Characterization of Bovine Calpastatin Gene in Nelore Cattle Using Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms

Problem statement: In beef cattle production, of meat quality and carc ass traits are important. Traditionally beef cattle breeding prog rams unfortunately are time consuming and also recording of carcass and growth traits need heavy c ost, Approach: Marker Assisted Selection (MAS) should be utilized in beef herds, along with econom ically important phenotypic traits, for genetic progress to made with respect to improving the unif ormity and consistency of beef. Blood samples were collected from 41 nelor cattle in Malaysia. Fo rward and reversed primers amplified a 1552 bp fragment from calpastatin gene. XmnI enzyme was use d for restriction analysis of PCR products. Result: Overall, the frequency of alleles A and B in the s tudied breeds were estimated as 0.42 and 0.58, respectively. In this study we calculated gen otype frequency AA, AB and BB 12.2, 58.53 and 29.27% respectively and also observed heterozygosit y, expected heterozygosity and average value of heterozygosity were 0.58, 0.49 and 0.48 respectivel y. Highest frequency of allele was B (0.58) and lowest was A (0.42) This Nelor cattle population wa s in the Hardy-Weinberg equilibrium. Conclusion: Perhaps, this molecular genetic information helps breeders for designing the proper genetic selection program in the development direct ion of this breed.


INTRODUCTION
In Malaysia, there is an increasing preference by meat consumers for high quality and tender meat. It is clear that genetic background of the slaughtered animals makes a significant contribution to the variation in meat tenderness as tenderness varies among and within breeds.
Recently, genetic tests for meat tenderness in beef, utilizing genetic polymorphisms in the Calpastatin(CAST) and/or Calpain gene, have been made available by many private companies. The Calpain/calpastatin system is an endogenous, calciumdependent proteinase system, theorized to mediate the proteolysis of key myofibrillar proteins during postmortem storage of carcass and meat cuts at refrigerated temperatures [3] . Calpain is responsible for the breakdown of myofibril proteins, which are closely related to meat tenderness [11] . Calpastatin (CAST) inhibits µ and m-Calpain activity and, therefore, regulates postmortem proteolysis. Increased postmortem CAST activity has been correlated with reduced meat tenderness [3,8] . The CAST gene, mapped to BTA 7 [1] , is considered a candidate gene for beef tenderness. The Nelore cattle is considered as a breed for economic beef production in the tropics and makes up 65% of the world's bovine population. The Nelore can efficiently convert poor quality forages into beef and withstand long periods without water. Due to their habit of feeding lightly but frequently, Nelore and Nelore hybrid cattle are highly resistant to bloating, which is a cause of death in other breeds. Excellent feed conversion ratio and good disposition allows the Nelore to be early finished in feedlots, with highly positive results [6] . Currently, Malaysia imported large number of this cattle breed from Brazil due to their high adaptation and productivity.
The objective of this study was to assess the allelic variation at the bovine calpastatin gene in the Nelore cattle in Malaysia using PCR-RFLP.

MATERIALS AND METHODS
DNA was extracted from the blood of 41 animals from the Nelore herd of the Department of veterinary services Malaysia. The DNA was amplified with primers for calpastatin gene Chung et al. [2] : Samples were amplified for 35 cycles (Thermocycle MJ Research PTC-200) with the following regime: denaturation step at 95°C for 1 min, annealing at 65°C for 30 sec and extension step at 74°C for 40 sec.
Products of amplification were checked by electrophoresis in 1% agarose gel. The gel stained with ethidium bromide and viewed under UV light.

PCR-RFLP:
The PCR products were digested by XmnI the restriction endonuclease. Digestion was conducted at 37°C for 24 h and in a 10 µL reaction solution contains 2.7 µL distilled H 2 O, 1.0 µL of specific Buffer, 0.3 µL (3 U) of restriction endonuclease and 6 µL of PCR product solution. The DNA fragments from the digested PCR products were separated 2.0% electrophoresis agarose gel containing in ethidium bromide (0.4 µg mL −1 ). Electrophoresis was performed in 1 × TBE buffer (108 g Tris, 55 g boric acid and 40 mL of 0.5 M EDTA in 1,000 mL of 10 × concentrated stock solution, pH 8.0) under 120 V for 45 min.

RESULTS
Three genotypes were observed in this population and these are shown in Fig. 1. Two alleles were observed. The frequency of alleles A and B in the population were 0.42 and 0.58, respectively. The genotypic frequencies of AA, AB and BB were 0.122, 0.585 and 0.293, respectively. Observed, expected heterozygosities were 0.58, 0.49 and 0.48, respectively. The Nelore population was in Hardy-Weinberg equilibrium for the locus (p>0.05). The information generated from this study may be utilized in designing selection program for this breed.

DISCUSSION
Shackelford et al. [10] showed that approximately 65% of the variation in tenderness among cattle of all breeds was due to genetic effects. In an experiment with Angus bulls Palmer et al. [7] observed three genotypes and two alleles for CASTI and CAST5 loci and six genotypes AA, BB, CC, AB, AC and BC, for CAST10 locus. Single Nucleotide Polymorphisms (SNPs) in the calpastatin (CAST) gene was studied in Bos taurus (Jersey × Limousin, Angus and Hereford-cross cattle) by Morris et al. [5] . Schenkel et al. [9] identified a SNP in the CAST gene (G to C substitution) in the crossbred commercial heifers, steers and bulls from the beef feedlots of the University of Guelph. They identified three genotypes (CC, CG and GG) and reported that the CAST's SNP allele C was more frequent (63%) than Gallele. Kurly et al. [4] identified the polymorphism of Calpastatin gene with three restriction enzymes (Hinf I, MspI, RsaI) in Stambeek (Dutch Large white× Dutch Landrrace) pig breed.

CONCLUSION
The results of this study showed that the frequency of allele B was higher than frequency of A in the Nelore population. This Nelor cattle population was in the Hardy-Weinberg equilibrium. This molecular genetic information may be used by breeders for designing the genetic selection program for the development of this breed.

ACKNOWLEDGMENT
The researchers would like to express their deep appreciation to the Department of Veterinary Services Malaysia for their support of this project. We thank the management and staff of farm for their assistance in sampling of animals.