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Effects of calpastatin and µ-calpain markers in beef cattle on tenderness traits^1,2

E. Casas^*,3, S. N. White^*, T. L. Wheeler^*, S. D. Shackelford^*, M. Koohmaraie^*, D. G. Riley, C. C. Chase, Jr., D. D. Johnson and T. P. L. Smith^*

^* USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933; and USDA, ARS, Subtropical Agricultural Research Station, Brooksville, FL 34601; and and University of Florida, Gainesville 32611

	Abstract

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The objective of this study was to assess the association of single nucleotide polymorphisms (SNP) developed at the calpastatin (CAST) and µ-calpain (CAPN1) genes with meat tenderness and palatability traits in populations with diverse genetic backgrounds. Three populations were used in the study. One population consisted of Bos taurus that included crossbred animals derived from Hereford, Angus, Red Angus, Limousin, Charolais, Gelbvieh, and Simmental (GPE7; n = 539). Another population consisted of Bos taurus with Bos indicus influence, including crossbred animals from Hereford, Angus, Brangus, Beefmaster, Bonsmara, and Romosinuano (GPE8; n = 580). The third population was Bos indicus and consisted of purebred Brahman (STARS; n = 444). Traits evaluated were meat tenderness measured as Warner-Bratzler shear force (WBSF; kg) at 14 d postmortem, and traits evaluated by trained sensory panels that included tenderness score, juiciness, and flavor intensity. A SNP at the CAST gene had a significant (P < 0.003) effect on WBSF and tenderness score in the GPE7 and GPE8 populations. Animals inheriting the TT genotype at CAST had meat that was more tender than those inheriting the CC genotype. The marker at the CAPN1 gene was significant (P < 0.03) for tenderness score in GPE7 and GPE8. Animals inheriting the CC genotype at CAPN1 had meat that was more tender than those inheriting the TT genotype. Markers at the CAST and CAPN1 genes were associated with flavor intensity in the GPE8 population. Animals inheriting the CC genotype at CAST and the TT genotype at CAPN1 produced steaks with an intense flavor when compared with the other genotypes. An interaction between CAST and CAPN1 was detected (P < 0.05) for WBSF on GPE8. The statistical significance of the interaction is questionable because of the limited number of observations in some cells. Markers developed at the CAST and CAPN1 genes are suitable for use in identifying animals with the genetic potential to produce meat that is more tender.

Key Words: µ-calpain • calpastatin • cattle • genetic marker • meat tenderness • shear force

	INTRODUCTION

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Meat tenderness is one of the most important factors leading to consumer satisfaction when eating beef. Among the factors that have been identified as responsible for the postmortem meat tenderization process is the calpain proteolytic system. Two enzymes responsible for this process are the micromolar calcium-activated neutral protease µ-calpain (CAPN1), which is encoded by the CAPN1 gene, and its inhibitor, calpastatin (CAST), which is encoded by the CAST gene (Koohmaraie, 1996).

To date several markers have been developed at the CAST gene (Barendse, 2002), and 3 markers have been developed at the CAPN1 gene (Page et al., 2002; White et al., 2005). Previous studies (Barendse, 2002; Page et al., 2002, 2004; White et al., 2005) have independently evaluated markers at the CAST and CAPN1 genes. These studies have shown an association of individual markers at CAST and CAPN1 with meat tenderness in beef cattle. However, there has been no simultaneous evaluation of both genes to assess their effect in meat tenderization. Thus, the objective of the study was to assess the association of markers at the CAST and CAPN1 genes with meat tenderness in populations with diverse genetic backgrounds.

	MATERIALS AND METHODS

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Populations
Three independent populations were studied. The first was a population of Bos taurus descent, the second was a population that included germplasm from Bos taurus and Bos indicus, and the third was a purebred Bos indicus population.

