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Polymorphisms of three neuroendocrine-correlated genes associated with growth and reproductive traits in the chicken
Polymorphisms of three neuroendocrine-correlated genes associated with growth and reproductive traits in the chicken J. T. Ou,1 S. Q. Tang,1 D. X. Sun, and Y. Zhang2 Department of Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China (P = 0.0061), and the C56072547T (C-968T) SNP of the insulin-like growth factor binding protein 3 gene was associated with BW at 8 and 10 wk of age (P = 0.0056 and P = 0.0016, respectively). The C4535156T (C-1591T), G4533815A (G-250A), and G4533675C (G110C) SNP of the STAT5B gene were associated with age at first egg (P = 0.0143, P = 0.0088, and P = 0.0114, respectively). Moreover, Lewontin’s D′ (|D′|) and r2 of C4535156T and G4533815A SNP, C4535156T and G4533675C SNP, and G4533815A and G4533675C SNP of the STAT5B gene were 0.939 and 0.852, 0.967 and 0.858, and 0.971 and 0.896, respectively. The 3 SNP were strong-linked with each other and lay within a haplotype block. Our results suggest that these SNP were significantly associated with early growth or with sexual maturation in chickens, or both, and may be potential molecular markers for MAS.
Key words: chicken, candidate quantitative trait loci gene, economic trait, single nucleotide polymorphism 2009 Poultry Science 88:722–727 doi:10.3382/ps.2008-00497
INTRODUCTION
important traits are becoming increasingly important in animal and plant breeding programs. Moreover, with the gradual progression of genetic genomics to systems genetics, candidate signaling pathway and gene networks linked to rich sources of biological annotation may be an attractive strategy to search for candidate QTL genes (Korstanje and Paigen, 2002; Borevitz and Chory, 2004; Kadarmideen et al., 2006; Beyer et al., 2007). The insulin-like growth factors (IGF) play essential roles in cell growth, metabolism, differentiation, proliferation, and survival under different physiological conditions. The IGF binding protein (IGFBP) superfamily includes 6 distinct high-affinity binding proteins, from IGFBP1 to IGFBP6. The 6 IGFBP can act as modulators by either enhancing or inhibiting the activity and bioavailability of IGF (Firth and Baxter, 2002). The biological functions of the IGF system in birds have been found to include stimulation of growth, protein synthesis, cell differentiation, and regulation of ovary development (McMurtry et al., 1997; Yun et al., 2005; Li et al., 2006).
Growth and reproduction that are under the control of multiple genes are the 2 most economically important characteristics for the poultry industry. Integrating emerging technologies, identifying the related genes, and uncovering the molecular mechanisms governing their activity will provide an opportunity for more efficient selection for growth and reproductive performance in chickens (Williams et al., 2002; Andersson and Georges, 2004; Soller et al., 2006). The QTL mapping for chicken growth and reproductive traits such as BW and age at first egg was widely studied in the past decade (Abasht et al., 2006; Hu et al., 2007). The identification and utilization of potential candidate genes for QTL with significant effects on economically
©2009 Poultry Science Association Inc. Received November 17, 2008. Accepted December 11, 2008. 1 The first 2 authors contributed equally to this work. 2 Corresponding author: [email protected]
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ABSTRACT The identification and utilization of potential candidate genes for QTL with significant effects on economically important traits are becoming increasingly important in poultry breeding programs. Chicken insulin-like growth factor binding protein 1 and 3 and signal transducers and activators of transcription 5B (STAT5B) genes are 3 essential nodes for signaling pathways and gene networks of growth and reproduction. The pooled DNA sequencing approach was used for identification of 9 SNP of the 5′ upstream region of the 3 genes. A total of 826 individuals from Beijing You chicken were genotyped for 5 SNP using a modified PCR-RFLP method and the association with chicken growth and reproductive traits was studied using the GLM procedure. The T56039403C (T808C) SNP of the insulin-like growth factor binding protein 1 gene was associated with BW at 10 wk of age
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MATERIALS AND METHODS Chicken Populations and Phenotypic Traits A total of 826 individuals from Beijing You chicken, including 316 males and 510 females, were developed by crossing 29 sires and 224 dams, which were reared
in cages under the same conditions. Nine growth traits [BW at hatch (BW0), 8, 10, 13, 17, and 40 wk of age; shank length at 13 wk of age; shank circumference at 13 of age; and chest bone length at 13 wk of age] and 10 reproductive traits [BW at first egg (BWAFE); egg weight at first egg; age at first egg (AFE); number of eggs at 24, 28, 32, 36, and 40 wk of age; and egg weight at 36 and 40 wk of age] were measured.
Discovery of Polymorphisms and Genotyping Genomic DNA was extracted from blood samples by TIANamp Blood DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). Each DNA pool was constructed from 30 chicken samples containing 100 ng of DNA from each individual. Polymerase chain reactions were performed in a total of 25 µL, which included 2 µL of pooled DNA, 5 mM of each primer, 200 µM of each deoxynucleoside triphosphate, 2.5 µL of 10 × PCR buffer, and 1.5 U of Taq DNA polymerase (Takara Biotechnology Co. Ltd., Dalian, China). The normal reaction conditions were as follows: 95°C for 5 min, followed by 34 cycles of 94°C for 30 s, annealing from 50 to 60°C for 45 s, 72°C for 50 s, and a final extension at 72°C for 5 min. Polymerase chain reaction products of the pooled DNA samples including each 2-kb fragments of the 5′ upstream region of chicken IGFBP1, IGFBP3, and STAT5B genes were sequenced, respectively, and SNP were identified by sequence traces. For individual genotyping, 5 pairs of primers were designed using Oligo6.0 and primer-introduced restriction analysis software available online (http://cedar. genetics.soton.ac.uk/public_html/primer2.html; Ke et al., 2001). The PCR products were digested with restriction enzymes and the digestion products were electrophoresed on a 4% agarose gel stained with ethidium bromide (Ota et al., 2007).The primers are shown in Table 1.
