The Association of Bovine T1R Family of Receptors Polymorphisms with Cattle Growth Traits ⇑ C.L

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Research in Veterinary Science xxx (2012) xxx–xxx

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Research in Veterinary Science

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The association of bovine T1R family of receptors polymorphisms with cattle growth traits ⇑ C.L. Zhang a, J. Yuan a, Q. Wang a, Y.H. Wang a, X.T. Fang a, C.Z. Lei b, D.Y. Yang c, H. Chen a, a Institute of Cellular and Molecular Biology, Xuzhou Normal University, Xuzhou, Jiangsu, PR China b College of Animal Science and Technology, Northwest Agriculture and Forestry University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling, Shaanxi, PR China c College of Life Science, Dezhou University, Dezhou, Shandong 253023, PR China article info abstract

Article history: The three members of the T1R class of taste-specific G protein-coupled receptors have been proven to Received 12 August 2011 function in combination with heterodimeric sweet and umami taste receptors in many mammals that Accepted 20 January 2012 affect food intake. This may in turn affect growth traits of livestock. We performed a comprehensive eval- Available online xxxx uation of single-nucleotide polymorphisms (SNPs) in the bovine TAS1R gene family, which encodes receptors for umami and sweet tastes. Complete DNA sequences of TAS1R1-, TAS1R2-, and TAS1R3-cod- Keywords: ing regions, obtained from 436 unrelated female cattle, representing three breeds (Qinchuan, Jiaxian Red, Taste receptors Luxi), revealed substantial coding and noncoding diversity. A total of nine SNPs in the TAS1R1 gene were SNP identified, among which seven SNPs were in the coding region, and two SNPs were in the introns. All five Cattle Body height SNPs in the TAS1R2 gene and all three SNPs in the TAS1R3 gene were identified in the coding region. Four SNPs (TAS1R1 g.5081C > T, TAS1R1 g.5110C > A, TAS1R2 g.288A > G, TAS1R2 g.2552T > C) were significantly associated with body height of Qinchuan cattle (P < 0.05). The heterozygous genotypes of the four SNPs showed a molecular heterosis on cattle heights at hip cross and sacra. The individuals with different genotypic combinations of the four SNPs had significant association with heights at hip cross and sacra (P < 0.05). Ó 2012 Published by Elsevier Ltd.

1. Introduction may promote selection and ingestion of a diet containing an ade- quate level of protein (Gietzen et al., 2007). The sensory capabili- The T1R family of Class C G protein-coupled receptors is respon- ties of normal individuals with normal weight appear to be sible for the generation of the signals that are ultimately inter- higher than that of overweight subjects (Simchen et al., 2006). preted as ‘‘sweetness’’ and those leading to the so called The extent of variation in human TAS1R genes has been well ‘‘umami’’ taste associated with some L-amino acids, especially L- characterized, 17 SNPs in TAS1R1 (including three synonymous glutamate. There are three proteins in the family, T1R1, T1R2, and 14 nonsynonymous variants) and 12 SNPs in TAS1R3 (six and T1R3, encoded by their respective genes, Tas1r1, Tas1r2, and nonsynonymous and six synonymous SNPs) were detected Tas1r3. T1R2 and T1R3 form a heterodimer that can bind sugars (Kim et al., 2006). The frequencies of two nsSNPs, C329T in and other sweeteners (Li et al., 2002; Zhao et al., 2003; Nie et al., TAS1R1 and C2269T in TAS1R3, were significantly higher in 2005). T1R3 also combines with T1R1 to form a heterodimer that nontasters than expected, whereas G1114A in TAS1R1 was more binds with L-amino acids (Nelson et al., 2002; Damak et al., frequent in tasters (Raliou et al., 2009). The TAS1R1-372T creates 2003; Taylor-Burds et al., 2004; Scott, 2004). a more sensitive umami receptor than -372A, while TAS1R3- The ability to detect orexigenic tastes had significant affect on 757C creates a less sensitive one than -757R for MSG and MSG dietary behavior and growth of humans and other mammals plus IMP, and showed a strong correlation between the recogni- (Golding et al., 2009). The bone resorption ability was significantly tion thresholds and in vitro dose response relationships (Shigem- higher in the high sugar preference group compared with the low ura et al., 2009). The amino acid substitutions (A110V and preference group (Coldwell et al., 2009). It was reported that the R507Q) in the N-terminal ligand-binding domain of T1R1, and absorption of sugars, peptides and amino acids is coordinated by the two amino acid substitutions (F749S and R757C), located signaling of sweet and amino acid taste receptors to target a in the transmembrane domain of T1R3, severely impaired common enterocytic pool of PKCbII (Mace et al., 2009). Taste in vitro T1R1/T1R3 response to MSG (Raliou et al., 2011). A genetic variation in TAS1R2 (Ile191Val) affected the habitual con-