Cycle 7 of the Germplasm Evaluation project (GPE7) included 539 crossbred steers of Bos taurus descent that were used in this study (Page et al., 2004; Wheeler et al., 2005). In brief, approximately equal numbers of calves were produced from 149 purebred sires representing the 7 beef breeds in the United States with the highest numbers of annual registrations (Hereford, Angus, Red Angus, Simmental, Gelbvieh, Limousin, and Charolais). These sires were mated to Angus, Hereford, or MARCIII (composite of ¹/3 Hereford, ¹/3 Angus, ¹/3 Pinzgauer, and ¹/3 Red Poll) cows. Management of cattle and collection of phenotypic data have been recently described by Wheeler et al. (2005).

Cycle 8 of the Germplasm Evaluation project (GPE8) included 580 crossbred steers that were used in this study (T. L. Wheeler, personal communication). Briefly, approximately equal numbers of calves were produced from 127 purebred sires representing tropically adapted breeds, including Beefmaster, Brangus, Bonsmara, and Romosinuano, as well as Hereford and Angus. All dams were Angus or MARCIII cows. Management of these animals and collection of phenotypes were similar to GPE7 (T. L. Wheeler, personal communication).

A population of 504 Brahman calves managed by the Subtropical Agricultural Research Station (Brooksville, FL) and collection of phenotypic data have been previously described (Riley et al., 2002) and will be referred herein as the STARS population. Briefly, 22 sires were used over 5 yr to produce Brahman calves in 1996 through 2000 (246 steers; 258 heifers). Calves were fed on site and were slaughtered at a commercial facility in Florida.

Traits Evaluated
Traits analyzed were meat tenderness measured as Warner-Bratzler shear force (WBSF), tenderness score, juiciness, and flavor intensity. Warner-Bratzler shear force data were collected on LM samples from steers at d 14 postmortem for GPE7 and GPE8 (Wheeler et al., 2005) and from steers and heifers at d 14 postmortem for STARS (Riley et al., 2003). Wheeler et al. (2005) and Riley et al. (2003) describe the method for obtaining tenderness scores, juiciness, and flavor from the steaks. In brief, 2.54 cm thick frozen steaks were thawed between 4° and 5°C during 18 to 24 h. Steaks were cooked, and samples were given to trained sensory panel members (AMSA, 1995). The panel members evaluated the steaks for tenderness, juiciness, and beef flavor on scales of 1 through 8 (1 = extremely tough, extremely dry, extremely bland; 8 = extremely tender, extremely juicy, extremely intense).

Markers Used
The single nucleotide polymorphism (SNP) developed at the CAST gene was reported by Barendse (2002). The marker is a transition from a guanine to an adenine at the 3' untranslated region of the gene. The marker will be referred to as CAST.

The marker developed at the CAPN1 gene was reported by White et al. (2005). The marker is a transition from a cytosine to a thymine at position 6545 of the GenBank accession AF248054 from the gene. The marker will be referred to as CAPN1 (White et al., 2005).

Genotyping
For the GPE7 and GPE8 populations, a saturated salt procedure (Miller et al., 1988) was used to obtain DNA from white blood cells. For the STARS population, DNA was obtained using a Qiagen QIAmp DNA blood mini kit (Valencia, CA). Blood samples were collected in 60-mL syringes with 4% EDTA. Blood was spun at 2,500 rpm for 25 min, and buffy coats were aspirated, cleaned, and frozen until DNA was extracted (Casas et al., 2005; White et al., 2005).

Genotyping was performed using a primer extension method with mass spectrometry-based analysis of the extension products on a MassArray system as suggested by the manufacturer (Sequenom, Inc., San Diego, CA) and as described by Stone et al. (2002). A universal mass tag sequence was added to the 5' end of each gene-specific amplification primer sequence as recommended by the manufacturer. Genotypes for each animal were collected, and the automated calls were checked by visualization of the spectrographs to minimize errors. Limited availability of tissue samples and problems with degradation of existing DNA samples hampered the collection of a complete dataset of all animals for the markers. When necessary, genotype assays were performed a second time to increase the number of successful genotypes, but samples were not tried a third time.