Table 1. Summary of genes, SNP markers, and primers Gene1 IGFBP1 IGFBP3 STAT5B
1
Chromosome, location2(nt)
Primer sequence
2, T-808C T56039403C 2, C-968T C56072547T 27, C-1591T C4535156T 27, G-250A G4533815A 27, G-110C G4533675C
5′-CCTCACACAACCAAAAATCAGGC-3′ 5′-ATGCAAAAGCATTCAAACTTGACTGT-3′ 5′-TATCGTCTACCGGTGCCACG-3′ 5′-GTTTACGGGCTCTTCGAGATCG-3′ 5′-TGGAGCTACTGGCATCTCTCA- 3′ 5′-TGCTGCAGTTGCTGTGGTCT-3′ 5′-CCATCCCTTCCTGGTGCAGT-3′ 5′-ACTGCTGCCATTTCCCTTTG-3′ 5′-GCTCTCCAATGGCTTTTCCTAGAGTA-3′ 5′-GAAGTGATGCCCATAGAGTGCCTG-3′
Annealing temperature (°C)
Genotyping method3
Enzyme
54
PIRA-RFLP
HaeIII
58
PIRA-RFLP
Eco72I
59
PCR-RFLP
HhaI
60
PCR-RFLP
MspI
58
PIRA-RFLP
RsaI
IGFBP1 = insulin-like factor binding protein 1; IGFBP3 = insulin-like factor binding protein 3; STAT5B = signal transducers and activators of transcription 5B. 2 Location on chromosome where gene is found (http://mgc.ucsc.edu/cgi-bin/hgBlat); location on gene where nucleotides are numbered relative to initiation codon ATG. 3 PIRA = primer-introduced restriction analysis.
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Signal transducers and activators of transcriptions (STAT), consisting of 7 members, are a family of cytoplasmic proteins that are involved in the signal transduction pathways of numerous cytokines, growth factors, and hormones (Darnell, 1997; Levy and Darnell, 2002). They participate in a series of cellular processes such as cell growth, differentiation, survival, and apoptosis. The STAT5 proteins, including STAT5A and STAT5B, which are 96% similar at the amino acid level, are the important modulators of the growth hormone, growth hormone receptor, IGF, prolactin, and insulin signaling pathways, which are involved in growth, reproduction, lactation, and metabolism (Bachelot and Binart, 2007; Pilecka et al., 2007; Hennighausen and Robinson, 2008). The chicken IGFBP1 and IGFBP3 genes were localized to the growth QTL regions on chromosome 2, which may affect white meat percentage and other growth traits (Tatsuda and Fujinaka, 2001; McElroy et al., 2006; Park et al., 2006), and furthermore, chicken IGFBP1, IGFBP3, and STAT5B genes are 3 essential nodes for the signaling pathways and gene networks of growth and reproduction (Kanehisa and Goto, 2000; Cogburn et al., 2003; Kuhn et al., 2008). Hence, the IGFBP1, IGFBP3, and STAT5B genes may be investigated as potential candidate genes for QTL. However, few studies on association with growth and reproduction have been reported in chickens. In this paper, our objectives were to explore the association of the 3 genes of the neuroendocrine growth axis on growth or reproductive traits, or both, in chickens.
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Table 2. Frequency of alleles and genotypes and P-values for the Hardy-Weinberg equilibrium test Gene1
SNP
IGFBP1
T56039403C
IGFBP3
C56072547T
STAT5B
C4535156T G4533815A G4533675C
Count
Frequency
Allele
Frequency
P-value
TT TC CC CC CT TT CC CT TT GG GA AA GG GC CC
156 434 233 382 359 62 270 385 156 258 409 154 164 399 249
0.1896 0.5273 0.2831 0.4757 0.4471 0.0772 0.3329 0.4747 0.1924 0.3143 0.4982 0.1876 0.2020 0.4914 0.3067
T C
0.4532 0.5468
0.0664
C T
0.6993 0.3007
0.0744
C T
0.5703 0.4297
0.3710
G A
0.5633 0.4367
0.7182
G C
0.4477 0.5523
0.8563
1 IGFBP1 = insulin-like factor binding protein 1; IGFBP3 = insulin-like factor binding protein 3; STAT5B = signal transducers and activators of transcription 5B.
Analysis of the 5′-Upstream Sequence On the basis of Ensembl GeneSeqView (http://www. ensembl.org/Gallus_gallus/gene), sequences of IGFBP1, IGFBP3, and STAT5B genes were retrieved in chickens. The DNA sequencing chromatograms of the 2-kb fragment of the 5′ upstream region from the 3 genes were used to confirm the correct sequence and size. Potential transcription factor binding sites of the 5′ upstream region of 3 genes were analyzed by the Web site http://molsun1.cbrc.aist.go.jp/research/db/ TFSEARCH.html and with 85% similarity as a threshold.
Statistical Analysis The above 5 SNP allele and genotype frequencies for the Hardy-Weinberg equilibrium test were estimated and tested by using the ALLELE procedure in SAS/ Genetics 9.1.3 (SAS Institute, Cary, NC). The linkage disequilibria D′ value and r2 of the 5 SNP were estimated by Haploview (Barrett et al., 2005). With the GLM procedure in SAS 9.1.3 software, the associations between SNP of the 3 genes and chicken growth and reproductive traits were respectively analyzed using the following models: Y = µ + S +F +M (F) + G + BW0 + e or Y = µ + S +F +M (F) + G + BWAFE + e, where Y was the dependent variable, µ was the overall mean, S was the fixed effect of sex, G was the fixed effect associated with the genotype, F was the fixed effect of sire, M (F) was the fixed effect of dam nested within sire, BW0 and BWAFE were covariables, and e was the random error. The data were presented as probability values and least squares means ± SEM. The significant differences of least squares means were tested with Duncan’s multiple range tests, and a P-value of ≤ 0.05 was considered statistically significant.
RESULTS Identified SNP and Allele and Genotype Frequencies Nine SNP of 5′ upstream region of chicken IGFBP1, IGFBP3, and STAT5B genes were identified by the pooled DNA sequencing and sequence traces. Of them, 5 SNP were examined for associations with traits. These 5 SNP, their corresponding frequencies of allele and genotype, and P-values for the Hardy-Weinberg equilibrium test are presented in Table 2. The result showed that these 5 SNP were in Hardy-Weinberg equilibrium (P > 0.05). Genotype frequencies of TC (T56039403C), CC (C56072547T), CT (C4535156T), GA (G4533815A), and GC (G4533675C) were the highest for 0.5273, 0.4757, 0.4747, 0.4982, and 0.4914, respectively.
