
from the jugular vein in vacuum tubes containing EDTA as anticoagulant.
Genomic DNA was extracted from whole blood using a commercial purification kit
according to the manufacturer’s instruction (Illustra Blood Genomic Prep DNA Purification
Kit, GE Healthcare). DNA concentration and purity were determined using
spectrophotometer Biodrop and agarose electrophoresis on 1% agarose gel (Bioline) and
TBE buffer (Jena Bioscience).
PCR reactions were performed in total volume of 10 µl containing 40 ng genomic DNA,
0.2 μl dd H2O, 20 pM of each primer and 5 μl of ready-to-use 2×(1.5 mM MgCl2)
MyTaqTM HS Red Mix (Bioline). For the amplification of CAST gene was used primer set
suggested by Palmer et al., [15]:
forward primer: 5’-TGG GGC CCA ATG ACG CCA TCG ATG-3’
reversed primer: 5’-GGT GGA GCA GCA CTT CTG ATC ACC-3’.
Amplification process was performed on thermal cycler QB-96 r (Quanta Biotech). The
specific PCR conditions were shown on tablе 1.
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Table 1. Specific PCR conditions for amplification of ovine CAST gene
Locus
Primary
Denaturation
Cycles
Denaturation
Annealing
Elongation
Final
elongation
CAST
94°C for 5 min
30
94°C for 30 s
62°C for 45 s
72°C for 1
min
72°C for 10
min
The genotypes of tested individuals were determined using RFLP method. The
restriction reactions were carried out in 10 µl final volume containing 6 µl PCR product, 10
U/µl speed enzyme MspI (Jena Bioscience), buffer and ddH2O. The incubation was
performed in heat-block at 370C for 15-20 min. The fragment sizes were validated by
agarose gel electrophoresis using 50 bp DNA Ladder (Thermo) on 2,5% agarose gel
(Bioline) stained by 10000x RedGelTM Nucleic Acid Stain (Biotuim) and 1x TBE buffer
(Jena Bioscience). The results were visualized under UV light.
All steps in the present experiment were carried out in Laboratory of Genetics, part of
Agronomy Faculty in University of Forestry, Sofia, Bulgaria.
3 Results and Discussion
A 622 bp fragments from exon 1 and intron 1 of the CAST gene of the investigated animals
were amplified by the described technique. All PCR products were subsequently subjected
to enzymatic digestion with restriction endonuclease MspI. According to authors, this
enzyme specifically cleaves the particular site with sequence 5' C↓CGG 3' and as a result
produces two fragments with sizes of 336 bp and 286 bp, which define the allele M. The
nucleotide sequences of 622 bp where the specific site 5'C↓CGG3' is absent remains uncut
and determines the N allele. As a result, three genotypes can be observed - MM (with
fragment size of 336 and 286 bp), MN (with 622, 336 and 286 bp) and NN (with 622 bp)
[15].
Fig. 1. DNA electrophoresis of CAST amplicons after digestion with MspI restriction enzyme: lane 7
- 50 bp DNA ladder, lanes 1, 2, 4 ,5, 6, 8, 9 and 10 - genotype MM, lanes 3 and 11 - MN genotype
In present experiment were determined two genotypes – homozygous genotype MM
and heterozygous genotype MN (Figure 1). The most common allele in both breeds was the
M allele. In the local Karnobat breed, the M allele was found with a frequency of 0.98 and
the N allele with a frequency of 0.02. The Karnobat merino breed shows the following
frequencies - of the allele M - 0.97 and of the N allele - 0.03 (Table 2). Both MM and MN
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genotypes were identified in both studied breeds, while the NN genotype was not
established. In the local Karnobat breed, we observed that the MM genotype occurred with
a frequency of 0.97, and the heterozygous MN genotype - with a frequency of 0.03. The
observed value of the MN genotype was slightly higher in the Karnobat merino breed -
0.07, while the frequency of the MM genotype was 0.93. According to the statistical
analysis both breeds were in HWE equilibrium.
