Associations between LEP, DGAT1 and FABP4 gene polymorphisms and carcass and meat traits in Nelore and crossbred beef cattle

Livestock Science 135 (2011) 244–250

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Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i

Associations between LEP, DGAT1 and FABP4 gene polymorphisms and carcass and meat traits in Nelore and crossbred beef cattle R.A. Curi a,⁎, L.A.L. Chardulo b, M.D.B. Arrigoni a, A.C. Silveira a, H.N. de Oliveira c a b c

Departamento de Melhoramento e Nutrição Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista, Botucatu, SP 18618-000, Brazil Departamento de Química e Bioquímica, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP 18618-000, Brazil Departamento de Zootecnia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, SP 14884-900, Brazil

a r t i c l e

i n f o

Article history: Received 10 December 2009 Received in revised form 13 July 2010 Accepted 16 July 2010 Keywords: Molecular markers Candidate gene Fat deposition Meat tenderness Zebu

a b s t r a c t The aim of this study was to estimate the segregation of leptin (LEP), diacylglycerol Oacyltransferase (DGAT1) and fatty acid-binding protein 4 (FABP4) gene polymorphisms in Nelore (Bos indicus) and Nelore × Bos taurus beef cattle, and to evaluate their effects on carcass and meat traits. A total of 300 animals were genotyped for the LEP/BsaAI (Y_11369.1: g.1620G N A), DGAT1-VNTR (18-bp repeat element in the promoter region) and FABP4/MspA1I (AAFC_01136716.1:g.7516G N C) polymorphisms and phenotyped for rib eye area, backfat thickness (BT), intramuscular fat, shear force, and myofibrillar fragmentation index. The allele substitution effects for each of the polymorphisms on the traits of interest were estimated by regression on the number of copies of a particular allele using the General Linear Model procedure. To keep the experiment wise error rate to the specified level, the Bonferroni adjustment was applied. Although the LEP/BsaAI polymorphism has shown segregation for association studies in Nelore cattle [f(A) = 0.351], no associations were observed between its alleles and the traits analyzed. The DGAT1-VNTR was found to be polymorphic in Nelore cattle, as well as a potential association with BT. In addition to being non-informative in Nelore animals (allele C was found to be fixed), the FABP4/MspA1I polymorphism showed no association with the studied traits in crossbred animals. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Fat deposition directly influences meat quality and therefore affects the choice of consumers. Marbling resulting from intramuscular fat deposition confers juiciness and flavor to meat, influencing consumption habits and the final price of the product. Subcutaneous fat deposition is necessary to guarantee carcass quality after post-slaughter cooling (Fortes et al., 2009). In view of the physiological function of their protein products (Zhang et al., 1994; Cases et al., 1998; Shen et al., 1999) and physical proximity to QTLs identified in cattle (Cattle QTL Database, 2009), the leptin (LEP) gene, mapped to

⁎ Corresponding author. Tel.: +55 14 38117187; fax: +55 14 38117197. E-mail address: [email protected] (R.A. Curi). 1871-1413/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2010.07.013

bovine chromosome 4, and the diacylglycerol O-acyltransferase (DGAT1) and the fatty acid-binding protein 4 (FABP4) genes, mapped to bovine chromosome 14, are functional and positional candidate genes for traits related to fat synthesis in both dairy and beef cattle. A single nucleotide polymorphism (SNP) in exon 2 of the bovine LEP gene, discovered by Buchanan et al. (2002), has been the most extensively investigated in relation to association with carcass fat content. However, Choudhary et al. (2005) and Fortes et al. (2009) demonstrated that, whereas this polymorphism is absent or segregate with low minor allele frequency in Bos indicus breeds, the substitution of a guanine for adenine in intron 2 of the gene (Y_11369.1: g.1620G N A) described by Lien et al. (1997) shows high minor allele frequency in this subspecies, permitting association studies between gene variants and traits of interest in zebu breeds.