⇑ Corresponding author. sumption of sugars, and it may have contributed to difference E-mail address: [email protected] (H. Chen). of human body weight (Eny et al., 2010). This suggested that

0034-5288/$ - see front matter Ó 2012 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2012.01.014

Please cite this article in press as: Zhang, C.L., et al. The association of bovine T1R family of receptors polymorphisms with cattle growth traits. Res. Vet. Sci. (2012), doi:10.1016/j.rvsc.2012.01.014 2 C.L. Zhang et al. / Research in Veterinary Science xxx (2012) xxx–xxx the variation of TAS1R genes might affect their function. For live- three samples containing identical genotypes confirmed that they stock, this may in turn affect tissue accretion and growth. In an displayed strictly identical sequences. effort to obtain thorough understanding of genetic and func- tional variation in bovine taste perception, we performed a sur- 2.5. Statistical analysis vey of polymorphisms in the three taste receptor genes. Gene frequencies were determined for each breed by direct counting. Statistical analysis of associations between genotypes 2. Materials and methods and growth traits of Qinchuan heifers was performed, using SPSS (version 13.0). The model applied was: 2.1. Sampling and DNA extraction Yijk = l + Ai + Bj + Gk + eijkl, where Yijk was the trait measured on each of the th animal, was the overall population mean, A was In this study, 436 unrelated female cattle, representing Qinch- ijk l i fixed effect due to the th age, B was fixed effect due to the breed, uan (228), Jiaxian Red (143), and Luxi (65) were included. Qinch- i j G was the fixed effect associated with th genotype and e was uan, Jiaxian Red, Luxi are important breeds for beef production k k ijkl the random error (animal as a random effect with pedigree rela- in China. The animals of each breed were selected to be unre- tionships between animals). The least square means estimates lated for at least three generations for the purpose of having di- with standard errors for genotypes and growth traits were used. verse lineages within each breed. Blood samples were obtained from the jugular veins of cattle. Blood genomic DNA was ex- tracted by standard methods. 3. Results The 228 cows of Qinchuan breed used for the association study came from a common ancestor, and pedigrees of core breeding In the present study, a total of nine SNPs of TAS1R1 gene were population animals were traced back three generations. Calves identified, among which seven were in the coding region, and two were weaned at the average age of 6 months and raised from were in the introns. Five SNPs of TAS1R2 gene and three SNPs of weaning to slaughter on a corn–corn silage diet, according to nutri- TAS1R3 gene were detected in the coding region (Fig. 1). The nat- ent requirements of growing heifers (NRC, 2000). Heifers have free ure and the distribution of mutations in the three genes are shown access to the same diet and fresh water. All heifers were tethered in Table 1. individually in stalls and housed in a large barn. They were daily We investigated whether TAS1R genes variations associated released to sport field for three hours to ensure their health if with the growth traits of cattle. Results of the association analysis weather permitted. Heights at hip cross and sacra, chest circumfer- are summarized in Table 2. Four SNPs have an effect on body ence and body lengh, which were key parameters of growth traits, height (P < 0.05, Table 2), but they have no effect on chest circum- were recorded at 24 months. ference and body lengh of Qinchuan cattle. Cattle with the TAS1R1 g.5081CT genotype had lower heights at hip cross and sacra than the TAS1R1 g.5081CC animals, and