Linkage Disequilibrium of SNP Linkage disequilibrium (LD) of the 5 SNP were measured by Lewontin’s D′ (|D′|) and r2 with Haploview version 4.1 software. Lewontin’s D′ (|D′|) and r2 of C4535156T and G4533815A SNP, C4535156T and G4533675C SNP, G4533815A and G4533675C SNP of the STAT5B gene were 0.939 and 0.852, 0.967 and 0.858, and 0.971 and 0.896, respectively. When r2 is higher than 0.3, two loci are considered in LD, and when r2 is higher than 0.6, two loci are considered in strongly LD (Ardlie et al., 2002). Moreover, 1 haplotype block has been defined as 3 or more markers for which all estimated values of |D′| exceeded 0.9 (Phillips et al., 2003). The results indicated that the 3 SNP of the STAT5B gene were strong-linked with each other and lay within a haplotype block; thereinto, the G4533815A SNP was tagSNP. In addition, D′ and r2 of T56039403C and C56072547T SNP of IGFBP1 and IGFBP3 genes were 0.949 and 0.465, respectively, and the 2 SNP were in LD.
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Genotype
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Trait Associations and Effects of SNP
Analysis of the 5′-Upstream Sequence By sequencing analysis, we also checked whether there are any transcriptional binding sites on these SNP and found the transcription factor response elements associated with 3 SNP, including T56039403C, C56072547T, and G4533815A, the homeodomain factor
DISCUSSION It is known that the neuroendocrine growth axis is involved in the activity of many hormones, cytokines, and chemokines and their receptors play crucial roles in the regulation of animal growth, reproduction, immunity, and metabolism. At present, it has become a hot topic in research to probe into balanced selection and breeding for growth, reproduction, immunity, and metabolism in chickens for optimum performance. Combining the relative information with comparative omics (such as genomics, transcriptomics, proteomics, and metabonomics), physiological functions, mapping and fine mapping of QTL and expression QTL, signaling pathways, and gene networks, the method of the candidate gene approach is becoming increasingly economical and effective in finding candidate QTL gene and quantitative trait nucleotides with significant effects on economically important traits (Kanehisa and Goto, 2000; Cogburn et al., 2003; Zhu and Zhao, 2007; Kuhn et al., 2008). According to the above integration strategies, the IGFBP1, IGFBP3, and STAT5B genes were effectively selected and used to study the association with growth and reproductive traits in chickens. As the main component of gene expression regulation, the 5′ upstream region plays an important role in affecting the phenotypes of traits (Liu et al., 2006). In this study, 5 SNP selected from 9 SNP of the 5′upstream region of the 3 genes were associated with growth or reproductive traits, or both, in chickens. The results showed that these 5 SNP were in Hardy-Weinberg equilibrium
Table 3. Effects (P-value and least squares means ± SEM) of the 5 SNP on growth and reproductive traits SNP T56039403C P-value C56072547T P-value C4535156T P-value G4533815A P-value G4533675C P-value a,b
Genotype TT TC CC CC CT TT CC CT TT GG GA AA GG GC CC
BW 81 (g) 612 ± 8.1 612 ± 4.1 631 ± 6.3 0.0504 628 ± 4.6A 605 ± 4.7B 612 ± 11.9AB 0.0056** 618 ± 6.3 613 ± 4.6 634 ± 8.1 0.0664 615 ± 6.6 616 ± 4.6 628 ± 8.4 0.4450 638 ± 8.1a 615 ± 4.6b 611 ± 6.6b 0.0319*
Means within a row with no common superscript differ significantly (P < 0.05). Means within a row with no common superscript differ significantly (P < 0.01). 1 BW 8 = 8 wk of age; BW 10 = 10 wk of age; AFE = age at first egg. *P < 0.05; **P < 0.01. A,B
BW 10 (g) b
783 ± 9.3 784 ± 4.8b,B 812 ± 7.3a,A 0.0061** 806 ± 5.4A 777 ± 5.5B 780 ± 13.8AB 0.0016** 797 ± 7.3 784 ± 5.3 805 ± 9.4 0.0815 790 ± 7.7 788 ± 5.4 800 ± 9.9 0.5832 808 ± 9.5 789 ± 5.4 788.85 ± 7.63 0.1883
AFE (d) 149 ± 3.2 148 ± 1.6 151 ± 2.6 0.7443 150 ± 2.0 148 ± 1.7 151 ± 5.2 0.8239 154 ± 2.63a,A 150 ± 1.9a 139 ± 3.7b,B 0.0143* 156 ± 3.0a,A 148 ± 1.9a 140 ± 3.3b,B 0.0088** 140 ± 3.2b,B 149 ± 1.8a 155 ± 3.0a,A 0.0114*
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Trait associations (P-values) and effects (least squares means) of the 5 SNP with growth and reproductive traits are shown in Table 3. The C56072547T and T56039403C SNP of the IGFBP3 and IGFBP1 genes were significantly associated with BW at 8 or10 wk of age, or both weeks of age (P = 0.0056 and P = 0.0016, P = 0.0061, respectively). The C4535156T, G4533815A, and G4533675C SNP of the STAT5B gene were associated with age at first egg for P = 0.0143, P = 0.0088, and P = 0.0114, respectively. At the same time, the G4533675C SNP was associated with BW at 8 wk of age (P = 0.0319). There were higher (P < 0.01) BW at 8 or 10 wk of age, or both, in the birds that were of the CC genotype of the C56072547T and T56039403C SNP than those of the TT and TC genotypes. There were higher (P < 0.05) BW at 8 wk of age that were of the GG genotype of the G4533675C SNP than those of the GC and CC genotype. There were shorter AFE (P < 0.01) in the birds that were of the TT genotype of the C4535156T SNP than those of the CC and CT genotypes, of the AA genotype of the G4533815A SNP than those of the GG and GA genotypes, and of the GG genotype of the G4533675C SNP than those of the GC and CC genotypes.
