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ANIMAL GENETICS |
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* ARS, USDA, U.S. Meat Animal Research Center, Clay Center, NE 68933;
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ARS, USDA, Subtropical Agricultural Research Station, Brooksville, FL 34601; and
and
Department of Animal Sciences, University of Florida, Gainesville 32611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Brahman • Carcass Traits • DGAT1 • µ-Calpain • Thyroglobulin
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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, 2004). However, conflicting results on its effect on fat deposition in beef cattle have been reported (Moore et al., 2003; Thaller et al., 2003b). The SNP reported in TG, which encodes the glycoprotein precursor to the thyroid hormones, lies in a repetitive element upstream from the promoter of the gene, and has been associated with differences in marbling score in beef cattle (Barendse, 1999). The CAPN1 gene, encoding a cysteine protease (µ-calpain) that seems to be the primary enzyme in the postmortem tenderization process (Koohmaraie, 1996), has been mapped to chromosome 29. Several SNP in this gene have been reported to be associated with meat tenderness in beef cattle (Page et al., 2002, 2004), two of which produce glycine-to-alanine and valine-to-isoleucine substitutions in exons 9 and 14, respectively. Association studies of SNP in these three genes with carcass traits have primarily been performed in Bos taurus cattle. There is no publicly available evaluation of the association of these markers with carcass composition traits in Bos indicus cattle. Thus, the objective of this study was to assess the association of reported SNP in the DGAT1, TG, and CAPN1 genes with carcass composition and meat quality traits in Bos indicus cattle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. In brief, a progeny test of 22 Brahman sires was conducted by mating them to Brahman cows from 1995 to 1999. Calves (n = 504, including 258 heifers and 246 steers) were fed on site and were slaughtered at a commercial facility in Florida.
Traits Evaluated
Traits analyzed were ADG, hip height, slaughter weight, 12th-rib adjusted fat thickness (USDA, 1990), HCW, marbling score, LM area (LMA), estimated KPH fat, yield grade, retail yield, carcass hump height, and cooked meat tenderness measured as Warner-Bratzler shear force at 7, 14, and 21 d postmortem and sensory panel tenderness score at 14 d postmortem (Riley et al., 2002, 2003). Marbling score was evaluated on a cross section of the longissimus muscle at the 12th-to 13th-rib interface as follows: Devoid = 100 to 199; Traces = 200 to 299; Slight = 300 to 399; Small = 400 to 499; Modest = 500 to 599; and Moderate = 600 to 699. The yield percent of boneless, closely trimmed retail cuts from the round, loin, rib, and chuck (cutability) was estimated using the equation originally proposed by Murphey et al. (1963): 51.34 – (2.277 x adjusted fat thickness over the 12th rib) – (0.462 x % kidney, pelvic, and heart fat) – (0.0205 x hot carcass weight) + (0.1147 x LMA). Retail product yield was then estimated as the product of cutability and HCW. Carcass hump height was measured from the most dorsal point of the hump to the dorsal edge of the ligamentum nuchae.
Markers Used
Four SNP in the bovine CAPN1 gene (GenBank accession AF248054 and AF252504) were genotyped. Marker CAPN316 is a cytidine/guanosine (C/G) polymorphism in exon 9 of the gene (base 5709 of AF252504) that produces an AA substitution (C allele codes for alanine, G allele codes for glycine; Page et al., 2002). Marker CAPN530 is an adenosine/guanosine (A/G) polymorphism in exon 14 of the gene (base 4558 of AF248054) that produces an amino acid substitution (A allele codes for isoleucine, G allele codes for valine; Page et al., 2002). Amplification primers and probes for these two markers have been described previously (Page et al., 2004). Marker CAPN4753 is an adenosine/cytidine polymorphism that lies in intron 21 of the gene (position 8676 of AF248054), and CAPN5331 is an adenosine/thymidine (A/T) polymorphism that lies in intron 1 of the gene (position 327 of AF252504). Gene-specific primer sequences for amplification and primer-extension genotyping of markers CAPN4753 and CAPN5331 were as follows: CAPN4753 forward amplification: 5'-TCTCTGGTTTCTGAGGGTGG-3'; CAPN4753 reverse amplification: 5'-GGCATAGAGAGCAGTCAGCC-3'; CAPN4753 probe primer: 5'-CTCTCCCTCCTGCCCTT GA-3'; CAPN5331 forward amplification: 5'-GGGCCG AGGAGATACCGTGAA-3'; CAPN5331 reverse amplification: 5'-GCTTCCCGGGTGGCAACTG-3'; and CAPN5331 probe primer: 5'-AACCAGGAAGAGCGCT CC-3'.