cattle with the TAS1R1 2.2. Primer design and PCR amplification g.5110CA genotype had lower heights at hip cross and sacra mean than the TAS1R1 g.5110CC and g.5110AA animals (P < 0.05) (Table Thirty-one pairs of PCR primers (Supplementary Table 1) were 2). Cattle with the TAS1R2 g.2552TC genotype had higher heights designed to amplify the coding and flanking region of bovine at hip cross and sacra than the TAS1R2 g.2552TT and TAS1R2 TAS1R1 (NW_001493427), TAS1R2 (NC_007300) and TAS1R3 g.2552CC animals, but cattle with the TAS1R2 g.12375CG genotype (NW_001493430) by using Primer V5.0 software. The 20 ll PCR had lower heights at hip cross and sacra than the TAS1R2 reaction volume contained 50 ng DNA template, 0.20 mM dNTP, g.12375CC and TAS1R2 g.12375GG animals (P < 0.05, Table 2). 2.5 mM MgCl2 and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, The four SNPs that associated with growth traits produced China). The PCR protocol was performed using the following pro- seven types of genotypic combinations (Table 3). The association gram: 94 °C for 5 min followed by 35 cycles of 94 °C for 40 s, analysis suggested that the individuals with different genotypic annealing for 40 s, and 72 °C for 1 min and a final extension at combinations had different heights at hip cross and sacra 72 °C for 10 min. (P < 0.05) (Table 3). Cattle with TAS1R1 g.5081CT/TAS1R1 g.5110AA/TAS1R2 g.2552TC/TAS1R2 g.12375CC (CT/AA/TC/CC) 2.3. Single stranded conformation polymorphism (SSCP) had the highest heights at hip cross and sacra. The animals with CT/AA/TC/CC genotypic combination were significant taller than Aliquots of 5 ll of above PCR products were mixed with 5 llof that of with CC/CC/TC/CC, TT/CC/TC/CC, CC/CA/TC/CC and CC/CC/ the denaturing solution (95% formamide, 25 mM EDTA, 0.025% xy- TC/CG genotypic combination (P < 0.05). lene-cyanole and 0.025% bromophenol blue), heated for 10 min at 98 °C and chilled on ice. Denatured DNA was subjected to 10% 4. Discussion PAGE (80 Â 73 Â 0.75 mm) analysis which was run with 1 Â TBE buffer (89 mM Tris–Borate, 2 mM EDTA, pH 8.3) for 2 h at room It has been well documented that variation in the TAS1R gene temperature under a constant voltage (150 V). The gel was stained family has been under positive natural selection, so many of the with silver nitrate and visualized with 2% NaOH solution (contain- variants detected might be functionally significant in taste percep- ing 0.1% formaldehyde) according to Zhang et al. (2007). tion (Kim et al., 2006; Shi and Zhang, 2006). Our findings suggested that most of the SNPs in exon of bovine TAS1R genes (10 of 15) re- 2.4. DNA sequencing analysis sulted to amino acid variation. This is consistent with the observa- tion in humans (Kim et al., 2006). The PCR products from different PCR-SSCP genotypes were puri- Four SNPs in the coding region of TAS1R1 gene were nonsynony- fied with the GenElute PCR DNA Purification Kit (Sigma–Aldrich mous. The TAS1R1 g.4491G > C (405G > R) and TAS1R1 g.4522G > A Corporation, USA) and sequenced by the ABI 377 sequencer from (415G > Q) located in exon3. The TAS1R1 g.4491 G > C leads to a both directions (Applied Biosystems, USA). Follow-up sequencing substitution of glycine for arginine at 405 position (405G > R). This of all different genotypes confirmed that they differed in one or mutation induced a change of the pI from 5.97 to 10.76 which in more mutations; while on the other hand, sequencing of up to turn resulted in the electric charge variation. The TAS1R1

Fig. 1. The distribution of SNPs and amino acid variations in TAS1R genes. Solid boxes correspond to exons, and lines correspond to introns.

Table 1 The SNPs identiﬁed in the TAS1R genes in Chinese cattle and allele frequencies of the SNPs.