(Nkx-2.5) identified on T56039403C and C56072547T, and cyclic adenosine monophosphate response elementbinding proteins (CREB) on G4533815A SNP.
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2006). By using DNA pooling as an initial screening process combined with modified PCR-RFLP, researchers can be more specific for candidate genes analysis and can then reduce labor time and costs (Ke et al., 2001; Zhang et al., 2005). In summary, association of the 5 SNP from the 3 genes with chicken growth and reproductive traits was analyzed in the present study. These SNP were found to be significantly associated with early growth or with sexual maturation in chickens, or both. Therefore, in conclusion, the genes of the neuroendocrine growth axis not only affected chicken growth but also were associated with reproductive traits.
ACKNOWLEDGMENTS We thank the members of the animal genetics group and poultry genetics group at China Agricultural University for managing the birds and teaching experimental techniques and Jiuzhou Song at University of Maryland, College Park, for giving suggestions on the manuscript. The work was supported by the State Basic Research and Development Project of China (No. 2006CB102107) and partly supported by National Key Technologies R & D Program (2006BAD04A01), National High-Tech R & D Program (2007AA10Z157), and National “948” Project (2006-G48).
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and were linked significantly with chicken growth or reproductive traits, or both. The 2 SNP of the IGFBP1 and IGFBP3 gene were significantly related with BW. The 3 SNP of the STAT5B gene were associated with AFE. Meanwhile, the 3 SNP of the STAT5B gene appeared to be strongly linked with each other and lay within a haplotype block because their D′ (|D′|) and r2 statistics were higher than 0.93 and 0.85. Moreover, STAT5B was activated by differentiation-dependent transcriptional regulation of distinct cytokine signaling pathway components and mediated distinct functional processes in the rat ovary for early follicle growth, atresia, and luteinization (Russell et al., 1996; Dajee et al., 1998). Hence, our findings in the present study implied that the chicken STAT5B gene may affect sexual maturation of chicken by regulating ovary development. In addition, we found that the G4533675C SNP of the STAT5B gene was related to growth traits besides reproductive traits, indicating such polymorphism might provide the basis for balanced selection and may be used in MAS to improve growth and reproductive production simultaneously. In the current study, 2 kinds of the transcription factor response elements were identified that included Nkx-2.5 and CREB proteins. It has been found that Nkx-2.5 plays critical roles in regulating tissue-specific gene expression and induces chicken cardiac myocyte differentiation in tissues (Schultheiss et al., 1995; Harris et al., 2006). Cyclic adenosine monophosphate response element-binding protein, which binds to the cyclic adenosine monophosphate response elements and increases or decreases the transcription of genes, is critical in a variety of cellular processes such as cell proliferation, differentiation, and adaptive responses in chickens (Sirotkin and Grossmann, 2003; Kim et al., 2005). The presence of Nkx-2.5 and CREB protein binding sites suggested the possible transcriptional regulation of the chicken IGFBP1, IGFBP3, and STAT5B genes by Nkx-2.5 and CREB proteins. The association studies might provide the basis for uncovering the molecular mechanisms of the 3 genes on growth and reproduction performances. Recently, numerous QTL have been detected in chickens using different DNA markers. Several QTL for growth are located on GGA2 or GGA7, which contain chicken IGFBP1 and IGFBP3 genes or IGFBP2 and IGFBP5 genes, respectively, and may affect white meat percentage and other growth traits (Tatsuda and Fujinaka, 2001; McElroy et al., 2006; Park et al., 2006). In the present study, our results were consistent with the former that focused on QTL mapping on GGA2, which indicated that chicken IGFBP1 and IGFBP3 genes should be important candidate genes for chicken growth. In the current study, the pooled DNA sequencing approach, which proved to be a powerful SNP discovery tool, was used for the effective identification of 9 SNP of the 5′ upstream region of the chicken IGFBP1, IGFBP3, and STAT5B genes (Amos et al., 2000; Ye et al.,
ASSOCIATION OF GROWTH-CORRELATED GENES WITH ECONOMIC TRAITS
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of cardiac Purkinje fibers requires precise spatiotemporal regulation of Nkx2-5 expression. Dev. Dyn. 235:38–49. Hennighausen, L., and G. W. Robinson. 2008. Interpretation of cytokine signaling through the transcription factors STAT5A and STAT5B. Genes Dev. 22:711–721. Hu, Z. L., E. R. Fritz, and J. M. Reecy. 2007. AnimalQTLdb: A livestock QTL database tool set for positional QTL information mining and beyond. Nucleic Acids Res. 35:604–609. Kadarmideen, H. N., P. von Rohr, and L. L. Janss. 2006. From genetical genomics to systems genetics: Potential applications in quantitative genomics and animal breeding. Mamm. Genome 17:548–564. Kanehisa, M., and S. Goto. 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28:27–30. Ke, X., A. Collins, and S. Ye. 2001. PIRA PCR designer for restriction analysis of single nucleotide polymorphisms. Bioinformatics 17:838–839. Kim, D. W., J. H. Chang, S. W. Park, G. S. Jeon, J. H. Seo, and S. S. Cho. 2005. Activated cyclic AMP-response element binding protein (CREB) is expressed in a myelin-associated protein in chick. Neurochem. Res. 30:1133–1137. Korstanje, R., and B. Paigen. 2002. From QTL to gene: The harvest begins. Nat. Genet. 31:235–236. Kuhn, M., C. von Mering, M. Campillos, L. J. Jensen, and P. Bork. 2008. STITCH: Interaction networks of chemicals and proteins. Nucleic Acids Res. 36:684–688. Levy, D. E., and J. E. Darnell Jr. 2002. Stats: Transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol. 3:651–662. Li, Z. H., H. Li, H. Zhang, S. Z. Wang, Q. G. Wang, and Y. X. Wang. 2006. Identification of a single nucleotide polymorphism of the insulin-like growth factor binding protein 2 gene and its association with growth and body composition traits in the chicken. J. Anim. Sci. 84:2902–2906. Liu, R., Y. C. Wang, D. X. Sun, Y. Yu, and Y. Zhang. 2006. Association between polymorphisms of lipoprotein lipase gene and chicken fat deposition. Asian-australas. J. Anim. Sci. 19:1011–2367. McElroy, J. P., J. J. Kim, D. E. Harry, S. R. Brown, J. C. Dekkers, and S. J. Lamont. 2006. Identification of trait loci affecting white meat percentage and other growth and carcass traits in commercial broiler chickens. Poult. Sci. 85:593–605. McMurtry, J. P., G. L. Francis, and Z. Upton. 1997. Insulin-like growth factors in poultry. Domest. Anim. Endocrinol. 14:199– 229. Ota, M., H. Fukushima, J. K. Kulski, and H. Inoko. 2007. Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism. Nat. Protocols 2:2857–2864. Park, H. B., L. Jacobsson, P. Wahlberg, P. B. Siegel, and L. Andersson. 2006. QTL analysis of body composition and metabolic traits in an intercross between chicken lines divergently selected for growth. Physiol. Genomics 25:216–223.