The two SNP in the DGAT1 gene lie immediately adjacent to one another in exon 8 (position 6829 and 6830 of accession AY065621) and were described by Grisart et al. (2001). Gene-specific primer sequences for this locus were as follows: DGAT6829 forward amplification: 5'-CTACCGGGACGTCAACCTC-3'; DGAT6829 reverse amplification: 5'-GGTTGTCGGGG TAGCTCA-3'; and DGAT6829 probe primer: 5'-AGCT CCCCCGTTGGCC-3'.
The two alleles at this locus are AA and CG, encoding lysine (K) and alanine (A) at amino acid 232 of the acyl-CoA:diacylglycerol acyltransferase enzyme, respectively. Due to the dual-SNP nature of this locus, the genotypes corresponding to the K232A DGAT1 alleles are herein referred to as KK, KA, and AA (AA indicates homozygous animals that encode alanine) as described by Thaller et al. (2003a).
The SNP in the TG gene is a C/T polymorphism in a repetitive element upstream from the promoter (position 422 of accession X05380; Barendse, 1999). Gene-specific primer sequences for this locus were as follows: TG422 forward amplification: 5'-ATTCTTGCCTGG-GAAATCCC-3'; TG422 reverse amplification: 5'-AGTC-GTATCTGACTCTTTC-3'; and TG422 probe primer: 5'-TGGGTTGGGAAGAT-3'.
Genotyping
Blood (36 mL) from each animal was collected into four 9-mL Sarstedt Monovette EDTA KE tubes (Sarstedt, Inc., Newton, NC). The white blood cell layers, or buffy coats, were isolated and cleaned by centrifugation through Sigma Diagnostics Accuspin System using Sigma Diagnostics Histopaque-1077 solution (Sigma, St. Louis, MO) as prescribed in the accompanying protocol, and stored at –80°C. The DNA was extracted from a 200-µL aliquot of white blood cells using a Qiagen (Valencia, CA) QIAmp DNA blood mini kit as described in the accompanying protocol, and stored at –80°C.
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 all 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 following model was used:
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where Yijkl = phenotypic observations, µ= overall mean, Ci = fixed effect of contemporary group (1 through 44), Sj = random effect of sire, Mk = fixed effect of marker genotypes (CAPN316, CAPN4753, CAPN5331, TG, or DGAT1), ßage = linear effect of age of calf as covariate, and eijkl = random error. 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 for all traits in the study. Each marker was fitted independently in the model. Probability values were nominal and do not correct for multiple testing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. All traits displayed substantial variation within the population, including meat tenderness characteristics. A total of 479 individuals were used in the study. Samples from 25 of the 504 animals in the population were either not collected or were unable to amplify any product to be genotyped, and were thus excluded from the study. The statistics presented in Table 1 are for the 479 calves in the population with DNA samples, regardless of whether the samples produced successful genotypes for all markers.
