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A novel SNP of the GHRL gene in goat and its association with growth traits
Small Ruminant Research 90 (2010) 150–152
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
A novel SNP of the GHRL gene in goat and its association with growth traits Q.J. Jin a,1 , X.T. Fang a,1 , C.L. Zhang a , L. Yang a , J.J. Sun a , D.X. Chen a , X.Y. Shi a , Y. Du a , X.Y. Lan b , H. Chen a,∗ a
Institute of Cellular and Molecular Biology, Xuzhou Normal University, Xuzhou, Jiangsu 221116, PR China College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling, Shaanxi 712100, PR China
b
a r t i c l e
i n f o
Article history: Received 20 September 2009 Received in revised form 2 November 2009 Accepted 4 January 2010 Available online 20 January 2010 Keywords: GHRL gene Single nucleotide polymorphism (SNP) PCR–SSCP Goat
a b s t r a c t Ghrelin, an endogenous gland for growth hormone secretagogue receptor, has been shown to stimulate food intake and control energy homeostasis and lipid metabolism. So, ghrelin precursor (GHRL) gene is a potential candidate gene for caprine growth traits. In this study, we detected the polymorphism of the caprine GHRL gene by PCR–SSCP and DNA sequencing methods in 459 individuals from four goat breeds. A novel single nucleotide polymorphism (SNP) (IVS2 + 147G > A) was detected. Frequencies of IVS2 + 147G allele varied from 0.842 to 1.000. The association of IVS2 + 147G > A with growth traits was analyzed and IVS2 + 147G > A was shown to be associated with growth traits. Individuals with genotype AG were significantly higher than those of individuals with genotype GG in circumference of chest and cannon and trunk index (P < 0.05 or P < 0.01). Moreover, there was a tendency that genotype AG individuals had better performance in other aspects such as body height and body length than genotype GG individuals although no significant differences appeared (P > 0.05). We suggested that IVS2 + 147G > A could be a perfect molecular marker in marker-assisted selection (MAS). © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin has been shown to exert very potent and specific GH-releasing activity in rats (Yamazaki et al., 2002) and humans (Arvat et al., 2000). It is secreted from neuroendocrine cells, particularly of the gastrointestinal tract, and plays a role in stimulating eating and regulating energy balance, insulin signaling, and glucose levels (Kojima et al., 1999; Date et al., 2000; Murata et al., 2002). It is reported that plasma ghrelin levels decrease 1 h after feeding and then return to prefeeding levels in cows (Hayashida et
al., 2001). A recent study showed that injection of bovine ghrelin at physiological concentration resulted in elevated plasma GH concentrations and length of time spent eating was greater (Wertz-Lutz et al., 2006). Considering the effects of ghrelin on the growth hormone axis and the control of enteric nutrition, this study was conducted to detect the polymorphisms within caprine GHRL gene, and then analyzed the association between each genotype and its growth traits among four goat breeds. This will be helpful for conserving, utilizing, and exploiting the genetic resources of goat. 2. Material and methods
∗ Corresponding author at: Institute of Cellular and Molecular Biology, Xuzhou Normal University, Xuzhou, Jiangsu 221116, PR China. Tel.: +86 516 83500087; fax: +86 516 83500087. E-mail address: [email protected] (H. Chen). 1 These authors contributed equally to this paper. 0921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2010.01.001
2.1. Goats and DNA sources Genomic DNA samples were obtained from 459 unrelated goats belonging to four breeds: Boer goat (BE, n = 111), Chinese Xuhuai white goat (XH, n = 108) and Chinese Haimen goat (HM, n = 136), crossing pop-
Q.J. Jin et al. / Small Ruminant Research 90 (2010) 150–152
151
Fig. 1. PCR–SSCP patterns and the corresponding sequencing map of the GHRL gene P1 locus. ulation (BE × XH, n = 104). They were reared in Jiangsu Province of China. DNA samples were extracted from leucocytes according to Mullenbach et al. (1989).
