Polymorphisms of calpain gene in Colombian Creole cattle

M A Moreno*,**, A M Gallón*,** W A Mesa*,**, A E Montoya*,** and M F Cerón-Muñoz*,***

* Research Group in Genetics and Animal Breeding,
^** Institute of Biology,
^*** Faculty of Agricultural Sciences, University of Antioquia, Medellín-Colombia. Instituto de Biología. Universidad de Antioquia, Calle 67 N 53-108. (7-106), Medellín, Colombia
mmoreno@matematicas.udea.edu.co

Abstract

The objective of this study was to determine polymorphisms in calpain (Capn2) gene which has been known as candidate gene in meat tenderization, in breeds with different genetic backgrounds. Genomic DNA was isolated from 323 individuals from four Creole breeds: Hartón del Valle, Romosinuano, Blanco Orejinegro and Sanmartinero and one Colombian Breed: Velásquez. Capn2 polymorphisms were characterized using polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis.

Two alleles, A and B were found in all breeds analyzed in this study. All populations showed higher frequencies of heterozygote genotype and allele frequencies did not showed big differences, except Romosinuano and Sanmartinero, which exhibited marked differences on their alellic frequencies, being A allele in a lower frequency. Our results could be used to guide association studies between these polymorphisms and meat traits in these breeds.

Key words: Breeds, meat quality, tenderness

Introduction

Skeletal muscles of different vertebrates are composed of approximately 75% water, 20% protein, various amounts of lipids and carbohydrates, and a small amount of soluble organic compounds (Jiang 1998). In recent years there has been a shift in emphasis in livestock production away from increased muscle growth towards improved meat quality, as muscle is converted to meat a number of metabolic and structural changes occur, the final eating quality of meat depends on a number of organoleptic properties including appearance, color, fat content, taste, texture and tenderness (Sensky et al 2001).  Some studies have shown that is the degree to which muscle tenderizes after slaughter that is one of the most critical factors contributing to overall meat quality and leading to costumer satisfaction when eating beef (Tarrant 1998; Koohmaraie 1995; Jiang 1998). Tenderization process involves complex changes in muscle metabolism after slaughter depending on genetic background, protein complement, metabolic status and environmental factor (Koohmaraie and Geesink 2006; Takahashi 1996; Goll et al 1995). However, the main determinant of ultimate tenderness appears to be the extent of proteolysis of the key target proteins within muscle fibers (Koohmaraie and Geesink 2006; Hwang et al 2003; Taylor et al 1995). Myofibrilar toughness is affected by the development of rigor mortis meanwhile tenderness is caused by enzymatic break-down of contractile proteins (Koohmaraie 1996). Unlike the toughening process, tenderization does not occur equally in all the animals (Taylor et al 1995; Koohmaraie 1992a, 1992b). Many biochemical changes during post-mortem tenderization have been assumed to arise from the release of endogenous muscle proteases which are active at the post-mortem pH of muscle (Robbins et al 1979).

Although several proteolytic systems related to the tenderization process have already been described, most of the research in livestock species has pointed to the two of them, calpains and lysosomal proteinases, suggesting that tenderization is primarily a result of calpain-mediated degradation of myofibrilar and cytoskeletal proteins (Sensky et al 2001; Luciano et al 2007; Costello et al 2007; Casas et al 2006; Veiseth et al 2004; Maltin et al 2003; Wheeler et al 2000; Boehm et al 1998; Koohmaraie 1996, 1992b; Taylor et al 1995; Calkins and Seideman 1988).  