Gene Region SNP Amino acid coded Allelic variant frequency QC (n = 228) JX (n = 143) LX (n = 65) TAS1R1 Exon3 4491G > C 405G > R 0.24 0.30 0.00 4522G > A 415G > Q 0.08 0.23 0.00 Intron3 4593C > T – 0.13 0.16 0.00 Exon4 5081C > T 452I > I 0.17 0.39 0.08 5096C > T 457S > S 0.43 0.26 0.49 5110C > A 462T > N 0.17 0.01 0.08 Exon5 5517G > A 499R > R 0.30 0.36 0.40 5610A > G 530 K > R 0.40 0.49 0.45 Intron5 5624G > A – 0.30 0.36 0.40 TAS1R2 Exon1 288A > G 46 K > K 0.32 0.33 0.28 Exon2 2552T > C 111 V > A 0.25 0.23 0.25 Exon4 6198A > C 445 N > H 0.09 0.13 0.05 Exon6 12004C > T 614T > M 0.38 0.43 0.32 Exon6 12375C > G 738L > V 0.38 0.36 0.27 TAS1R3 Exon3 598C > T 200R > W 0.13 0.16 0.08 628G > A 210 V > M 0.13 0.16 0.08 Exon6 2217C > T 739A > A 0.29 0.38 0.00

Nucleotides numbered in relation to DNA nucleotide positions (GenBank accession No. NW_001493427, NC_007300 and XM_588865); QC = Qinchuan cattle, JX = Jiaxian cattle, LX = Luxi cattle. g.5110C > A in exon4 leads to a substitution of threonine for aspar- and genetic variations in human TAS1R1 were observed, where agine at 462 position (462T > N), which induces a change of pI from TAS1R1-372T creates a more sensitive umami receptor than - 6.53 to 5.41. The TAS1R1 g.5610A > G in exon5 leads to a substitu- 372A (Shigemura et al., 2009). The amino acid residuals at position tion of lysine for arginine at 530 position (530 K > R), which induces 405, 415 and 462 are located at ligand-binding domain of the bovine a change of pI from 9.74 to 10.76. The N-terminal extracellular do- T1R taste receptor, which may affect the amino acid taste. main was ligand-binding domain, it determined the ligand speciﬁc- Four SNPs in the coding region of bovine TAS1R2 gene resulted ity of T1R taste receptor (Zhang et al., 2008, 2010). Moreover, the in the variation of coding amino acid. The TAS1R2 g.6198A > C lead association between recognition thresholds for umami substances to a substitution of valine for alanine at 111 position (111V > A),

Table 2 The effect of different genotypes on growth traits in Qinchuan cattle.

Gene Genotype Number Height at hip cross (cm) Height at sacra (cm) TAS1R1 5081CC 20 132.00 ± 6.86a 135.90 ± 8.15 5081CT 208 127.25 ± 4.19 130.60 ± 4.52 P value 0.026 0.024 5110CC 188 127.49 ± 4.19 130.93 ± 4.58 5110CA 20 125.00 ± 3.82 127.60 ± 2.75 5110AA 20 132.00 ± 6.87 135.90 ± 4.15 P value 0.043 0.028 TAS1R2 2552TT 136 126.38 ± 4.37 129.76 ± 4.80 2552TC 64 129.88 ± 3.15 133.72 ± 3.71 2552CC 28 128.86 ± 6.65 131.36 ± 7.06 P value 0.030 0.033 12375CC 116 127.17 ± 4.51 130.21 ± 4.87 12375CG 72 126.56 ± 3.90 130.44 ± 4.63 12375GG 40 131.10 ± 4.87 134.70 ± 5.24 P value 0.028 0.041

a Means ± standard deviation.

Table 3 Genotypic combinations of TAS1R1 and TAS1R2 gene and their association with height at hip cross and height at sacra of Qinchuan cattle.