2.2. PCR amplification As there was no available sequence information of caprine GHRL gene, comparative alignments of amino-acid sequences and nucleotide sequences from cattle and sheep were used to design PCR primers that would amplify most exons and some introns of caprine GHRL gene. The sequences of these primers were: P1: forward 5 -GTCCATCTGCCTCCAGCCA-3 and reverse 5 -CACCTTCCTCCTGACTTCCCA-3 ; P2: forward 5 -TTCAATGCCCCCTTTAAC-3 and reverse 5 -CGTGGTCTCGGAAGTGTC-3 .
2.3. SSCP and DNA sequencing Aliquots of 5 l PCR products were mixed with 5 l denaturing solution, heated for 10 min at 98 ◦ C and chilled in ice. Denatured DNA was subjected to 10% PAGE (polyacrylamide gel electrophoresis) in 1× TBE buffer and constant voltage (150 V) for 15 h at a constant temperature of 4 ◦ C. The gel was stained with 0.1% silver nitrate and visualized with 2% NaOH solution (containing 0.1% formaldehyde) (Zhang et al., 2007). The PCR fragments from different SSCP patterns were sequenced in both directions.
2.4. Association studies and statistical analysis Gene frequencies were determined for each breed by direct counting. A 2 -test was applied to assess statistical significance using the software of SPSS V17.0 (SPSS Inc., USA). The effects of genotype on the traits were analyzed by the least-squares method as applied in the general linear model (GLM) procedure of SPSS according to the following statistical model: Yij = + Bi + Gj + Eij , where Yij was the trait measured on each of the ijth animal, the overall mean, Bi the type of the ith breed; Gj the type of the jth genotype and Eij was the random error.
3. Results and discussions According to the strong similarity between cattle, sheep and goat, in the present study, we detected the exons 2, 3, 4, 5 and introns 2, 4 of the caprine GHRL gene. No polymorphism was detected in the region of exons 4, 5 and intron 4 (P2 locus), and two unique SSCP banding patterns (GG/AG) were detected in the region of exons 2, 3 and intron 2 (P1 locus) after PCR–SSCP analysis (Fig. 1). Then seven DNA amplification fragments of different SSCP patterns of four goat breeds were sequenced. The comparisons among these sequences revealed a novel mutation: A to G at nucleotide 147 of intron 2 (IVS2 + 147G > A) (Fig. 1). The frequency of genotype AG is low in four breeds (BE, 0.315; HM, 0.000; XH, 0.250; BE × XH, 0.288) (Table 1). Homozygote genotype AA was not observed in the four goat breeds in this study. It is an interesting phenomenon worth of further investigation on mutations that influence growth variation. According to Nei and Roychoudhury’s (1974) methods, the population genetic indexes (namely, gene homozygosity, gene heterozygosity, effective allele numbers (Ne) and polymorphism information content (PIC)) were calculated (Table 1). Both loci of the four populations showed low polymorphism. The values of PIC, He of Boer breed in the P1 locus were higher than that of other populations, which implied that the polymorphism and genetic variation of Boer breed were higher than that of other populations. Gene homozygosity varied from 0.685 (BE) to 1.000 (HM) and Ne ranged from 1.000 to 1.362. The minimum and maximum PIC values were 0.195 and 0.230. This reflected that there was not a very high genetic diversity within caprine GHRL gene in analyzed populations.