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shows the genotype frequencies for the markers used. Some genotypes were present at low frequency in these Brahman animals. For example, no animals were homozygous CC at marker CAPN316. In addition, only seven and five animals had the genotypes TT and AA, respectively, at the TG and DGAT1 markers. The low frequency of these genotypes interferes with accurate assessment of their association with carcass traits, especially if any of the effects have a dominant/recessive mode of action or the additive effects are relatively small. In contrast, genotypes CC and TT at markers CAPN4753 and CAPN5331, respectively, were in low frequency, but adequate number of animals had these genotypes to estimate their association with phenotypic traits.
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Marker Associations
Levels of significance, least squares means, and standard errors are reported in Table 3 for the effects of CAPN316, CAPN4753, CAPN5331, TG, and DGAT1 on fat thickness, marbling score, LMA, sensory panel tenderness score, and hump height. The marker CAPN316 was associated (P < 0.05) with sensory panel tenderness score. Animals with the GG genotype were tenderer than those with the CG genotype.
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). This could indicate that both markers are in linkage disequilibrium and segregating as a haplotype.
Thyroglobulin had a significant effect on fat thickness and LMA (P < 0.05; Table 3). For both traits, heterozygous animals were leaner but with a smaller LMA than either homozygous genotype.
No association was detected between any of the markers with ADG, hip height, slaughter weight, HCW, marbling score, estimated KPH fat, yield grade, retail yield, and shear force measured as Warner-Bratzler shear force at 7, 14, and 21 d postmortem.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Moore et al., 2003). Both DGAT1 and TG have been mapped to the region of the QTL, and markers in these genes have been reported to be associated with fat deposition (Barendse, 1999; Grisart et al., 2001). They represent attractive potential candidate genes to explain the variation observed in the QTL experiments. Similarly, the CAPN1 gene has been mapped to a QTL affecting shear force on chromosome 29 (Smith et al., 2000), and markers in this gene have been found to be associated with shear force in several populations (Page et al., 2002, 2004).
The first goal was to determine whether the markers were informative in this Brahman population. One potential SNP marker with a reported effect on meat tenderness, specifically marker CAPN530 in exon 14 of the bovine CAPN1 gene, which produces a substitution of isoleucine for valine at position 530 of the µ-calpain protein, was homozygous in this population (data not shown). In addition, the CAPN316 marker producing a substitution of alanine for glycine at position 316 had low polymorphism (C allele frequency 1%; Table 2), leading us to apply two previously unreported polymorphisms (CAPN4753 and CAPN5331) known to be polymorphic in Brahman cattle (S. N. White and B. Page, unpublished data). The only reported TG marker was polymorphic but with very low frequency (3%) of the favorable T allele reportedly associated with increased marbling (Barendse, 1999). The DGAT1 marker is distinct from those in the other two genes in that the sequence difference represented by the marker is reported to be the causative mutation leading to the observed variation in fat deposition (Grisart et al., 2004). Both alleles of this marker are segregating in the Brahman population, but the unfavorable alanine-encoding allele, reported to be associated with reduced fat in milk and decreased marbling in beef, is observed at low frequency (10%).
Thyroid hormones play an important role in regulating metabolism and can affect homeostasis of fat depots. Variation in the TG gene, producing the precursor for thyroid hormones, has been proposed to account for some of the genetic variation in marbling score in beef cattle (Barendse, 1999). A separate study of beef cattle in Germany (Thaller et al., 2003b) found that the same TG marker used in the present study was associated with differences in the expression of i.m. fat in LM of German Holstein cattle, but the authors were unable to detect this association in Charolais. It should be noted that the sample size in the German study was extremely small (28 German Holstein and 27 Charolais animals), that the data indicated a recessive mode of effect with the T allele homozygotes having higher marbling, and that the association was not detected for marbling in semitendinosus muscles. In the present study, there was only a tendency (P < 0.10) in the association between the TG marker and marbling score in Bos indicus cattle, but the number of homozygous T allele animals (7; Table 2) may have been too small for detection of a recessive mode of action. Previous studies have reported T allele frequencies of this marker at 22 to 25% (Moore et al., 2003; Thaller et al., 2003b). Thus, support for the association of variation in the TG gene and marbling score is inconclusive in Bos indicus; however, an association was detected between the Thyroglobulin gene with fat thickness and LMA in the present study. In both traits, the mean of heterozygous animals was significantly lower than either of the alternate homozygous classes. Evidence for a QTL affecting fat thickness on chromosome 14 in Bos taurus cattle has been presented for several populations (Casas et al., 2000, 2003; Moore et al., 2003), although the TG marker has not been associated with this quantitative trait locus (Moore et al., 2003). It is possible that alleles of the TG marker are in linkage disequilibrium with functional alleles affecting fat thickness in Bos indicus cattle, regardless of whether the variation acts through thyroglobulin.