Combined genotype Sequence Number Height at hip cross (cm) Height at sacra (cm) 1 CC/CC/TC/CC 63 126.39 ± 3.07* 129.29 ± 3.29** 2 TT/CC/TC/CC 18 125.38 ± 5.96* 128.13 ± 6.36** 3 CT/CC/TC/CC 27 129.67 ± 2.99 132.92 ± 3.99 4 CT/AA/TC/CC 23 131.00 ± 3.81# 136.00 ± 3.45# 5 CC/CA/TC/CC 54 125.17 ± 2.96** 129.13 ± 3.50** 6 CT/CA/TC/CC 14 127.50 ± 2.78 130.83 ± 3.25 7 CC/CC/TC/CG 22 122.88 ± 1.03** 125.75 ± 0.50**

# The value of combined genotype 4 served as reference. * P < 0.05. ** P < 0.01. which was ligand-binding domain of the T1R taste receptor. An TAS1R gene polymorphisms and cattle body height should be con- SNP in the human TAS1R2 gene located in this homologous region firmed in larger population and more cattle breeds before TAS1R was detected to be associated with consumption of sugars (Eny SNPs be applied to molecular breeding. et al., 2010). Two SNPs (TAS1R3 g.598C > T and TAS1R3 g.628G > A) in the Acknowledgements exon3 of TAS1R3 gene were nonsynonymous. The TAS1R3 g.598C > T leads to a substitution of arginine for tryptophan at This work was funded by the National Natural Science Founda- 200 position (200R > W). This mutation induced a change of the tion of China (30972080, 30901023, 31101703), Natural Science pI from 10.76 to 5.89 which in turn resulted in the electric charge Foundation of Jiangsu Province (BK2011206), National Key Tech- variation. The TAS1R3 g.628G > A mutation leads to a substitution nology R&D Program (2008BADB2B03-19), Keystone Project of of valine for methionine at position 210 (210 V > M). The two Transfergene in China (2009ZX08009-157B, 2008ZX08007-002 amino acid variations are located in the monosaccharides ligand and 2009ZX08007-005B-07), MaTS-Beef Cattle System (CARS-38), binding domains, so they may affect the sweet taste, but may not Graduate Students Research and Innovation Plan of Jiangsu Prov- affect the amino acid taste. ince (CX09S-044Z), Graduate Student Research Program of Xuzhou Interestingly, heterozygous genotypes of the four SNPs showed Normal University (NO. 09YLA006). a molecular heterosis. Molecular heterosis occurs when subjects heterozygous for a specific genetic polymorphism show a significantly greater effect (positive heterosis) or lesser effect (negative Appendix A. Supplementary data heterosis) for a quantitative or dichotomous trait than subjects homozygous for either allele (Comings and MacMurray, 2000). Supplementary data associated with this article can be found, in Many examples of molecular heterosis in humans and rodents the online version, at doi:10.1016/j.rvsc.2012.01.014. have been reported, including those for the following genes: ADRA2C, C3 complement, DRD1, DRD2, DRD3, DRD4, ESR1, HP, References HBB, HLA-DR DQ, HTR2A, properdin B, SLC6A4, PNMT, and secretor (Gosso et al., 2008; Tsuchimine et al., 2009; May et al., 2010). But Coldwell, S.E., Oswald, T.K., Reed, D.R., 2009. A marker of growth differs between adolescents with high vs. low sugar preference. Physiology & Behavior 96, 574– no examples of molecular heterosis have been reported in live- 580. stock. It was thought that allelic variants at a large number of loci Comings, D.E., MacMurray, J.P., 2000. Molecular heterosis: a review. Molecular acting through partial to complete dominance would provide Genetics and Metabolism 71, 19–31. Damak, S., Rong, M., Yasumatsu, K., Kokrashvili, Z., Varadarajan, V., Zou, S., Jiang, P., favorable combinations resulting in superior hybrid phenotypes, Ninomiya, Y., Margolskee, R.F., 2003. Detection of sweet and umami taste in the but the molecular basis of heterosis is not fully understood absence of taste receptor T1r3. Science 301, 850–853. (Springer and Stupar, 2007). Eny, K.M., Wolever, T.M., Corey, P.N., El-Sohemy, A., 2010. Genetic variation in TAS1R2 (Ile191Val) is associated with consumption of sugars in overweight and However, because many parameters are involved in the quanti- obese individuals in 2 distinct populations. American Journal of Clinical tative characters as height of bones, the association between Nutrition 92, 1501–1510.

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