Molecular Biology Reports

, 36:2259

Associations of polymorphism within the GHSR gene with growth traits in Nanyang cattle

Authors

  • Bao Zhang
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
    • Institute of Cellular and Molecular BiologyXuzhou Normal University
  • Yikun Guo
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
  • Liangzhi Zhang
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
  • Miao Zhao
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
  • Xianyong Lan
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
  • Chunlei Zhang
    • Institute of Cellular and Molecular BiologyXuzhou Normal University
  • Chuanying Pan
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
  • Shenrong Hu
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
  • Juqiang Wang
    • Research Center of Cattle Engineering Technology in Henan
  • Chuzhao Lei
    • College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for AgricultureNorthwest A&F University
Article

DOI: 10.1007/s11033-008-9442-x

Cite this article as:
Zhang, B., Chen, H., Guo, Y. et al. Mol Biol Rep (2009) 36: 2259. doi:10.1007/s11033-008-9442-x

Abstract

GH secretagogue receptor (ghrelin receptor, GHSR) is known to be involved in the control of GH release by mediating the strong stimulatory effect of the endogenous ligand, ghrelin, on GH secretion. Associations between the GHSR gene polymorphism and the growth traits were revealed in Nanyang cattle. The mutations at nt456(G > A) and nt667(C > T) were complete linkage and located in exon 1 of the coding region of the GHSR gene. Least squares analysis revealed a significant statistical effect (P < 0.05) of the GHSR gene different genotypes on body weight and average daily gain at 6 months of age in Nanyang cattle. Individuals with GHSR-MM genotype showed higher body weight and average daily gain than individuals with GHSR-MN genotype.

Keywords

CattleGHSR genePolymorphismSNP (single nucleotide polymorphism)Growth traits

Introduction

The growth and development of animals are important for livestock production. In cattle, the growth and development have major effects on beef and milk production traits. These traits are controlled by polygene with pleiotropic effect. However, the major gene model suggests that a few genes may account for relatively large proportion of the genetic variation [1]. Moreover, these genes may be linked to some quantitative trait loci that may be associated with growth and production of animals.

The importance of ghrelin in the central regulation of feeding has been demonstrated in humans and animals [2, 3]. Ghrelin administration increases appetite and food intake in normal subjects and patients with decreased appetite, such as those suffering from cancer cachexia [2]. And ghrelin reduces insulin secretion and enhances energy intake in humans [4]. Moreover, plasma ghrelin levels had been shown to be lower in obese subjects [5, 6]. Recent evidence suggested that obesity was associated with an impairment of the entire ghrelin system [7]. GHSR is the sole receptor responsible for ghrelin’s acute orexigenic activity [8]. The GHSR gene was identified by expression cloning and found to be a previously unknown G protein coupled receptor expressed predominantly in brain, pituitary gland, and pancreas [9].

We were led to pursue the GHSR because the major physiological role of the GHSR appeared to be in the central regulation of food intake and body weight. In human, the GHSR gene is located within the QTL on chromosome 3q26–q29, which has been linked to phenotype of the metabolic syndrome. In one report, seven variants of the GHSR gene were identified. The frequency of the 171T allele of GHSR rs495225 was slightly, but not significantly, higher in obese subjects than in underweight individuals [10]. The 171T > C SNP mutation of the GHSR gene was a risk factor for bulimia nervosa [11]. The 611C > A transversion unveiled the critical importance of the GHSR-associated constitutive activity, and disclosed an unusual pathogenic mechanism of growth failure in humans [12].

So far, the associations between the GHSR gene polymorphism and growth traits have not been reported in cattle. Accordingly, our objective in this study was to examine the associations of the GHSR gene polymorphism with growth traits in Nanyang cattle.

Materials and methods

Animals

Genomic DNA samples were obtained from 649 individuals of five cattle breeds in China and an exotic breed (Nanyang cattle, 240; Qinchuan cattle, 141; Jiaxian cattle, 133; Chinese Holstein, 61; Jinnan cattle, 30; Angus, 44). These six cattle breeds were the same samples used in the previous work [13]. The records of growth traits and body sizes (birth weight, body weight, body height, body length, chest girth, hucklebone width, and average daily gain) from different growth periods (at the age of 6, 12, 18, and 24 months) in Nanyang breed were collected for statistical analysis. DNA samples were extracted from blood samples in compliance with standard procedures [14].

