Goat (Capra aegagrus), is one of the livestock, first domesticated in Asia. Goats live in different areas. Goat is one of the most adaptable animals with different environmental conditions. Calpastatin, initially identified in skeletal muscle (Busch et al. 1972), is the endogenous inhibitor of the calpains (Goll et al. 2003). Calpastatins are rich in proline and glutamate, but poor in aromatic amino acids (Murachi, 1983). The rate of protein degradation post mortem affects meat quality (Koohmaraie et al. 2002(. The calpastatin gene has also been found to have a large effect on pork quality (Rohrer et al. 2012). Genetic variation in calpastatin gene and the effect on meat quality traits both in cattle and sheep has been reported by many researchers (Zhou and Hickford, 2008). At the molecular level, calpastatin protein is composed of five-domains having molecular weight of 76 kDa which bind and inhibit the calpains system. The function of N terminal domain is to enhance the targeting of the inhibitory region of calpain system by other inhibitory domain but itself don’t play any inhibitory role (Averna et al. 2001). Marker assisted selection is one of the new DNA based methods that improves accuracy and progress in animal selection programs (Bastos et al. 2001). Genetic markers are important for the determination of allelic polymorphism at any specific locus. A two-allele system of polymorphic variants (M and N) of the ovine calpastatin gene by a PCR-RFLP method has been described (Palmer et al. 1998). Tenderness is one of the attributes of quality of beef that is most appreciated by consumers (Alfnes et al. 2008). PCR-RFLP is one of the most commonly used methods for polymorphism genotyping due to its simplicity. Initially, the RFLP analysis required a radio-actively labeled probe for detection, and now the method is coupled with PCR and simple agarose gel electrophoresis. In a study two allels (M and N) determined in region of the ovin calpastatin by PCR-RFLP method (Palmer et al. 1999).
MATERIALS AND METHODS
In this study, 150 Tali and 150 Raeni goats was sampled in the Kerman province. Blood samples were taken mainly from the jugular vein. In tubes containing EDTA and immediately transferred to ice, and the samples were stored at -20 ˚C until DNA extraction. DNA was extracted from 0.3 mL blood using the DNA purification kit (Fermentas, EU) according to manufacturer's instructions. Quality and quantity of DNA was measured by agarose gel (2%). The DNA amplification of the calpastatin gene was achieved by PCR. PCR-RFLP genotyping was used to detect the polymorphism in the region between exons 6 and 7 of the calpastatin gene. One pair of primers was used as follows.
Forward primer: 5'-AGCAGCCACCATCAGAGAAA-3'
Reversed primer: 5'-TCAGCTGGTTCGGCAGAT-3'
The final volume was 25 µL reactions, thermal program included: first denaturation temperature of 33 stages with 94 to 30 seconds, 56 ˚C connection temperature for 30 seconds, temperature 72 ˚C for 30 seconds and develop the final amplificon temperature of 72 ˚C for 4 minutes. The correctness of the resulting part of product on 1.5% agarose gel and noticeable with ethidium bromide voltage 82 and was established for 25 minutes. The PCR products by restriction endonucleases XmnI were digested. Cut pieces again on 2% agarose gel and stained with ethidium bromide voltage 72, after 2 hours determined genotypes. Gel documentation system was used for Photography.
For determine gene, genotypic frequency and Hardy-Weinberg equilibrium use Pop-Gene32 program, version 1.31, Canada (Yeh et al. 1997).
RESULTS AND DISCUSSION
The samples of PCR showed on the gel agarose that was area between 1500 and 1600 bp. PCR product without any nonspecific band (Figure 1). The allelic variation in the calpastatin gene was tested by PCR-RFLP. The PCR-RFLP method revealed two alleles; allele B was the not digested and had 1552 bp and allele A was the PCR product with the restriction site for XmnI which upon digestion produced two fragments of roughly 960 and 592 bp PCR product (Figure 2).