Calpains are calcium-dependent proteases consisting of at least three proteases, µ-calpain, m-calpain and skeletal muscle-specific calpain (Koohmaraie and Geesink 2006). The calpain system is highly sensitive to fluctuating levels of calcium ion, pH and temperature, all of which change rapidly in the immediate post-mortem period; both µ-calpain and m-calpain decline relatively rapidly post-mortem while tenderization continues for up to four weeks, depending on the species. Nevertheless, there is considerable evidence linking the calpains to tenderization in beef, lamb and pork (Koohmaraie et al 1991), is clear that both calpains are active at physiological levels of Ca²⁺, which are lower than those used to assay calpain activation in vitro, it is probable that these levels would be sufficient to fully activate m-calpain. In addition, as the enzyme loses little of its activity with post-mortem storage, it is conceivable that to date the relative contribution of m-calpain activity to post-mortem proteolysis has been underestimated (Maltin et al 2003).

Modern breeding strategies have been focused to achieve costumer’s expectations. Nowadays we can find a product labeled as “guaranteed tenderness” such as marinades and case-ready products (Koohmaraie et al 2002), and costumers are willing to pay more for this kind of products (Feldkamp et al 2005). Considering the hypothesis of a possible relationship of CAPN2-A allele with meat tenderization (Lara et al 2005), it will allow to identify precociously the desirable animals, favoring the genetic progress of cattle populations.

Colombia has seven Creole Cattle breeds, namely Romosinuano, Costeño con cuernos, Blanco Orejinegro, Sanmartinero, Hartón del Valle, Chino Santandereano and Casanareño and two Colombian cattle (Velásquez and Lucerna). Some genetic studies on Colombian Creole cattle has shown the value of these breeds in tropical production systems; nevertheless few studies have been carried out in order to determine association between gene polymorphisms and  some quality traits. Due to the high importance of the Creole cattle and their decreasing number during the last years, Colombia government established conservation policies in order to set up selection strategies based on morphologic, productive and genetic characterization (Barrera et al 2006). Selection for breed producing high quality carcasses would also be beneficial for producers who value the adaptability and productivity of Colombian Creole cattle.

Identifying the reasons for the variability of the rate and the extent of post-mortem tenderization, especially genetic polymorphisms, would make possible to manipulate crosses in order to predict tenderization process in carcasses and guarantee costumer satisfaction. 

The objective of this study was to determine the occurrence of polymorphisms in calpain gene of five Colombia Creole Cattle.

Materials and methods

Population sample

A total of 323 DNA samples were obtained from five Colombian Creole cattle: 60 samples of Romosinuano (RM) animals, located in Valle del Cauca, Meta and Antioquia states; 84 samples of Hartón del Valle (HV) animals, located at Valle del Cauca state; 69 samples of Sanmartinero (SM) animals, located in Meta state; 68 samples of Blanco Orejinegro (BON), located in Antioquia state and 42 samples of Velasquez (Vz) animals, located in Caldas state. All DNA samples were provided by the DNA Bank of the Laboratory of Animal Genetics at the University of Antioquia, Medellín-Colombia.

PCR reaction and DNA amplification

Primers described by Zhang et al (1996) were used to amplify a predicted 1800 b.p. fragment: Fwd: 5’-CCCCTCGCACACATTACTCCAAC-3’, and Rev: 5’-ATACGGCCTGCCACTTTTTGATG-3’. Amplification for capn2 gene was performed in a 20µl final reaction volume containing 1X PCR buffer (100mM Tris-HCL pH 8,8; 500mM KCl; 0,8 % Nonidet P40), 1.25mM MgCl₂, 200µM dNTPs mix, 0.53µM primer mix, 2.5U Taq polymerase and about 100ng genomic DNA. Amplification profile included an initial denaturation for 5 min at 95⁰ C; followed by 30 cycles of 1 min at 95⁰ C, 1 min at 62.6⁰ C, 2 min at 72⁰ C; 7 min at 72⁰ C. Amplification process was made on a T-Personal 48 thermocycler (Biometra GMBHD – 37079 Goettingen, Germany). After amplification, all samples were verified through 2% agarose gel electrophoresis, stained with ethidium bromide, and viewed under UV light.

RFLP (Restriction Fragment Length Polymorphism) reaction

After capn2 gene amplification, fragments were subjected to digestion by HhaI restriction endonuclease (Fermentas®), in a 20 µl final reaction volume, containing 8 microlitres of PCR product, 4 units of enzyme and buffer Tango, incubated at 37 ºC in water bath for 3 h.