Table 1 Genotype distribution and genetic diversity at the GHRL gene P1 locus. Breeds
Boer goat Haimen goat Xuhuai goat Boer × Xuhuai goat
Observed genotypes
Allele frequencies
GG
AG
A
G
76 136 81 73
35 0 27 31
0.158 0.000 0.125 0.149
0.842 1.000 0.875 0.851
Gene homozygosity (Ho)
Gene heterozygosity (He)
Effective allele numbers (Ne)
Polymorphism information content (PIC)
0.685 1.000 0.750 0.702
0.315 0.000 0.250 0.298
1.362 1.000 1.280 1.340
0.231 0.000 0.195 0.221
152
Q.J. Jin et al. / Small Ruminant Research 90 (2010) 150–152
Table 2 Association analysis of IVS2 + 147G > A with growth traits in goats (Boer goat, Xuhuai white goat and Boer goat × Xuhuai goat). Growth trait
Genotype (means ± standard error of means) GG (n = 230)
Body height (cm) Body length (cm) Cannon circumference (cm) Chest circumference (cm) Body length index (%) Chest circumference index (%) Cannon circumference index (%) Trunk index (%)
63.826 73.778 8.961 78.785 116.043 123.934 14.105 107.418
± ± ± ± ± ± ± ±
AG (n = 93)
0.418 0.529 0.627a 0.085a 0.800 0.965 0.135 0.833a
64.462 73.909 9.323 81.882 114.770 127.165 14.483 111.243
± ± ± ± ± ± ± ±
P value 0.541 0.825 0.933b 0.137b 1.003 1.177 0.191 1.129b
0.392 0.895 0.024 0.008 0.368 0.057 0.124 0.011
Means with a different superscript letter were significantly different (P < 0.05).
The relationships between genotypes and eight growth traits were done in 323 samples (Table 2). Significant statistical differences were found in chest circumference (P < 0.01), cannon circumference (P < 0.05) and trunk index (P < 0.05). Multiple comparisons results indicated that individuals with genotype AG were significantly higher than those of individuals with genotype GG in circumference of chest and cannon and trunk index (P < 0.05). Moreover, there was a tendency that genotype AG individuals had better performance in other aspects such as body height and body length than genotype GG individuals although no significant differences appeared (P > 0.05). The SNP studied here is in intron 2 of the caprine GHRL gene, so it may not be a causal mutation. Thus, one may suggest that it could be in linkage disequilibrium with another SNP in the GHRL gene with greater effects on the traits. A mutation of this kind could be present in, for example, promoter or intron areas that were not screened in this study. Moreover, introns have been revealed to affect transcriptional efficiency of numerous genes in a variety of organisms (Greenwood and Kelsoe, 2003; Le Hir et al., 2003). The present study revealed a novel SNP in intron 2 of caprine GHRL gene. The SNP is associated with chest circumference, cannon circumference and trunk index. Considering the G allele was recessive in four cattle breeds, we should reject the G allele in the breeding schemes of goat. This study was designed as the first step in detecting genetic markers for caprine GHRL gene, which would eventually provide useful information for the improvement of Chinese native goat. Acknowledgements This work was supported by the National “863” Program of the P.R. China (No. 2008AA10Z138), Natural Science Foundation of Jiangsu Province (No. BK2008120), the “13115” Sci-Tech Innovation Program of Shaanxi Province (2008ZDKG-11), Research Fund for the Doctor Program of Higher Education of China (No. 200807120012), Natural science fund for colleges and universities in Jiangsu