Primer sequences

Four primer pairs of the GHSR gene were designed based on bovine GHSR gene (GenBank accession no. LOC514203 and no. NW_001493715.1) [13].
Exon1

P1 F:5′-CACTCT TTTGCGCCTAACTAA-3′, R:5′-TCTCGCTGACAAACTGGAAG-3′. P2 F:5′-TTACCGGCCCTGGAACTTG-3′, R:5′-CAGCATCTTCACGGTCTGTTTG-3′

Exon2

P3 F:5′-ACTGACGTTCTCTTTCTCATTGT-3′, R:5′-CCGCTGTACTATGGCTTCTG-3′. P4 F:5′-AGTACAGCGGAACTTGGGA-3′, R:5′-ACAGCACTGATCTGGGACC-3′

Methods

The 15 μl volume contained: 50 ng genomic DNA, 10 pmol of each primer, 1× buffer (including 1.5 mmol/l MgCl2), 200 μmol dNTPs and 0.5 U of Taq DNA polymerase (TaKaRa, China). The PCR protocol was 95°C for 5 min followed by 35 cycles of 94°C for 35 s, annealing for 30 s, 72°C for 45 s and a final extension at 72°C for 10 min (56.3, 56.5, 63.5, and 56.5°C corresponding to four primer pairs). PCR products were analyzed by single-stranded conformation polymorphisms (SSCP). Aliquots of 5 μl of the PCR products were mixed with 5 μl of the denaturing solution, heated for 10 min at 98°C then chilled on ice. Denatured DNA was subjected to 10% PAGE (polyacrylamide gel, 80 × 73 × 0.75 mm) in 1 × TBE buffer and constant voltage (200 V) for 1.5–2.0 h. The gel was stained with 0.1% silver nitrate and visualized with 2% NaOH solution (supplied with 0.1% formaldehyde) [13]. After the polymorphism was detected, the PCR products of different electrophoresis patterns were sequenced and analyzed.

Statistical analysis

The linear model was applied to analyze the associations between the GHSR gene polymorphism and the growth traits in Nanyang breed. The following model for the PCR–SSCP marker effect was used for analysis: Y = μ + age + marker + e.

In this model, Y is phenotype of the animal, μ is the mean of the animal population, age is the age effect, marker is the marked genotype effect, e is the stochastic error.

The software SPSS (version 13.0) was used to analyze the associations between the genotypes and growth traits in cattle.

Results and discussion

Mutations of P1, P2, P3, and P4 loci were detected using PCR–SSCP and sequencing. In the previous study we found five SNPs [nt-7(A > C), nt456(G > A), nt667(C > T), nt3552(T > C), nt3566(A > G)] in the GHSR gene among 649 unrelated animals. And the discovered SNPs were already deposited in GenBank (Acc. No.: EU146105-EU146109). According to Acc. No.XM_592014 and NC_007299, the SNPs at nt-7 of the GHSR gene was in the 5′ untranslated region, nt3552 and 3566 were in the 3′ untranslated region. The SNPs at nt456 and 667 were located in exon 1 of the GHSR gene coding region, but caused no amino acid exchange [13]. Surprisingly, the nt456 and 667 were completely linked with each other. We firstly named the “G–C”mutation in the No. 456nt and 667nt of the exon1 as GHSR-M allele, while called the “A–T” mutation for GHSR-N allele. In the present study, the SNP at the No. 3566nt in complementary strand was T > C which hold an EcoRI endonuclease restriction site (G^AATTC), 383 bp PCR products digestion with EcoRI endonuclease produced different banding patterns which named as TT, TC, and CC genotypes (Fig.1).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-008-9442-x/MediaObjects/11033_2008_9442_Fig1_HTML.gif
Fig. 1

The SNP of nt3566 digested with EcoRI restriction enzyme. Representative EcoRI genotypes observed for 383 bp GHSR gene fragment in various banding patterns of cattle. In the present study, the SNP at No. 3566nt in the complementary strand was T > C which hold an EcoRI endonuclease restriction site (G^AATTC). Acodding to this picture, Lane M, molecular size marker (100 bp DNA ladder); lanes1, 3, 4, 5 were TT genotype; lanes 2, 6, 8, 9, 10 were TC genotype; lane 7 was CC genotype

We analyzed the associations of these five SNPs with growth traits in Nanyang cattle. Growth traits (birth weight, body weight, body height, body length, chest girth, hucklebone width, average daily gain) were analyzed at 6, 12, 18 and 24 months of the age of Nanyang cattle. Significant association of these SNPs with growth traits were not detected at nt-7, nt3552, nt3566 in tested populations (P > 0.05). At the exon1, individuals with GHSR-MM genotype had higher body weight and average daily gain than that of GHSR-MN genotype at the age of 6 months in Nanyang breed (P < 0.05). The individuals with GHSR-MM genotype also had higher birth weight, body height, body length, chest girth, hucklebone width than those of GHSR-MN genotype at the age of 6 months in Nanyang breed (P > 0.05). GHSR-NN genotype was not found in the present study. The results were shown in (Table 1). However, this association did not exist at 12, 18 and 24 months of the age of Nanyang cattle.
Table 1