Genotyping

After digestion, five microlitres of restriction reaction were subjected to 2% agarose gel electrophoresis, stained with ethidium bromide, and then visualized under UV light. Polymorphisms could be observed by the size change of the DNA fragment. Two, three or four fragments of projected sizes were expected to be generated according to enzyme cuts when it recognizes or not GCG’C sequences (Lara et al 2005). Evaluation of fragments size were made by comparing with pGEM® DNA marker.

Computation and statistical analysis

GENEPOP program, version 4.0 (Rousset 2008) was used to calculate the allele and genotypic frequencies, and to test for Hardy-Weinberg equilibrium departures.

Results and discussion 

Using designations from Zang et al (1996), genotypes were assigned according to the observed patterns of RFLP bands. Digestion of fragment capn2 by HhaI generated two or three fragments, based up on the presence of a restriction site. The RFLP analysis revealed the existence of two alleles; these results are similar to those found by Zhang et al (1996); Lara et al (2005), and Costello et al (2007), with a band pattern of 280, 620 and 900 bp, or 1520 and 280 bp, respectively for the two alleles A and B; those two observed alleles resulted in three genotypes in all cattle populations.

Genotype frequencies of capn2 gene fragment are presented in table 1, from which it can be noticed that AB heterozygous genotype frequency was higher in all cattle populations.

There were no significant differences among the most of the populations for allelic and genotypic frequencies for this locus. Otherwise, a substantial difference was noticed between Romosinuano and the rest breeds, for which AA genotype was found at a low frequency. All creole breeds showed relative high allelic frequencies, in relation to A allele, which is the desirable one, related to tenderness in meat acording to Lara et al (2005), and those values are similar to those from Aberdeen Angus cattle, a meat-specialized European breed (Chung et al 1999).

Hardy-Weinberg equilibrium test

Using GENEPOP program, as shown in table 2, all populations were found to be in HWE (p> 0.05), except for Romosinuano population which showed departure from H-W equilibrium (p=0.008).

Harton del Valle population showed a positive value for Fis, therefore a heterozygote  deficiency test was applied and it was found that it was not significant (p>0.05). Romosinuano, Blanco Orejinegro, Sanmartinero and Velasquez populations, with a negative value for Fis, were used to apply the heterozygote excess test, it showed no significance.

Comparing genotypic frequencies, it is possible to affirm that in this study it were found that the two alleles for Capn2 gene (A and B) were distributed in all possible genotypes, nevertheless  Romosinuano catlle exhibit the lowest frequency of homozygous AA genotype; whereas some breeds analyzed by Lara et al (2005) showed only two of the three genotypes (Table 3).

Knowing about the presence of alleles previously related to tenderness through those populations, would allow farm owners to decide which individuals to breed together. Such a way, selective breeding crosses programs could be carried out in order to enhance frequencies of the desired allele.

Conclusions 

• Colombian Creole cattle studies indicate that gentle differences exist between breeds of catlle regarding to capn2 gene allele frequencies, both alleles (A and B) were present in the three possible genotypes, which means that Colombian Creole Cattle breeds exhibit big opportunities for being object of artificial selection for this locus, related to meat quality.

  
  

• More studies need to be conducted on the capn2 gene to determine association between these polymorphisms and meat traits such as tenderness in these breeds.

  
  

• Special attention should be directed to Romosinuano breeding crosses programs in order to enhance AA genotype and probably reaching high quality carcasses.

Acknowledgements 

Authors would like to acknowledge all the farm owners (Vegas de La Clara, Hatoviejo, Corpoica el Nus, Corpoica La Libertad and África), and Secretaría de Agricultura del Meta who kindly supplied samples for this study, and to the Comité para el Desarrollo de la Investigación, CODI, at the University of Antioquia for sponsoring this work.

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Received 8 December 2008; Accepted 30 April 2009; Published 1 June 2009

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