R.A. Curi et al. / Livestock Science 135 (2011) 244–250

In view of its low frequency in some breeds or lack of segregation in others, association studies between the K232A polymorphism of the DGAT1 gene and dairy and meat traits in zebu breeds are not very promising (Winter et al., 2002; Lacorte et al., 2006). However, Kuhn et al. (2004), studying German Holstein cattle (Bos taurus), found an effect of a VNTR located in the regulatory gene region on milk fat percentage in animals homozygous (AA) for the K232A polymorphism, a finding indicating that the polymorphism in exon 8 may not be the only one exerting phenotypic effects. In a pioneering study, Michal et al. (2006) searched for polymorphisms in the bovine FABP4 gene in DNA pools of animals with high and low marbling scores and identified significant associations between AAFC_01136716.1:g.7516G N C SNP genotypes and backfat thickness and marbling in a Wagyu × Limousin F2 population (B. taurus). Recently, Hoashi et al. (2008) and Barendse et al. (2009) identified new gene polymorphisms that have potential application to beef cattle breeding. Nevertheless, we found no studies investigating the segregation and/or association between FABP4 polymorphisms and traits of interest in Nelore cattle (B. indicus). In view of the lack of information regarding the potential use of molecular markers for phenotypes of interest in zebu animals and the relevance of the Nelore breed (B. indicus) in Brazil, the objective of the present study was to estimate allele and genotype frequencies of the LEP gene (Y_11369.1: g.1620G N A), DGAT1 gene (DGAT1-VNTR), and FABP4 gene (AAFC_01136716.1:g.7516G N C) polymorphisms in beef cattle of diverse genetic backgrounds (Nelore and Nelore × B. taurus), and to evaluate the effect of these genotypes on carcass and meat traits. 2. Material and methods 2.1. Animals This experiment used 114 Nelore animals (B. indicus), 67 Angus × Nelore crosses (1/2 B. taurus + 1/2 B. indicus), 44 Rubia Gallega × Nelore crosses (1/2 B. taurus + 1/2 B. indicus), 41 Canchim animals (5/8 B. taurus + 3/8 B. indicus), 19 Brangus three-way crosses (9/16 B. taurus + 7/16 B. indicus), and 15 Braunvieh three-way crosses (3/4 B. taurus + 1/4 B. indicus). Except for the Rubia Gallega × Nelore, bred in a semiintensive cattle-raising system in 2006, these animals, originating from commercial herds of seven farms, were bred in 2003, 2005, 2006 and 2007 in the feedlot sector of the Department of Animal Nutrition and Genetic Improvement, School of Veterinary Medicine and Animal Science, São Paulo State University (Unesp), Botucatu/SP, Brazil, using an intensive system. The 300 animals used in the experiment, 32 females and 268 males, were bred according to the Brazilian legislation for animal well-being (protocol No. 89/ 2006 approved by the Ethics Committee on Animal Experimentation — CEEA, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu/SP, Brazil). The animals were slaughtered at 15, 17 and 19 months of age. 2.2. Sample collection and phenotyping After slaughter carried out at collaborating slaughterhouses according to the guidelines for humane slaughter of

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cattle, the carcasses were identified and chilled for 24 h. Next, two longissimus lumborum muscle samples (2.54 cm thick) were collected between the 11th and 13th rib from the left half carcasses. Samples collected between the 12th and 13th rib were used for the measurement of rib eye area (REA), backfat thickness (BT) and shear force (SF). Samples collected between the 11th and 12th rib were used for the measurement of the myofibrillar fragmentation index (MFI) and intramuscular fat (IF) (percentage of total lipids), as well as for the extraction of genomic DNA. REA was measured by the quadrant point method and BT was determined with a ruler, both according to the method described by the USDA Quality Grade (USDA, 1997). After these first measurements carried out at the slaughterhouses, the longissimus lumborum muscle samples were de-boned, vacuum wrapped, aged at 1 to 2 °C for 14 days, and then frozen at −20 °C. The other phenotypic parameters (SF, MFI and IF) were determined in the laboratory according to the methods described by Wheeler et al. (1995), Culler et al. (1978) and Bligh and Dyer (1959), respectively. 2.3. DNA extraction and genotyping Genomic DNA was extracted from 250-mg meat samples by a non-phenolic method after digestion with proteinase K and precipitation with NaCl and alcohol (Sambroock et al., 1989). The LEP and FABP4 gene polymorphisms were genotyped by PCR-RFLP and the DGAT1 gene polymorphism was genotyped by PCR. For identification of alleles A and G of the LEP gene polymorphism (Y_11369.1:g.1620G N A), a fragment of 522 bp located in intron 2 was amplified and digested with BsaAI as described by Choudhary et al. (2005). The four alleles (1, 2, 3 and 4) with 110, 128, 146 and 164 bp of the VNTR located in the regulatory region of the DGAT1 gene (DGAT1-VNTR) were identified as described by Kuhn et al. (2004). For identification of alleles C and G of the AAFC_01136716.1: g.7516G N C SNP of the FABP4 gene, a fragment of 452 bp was amplified and digested with MspA1I (Michal et al., 2006). From now on, the LEP, DGAT1 and FABP4 gene polymorphisms are referred to as LEP/BsaAI, DGAT1-VNTR and FABP4/MspA1I, respectively. The amplified and digested DNA fragments of the LEP and FABP4 genes were separated on 3% and 2% agarose gels, respectively. The amplified fragments of the DGAT1 gene were separated by electrophoresis on 4% high resolution agarose gel. A 100-bp molecular weight standard was included in each gel to permit the calculation of the size of the fragments produced. The genotypes of the individuals were determined for each polymorphism by analysis of the size of the fragments in base pairs (bp). 2.4. Statistical analysis Allele frequencies were calculated for each polymorphism based on the genotypes identified in the gels according to Weir (1996). Differences in allele frequencies within and between the genetic groups studied were evaluated using contingency tables (Curi and Moraes, 1981). The allele substitution effects of the different alleles of the polymorphisms on the traits of interest were estimated by regression on the number of copies of a particular allele using the General Linear Model (GLM) procedure of the