No previously reported studies have detected a quantitative trait locus for LMA on chromosome 14. The detailed description of the present Brahman population (Riley et al., 2002) reported that the genetic and phenotypic correlations between fat thickness and LMA in this population are 0.02 and 0.10, respectively. This suggested that it is unlikely that the same variation is affecting both traits. Several sources for the observed effect of TG marker genotype on LMA and fat thickness are possible. The region of chromosome 14 may harbor variation in two different genes, each influencing either trait independently in this Brahman population, and the marker is in linkage disequilibrium with the functional allele of both genes. Alternatively, a gene that influences both traits may reside in this region, with the same variation affecting both phenotypes, again postulating that the marker is in linkage disequilibrium with this functional difference. Finally, it is possible that the association of the marker allele with LMA is spurious or caused by unrecognized population stratification in this population.
The K232A mutation at the DGAT1 gene has been confirmed as being responsible for increased milk yield, fat yield, protein yield, fat content, and protein content in dairy cattle (Grisart et al., 2004). This suggests the possibility that it might affect intramuscular or subcutaneous fat depots as well (Thaller et al., 2003a). Indeed, a QTL for fat thickness has been detected on chromosome 14 in multiple populations of Bos taurus beef cattle as mentioned above (Casas et al., 2000, 2003; Moore et al., 2003). In one study, the K232A allele was directly tested but no association with fat thickness was observed (Moore et al., 2003). Moore et al. (2003) concluded that a gene on chromosome 14 other than DGAT1 must be responsible for the fat deposition QTL in Bos taurus. In the present study, no association of DGAT1 alleles was detected with any of the traits measured (P > 0.05). The low number of animals homozygous for the alanine-encoding allele (AA) decreases the power to detect association if this variant acts in a recessive fashion; however, the previously reported study of German Holsteins and Charolais suggested that it is the lysine-encoding, fat-increasing allele (KK) that is recessive (Thaller et al., 2003b). If there is variation on chromosome 14 affecting fat deposition in Brahman, it is improbable that the reported DGAT1 mutation is the cause of the effect, nor does it seem that the marker is in linkage disequilibrium with variation affecting fat thickness.
Single nucleotide polymorphisms CAPN316 and CAPN530 in the CAPN1 gene have been associated with meat tenderness, measured as Warner-Bratzler shear force, in Bos taurus cattle (Page et al., 2002, 2004). The CAPN530 marker was uninformative in the Brahman population studied. In contrast, the A allele of this marker has been observed to have 36% frequency across a breed panel of 147 bulls from seven Bos taurus beef cattle breeds (Page et al., 2004). The C allele of the CAPN316 marker had only a 1% frequency in this Brahman population, where the study by Page et al. (2004) had observed a frequency of 16%. As a result of these allele frequencies, the contrast in this Brahman population is between the two CAPN316 and CAPN530 haplotypes (C/G and G/G). No association was detected between CAPN316 genotype and meat tenderness measured as Warner-Bratzler shear force. That is, the contrast between C/G and G/G haplotypes was not large enough to detect a significant difference given the small number of C/G haplotypes (1%) in the population. However, this marker was a significant indicator of sensory panel tenderness score, an alternative measure of meat tenderness, despite the low frequency of the C/G haplotype. As in all other populations studied, the C/G haplotype was associated with tenderer meat than the G/G haplotype.