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Key words: chicken, candidate quantitative trait loci gene, economic trait, single nucleotide polymorphism 2009 Poultry Science 88:722–727 doi:10.3382/ps.2008-00497
INTRODUCTION
important traits are becoming increasingly important in animal and plant breeding programs. Moreover, with the gradual progression of genetic genomics to systems genetics, candidate signaling pathway and gene networks linked to rich sources of biological annotation may be an attractive strategy to search for candidate QTL genes (Korstanje and Paigen, 2002; Borevitz and Chory, 2004; Kadarmideen et al., 2006; Beyer et al., 2007). The insulin-like growth factors (IGF) play essential roles in cell growth, metabolism, differentiation, proliferation, and survival under different physiological conditions. The IGF binding protein (IGFBP) superfamily includes 6 distinct high-affinity binding proteins, from IGFBP1 to IGFBP6. The 6 IGFBP can act as modulators by either enhancing or inhibiting the activity and bioavailability of IGF (Firth and Baxter, 2002). The biological functions of the IGF system in birds have been found to include stimulation of growth, protein synthesis, cell differentiation, and regulation of ovary development (McMurtry et al., 1997; Yun et al., 2005; Li et al., 2006).
Growth and reproduction that are under the control of multiple genes are the 2 most economically important characteristics for the poultry industry. Integrating emerging technologies, identifying the related genes, and uncovering the molecular mechanisms governing their activity will provide an opportunity for more efficient selection for growth and reproductive performance in chickens (Williams et al., 2002; Andersson and Georges, 2004; Soller et al., 2006). The QTL mapping for chicken growth and reproductive traits such as BW and age at first egg was widely studied in the past decade (Abasht et al., 2006; Hu et al., 2007). The identification and utilization of potential candidate genes for QTL with significant effects on economically
©2009 Poultry Science Association Inc. Received November 17, 2008. Accepted December 11, 2008. 1 The first 2 authors contributed equally to this work. 2 Corresponding author: [email protected]
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ABSTRACT The identification and utilization of potential candidate genes for QTL with significant effects on economically important traits are becoming increasingly important in poultry breeding programs. Chicken insulin-like growth factor binding protein 1 and 3 and signal transducers and activators of transcription 5B (STAT5B) genes are 3 essential nodes for signaling pathways and gene networks of growth and reproduction. The pooled DNA sequencing approach was used for identification of 9 SNP of the 5′ upstream region of the 3 genes. A total of 826 individuals from Beijing You chicken were genotyped for 5 SNP using a modified PCR-RFLP method and the association with chicken growth and reproductive traits was studied using the GLM procedure. The T56039403C (T808C) SNP of the insulin-like growth factor binding protein 1 gene was associated with BW at 10 wk of age
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MATERIALS AND METHODS Chicken Populations and Phenotypic Traits A total of 826 individuals from Beijing You chicken, including 316 males and 510 females, were developed by crossing 29 sires and 224 dams, which were reared
in cages under the same conditions. Nine growth traits [BW at hatch (BW0), 8, 10, 13, 17, and 40 wk of age; shank length at 13 wk of age; shank circumference at 13 of age; and chest bone length at 13 wk of age] and 10 reproductive traits [BW at first egg (BWAFE); egg weight at first egg; age at first egg (AFE); number of eggs at 24, 28, 32, 36, and 40 wk of age; and egg weight at 36 and 40 wk of age] were measured.
Discovery of Polymorphisms and Genotyping Genomic DNA was extracted from blood samples by TIANamp Blood DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). Each DNA pool was constructed from 30 chicken samples containing 100 ng of DNA from each individual. Polymerase chain reactions were performed in a total of 25 µL, which included 2 µL of pooled DNA, 5 mM of each primer, 200 µM of each deoxynucleoside triphosphate, 2.5 µL of 10 × PCR buffer, and 1.5 U of Taq DNA polymerase (Takara Biotechnology Co. Ltd., Dalian, China). The normal reaction conditions were as follows: 95°C for 5 min, followed by 34 cycles of 94°C for 30 s, annealing from 50 to 60°C for 45 s, 72°C for 50 s, and a final extension at 72°C for 5 min. Polymerase chain reaction products of the pooled DNA samples including each 2-kb fragments of the 5′ upstream region of chicken IGFBP1, IGFBP3, and STAT5B genes were sequenced, respectively, and SNP were identified by sequence traces. For individual genotyping, 5 pairs of primers were designed using Oligo6.0 and primer-introduced restriction analysis software available online (http://cedar. genetics.soton.ac.uk/public_html/primer2.html; Ke et al., 2001). The PCR products were digested with restriction enzymes and the digestion products were electrophoresed on a 4% agarose gel stained with ethidium bromide (Ota et al., 2007).The primers are shown in Table 1.