Statistical Methods
Model was evaluated using the Mixed procedure of SAS (SAS Inst., Inc., Cary, NC). The model used for GPE7 and GPE8 included sire breed, dam breed, the interaction between sire breed and dam breed, year of birth, slaughter group within year, CAST genotype, CAPN1 genotype, and the interaction between CAST and CAPN1 genotypes as fixed effects (White et al., 2005). The interaction between the CAST and the CAPN1 genotypes was removed from the model when not significant. Weaning age was included as a linear covariate. Sire was included as a random effect nested within sire breed. The model for the STARS population included the random effect of sire, the fixed effects of contemporary group (1 through 44), CAST genotype, CAPN1 genotype, and the interaction between CAST and CAPN1 genotypes (Casas et al., 2005). The interaction between the CAST and the CAPN1 genotypes was removed from the model when not significant. Contemporary group was defined as a group of calves of the same gender, fed in the same pen, and slaughtered on the same date. There were 44 contemporary groups in the study. Probability values were not corrected for multiple testing.

	RESULTS

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Phenotypic Variation
Traits evaluated, number of records, and simple statistics are presented in Table 1. All traits displayed substantial variation within population. A total of 539, 580, and 444 individuals were used for GPE7, GPE8, and STARS, respectively, in the study (Table 2). Samples from 24, 28, and 60 individuals in GPE7, GPE8, and STARS, respectively, were unable to amplify any product to be genotyped for the CAST assay, the CAPN1 assay, or both. The statistics presented in Table 1 are for the total number of individuals with phenotypic information, regardless of whether the samples produced successful genotypes for both markers.

CAST Effect
Levels of significance, least squares means, and standard errors are reported in Table 3 for the effect of CAST on WBSF, tenderness score, juiciness, and flavor in the populations studied. The marker at the CAST gene was associated (P < 0.01) with WBSF and tenderness score in the GPE7 and GPE8 populations. Animals inheriting the CC and the CT genotypes produced tougher meat when compared with animals that inherited the TT genotype. There was an association (P < 0.01) of the CAST marker with juiciness in the GPE7 population. There was an unclear pattern because animals inheriting the CT genotype produced less juicy steaks than animals homozygous for either CC or TT genotypes. An association was observed (P < 0.01) between CAST and flavor in the GPE8 population. Animals inheriting the CC genotype produced blander steaks than animals inheriting the CT and TT genotypes.

View larger version (14K): [in this window] [in a new window]   Figure 1. Interaction of µ-calpain genotype and calpastatin genotype for Warner-Bratzler shear force (WBSF; kg).

	DISCUSSION

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Previous studies have suggested that genetic variation at the CAST locus contributes to variation in meat tenderness traits (Barendse et al., 2002), but the data presented here represent the first report in the scientific literature of the association of the CAST SNP with meat tenderness. (The original finding was patented in 2002.) In the original work, animals homozygous for the T allele were observed to produce meat with lower average shear force than animals homozygous for the C allele. A similar result was obtained in the present study for the 2 populations incorporating Bos taurus germplasm. It is possible that the lack of statistical significance in STARS reflects that the present marker system is not adequately matched to functional alleles to be useful in Bos indicus populations. Alternatively, the influence of the variation may be smaller in the STARS genetic background and fall below a detectable level. In total, the present data support the conclusion that the SNP at the CAST gene are associated with functional alleles of CAST that affect shear force and indicate that these effects extend to many, but perhaps not all, beef breeds.

A previous study documented the effect of CAPN1 genotype on shear force in GPE7, GPE8, and STARS; animals homozygous for the C allele had lower average shear force than animals of TT genotype (White et al., 2005). The magnitude of the observed effect on shear force is approximately the same as observed for alleles at CAST. The present study also examined tenderness as measured by an expert taste panel. Significant associations of genotype and taste panel tenderness were observed for GPE7 and GPE8, and the magnitude of effect was nearly identical for both markers. In contrast, the reduced tenderness of the unfavorable genotype in STARS did not reach significance because of the lack of CC homozygotes in STARS, which reduced the ability to detect significant association. Because of this constraint in the STARS data, we concluded that the data extend association of genotype at the CAPN1 SNP to include tenderness as measured by an expert taste panel.