Province (09KJD180002), Natural Science Foundation of Xuzhou Normal University (07XLA08; KY2007019), and the Young Topnotch Researcher Support Project of Northwest A&F University (No. QNGG-2009-007). References Arvat, E., Di Vito, L., Broglio, F., Papotti, M., Muccioli, G., Dieguez, C., Casanueva, F., Deghenghi, R., Camanni, F., Ghigo, E., 2000. Preliminary evidence that Ghrelin, the natural GH secretagogue (GHS)-receptor ligand, strongly stimulates GH secretion in humans. J. Endocrinol. Invest. 23, 493. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., Matsukura, S., Kangawa, K., Nakazato, M., 2000. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans 1. Endocrinology 141, 4255–4261. Greenwood, T.A., Kelsoe, J.R., 2003. Promoter and intronic variants affect the transcriptional regulation of the human dopamine transporter gene. Genomics 82, 511–520. Hayashida, T., Murakami, K., Mogi, K., Nishihara, M., Nakazato, M., Mondal, M.S., Horii, Y., Kojima, M., Kangawa, K., Murakami, N., 2001. Ghrelin in domestic animals: distribution in stomach and its possible role. Domest. Anim. Endocrin. 21, 17–24. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656–660. Le Hir, H., Nott, A., Moore, M.J., 2003. How introns influence and enhance eukaryotic gene expression. Trends Biochem. Sci. 28, 215–220. Mullenbach, R., Lagoda, P.J., Welter, C., 1989. An efficient salt-chloroform extraction of DNA from blood and tissues. Trends Genet. 5, 391. Murata, M., Okimura, Y., Iida, K., Matsumoto, M., Sowa, H., Kaji, H., Kojima, M., Kangawa, K., Chihara, K., 2002. Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J. Biol. Chem. 277, 5667–5674. Nei, M., Roychoudhury, A.K., 1974. Sampling variances of heterozygosity and genetic distance. Genetics 76, 379–390. Wertz-Lutz, A.E., Knight, T.J., Pritchard, R.H., Daniel, J.A., Clapper, J.A., Smart, A.J., Trenkle, A., Beitz, D.C., 2006. Circulating ghrelin concentrations fluctuate relative to nutritional status and influence feeding behavior in cattle. J. Anim. Sci. 84, 3285. Yamazaki, M., Nakamura, K., Kobayashi, H., Matsubara, M., Hayashi, Y., Kangawa, K., Sakai, T., 2002. Regulational effect of ghrelin on growth hormone secretion from perifused rat anterior pituitary cells. J. Neuroendocrinol. 14, 156. Zhang, C.L., Wang, Y., Chen, H., Lan, X.Y., Lei, C.Z., 2007. Enhance the efficiency of single-strand conformation polymorphism analysis by short polyacrylamide gel and modified silver staining. Anal. Biochem. 365, 286–287.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
A novel SNP of the GHRL gene in goat and its association with growth traits Q.J. Jin a,1 , X.T. Fang a,1 , C.L. Zhang a , L. Yang a , J.J. Sun a , D.X. Chen a , X.Y. Shi a , Y. Du a , X.Y. Lan b , H. Chen a,∗ a
Institute of Cellular and Molecular Biology, Xuzhou Normal University, Xuzhou, Jiangsu 221116, PR China College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling, Shaanxi 712100, PR China
b
a r t i c l e
i n f o
Article history: Received 20 September 2009 Received in revised form 2 November 2009 Accepted 4 January 2010 Available online 20 January 2010 Keywords: GHRL gene Single nucleotide polymorphism (SNP) PCR–SSCP Goat
a b s t r a c t Ghrelin, an endogenous gland for growth hormone secretagogue receptor, has been shown to stimulate food intake and control energy homeostasis and lipid metabolism. So, ghrelin precursor (GHRL) gene is a potential candidate gene for caprine growth traits. In this study, we detected the polymorphism of the caprine GHRL gene by PCR–SSCP and DNA sequencing methods in 459 individuals from four goat breeds. A novel single nucleotide polymorphism (SNP) (IVS2 + 147G > A) was detected. Frequencies of IVS2 + 147G allele varied from 0.842 to 1.000. The association of IVS2 + 147G > A with growth traits was analyzed and IVS2 + 147G > A was shown to be associated with growth traits. Individuals with genotype AG were significantly higher than those of individuals with genotype GG in circumference of chest and cannon and trunk index (P < 0.05 or P < 0.01). Moreover, there was a tendency that genotype AG individuals had better performance in other aspects such as body height and body length than genotype GG individuals although no significant differences