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Statistical Analysis System program (SAS, 2004) as follows: Yijk = μ + CGi + b × njk + eijk, where Yijk = trait of interest, μ = overall mean, CGi = fixed effect of ith contemporary group (i = 1,..., 13), b = regression coefficient, n = number of alleles of each polymorphism and eijk = random error. For the LEP/BsaAI and FABP4/MspA1I polymorphisms, the regression was done on A and C alleles, respectively. In the analyses of the DGAT1-VNTR, a multiple regression approach was used and, to avoid dependencies in the coefficient matrix, the effect of the allele 4 was set to zero. To keep the experiment wise error rate to the specified level, the Bonferroni adjustment was applied. In the DGAT1-VNTR analyses the total reduction in the sum of squares caused by the effect of the three alleles in the model was computed and used to obtain the nominal p-value for the locus. For the traits that, after adjustment by Bonferroni method, the effect of the locus was significant, the substitution effects of the alleles were compared by t-test. The contemporary groups considered animals of same genetic group, sex, age at slaughter, feedlot year, and farm of origin (these effects could not be considered separately in the model since there is a confounding among them). The bull effect was not included in the linear model since the number of genotyped offspring of individual bulls was very small. Thus, due to the large number of parents, the possibility of confounding effects of allele and bull on the traits was minimized. 3. Results 3.1. Allele and genotype frequencies The two genetic variants, A and G, of the LEP/BsaAI polymorphism were found in all genetic groups studied. Genotype AA was characterized by the presence of a single 522-bp fragment. Genotype GG presented two restriction fragments of 441 and 81 bp. Heterozygous (AG) individuals presented three fragments of 522, 441 and 81 bp. The frequency of allele G was significantly higher than that of allele A in Nelore, Angus × Nelore and Canchim animals (P b 0.05). No significant differences between alleles were observed for the other comparisons (P N 0.05). The frequency of allele G was significantly higher in Angus × Nelore and Canchim animals when compared to Brangus and Braunvieh three-way crosses (P b 0.05). Nelore animals presented intermediate frequency of allele G in relation to the groups