Table 2. Animal number, values of allele and genotype frequencies, average heterozygosity
(observed Ho and expected He) and degree of probability for CAST gene in investigated breeds.
Breeds
n
Allele
frequency
Genotype
frequency
Но
Не
Р*
M
N
MM
MN
NN
Local
Karnobat
30
0,98
0,02
0,97
0,03
0,00
0,033
0,039
>0,05
Karnobat
Merino
30
0,97
0,03
0,93
0,07
0,00
0,066
0,058
*non-significant difference
The results in this study showed that the heterozygous genotype MN is present in the local
Karnobat breed with very low frequency (0.02), while a previous study on the same sheep
breed showed the absence of the heterozygous genotype [16]. Studies in other native
Bulgarian breeds showed differences in the frequency of alleles of the CAST gene [17]. In
the Cooper-Red Shumen and Karakachan breeds, a lack of diversity and only the M allele
and the MM genotype were found [17, 18]. Two genotypes were identified in the Stara
Zagora and Breznik breeds - MM and MN. In the Stara Zagora sheep the frequency of the
mutant allele N is 0.03, of the genotype MM - 0.97 and of the heterozygous genotype MN -
0.03 [16], while in the Breznik breed there was a higher diversity - the frequency of the
allele N was 0.12, of the genotype MM - 0.77 and of the heterozygous MN - 0.23 [17]. In
studies of thin-tailed Turkish sheep breeds Kivircik, Imroz, and Karayaka, a null frequency
of the NN genotype was found similar to the results in the present study, and in the Imroz
breed the frequency of the M allele was 0.96 and of the heterozygous MN genotype was
also close to this established by us - 0.07 [8].
The current study of Karnobat merino sheep showed low genetic diversity of the studied
locus. The obtained results slightly, but statistically insignificant, exceeded the results
obtained for the local Karnobat breed. In a previous study of animals of this breed, two
alleles were found with a frequency of 0.94 for the M allele and 0.06 for the N allele and
two genotypes - with a frequency of 0.89 for the MM genotype and 0.11 for the MN
genotype [19]. Results with only two genotypes were reported in a study of other merino
breeds – Caucasian, Northeast Bulgarian Merino, Volgograd, Salskaya and Stavropol [17,
20, 21, 22]. Three genotype variants were identified in the Askanian, Soviet merino and
Altai Mountain breeds [17, 22, 23].
Many publications are devoted to the possible association of calpastatin polymorphism
with important traits of meat productivity in different sheep breeds. Only two genotypes,
MM and MN, were found in a study of Salsk sheep, and slaughter analyzes showed better
meat productivity results related to the MN genotype [5]. In other molecular studies in
sheep of the Volgograd breed also found the presence of two genotypes MM and MN. The
results in control slaughter, showed that sheep with genotype MN/CAST superiored their
peers with genotype MM/CAST by 3.7 kg in weight before slaughter [21]. Animals from
Pakistani breed Kajli of heterozygous MN genotype grown faster compare to animals with
MM genotype from birth to four months of age [24].
Identifying genes associated with productive traits would help improving the quality
and diversity of production in the livestock industry by optimizing breeding programs. The
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established polymorphism in the CAST gene shows that the study of this locus in Bulgarian
sheep breeds must be studied in detail in order to be included in future breeding programs.
4 Conclusions
The results of this study showed low genetic diversity in the studied animals of both local
Karnobat and Karnobat merino breeds. In both breeds, the presence of two allelic variants
(M and N) and two genotypes (MM and MN) was found, with the M allele and the MM
genotype predominance.
Acknowledgements
This research was part of the project КП-06-Н 26-9/18.12.2018 (2018–2022) "Investigation
of DNA markers associated with production in sheep breeds reared in Bulgaria" financed
by NSF - the Ministry of Education and Science, Republic of Bulgaria.
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