appeared (P > 0.05). We suggested that IVS2 + 147G > A could be a perfect molecular marker in marker-assisted selection (MAS). © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin has been shown to exert very potent and specific GH-releasing activity in rats (Yamazaki et al., 2002) and humans (Arvat et al., 2000). It is secreted from neuroendocrine cells, particularly of the gastrointestinal tract, and plays a role in stimulating eating and regulating energy balance, insulin signaling, and glucose levels (Kojima et al., 1999; Date et al., 2000; Murata et al., 2002). It is reported that plasma ghrelin levels decrease 1 h after feeding and then return to prefeeding levels in cows (Hayashida et
al., 2001). A recent study showed that injection of bovine ghrelin at physiological concentration resulted in elevated plasma GH concentrations and length of time spent eating was greater (Wertz-Lutz et al., 2006). Considering the effects of ghrelin on the growth hormone axis and the control of enteric nutrition, this study was conducted to detect the polymorphisms within caprine GHRL gene, and then analyzed the association between each genotype and its growth traits among four goat breeds. This will be helpful for conserving, utilizing, and exploiting the genetic resources of goat. 2. Material and methods
∗ Corresponding author at: Institute of Cellular and Molecular Biology, Xuzhou Normal University, Xuzhou, Jiangsu 221116, PR China. Tel.: +86 516 83500087; fax: +86 516 83500087. E-mail address: [email protected] (H. Chen). 1 These authors contributed equally to this paper. 0921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2010.01.001
2.1. Goats and DNA sources Genomic DNA samples were obtained from 459 unrelated goats belonging to four breeds: Boer goat (BE, n = 111), Chinese Xuhuai white goat (XH, n = 108) and Chinese Haimen goat (HM, n = 136), crossing pop-
Q.J. Jin et al. / Small Ruminant Research 90 (2010) 150–152
151
Fig. 1. PCR–SSCP patterns and the corresponding sequencing map of the GHRL gene P1 locus. ulation (BE × XH, n = 104). They were reared in Jiangsu Province of China. DNA samples were extracted from leucocytes according to Mullenbach et al. (1989).
2.2. PCR amplification As there was no available sequence information of caprine GHRL gene, comparative alignments of amino-acid sequences and nucleotide sequences from cattle and sheep were used to design PCR primers that would amplify most exons and some introns of caprine GHRL gene. The sequences of these primers were: P1: forward 5 -GTCCATCTGCCTCCAGCCA-3 and reverse 5 -CACCTTCCTCCTGACTTCCCA-3 ; P2: forward 5 -TTCAATGCCCCCTTTAAC-3 and reverse 5 -CGTGGTCTCGGAAGTGTC-3 .
2.3. SSCP and DNA sequencing Aliquots of 5 l PCR products were mixed with 5 l denaturing solution, heated for 10 min at 98 ◦ C and chilled in ice. Denatured DNA was subjected to 10% PAGE (polyacrylamide gel electrophoresis) in 1× TBE buffer and constant voltage (150 V) for 15 h at a constant temperature of 4 ◦ C. The gel was stained with 0.1% silver nitrate and visualized with 2% NaOH solution (containing 0.1% formaldehyde) (Zhang et al., 2007). The PCR fragments from different SSCP patterns were sequenced in both directions.
2.4. Association studies and statistical analysis Gene frequencies were determined for each breed by direct counting. A 2 -test was applied to assess statistical significance using the software of SPSS V17.0 (SPSS Inc., USA). The effects of genotype on the traits were analyzed by the least-squares method as applied in the general linear model (GLM) procedure of SPSS according to the following statistical model: Yij = + Bi + Gj + Eij , where Yij was the trait measured on each of the ijth animal, the overall mean, Bi the type of the ith breed; Gj the type of the jth genotype and Eij was the random error.