Table 1. Summary of genes, SNP markers, and primers Gene1 IGFBP1 IGFBP3 STAT5B
1
Chromosome, location2(nt)
Primer sequence
2, T-808C T56039403C 2, C-968T C56072547T 27, C-1591T C4535156T 27, G-250A G4533815A 27, G-110C G4533675C
5′-CCTCACACAACCAAAAATCAGGC-3′ 5′-ATGCAAAAGCATTCAAACTTGACTGT-3′ 5′-TATCGTCTACCGGTGCCACG-3′ 5′-GTTTACGGGCTCTTCGAGATCG-3′ 5′-TGGAGCTACTGGCATCTCTCA- 3′ 5′-TGCTGCAGTTGCTGTGGTCT-3′ 5′-CCATCCCTTCCTGGTGCAGT-3′ 5′-ACTGCTGCCATTTCCCTTTG-3′ 5′-GCTCTCCAATGGCTTTTCCTAGAGTA-3′ 5′-GAAGTGATGCCCATAGAGTGCCTG-3′
Annealing temperature (°C)
Genotyping method3
Enzyme
54
PIRA-RFLP
HaeIII
58
PIRA-RFLP
Eco72I
59
PCR-RFLP
HhaI
60
PCR-RFLP
MspI
58
PIRA-RFLP
RsaI
IGFBP1 = insulin-like factor binding protein 1; IGFBP3 = insulin-like factor binding protein 3; STAT5B = signal transducers and activators of transcription 5B. 2 Location on chromosome where gene is found (http://mgc.ucsc.edu/cgi-bin/hgBlat); location on gene where nucleotides are numbered relative to initiation codon ATG. 3 PIRA = primer-introduced restriction analysis.
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Signal transducers and activators of transcriptions (STAT), consisting of 7 members, are a family of cytoplasmic proteins that are involved in the signal transduction pathways of numerous cytokines, growth factors, and hormones (Darnell, 1997; Levy and Darnell, 2002). They participate in a series of cellular processes such as cell growth, differentiation, survival, and apoptosis. The STAT5 proteins, including STAT5A and STAT5B, which are 96% similar at the amino acid level, are the important modulators of the growth hormone, growth hormone receptor, IGF, prolactin, and insulin signaling pathways, which are involved in growth, reproduction, lactation, and metabolism (Bachelot and Binart, 2007; Pilecka et al., 2007; Hennighausen and Robinson, 2008). The chicken IGFBP1 and IGFBP3 genes were localized to the growth QTL regions on chromosome 2, which may affect white meat percentage and other growth traits (Tatsuda and Fujinaka, 2001; McElroy et al., 2006; Park et al., 2006), and furthermore, chicken IGFBP1, IGFBP3, and STAT5B genes are 3 essential nodes for the signaling pathways and gene networks of growth and reproduction (Kanehisa and Goto, 2000; Cogburn et al., 2003; Kuhn et al., 2008). Hence, the IGFBP1, IGFBP3, and STAT5B genes may be investigated as potential candidate genes for QTL. However, few studies on association with growth and reproduction have been reported in chickens. In this paper, our objectives were to explore the association of the 3 genes of the neuroendocrine growth axis on growth or reproductive traits, or both, in chickens.
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Table 2. Frequency of alleles and genotypes and P-values for the Hardy-Weinberg equilibrium test Gene1
SNP
IGFBP1
T56039403C
IGFBP3
C56072547T
STAT5B
C4535156T G4533815A G4533675C
Count
Frequency
Allele
Frequency
P-value
TT TC CC CC CT TT CC CT TT GG GA AA GG GC CC
156 434 233 382 359 62 270 385 156 258 409 154 164 399 249
0.1896 0.5273 0.2831 0.4757 0.4471 0.0772 0.3329 0.4747 0.1924 0.3143 0.4982 0.1876 0.2020 0.4914 0.3067
T C
0.4532 0.5468
0.0664
C T
0.6993 0.3007
0.0744
C T
0.5703 0.4297
0.3710
G A
0.5633 0.4367
0.7182
G C
0.4477 0.5523
0.8563
1 IGFBP1 = insulin-like factor binding protein 1; IGFBP3 = insulin-like factor binding protein 3; STAT5B = signal transducers and activators of transcription 5B.
Analysis of the 5′-Upstream Sequence On the basis of Ensembl GeneSeqView (http://www. ensembl.org/Gallus_gallus/gene), sequences of IGFBP1, IGFBP3, and STAT5B genes were retrieved in chickens. The DNA sequencing chromatograms of the 2-kb fragment of the 5′ upstream region from the 3 genes were used to confirm the correct sequence and size. Potential transcription factor binding sites of the 5′ upstream region of 3 genes were analyzed by the Web site http://molsun1.cbrc.aist.go.jp/research/db/ TFSEARCH.html and with 85% similarity as a threshold.
Statistical Analysis The above 5 SNP allele and genotype frequencies for the Hardy-Weinberg equilibrium test were estimated and tested by using the ALLELE procedure in SAS/ Genetics 9.1.3 (SAS Institute, Cary, NC). The linkage disequilibria D′ value and r2 of the 5 SNP were estimated by Haploview (Barrett et al., 2005). With the GLM procedure in SAS 9.1.3 software, the associations between SNP of the 3 genes and chicken growth and reproductive traits were respectively analyzed using the following models: Y = µ + S +F +M (F) + G + BW0 + e or Y = µ + S +F +M (F) + G + BWAFE + e, where Y was the dependent variable, µ was the overall mean, S was the fixed effect of sex, G was the fixed effect associated with the genotype, F was the fixed effect of sire, M (F) was the fixed effect of dam nested within sire, BW0 and BWAFE were covariables, and e was the random error. The data were presented as probability values and least squares means ± SEM. The significant differences of least squares means were tested with Duncan’s multiple range tests, and a P-value of ≤ 0.05 was considered statistically significant.
RESULTS Identified SNP and Allele and Genotype Frequencies Nine SNP of 5′ upstream region of chicken IGFBP1, IGFBP3, and STAT5B genes were identified by the pooled DNA sequencing and sequence traces. Of them, 5 SNP were examined for associations with traits. These 5 SNP, their corresponding frequencies of allele and genotype, and P-values for the Hardy-Weinberg equilibrium test are presented in Table 2. The result showed that these 5 SNP were in Hardy-Weinberg equilibrium (P > 0.05). Genotype frequencies of TC (T56039403C), CC (C56072547T), CT (C4535156T), GA (G4533815A), and GC (G4533675C) were the highest for 0.5273, 0.4757, 0.4747, 0.4982, and 0.4914, respectively.