Rubia Gallega × Nelore, Brangus and Braunvieh three-way crosses and the groups Angus × Nelore and Canchim (Fig. 1). The DGAT1-VNTR polymorphism was characterized by four alleles (1, 2, 3 and 4) of 110, 128, 146 and 164 bp, respectively, which were present in all genetic groups studied. Alleles 2, 3 and 4 predominated in the Nelore group (P b 0.05). In Angus × Nelore animals, allele 4 was the most frequent, followed by allele 3 (P b 0.05). Alleles 2 and 3 were the most frequent in Rubia Gallega × Nelore crosses (P b 0.05). Allele 2 was the most frequent in the Canchim group, followed by alleles 3 and 4 (P b 0.05). No significant differences between the four alleles were observed in Brangus and Braunvieh threeway crosses (P N 0.05). Comparison of allele frequencies among genetic groups showed a significantly higher frequency of allele 1 in Brangus and Braunvieh three-way crosses compared to the Nelore and Canchim groups (P b 0.05). Allele 2 was less frequent only in the Angus × Nelore group (P b 0.05). Allele 3 was more frequent in Rubia Gallega × Nelore animals compared to Canchim animals and Braunvieh three-way crosses (P b 0.05). Allele 4 was the most frequent in Angus × Nelore crosses, followed by Nelore, Canchim and Brangus three-way animals (P b 0.05) (Fig. 2). With respect to the FABP4/MspA1I SNP, genotype CC was characterized by the presence of a 452-bp restriction fragment, whereas genotype GG presented two fragments of 352 and 100 bp. The heterozygous (CG) genotype was characterized by the presence of three fragments corresponding to the combination of the two homozygous patterns. Allele C was fixed in the Nelore group, whereas both alleles were present in the other groups, with the frequency of allele C being significantly higher than that of allele G (P b 0.05). The frequency of allele C was significantly higher in the Nelore, Angus × Nelore and Rubia Gallega × Nelore groups when compared to Canchim animals (P b 0.05). Brangus and Braunvieh three-way crosses presented intermediate frequencies of allele C in relation to the groups Nelore, Angus × Nelore and Rubia Gallega × Nelore and the group Canchim (Fig. 3). 3.2. Allele substitution effect The analysis of allele substitution effect, which results are presented in Table 1, shows no significant effects of allele substitution of LEP/BsaAI and FABP4/MspA1I polymorphisms on the traits of interest (P = 1). For DGAT1-VNTR polymor-

Fig. 1. Frequencies of alleles A and G of LEP/BsaAI polymorphism in the genetic groups studied. Frequencies with equal letters, uppercase among genetic groups and lowercase within genetic groups, do not differ at 5% probability. The number between parentheses indicates the number of animals in each genetic group.

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Fig. 2. Frequencies of alleles 1, 2, 3 and 4 of DGAT1-VNTR polymorphism in the genetic groups studied. Frequencies with equal letters, uppercase among genetic groups and lowercase within genetic groups, do not differ at 5% probability. The number between parentheses indicates the number of animals in each genetic group.

phism it was found significant effects on BT (P = 0.0092). On the other traits (REA, IF, SF and MFI) were not significant the effects of this polymorphism (P = 1). Table 2 shows no significant effect of substitution of alleles A for G at LEP/BsaAI polymorphism and C for G at FABP4/ MspA1I polymorphism on the analyzed traits (P N 0.05). Table 3 shows the significant effect of substitution of one random allele by alleles 2 (P b 0.01), 3 (P b 0.05) and 4 (P b 0.01) of DGAT1-VNTR polymorphism on BT, with negative effects of alleles 2 and 3 (−0.541 and −0.353 mm, respectively) and positive effect of allele 4 (0.402 mm). 4. Discussion 4.1. LEP/BsaAI polymorphism The LEP/BsaAI polymorphism has shown high minor allele frequency in Nelore cattle, with allele A frequency of 0.351, supporting association studies in Nelore and Nelore × B. taurus. However no associations were observed between its alleles and the traits analyzed. Among the polymorphisms described for the bovine LEP gene, those present in exon 2, E2FB (Buchanan et al., 2002) and E2JW (Lagonigro et al., 2003), and in the regulatory region of the gene, UASMS1 and UASMS2 (Nkrumah et al., 2005), have been extensively studied. However, little is known about their occurrence in B. indicus populations. Although Barendse et al.

(2005) reported a frequency of allele T of 19% in Brahman animals (B. indicus), the E2FB polymorphism (AJ_236854: c.73 T N C) located in exon 2 described by Buchanan et al. (2002) does not occur in Hariana, Sahiwal, Gyr or Nimari breeds (Choudhary et al., 2005) or segregates with low minor allele frequency in Nelore animals (Fortes et al., 2009). The study of Choudhary et al. (2005) investigating the Y_11369.1: g.1620GN A SNP (LEP/BsaAI) was the first report of appropriate segregation of the leptin gene polymorphism in different purebred B. indicus populations. These authors found a frequency of allele A ranging from 25 to 34% in B. indicus breeds (close to that observed in the present study for Nelore cattle, 35%) and suggested that, in contrast to other LEP gene polymorphisms, this polymorphism might be the result of a mutation in the bovine genome that occurred before the separation of the species that culminated in the formation of subspecies. Despite the lack of information, comparison of the present results regarding the allele distribution of the LEP/ BsaAI polymorphism in crossbred genetic groups with those reported by Choudhary et al. (2005) for B. taurus breeds and B. taurus × B. indicus crosses indicates the lack of obviously different allele frequencies between taurine and zebu breeds. The fact that the polymorphisms located in exon 2 and in the regulatory region of the bovine LEP gene are found in gene regions with a greater potential of causing phenotypic variations has led to the extensive investigation of these polymorphisms in B. taurus populations (Buchanan et al.,

Fig. 3. Frequencies of alleles C and G of FABP4/MspA1I polymorphism in the genetic groups studied. Frequencies with equal letters, uppercase among genetic groups and lowercase within genetic groups, do not differ at 5% probability. The number between parentheses indicates the number of animals in each genetic group.