3. Results and discussions According to the strong similarity between cattle, sheep and goat, in the present study, we detected the exons 2, 3, 4, 5 and introns 2, 4 of the caprine GHRL gene. No polymorphism was detected in the region of exons 4, 5 and intron 4 (P2 locus), and two unique SSCP banding patterns (GG/AG) were detected in the region of exons 2, 3 and intron 2 (P1 locus) after PCR–SSCP analysis (Fig. 1). Then seven DNA amplification fragments of different SSCP patterns of four goat breeds were sequenced. The comparisons among these sequences revealed a novel mutation: A to G at nucleotide 147 of intron 2 (IVS2 + 147G > A) (Fig. 1). The frequency of genotype AG is low in four breeds (BE, 0.315; HM, 0.000; XH, 0.250; BE × XH, 0.288) (Table 1). Homozygote genotype AA was not observed in the four goat breeds in this study. It is an interesting phenomenon worth of further investigation on mutations that influence growth variation. According to Nei and Roychoudhury’s (1974) methods, the population genetic indexes (namely, gene homozygosity, gene heterozygosity, effective allele numbers (Ne) and polymorphism information content (PIC)) were calculated (Table 1). Both loci of the four populations showed low polymorphism. The values of PIC, He of Boer breed in the P1 locus were higher than that of other populations, which implied that the polymorphism and genetic variation of Boer breed were higher than that of other populations. Gene homozygosity varied from 0.685 (BE) to 1.000 (HM) and Ne ranged from 1.000 to 1.362. The minimum and maximum PIC values were 0.195 and 0.230. This reflected that there was not a very high genetic diversity within caprine GHRL gene in analyzed populations.
Table 1 Genotype distribution and genetic diversity at the GHRL gene P1 locus. Breeds
Boer goat Haimen goat Xuhuai goat Boer × Xuhuai goat
Observed genotypes
Allele frequencies
GG
AG
A
G
76 136 81 73
35 0 27 31
0.158 0.000 0.125 0.149
0.842 1.000 0.875 0.851
Gene homozygosity (Ho)
Gene heterozygosity (He)
Effective allele numbers (Ne)
Polymorphism information content (PIC)
0.685 1.000 0.750 0.702
0.315 0.000 0.250 0.298
1.362 1.000 1.280 1.340
0.231 0.000 0.195 0.221
152
Q.J. Jin et al. / Small Ruminant Research 90 (2010) 150–152
Table 2 Association analysis of IVS2 + 147G > A with growth traits in goats (Boer goat, Xuhuai white goat and Boer goat × Xuhuai goat). Growth trait
Genotype (means ± standard error of means) GG (n = 230)
Body height (cm) Body length (cm) Cannon circumference (cm) Chest circumference (cm) Body length index (%) Chest circumference index (%) Cannon circumference index (%) Trunk index (%)
63.826 73.778 8.961 78.785 116.043 123.934 14.105 107.418
± ± ± ± ± ± ± ±
AG (n = 93)
0.418 0.529 0.627a 0.085a 0.800 0.965 0.135 0.833a
64.462 73.909 9.323 81.882 114.770 127.165 14.483 111.243
± ± ± ± ± ± ± ±
P value 0.541 0.825 0.933b 0.137b 1.003 1.177 0.191 1.129b
0.392 0.895 0.024 0.008 0.368 0.057 0.124 0.011
Means with a different superscript letter were significantly different (P < 0.05).