To more thoroughly examine potential association of CAPN1 variation and shear force, additional SNP with higher minor allele frequency in Brahmans were required. More than 100 other SNP have been detected in the gene (S. N. White and T. Smith, unpublished data), although no effort has been made to identify markers specifically in Brahman. Markers CAPN4753 and CAPN5331 were chosen for this study and had 32 and 29% minor allele frequency in the present population, respectively; however, none of the markers showed an association with shear force or with tenderness score. Potential explanations for this result are that there is no functional variation at the CAPN1 locus in this population of Brahman cattle, that the functional variants are too rare in the Brahman population to give statistically significant results, or that the markers used do not effectively divide functional haplotypes. To address these hypotheses, other SNP in the CAPN1 gene or neighboring genes need to be assessed in Bos indicus populations to determine their effect on carcass composition and meat quality traits.
All three CAPN1 markers showed an association with hump height (P < 0.02). The mean hump height for alternate homozygote and the heterozygous classes of the CAPN4753 and CAPN5331 markers are very similar, and genotype frequencies of these two markers are also closely matched. Because there is only approximately 30 kb separating the two markers, this raises the possibility that their alleles are in complete linkage disequilibrium and are segregating as a haplotype. Two-marker genotype frequencies for the CAPN4753 and CAPN5331 markers (not shown) indicate that all four possible two-marker haplotypes (A/A, A/T, C/A, C/T) are segregating in the population with frequencies of 65, 5, 6, and 23%, respectively. The extremely low frequency of the C allele of the CAPN316 marker did not permit meaningful evaluation of this marker in a similar three-marker haplotype analysis. For the majority (88%) of the population, the genotype at one marker is sufficient to identify the haplotype, which may be the cause of the observed concordance between the mean hump height measurements, even though the two markers are not in complete linkage disequilibrium. No connection between the biological function of the µ-calpain protease and a developmental trait like hump height is obvious, but it is possible that the markers are in linkage disequilibrium with functional variation in a nearby gene. Examination of a one million-bp region of the human genome surrounding the CAPN1 gene reveals more than 30 known or predicted genes, some of which have known roles in growth or development that are likely to have orthologs in the bovine genome.
The results reported here do not support the use of previously identified DNA markers, with demonstrated value in Bos taurus cattle, in Bos indicus populations. This does not necessarily indicate that variations in the genes identified do not exist or influence traits of economic importance, but that suitable markers need to be developed for Bos indicus cattle due to differences in allele frequencies between Bos taurus and Bos indicus cattle. Another possibility is that associations found in Bos taurus could be false positives. Association of marker information with traits of economic importance could be missed or misinterpreted if markers developed for Bos taurus are used in Bos indicus populations. Genetic and phenotypic parameters for carcass composition and meat quality traits for this population have been previously reported (Riley et al., 2002), and the results indicate that heritabilities and the genetic and phenotypic correlations for carcass traits were similar in this Brahman population to estimates of these parameters in other Bos taurus and Bos indicus studies. This suggests that similar sources of variation may exist in these cattle, and could be detected if suitable marker systems are developed.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 The authors thank E. Bowers, D. Brinkerhoff, L. Flathman, R. Godtel, M. Rooks, D. Sartain, S. Simcox, K. Simmerman, K. Tennill for technical assistance, L. Adams, E. Rooks, Subtropical Agricultural Research Station staff for animal care, B. Page for providing sequence data, and J. Watts for secretarial support.
4 Current address: Omaha’s Henry Doorly Zoo, Omaha, NE 68107.
3 Correspondence: P.O. Box 166 (phone: 402-762-4168; fax: 402-762-4173; e-mail: casas{at}email.marc.usda.gov).
Received for publication June 24, 2004. Accepted for publication September 27, 2004.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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