The LSM and SE among the mutations at No. 456nt and 667nt of the GHSR gene in Nanyang breed

Traits

6 months of age

GHSR-MM

GHSR-MN

P value

Mean ± SE

Mean ± SE

 

Body weight (kg)

162.984 a ± 2.318

147.200 b ± 5.865

0.015

Body height (cm)

106.688 ± 0.627

105.000 ± 1.586

0.326

Body length (cm)

106.563 ± 0.702

103.000 ± 1.775

0.066

Chest girth (cm)

130.359 ± 0.926

125.900 ± 2.343

0.081

Hucklebone width (cm)

18.461 ± 0.173

17.900 ± 0.439

0.238

Average daily gain (kg)

0.738 a ± 0.012

0.654 b ± 0.031

0.015

Birth weight (kg)

30.102 ± 0.331

29.500 ± 0.838

0.507

Notes: Data with a different letter (a, b) within the same line differ significantly at P < 0.05. Mean ± SE: means ± standard error of means

At No. 456nt and 667nt of the GHSR gene, individuals with GHSR-MM genotype had higher body weight and average daily gain than those of GHSR-MN genotype at the age of 6 months in Nanyang breed (P < 0.05)

Body weight is a multi-gene cooperation. Expressions of these genes were modulated not only by environmental factors but above all by a number of modified genes interacting with each other. Among candidate genes, the GHSR gene is related to body weight phenotype. The GHSR gene major physiological role, however, appears to be in regulating food intake and energy homeostasis by partaking in neuronal mechanisms involving neuropeptide Y and agouti-related protein [1518].

As the target of the endogenous ligand of ghrelin, GHSR integrates two anabolic actions: first, it mediates the stimulatory effect of ghrelin on GH release, and second, it communicates the orexigenic and adipogenic activity of ghrelin, contributing to energy balance [19]. GHSs stimulate GH secretion via GHSR distinctly from GH-releasing factor and somatostatin [20]. A positive energy balance, in turn, is necessary to maximize the anabolic actions of GH. And GH could influence the growth of peripheral organs [21].

The frequency of the GHSR gene 171T allele was higher in obese subjects than in underweight individuals and was a risk factor for bulimia nervosa in the previous studies [10, 11]. It was shown that rs495225 mutation of the GHSR gene might influence the food intake and energy homeostasis. Also the 611 C > A transversion of the GHSR gene was association with growth failure in humans. As GHSR was the sole receptor responsible for ghrelin’s acute orexigenic activity. It was that genetic variations in the GHSR gene might change either GHSR expression or receptor properties and had an effect on appetite regulation by altered signaling, altered response to ghrelin, or an impaired capability to counterbalance inhibitory signals [22].

The present study investigated the relationship between common sequence variants of the GHSR gene with growth traits in an independent sample of Nanyang breed. Five SNPs were found. At nt456 and 667, only GHSR-MM and GHSR-MN genotypes were found. The SNPs at nt456 and 667 were located in exon 1 of the coding sequence, but caused no amino acid exchange. Thus, it was rather difficult to conclude about a direct effect of the GHSR genotypes typed on growth traits involved. It might suggest a linkage to another mutation being a causal mutation in the coding or regulatory regions of the gene. The SNPs at nt-7, nt3552, nt3566 were not detected significant association with growth traits in Nanyang breed.

This study also revealed that the polymorphism of the GHSR gene was significantly associated with growth traits at 6 months of age of Nanyang breed. And no significance existed at the age of 12, 18, and 24 months of Nanyang breed. The mutational population had inferior phenotype in this study. So the results suggested that the GHSR gene could play an important role in the process of growth in Nanyang breed. Genotyping of larger sample size should increase power and provide more challenges for future studies.

Acknowledgments

This study was supported by the National 863 Program of China (No. 2006AA10Z197), National Natural Science Foundation of China (No. 30771544), National Key Technology R&D Program (No. 2006BAD01A10-5), Innovative Foundation of Outstanding Talent from Henan Province (No. 0521001900), Sustaining Program for Topnotch Persons of Northwest A&F University (No. 01140101), and Natural Science Foundation of Xuzhou Normal University and Talent Foundation of Northwest A&F University, Basic and Foreland Technology Study Program of Henna Province (No. 072300430160), “13115” Sci-Tech Innovation Program of Shaanxi Province (2008ZDKG-11).

Copyright information

© Springer Science+Business Media B.V. 2009