Linkage Disequilibrium of SNP Linkage disequilibrium (LD) of the 5 SNP were measured by Lewontin’s D′ (|D′|) and r2 with Haploview version 4.1 software. Lewontin’s D′ (|D′|) and r2 of C4535156T and G4533815A SNP, C4535156T and G4533675C SNP, G4533815A and G4533675C SNP of the STAT5B gene were 0.939 and 0.852, 0.967 and 0.858, and 0.971 and 0.896, respectively. When r2 is higher than 0.3, two loci are considered in LD, and when r2 is higher than 0.6, two loci are considered in strongly LD (Ardlie et al., 2002). Moreover, 1 haplotype block has been defined as 3 or more markers for which all estimated values of |D′| exceeded 0.9 (Phillips et al., 2003). The results indicated that the 3 SNP of the STAT5B gene were strong-linked with each other and lay within a haplotype block; thereinto, the G4533815A SNP was tagSNP. In addition, D′ and r2 of T56039403C and C56072547T SNP of IGFBP1 and IGFBP3 genes were 0.949 and 0.465, respectively, and the 2 SNP were in LD.
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Genotype
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Trait Associations and Effects of SNP
Analysis of the 5′-Upstream Sequence By sequencing analysis, we also checked whether there are any transcriptional binding sites on these SNP and found the transcription factor response elements associated with 3 SNP, including T56039403C, C56072547T, and G4533815A, the homeodomain factor
DISCUSSION It is known that the neuroendocrine growth axis is involved in the activity of many hormones, cytokines, and chemokines and their receptors play crucial roles in the regulation of animal growth, reproduction, immunity, and metabolism. At present, it has become a hot topic in research to probe into balanced selection and breeding for growth, reproduction, immunity, and metabolism in chickens for optimum performance. Combining the relative information with comparative omics (such as genomics, transcriptomics, proteomics, and metabonomics), physiological functions, mapping and fine mapping of QTL and expression QTL, signaling pathways, and gene networks, the method of the candidate gene approach is becoming increasingly economical and effective in finding candidate QTL gene and quantitative trait nucleotides with significant effects on economically important traits (Kanehisa and Goto, 2000; Cogburn et al., 2003; Zhu and Zhao, 2007; Kuhn et al., 2008). According to the above integration strategies, the IGFBP1, IGFBP3, and STAT5B genes were effectively selected and used to study the association with growth and reproductive traits in chickens. As the main component of gene expression regulation, the 5′ upstream region plays an important role in affecting the phenotypes of traits (Liu et al., 2006). In this study, 5 SNP selected from 9 SNP of the 5′upstream region of the 3 genes were associated with growth or reproductive traits, or both, in chickens. The results showed that these 5 SNP were in Hardy-Weinberg equilibrium
Table 3. Effects (P-value and least squares means ± SEM) of the 5 SNP on growth and reproductive traits SNP T56039403C P-value C56072547T P-value C4535156T P-value G4533815A P-value G4533675C P-value a,b
Genotype TT TC CC CC CT TT CC CT TT GG GA AA GG GC CC
BW 81 (g) 612 ± 8.1 612 ± 4.1 631 ± 6.3 0.0504 628 ± 4.6A 605 ± 4.7B 612 ± 11.9AB 0.0056** 618 ± 6.3 613 ± 4.6 634 ± 8.1 0.0664 615 ± 6.6 616 ± 4.6 628 ± 8.4 0.4450 638 ± 8.1a 615 ± 4.6b 611 ± 6.6b 0.0319*
Means within a row with no common superscript differ significantly (P < 0.05). Means within a row with no common superscript differ significantly (P < 0.01). 1 BW 8 = 8 wk of age; BW 10 = 10 wk of age; AFE = age at first egg. *P < 0.05; **P < 0.01. A,B
BW 10 (g) b
783 ± 9.3 784 ± 4.8b,B 812 ± 7.3a,A 0.0061** 806 ± 5.4A 777 ± 5.5B 780 ± 13.8AB 0.0016** 797 ± 7.3 784 ± 5.3 805 ± 9.4 0.0815 790 ± 7.7 788 ± 5.4 800 ± 9.9 0.5832 808 ± 9.5 789 ± 5.4 788.85 ± 7.63 0.1883
AFE (d) 149 ± 3.2 148 ± 1.6 151 ± 2.6 0.7443 150 ± 2.0 148 ± 1.7 151 ± 5.2 0.8239 154 ± 2.63a,A 150 ± 1.9a 139 ± 3.7b,B 0.0143* 156 ± 3.0a,A 148 ± 1.9a 140 ± 3.3b,B 0.0088** 140 ± 3.2b,B 149 ± 1.8a 155 ± 3.0a,A 0.0114*
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Trait associations (P-values) and effects (least squares means) of the 5 SNP with growth and reproductive traits are shown in Table 3. The C56072547T and T56039403C SNP of the IGFBP3 and IGFBP1 genes were significantly associated with BW at 8 or10 wk of age, or both weeks of age (P = 0.0056 and P = 0.0016, P = 0.0061, respectively). The C4535156T, G4533815A, and G4533675C SNP of the STAT5B gene were associated with age at first egg for P = 0.0143, P = 0.0088, and P = 0.0114, respectively. At the same time, the G4533675C SNP was associated with BW at 8 wk of age (P = 0.0319). There were higher (P < 0.01) BW at 8 or 10 wk of age, or both, in the birds that were of the CC genotype of the C56072547T and T56039403C SNP than those of the TT and TC genotypes. There were higher (P < 0.05) BW at 8 wk of age that were of the GG genotype of the G4533675C SNP than those of the GC and CC genotype. There were shorter AFE (P < 0.01) in the birds that were of the TT genotype of the C4535156T SNP than those of the CC and CT genotypes, of the AA genotype of the G4533815A SNP than those of the GG and GA genotypes, and of the GG genotype of the G4533675C SNP than those of the GC and CC genotypes.
(Nkx-2.5) identified on T56039403C and C56072547T, and cyclic adenosine monophosphate response elementbinding proteins (CREB) on G4533815A SNP.