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Table 1 Degrees of freedom (DF), mean squares (MS), F-test (F), nominal p-values (P) and p-values after adjustment by Bonferroni method (P*) for regression of the traits analyzed in the number of alleles for each polymorphism. Polymorphism

Trait

DF

MS

F

P

P*

LEP/BsaAI

REA (cm2) BT (mm) IF (%) SF (Kg) MFI REA (cm2) BT (mm) IF (%) SF (Kg) MFI REA (cm2) BT (mm) IF (%) SF (Kg) MFI

1 1 1 1 1 1 1 1 1 1 3 3 3 3 3

1.5884 5.6642 0.0005 1.1158 77.9006 0.1723 0.0278 0.8148 0.0047 39.753 100.2519 10.9561 0.2556 0.4370 73.8864

0.0215 3.0061 0.0007 2.0524 0.2726 0.0024 0.0146 1.1415 0.0085 0.0139 0.0199 9.1735 0.0176 0.3559 0.3002

0.8835 0.0840 0.9795 0.1531 0.6020 0.9611 0.9039 0.2862 0.9264 0.7095 0.2397 0.0006 0.7843 0.4955 0.8559

1 1 1 1 1 1 1 1 1 1 1 0.0092 1 1 1

FABP4/MspA1I

DGAT1-VNTR

2002; Lagonigro et al., 2003; Barendse et al., 2005; Choudhary et al., 2005; Kononoff et al., 2005; Nkrumah et al., 2005; Schenkel et al., 2005; Di Stasio et al., 2007). Among these polymorphisms, E2FB (C N T transition in exon 2 of the gene), described by Buchanan et al. (2002), has been the main focus since it results in a nonconservative change from arginine to cysteine at position 25 of the amino acid chain (R25C), a change potentially affecting protein function. However, the results regarding its effect on phenotypes related to fat deposition in B. taurus are inconsistent (Di Stasio et al., 2007). The inadequate segregation of the E2FB polymorphism in Nelore cattle requires the use or development of other markers for the gene or chromosome region in order to permit association studies in this breed. Among the polymorphisms described for the LEP gene, the only one that is known to segregate with high minor allele frequency in different B. indicus breeds is LEP/BsaAI. However, probably because of the location of this polymorphism in an intron region, there is only one report investigating its relationship with phenotypes of interest in beef cattle. Similar to the present results showing the absence of an effect of the LEP/ BsaAI SNP on BT and IF, Mercadante et al. (2008) found no significant associations between the genotypes of this polymorphism and subcutaneous backfat thickness measured by ultrasound in three lines of Nelore heifers selected for yearling weight. We found no association studies between the polymorphism and carcass traits in B. taurus. Although associations between genotypes of the LEP gene polymorphism and REA can be expected because of the interaction of

Table 2 Effect of substitutions of alleles A for G at LEP/BsaAI polymorphism and C for G at FABP4/MspA1I polymorphism (α) on rib eye area (REA), backfat thickness (BT), intramuscular fat (IF), shear force (SF) and myofibrillar fragmentation index (MFI) and standard errors (SE). Polymorphism LEP/BsaAI FABP4/MspA1I

α SE α SE

REA (cm2)

BT (mm)

IF (%)

SF (Kg)

MFI

0.122 0.833 −0.077 1.577

−0.233 0.134 0.031 0.255

0.002 0.083 0.167 0.156

0.103 0.072 −0.013 0.137

−0.863 1.654 1.167 3.130

Table 3 Effect of substitution of one random allele by alleles 1, 2, 3 and 4 of DGAT1VNTR (α) on rib eye area (REA), backfat thickness (BT), intramuscular fat (IF), shear force (SF) and myofibrillar fragmentation index (MFI) and standard errors (SE). Allele 1 2 3 4