The relationships between genotypes and eight growth traits were done in 323 samples (Table 2). Significant statistical differences were found in chest circumference (P < 0.01), cannon circumference (P < 0.05) and trunk index (P < 0.05). Multiple comparisons results indicated that individuals with genotype AG were significantly higher than those of individuals with genotype GG in circumference of chest and cannon and trunk index (P < 0.05). Moreover, there was a tendency that genotype AG individuals had better performance in other aspects such as body height and body length than genotype GG individuals although no significant differences appeared (P > 0.05). The SNP studied here is in intron 2 of the caprine GHRL gene, so it may not be a causal mutation. Thus, one may suggest that it could be in linkage disequilibrium with another SNP in the GHRL gene with greater effects on the traits. A mutation of this kind could be present in, for example, promoter or intron areas that were not screened in this study. Moreover, introns have been revealed to affect transcriptional efficiency of numerous genes in a variety of organisms (Greenwood and Kelsoe, 2003; Le Hir et al., 2003). The present study revealed a novel SNP in intron 2 of caprine GHRL gene. The SNP is associated with chest circumference, cannon circumference and trunk index. Considering the G allele was recessive in four cattle breeds, we should reject the G allele in the breeding schemes of goat. This study was designed as the first step in detecting genetic markers for caprine GHRL gene, which would eventually provide useful information for the improvement of Chinese native goat. Acknowledgements This work was supported by the National “863” Program of the P.R. China (No. 2008AA10Z138), Natural Science Foundation of Jiangsu Province (No. BK2008120), the “13115” Sci-Tech Innovation Program of Shaanxi Province (2008ZDKG-11), Research Fund for the Doctor Program of Higher Education of China (No. 200807120012), Natural science fund for colleges and universities in Jiangsu
Province (09KJD180002), Natural Science Foundation of Xuzhou Normal University (07XLA08; KY2007019), and the Young Topnotch Researcher Support Project of Northwest A&F University (No. QNGG-2009-007). References Arvat, E., Di Vito, L., Broglio, F., Papotti, M., Muccioli, G., Dieguez, C., Casanueva, F., Deghenghi, R., Camanni, F., Ghigo, E., 2000. Preliminary evidence that Ghrelin, the natural GH secretagogue (GHS)-receptor ligand, strongly stimulates GH secretion in humans. J. Endocrinol. Invest. 23, 493. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., Matsukura, S., Kangawa, K., Nakazato, M., 2000. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans 1. Endocrinology 141, 4255–4261. Greenwood, T.A., Kelsoe, J.R., 2003. Promoter and intronic variants affect the transcriptional regulation of the human dopamine transporter gene. Genomics 82, 511–520. Hayashida, T., Murakami, K., Mogi, K., Nishihara, M., Nakazato, M., Mondal, M.S., Horii, Y., Kojima, M., Kangawa, K., Murakami, N., 2001. Ghrelin in domestic animals: distribution in stomach and its possible role. Domest. Anim. Endocrin. 21, 17–24. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656–660. Le Hir, H., Nott, A., Moore, M.J., 2003. How introns influence and enhance eukaryotic gene expression. Trends Biochem. Sci. 28, 215–220. Mullenbach, R., Lagoda, P.J., Welter, C., 1989. An efficient salt-chloroform extraction of DNA from blood and tissues. Trends Genet. 5, 391. Murata, M., Okimura, Y., Iida, K., Matsumoto, M., Sowa, H., Kaji, H., Kojima, M., Kangawa, K., Chihara, K., 2002. Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J. Biol. Chem. 277, 5667–5674. Nei, M., Roychoudhury, A.K., 1974. Sampling variances of heterozygosity and genetic distance. Genetics 76, 379–390. Wertz-Lutz, A.E., Knight, T.J., Pritchard, R.H., Daniel, J.A., Clapper, J.A., Smart, A.J., Trenkle, A., Beitz, D.C., 2006. Circulating ghrelin concentrations fluctuate relative to nutritional status and influence feeding behavior in cattle. J. Anim. Sci. 84, 3285. Yamazaki, M., Nakamura, K., Kobayashi, H., Matsubara, M., Hayashi, Y., Kangawa, K., Sakai, T., 2002. Regulational effect of ghrelin on growth hormone secretion from perifused rat anterior pituitary cells. J. Neuroendocrinol. 14, 156. Zhang, C.L., Wang, Y., Chen, H., Lan, X.Y., Lei, C.Z., 2007. Enhance the efficiency of single-strand conformation polymorphism analysis by short polyacrylamide gel and modified silver staining. Anal. Biochem. 365, 286–287.