Figure 1 PCR products for calpastatin gene in Raini and Tali goats
Line M is the 100 bp molecular weight marker
Figure 2 RFLP polymorphism of caprine CAST gene
M shows the 100 bp molecular weight marker
Three different PCR-RFLP patterns (genotype) were identified (AA, AB and BB)
In this study, a total of three genotypes were observed in the test population. The frequencies of the observed genotypes to Tali goat were 38.4, 9.6 and 52.0 for AA, BB and AB, respectively. Allele frequencies were 0.47 and 0.53 for A and B, and for Raini goat also genotypes were 0.17, 0.71 and 0.11 for AA, BB and AB respectively. Allele frequencies were 0.12 and 0.88 for A and B (Table 1). The chi-square test showed significant (P<0.01) deviation from the Hardy-Weinberg equilibrium for this locus in the investigated these two populations. In the present study two alleles (A and B) and three genotypes (AA, BB and AB) were observed for calpastatin gene in Tali and Raini goat in kerman province, Iran. The most frequent allele and genotype in the Tali goat were 53.0 and 67.0% for allele A and allele B, respectively (Table 2) and to Raini goat were 12.0 and 88.0 respectively. The results obtained from this study revealed the polymorphism pattern of the calpastatin gene in Tali and Raini goats. Several methods including PCR-RFLP a good method to determine gene polymorphism is Calpastatin gene in domestic animals (Palmer et al. 1998). Study on calpastatin gene in goat is very limited (Zhou and Hickford, 2008), this result shows that the polymorphism was detected in CASTI segment, as previously observed (Palmer et al. 1998) and (Casas et al. 2006).
Table 1 Observed alleles and genotypic frequencies for CAST gene in Tali and Raeini goats
Table 2 Estimated statistically parameters for CAST gene in Tali and Raini gots
Small ruminants calpastatin gene mRNA transcript variants 2 and 4 have also been reported (Zhang et al. 2012). Who observed frequencies of 61, 36 and 3% of the MM, MN, and NN genotypes respectively in Iranian karakul sheep and Iranian kurdi sheep, the frequencies of 76, 24 and 0% were observed for MM, MN and NN genotype respectively, showing the existence of M allele more frequent then N allele (Nassiry et al. 2006). Similar data were found by (Mohammadi et al. 2008). There are several studies on the association of CAST gene polymorphism with meat quality by PCR-RFLP analysis in animals (Schenkel et al. 2006). The polymorphism in the CAST in goat was also reported by other researchers using PCR-RFLP technique (Zhou and Hickford, 2008). Higher frequencies of CAST gene A allele compared to the B allele have been reported in Brangus (0.78) and Pardo Suico (0.80) cattle (Asadi and Khederzadeh, 2015). Reported a significant association between A allele of bovine CAST gene and meat tenderness (Kuryl et al. 2003). Association between the D and F alleles of porcine CAST gene and meat quality traits was also reported (Kapelanski et al. 2004). The polymorphism in the exon 1 of the CAST in sheep was also reported by other researchers using PCR-RFLP technique (Mohammadi et al. 2008). In goats and bovine the exon 6 of CAST gene were investigated for polymorphisms and a number of allelic variants were identified in these species (Zhou and Hickford, 2008). Two allelic systems of polymorphic variants (M and N) in the region of ovine CAST locus have been described by PCR-RFLP method (Shahroodi et al. 2005). The present study was the first attempt for identification of CAST gene variation in Tali and Raini goats. Further studies are required to investigate the relationship between CAST gene polymorphisms and performance traits in Tali and Raini goat.
The PCR-RFLP analysis of calpastatingene in Tali and Raini goats revealed high level of polymorphism. This information could be utilized in future breeding plan to exploit the genetic potential of tali and raeni goats. The Tali and Raini goats breed showed genetic diversity for the calpastatin gene, but the polymorphism found in the CAST gene may be helpful in selection programs for genetic improvement of meat traits. Though, previous to submission in the genetic development of the indigenous goat breeds, the relationship of these polymorphisms with meat traits needs to be recognized.