The association of both CAST and CAPN1 markers with tenderness in 2 of the 3 populations of widely varied breed makeup supports the investigation of potential genetic interaction between the 2 loci. This analysis depends on the frequency of 2-marker genotypes to compare individual "cells" of genotype class. Allele frequency of the 2 markers in the 3 sets of animals was quite variable. Favorable T allele frequency for the CAST SNP was much higher in GPE7 (80%), GPE8 (83%), and STARS (72%) than the favorable C allele of CAPN1 (58, 64, and 10%, respectively). As a result, some of the 2-genotype cells had very few or no individuals, reducing the power to detect interaction between the 2 loci. Specifically, there was severe under-representation of animals that were homozygous for unfavorable alleles at both markers (CC at CAST and TT at CAPN1), an important class for detecting interaction in all 3 groups of animals. Nevertheless, the data from GPE7 and GPE8 provide an opportunity to investigate the potential for interaction among most of the possible genotypes.

Analysis of the 2 markers independently suggests that if the loci do not display genetic interaction and act in an additive fashion, the 2-marker genotype with lowest shear force would tend to be TT at CAST and CC at CAPN1. An apparent interaction was manifest in GPE8 because animals with CC genotype at both markers had the lowest average shear force and the highest tenderness rating. It must be emphasized that this genotype cell had only 4 animals; therefore, the mean effect of this combination of genotypes is underestimated, and the result should be considered inconclusive. A more stringent test for the specific effect of the rare 2-marker genotype class would require identification of a population or populations segregating a higher frequency of respective homozygotes at the 2 SNP. The failure to detect interaction in GPE7, in which there were more than twice as many animals in the same genotype cell than in GPE8, further weakens the hypothesis of interaction and suggests that despite the physical interaction of the gene products within the cell, the alleles of the CAST and CAPN1 loci defined by the SNP used in this study probably do not have significant genetic interaction in determining shear force. Therefore, the markers appear to act in an additive fashion in predicting changes in mean shear force in cattle populations.

Data on 2 other measures of meat quality were also evaluated in this study to provide some insight into the possibility that selection based on genotype at these markers might have unintended consequences on other traits. There was no clear evidence for detrimental effects of SNP alleles on juiciness or flavor. There was a statistically significant but small decrease in juiciness among the GPE7 steers with the favorable TT genotype at CAST and an increase in flavor of this genotype in GPE8, but because these effects were small and not consistently observed among populations, it is unlikely that they would result in a major impact on phenotype during marker-assisted selection. Similarly, the only significant effect of CAPN1 genotype was an increase in flavor in the favorable CC genotype in GPE8 animals. The only change in mean with a magnitude > 0.23 units for flavor or juiciness in any genotypic class was a statistically nonsignificant increase in juiciness in GPE7 steers with the favorable CC genotype at CAPN1. These results suggest that selection on genotype at these 2 loci will have negligible effects on these 2 meat quality traits. We conclude that selection for favorable alleles at CAST and CAPN1 as defined by the SNP genotypes described so far would be likely to improve mean shear force values without genetic interaction between the 2 loci to complicate selection procedures and without discernable effect on meat quality parameters of juiciness and flavor.

	IMPLICATIONS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 
Markers at the bovine calpastatin and µ-calpain loci that were previously described have been associated with meat tenderness in cattle. This report is the first evaluation of the calpastatin marker and demonstrates that the effects of the 2 loci, as identified by the single nucleotide polymorphism used in the study, appear to act in additive fashion to influence shear force. The impact on tenderness and apparent lack of interaction were observed in a wide range of beef breeds including animals of Bos taurus, Bos taurus, and Bos indicus influence and Bos indicus descent. There was no indication that selection for the single nucleotide polymorphism genotype at either locus would have a significant influence on other meat quality traits such as juiciness or flavor.

	Footnotes

 
¹ Mention of trade name, proprietary product, or specified equipment does not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of other products that may be suitable.