α SE α SE α SE α SE

REA (cm2)

BT (mm)

IF (%)

SF (Kg)

MFI

−2.519 1.423 − 0.768 1.009 0.717 0.992 0.857 0.953

−0.312 0.229 − 0.541** 0.163 − 0.353* 0.160 0.402** 0.153

−0.099 0.143 0.049 0.101 − 0.059 0.099 0.036 0.096

0.174 0.125 0.024 0.089 0.054 0.087 − 0.084 0.083

0.436 2.859 − 1.683 2.025 − 0.480 1.994 0.576 1.914

*P b 0.05; **P b 0.01.

leptin with growth hormone (Tannenbaum et al., 1998) and its participation in the regulation of feed intake, energy metabolism and growth (Choudhary et al., 2005), no such associations were observed. 4.2. DGAT1-VNTR polymorphism The DGAT1-VNTR was found to be polymorphic in Nelore cattle, as well as potentially associated with backfat thickness. Prospecting studies have revealed 19 polymorphic sites in the bovine DGAT1 gene (Winter et al., 2002). Seventeen of these sites are found in intron, regulatory and non-translated regions, whereas an AA/GC substitution at positions 10433 and 10434 (AJ318490) of exon 8 results in a nonconservative change from lysine to alanine at position 232 (K232A) of the predicted amino acid sequence (Kuhn et al., 2004). According to Kaupe et al. (2004), the K232A substitution probably occurred after the separation of B. taurus from B. indicus, with the K allele being an ancestral allele. Allele A, the mutant allele, would only be common in B. taurus breeds. In this respect, Kaupe et al. (2004), Lacorte et al. (2006) and Fortes et al. (2009) did not detect allele A or found only extremely low frequencies of this allele (1 to 5%) in Nelore animals (B. indicus). On the other hand, among the five alleles (1 to 5) initially described for the VNTR located in the regulatory region of the DGAT1 gene, which consists of three to seven repeat sequences, respectively (Winter et al., 2002), four (alleles 1, 2, 3 and 4) were identified in all genetic groups studied here, including Nelore animals (B. indicus). Kuhn et al. (2004) detected five alleles in 34 halfsib families of the German Holstein breed (B. taurus). Similarly to the set of animals analyzed here, allele 3 with five repeat sequences was the most frequent. Alleles 5 and 3 were the most frequent in the German Angeln dairy cattle population (B. taurus) (Sanders et al., 2006). In the study of Gautier et al. (2007), alleles 3, 2, and 3 and 2 presented the highest frequency in French Holstein, Normande and Montbéliarde populations (all B. taurus breeds), respectively. There are no studies investigating the DGAT1-VNTR polymorphism in B. indicus or taurine beef cattle breeds. Genetic and biochemical studies identified the K232A polymorphism of the DGAT1 gene as the causal mutation responsible for the QTL located in the centromeric region of bovine chromosome 14 (BTA14), which exerts an effect on milk fat percentage (Grisart et al., 2002; Winter et al., 2002;