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2006). By using DNA pooling as an initial screening process combined with modified PCR-RFLP, researchers can be more specific for candidate genes analysis and can then reduce labor time and costs (Ke et al., 2001; Zhang et al., 2005). In summary, association of the 5 SNP from the 3 genes with chicken growth and reproductive traits was analyzed in the present study. These SNP were found to be significantly associated with early growth or with sexual maturation in chickens, or both. Therefore, in conclusion, the genes of the neuroendocrine growth axis not only affected chicken growth but also were associated with reproductive traits.
ACKNOWLEDGMENTS We thank the members of the animal genetics group and poultry genetics group at China Agricultural University for managing the birds and teaching experimental techniques and Jiuzhou Song at University of Maryland, College Park, for giving suggestions on the manuscript. The work was supported by the State Basic Research and Development Project of China (No. 2006CB102107) and partly supported by National Key Technologies R & D Program (2006BAD04A01), National High-Tech R & D Program (2007AA10Z157), and National “948” Project (2006-G48).
REFERENCES Abasht, B., J. C. Dekkers, and S. J. Lamont. 2006. Review of quantitative trait loci identified in the chicken. Poult. Sci. 85:2079– 2096. Amos, C. I., M. L. Frazier, and W. Wang. 2000. DNA pooling in mutation detection with reference to sequence analysis. Am. J. Hum. Genet. 66:1689–1692. Andersson, L., and M. Georges. 2004. Domestic-animal genomics: Deciphering the genetics of complex traits. Nat. Rev. Genet. 5:202–212. Ardlie, K. G., L. Kruglyak, and M. Seielstad. 2002. Patterns of linkage disequilibrium in the human genome. Nat. Rev. Genet. 3:299–309. Bachelot, A., and N. Binart. 2007. Reproductive role of prolactin. Reproduction 133:361–369. Barrett,, J. C., B. Fry, J. Maller, and M. J. Daly. 2005. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics 21:263–265. Beyer, A., S. Bandyopadhyay, and T. Ideker. 2007. Integrating physical and genetic maps: From genomes to interaction networks. Nat. Rev. Genet. 8:699–710. Borevitz, J. O., and J. Chory. 2004. Genomics tools for QTL analysis and gene discovery. Curr. Opin. Plant Biol. 7:132–136. Cogburn, L. A., X. Wang, W. Carre, L. Rejto, T. E. Porter, S. E. Aggrey, and J. Simon. 2003. Systems-wide chicken DNA microarrays, gene expression profiling, and discovery of functional genes. Poult. Sci. 82:939–951. Dajee, M., G. H. Fey, and J. S. Richards. 1998. Stat5b and the orphan nuclear receptors regulate expression of the α2-macroglobulin (α2M) gene in rat ovarian granulosa cells. Mol. Endocrinol. 12:1393–1409. Darnell, J. E. 1997. STATs and gene regulation. Science 277:1630– 1635. Firth, S. M., and R. C. Baxter. 2002. Cellular actions of the insulinlike growth factor binding proteins. Endocr. Rev. 23:824–854. Harris, B. S., L. Spruill, A. M. Edmonson, M. S. Rackley, D. W. Benson, T. X. O’Brien, and R. G. Gourdie. 2006. Differentiation
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and were linked significantly with chicken growth or reproductive traits, or both. The 2 SNP of the IGFBP1 and IGFBP3 gene were significantly related with BW. The 3 SNP of the STAT5B gene were associated with AFE. Meanwhile, the 3 SNP of the STAT5B gene appeared to be strongly linked with each other and lay within a haplotype block because their D′ (|D′|) and r2 statistics were higher than 0.93 and 0.85. Moreover, STAT5B was activated by differentiation-dependent transcriptional regulation of distinct cytokine signaling pathway components and mediated distinct functional processes in the rat ovary for early follicle growth, atresia, and luteinization (Russell et al., 1996; Dajee et al., 1998). Hence, our findings in the present study implied that the chicken STAT5B gene may affect sexual maturation of chicken by regulating ovary development. In addition, we found that the G4533675C SNP of the STAT5B gene was related to growth traits besides reproductive traits, indicating such polymorphism might provide the basis for balanced selection and may be used in MAS to improve growth and reproductive production simultaneously. In the current study, 2 kinds of the transcription factor response elements were identified that included Nkx-2.5 and CREB proteins. It has been found that Nkx-2.5 plays critical roles in regulating tissue-specific gene expression and induces chicken cardiac myocyte differentiation in tissues (Schultheiss et al., 1995; Harris et al., 2006). Cyclic adenosine monophosphate response element-binding protein, which binds to the cyclic adenosine monophosphate response elements and increases or decreases the transcription of genes, is critical in a variety of cellular processes such as cell proliferation, differentiation, and adaptive responses in chickens (Sirotkin and Grossmann, 2003; Kim et al., 2005). The presence of Nkx-2.5 and CREB protein binding sites suggested the possible transcriptional regulation of the chicken IGFBP1, IGFBP3, and STAT5B genes by Nkx-2.5 and CREB proteins. The association studies might provide the basis for uncovering the molecular mechanisms of the 3 genes on growth and reproduction performances. Recently, numerous QTL have been detected in chickens using different DNA markers. Several QTL for growth are located on GGA2 or GGA7, which contain chicken IGFBP1 and IGFBP3 genes or IGFBP2 and IGFBP5 genes, respectively, and may affect white meat percentage and other growth traits (Tatsuda and Fujinaka, 2001; McElroy et al., 2006; Park et al., 2006). In the present study, our results were consistent with the former that focused on QTL mapping on GGA2, which indicated that chicken IGFBP1 and IGFBP3 genes should be important candidate genes for chicken growth. In the current study, the pooled DNA sequencing approach, which proved to be a powerful SNP discovery tool, was used for the effective identification of 9 SNP of the 5′ upstream region of the chicken IGFBP1, IGFBP3, and STAT5B genes (Amos et al., 2000; Ye et al.,
ASSOCIATION OF GROWTH-CORRELATED GENES WITH ECONOMIC TRAITS
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