² The authors thank E. Bowers, D. Brinkerhoff, L. Flatham, R. Godtel, M. Rooks, D. Sartain, S. Simcox, K. Simmerman, K. Tennill for technical assistance, the U.S. Meat Animal Research Center and Subtropical Agricultural Research Station staff for outstanding husbandry and animal care, and J. Watts for secretarial support.

³ Corresponding author: casas{at}email.marc.usda.gov

Received for publication September 8, 2005. Accepted for publication October 27, 2005.

	LITERATURE CITED

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AMSA. 1995. Research Guidelines for Cookery, Sensory Evaluation, and Instrumental Tenderness Measurements of Fresh Meat. Natl. Livestock and Meat Board, Chicago, IL.

Barendse, W. J. 2002. DNA markers for meat tenderness. International patent application PCT/AU02/00122. International patent publication WO 02/064820 A1.

Casas, E., S. N. White, D. G. Riley, T. P. L. Smith, R. A. Brenneman, T. A. Olson, D. D. Johnson, S. W. Coleman, G. L. Bennett, and C. C. Chase, Jr. 2005. Assessment of single nucleotide polymorphisms in genes residing on chromosomes 14 and 29 for association with carcass composition traits in Bos indicus cattle. J. Anim. Sci. 83:13–19.[Abstract/Free Full Text]

Koohmaraie, M. 1996. Biochemical factors regulating the toughening and tenderization process of meat. Meat Sci. 43:S193–S201.

Miller, S. A., D. D. Dykes, and H. F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215.[Free Full Text]

Page, B. T., E. Casas, M. P. Heaton, N. G. Cullen, D. L. Hyndman, C. A. Morris, A. M. Crawford, T. L. Wheeler, M. Koohmaraie, J. W. Keele, and T. P. L. Smith. 2002. Evaluation of single nucleotide polymorphisms in CAPN1 for association with meat tenderness in cattle. J. Anim. Sci. 80:3077–3085.[Abstract/Free Full Text]

Page, B. T., E. Casas, R. L. Quaas, R. M. Thallman, T. L. Wheeler, S. D. Shackelford, M. Koohmaraie, S. N. White, G. L. Bennett, J. W. Keele, M. E. Dikeman, and T. P. L. Smith. 2004. Association of markers in the bovine CAPN1 gene with meat tenderness in large crossbred populations that sample influential industry sires. J. Anim. Sci. 82:3474–3481.[Abstract/Free Full Text]

Riley, D. G., C. C. Chase, Jr., A. C. Hammond, R. L. West, D. D. Johnson, T. A. Olson, and S. W. Coleman. 2002. Estimated genetic parameters for carcass traits of Brahman cattle. J. Anim. Sci. 80:955–962.[Abstract/Free Full Text]

Riley, D. G., C. C. Chase, Jr., T. D. Pringle, R. L. West, D. D. Johnson, T. A. Olson, A. C. Hammond, and S. W. Coleman. 2003. Effect of sire on µ- and m-calpain activity and rate of tenderization as indicated by myofibril fragmentation indices of steaks from Brahman cattle. J. Anim. Sci. 81:2440–2447.[Abstract/Free Full Text]

Stone, R. T., W. M. Grosse, E. Casas, T. P. L. Smith, J. W. Keele, and G. L. Bennett. 2002. Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers. Mamm. Genome 13:211–215.[Medline]

Wheeler, T. L., L. V. Cundiff, S. D. Shackelford, and M. Koohmaraie. 2005. Characterization of biological types of cattle (Cycle VII): Carcass, yield, and longissimus palatability traits. J. Anim. Sci. 83:196–207.[Abstract/Free Full Text]

White, S. N., E. Casas, T. L. Wheeler, S. D. Shackelford, M. Koohmaraie, D. G. Riley, C. C. Chase, Jr., D. D. Johnson, J. W. Keele, and T. P. L. Smith. 2005. A new SNP in CAPN1 is associated with tenderness in cattle of Bos indicus, Bos taurus, and crossbred descent. J. Anim. Sci. 83:2001–2008.[Abstract/Free Full Text]

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