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Grisart et al., 2004). In contrast to the consistent results regarding the effect of the K232A polymorphism on dairy cattle traits, discrepant results regarding the association between DNA mutations and carcass fat deposition (intramuscular and subcutaneous) have been described in the literature (Fortes et al., 2009). Initial evidence for the existence of additional polymorphisms contributing to the effect of the QTL on milk fat percentage has been provided by Kuhn et al. (2004) and Bennewitz et al. (2004). In view of the numerous factors reported by Fürbass et al. (2006), the VNTR polymorphism in the regulatory region of the DGAT1 gene (DGAT1-VNTR) is a strong candidate. Despite recent disagreements (Sanders et al., 2006; Gautier et al., 2007), this is supported by the observation of an association between DGAT1-VNTR alleles and variations in milk fat content in animals homozygous (AA) for the K232A polymorphism (Kuhn et al., 2004). Furthermore, the DGAT1-VNTR sequence comprises two to eight 18-bp repeat elements rich in GC, including a GC box (CCCGCCC), a potential binding site for transcription factor Sp1. Although Fürbass et al. (2006) confirmed the Sp1-VNTR interaction, in vitro reporter gene assays showed no differential expression between the VNTR alleles. However, according to these authors, this situation might be different in the chromatin context existing in vivo. The present results showed negative effect of the alleles with four and five repeats and positive effect of the allele with six repeats of the Sp1-binding on backfat thickness. No studies are available investigating the effect of this VNTR on traits related to fat deposition in beef cattle. Associations between DGAT1-VNTR genotypes and REA, SF and MFI are not expected since they can so far not be explained physiologically or based on the physical proximity between the DGAT1 gene and QTLs already identified at the terminal end of the centromeric region of BTA14. 4.3. FABP4/MspA1I polymorphism In addition to being non-informative in Nelore animals with allele C was found to be fixed, the FABP4/MspA1I polymorphism showed no association with the studied traits in crossbred animals. In the only study investigating the FABP4/MspA1I polymorphism, Michal et al. (2006) observed a frequency of allele C of 75% in an F2 population of Wagyu×Limousin animals (B. taurus). The allele distributions found here for crossbred genetic groups were within those observed for Nelore animals (B. indicus) and Wagyu× Limousin F2 crosses. The possible absence of the AAFC_01136716.1:g.7516GN C SNP in the 3′-UTR region of the bovine FABP4 gene in Nelore cattle, a situation that may extend to other B. indicus breeds, indicates the use of other recently identified polymorphisms of the gene for studies involving this subspecies. Analyzing gene sequences in eight Japanese Black and Holstein animals (B. taurus), Hoashi et al. (2008) identified four SNPs (NC_007312:c.220Ab G, c.328Gb A, c.348Gb C, and c.396Ab G). The first two SNPs cause conservative substitutions (isoleucine for valine — I74V, and valine for methionine V110M) in the predicted amino acid sequence of the protein. In an attempt to identify nonconservative variants of the bovine FABP4 gene, Barendse et al. (2009) sequenced the coding sequence of the gene in 10 Angus and Shorthorn animals (B. taurus). The authors identified 14 SNPs, nine in the intron region, two in the

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3′-UTR region, one at the 3′-end of the poly-A site, one synonymous variant in exon 4, and one (AAFC_01136716.1: g.2502CN G) at the splice site between exon 3 and the subsequent intron. In contrast to the findings of Michal et al. (2006) who demonstrated an effect of the FABP4/MspA1I genotypes on marbling score and subcutaneous backfat thickness in Wagyu×Limousin F2 animals (B. taurus), the present results failed to show any association between this SNP and fat deposition (BT and IF) in Nelore×B. taurus animals. According to Barendse et al. (2009), mutations in the bovine FABP4 gene have not always shown associations with intramuscular or subcutaneous fat in beef cattle, suggesting that the effect of the gene is small. This fact impairs its detection in experiments involving a small number of individuals. Supporting this hypothesis, the authors found a weak association (0.3% of the overall phenotypic variance) between the AAFC_01136716.1:g.2502CN G SNP and intramuscular fat in a sample of 1409 animals of Angus, Hereford, Murray Grey, Shorthorn (B. taurus), Brahman (B. indicus), Belmont Red and Santa Gertrudis (B. taurus×B. indicus) populations. In addition, the effect was significant in the sample of animals as a whole and in Angus cattle (the largest breed sample), but not in the other breeds analyzed. In this respect, the small number of animals used here for the analysis of the association between the FABP4/ MspA1I polymorphism and traits of interest did not permit the detection of any effect, if present. In addition to the magnitude of the effect, the possibility of different epistatic interactions between the candidate gene and the genetic basis of the populations, breeds and distinct subspecies cannot be ruled out. Taken together, the results of the association analysis obtained for the FABP4 gene are unexpected since, according to Michal et al. (2006), the gene is situated in the QTL interval for marbling identified in three distinct cattle populations. In view of the physiological role of its protein product which, so far, is known to be closely related to lipid metabolism and adipocyte homeostasis, associations between the FABP4 gene SNP and REA, SF and MFI were not expected. 5. Conclusion The present results led us to conclude that 1) although the LEP/BsaAI polymorphism presented adequate segregation for association studies in Nelore animals, it does not seem to be a promising selection tool for Nelore and Nelore × B. taurus animals. 2) The DGAT1-VNTR was found to be polymorphic in Nelore animals, permitting association studies between gene variants and traits of interest in this breed and in crosses with B. taurus. A potential relationship between the polymorphism and backfat thickness was demonstrated for the first time in beef cattle. 3) The possible absence of the FABP4/MspA1I polymorphism in Nelore animals, together with the small effect of FABP4 on traits related to fat deposition, should discourage future initiatives involving this SNP in most Brazilian herds. Acknowledgments The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) for financial support.

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