- Email: [email protected]
A single nucleotide polymorphism in the parathyroid hormone gene and effects on eggshell quality in chickens
Rapid Communication A single nucleotide polymorphism in the parathyroid hormone gene and effects on eggshell quality in chickens R. S. Jiang, Z. Xie, X. Y. Chen, and Z. Y. Geng1 College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, P.R. China ing strength for genotypes GG and AG were greater (P < 0.05) than for AA, respectively; the total serum calcium for genotype GG was higher than for genotypes AA and AG by 11.8 and 10.1%, respectively; the serum phosphate for genotype GG was greater than genotype AA and AG by 16.7 and 12%, respectively. All genotypes shared the same calcium:phosphate ratio. For serum PTH, genotype GG was approximately 30% higher than genotype AA. Therefore, the SNP A2205G in PTH affected the eggshell percentage and breaking strength, and it may be associated with the variation of serum calcium and PTH level, indicating that the SNP in PTH has the potential for utilization in a MAS program for eggshell quality in chicken.
Key words: chicken, parathyroid hormone, single nucleotide polymorphism, eggshell quality 2010 Poultry Science 89:2101–2105 doi:10.3382/ps.2010-00888
INTRODUCTION Broken and cracked eggshells cause significant economic losses to the egg production industry (Hamilton et al., 1979; Dhawale, 2008). Poor shell strength enhances the occurrence of hairline cracks and therefore increases the risks of bacterial contamination of eggs (Messens et al., 2007). The successful development of avian embryos is dependent upon a robust eggshell for mechanical protection, for protection from infection, for prevention of excessive water loss from egg, and as a primary source of calcium for the embryonic skeleton (Karlsson and Lilja, 2008). Therefore, eggshell quality is important in chicken breeding. Eggshell quality is affected by many factors involving genetics, disease, nutrition, and environment (Roberts, 2004). The formation of eggshell requires calcium from blood, bones, and the gastrointestinal tract (Eastin and Paziani, 1978). Egg laying and shell calcification
©2010 Poultry Science Association Inc. Received May 10, 2010. Accepted July 17, 2010. 1 Corresponding author: [email protected]
impose severe extra demands on calcium homeostasis, and parathyroid hormone (PTH) is involved in this homeostasis (Potts, 2005). Parathyroid hormone, a protein hormone released by the parathyroid gland, controls the level of blood calcium and phosphate (Brommage et al., 1999; Murray et al., 2005) and maintains the calcium homeostasis by regulating the calcium liberation from the bone and resorption from the kidney (Ogawa et al., 2000; Tenne et al., 2010). In avians, PTH acts either directly or indirectly on the oviduct, limiting eggshell calcification (van de Velde, 1984). During the period of eggshell calcification, PTH bioactivity is elevated; after formation of the shell, plasma PTH decreases to a low level (van de Velde et al., 1984), indicating that changes in bioactive PTH play an important role in eggshell calcification during eggshell formation. Nucleotide variations in the PTH gene and the genetic effects on hypoparathyroidism and bone mineral density have been reported in humans (Hosoi et al., 1999; Sunthornthepvarakul et al., 1999; Goswami et al., 2004; Tenne et al., 2010). Chicken PTH has been cloned and the analysis of nucleotide sequence showed that the gene contains 3 exons and the encoding region consists of 357 nucleotides, which predicts a mature
2101
Downloaded from http://ps.oxfordjournals.org/ by guest on June 3, 2015
ABSTRACT Parathyroid hormone (PTH), released by the parathyroid gland of animals, plays an important role in regulating the metabolism of calcium and phosphate. As a candidate gene for eggshell quality traits, the SNP was screened and its genetic effects on eggshell qualities and levels of serum calcium, phosphate, and PTH were analyzed in this study. Three hundred Houdan hens, an indigenous breed of chicken in France, were used for genotyping and data recording. Of the 3 sets of primers used to amplify the exons, exon 3 was polymorphic and 3 genotypes were identified. Sequencing revealed a nucleotide transition, A2205G (GenBank accession no. NC_006092), which was a synonymous mutation and caused a codon for lysine to change from AAA to AAG. Eggshell percentage and break-
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chicken PTH of 88 amino acids in contrast to the 84 amino acids of the mammalian hormones (Khosla et al., 1988). However, the nucleotide variation in chicken PTH has not been reported. In the present study, the SNP was identified and the genetic effects of SNP on eggshell quality were evaluated.
MATERIALS AND METHODS Populations and Management
Measurements Traits related to eggshell were individually recorded from 275 to 279 d of age. During that period, only hens that produced 3 or more eggs were tested for the traits including egg weight, egg shape index, eggshell weight, and eggshell breaking strength. All of these traits were tested 24 h after laying. Eggshell weight was measured after washing the interior egg membrane and after drying overnight at 80°C; egg shape index was represented as egg length:breadth; the eggshell percentage was expressed as (100 × eggshell weight/egg weight); eggshell breaking strength was measured using Nabel DET6000 (Nabel Co. Ltd., Kyoto, Japan) by applying increased pressure to the broad pole of the shell. At 20 wk of age, 1 mL of blood was collected individually for DNA isolation. At 40 wk of age, 5 mL of blood was collected by wing vein puncture for each hen whose eggshell traits were recorded. Blood biochemical indices included the concentrations of total serum calcium, phosphate, and PTH. Total serum calcium and phosphate levels were done by standard methods using a Hitachi 917 Multianalyser (Hitachi Co. Ltd., Tokyo, Japan). The serum PTH was assayed using RIA ac-
Genotyping for PTH Genomic DNA was isolated from blood samples by the phenol-chloroform method. The 3 exons of PTH were amplified by PCR. The PCR primers (Table 1) were designed according to the sequence of PTH (GenBank accession no. NC_006092) using the online primer design procedure Primer 5.0 (http://www.premierbiosoft. com). The 20-μL PCR volume included 50 ng of DNA templates, 0.20 mM deoxynucleoside triphosphate, 2.5 mM MgCl2, 0.20 mM primers, and 0.5 U of Taq DNA polymerase. The PCR protocol was 94°C for 5 min followed by 35 cycles of 94°C for 45 s, 57°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 10 min. The PCR products were genotyped by the method of single-stranded conformational polymorphism. Two microliters of PCR product of each individual was mixed with 5 μL of denaturing buffer (98% formamide, 0.09% xylene cyanole FF, and 0.09% bromophenol blue) and denatured at 94°C for 5 min, followed by a rapid chill on ice for 10 min. The denatured PCR products were electrophoresed for 14 h at 8 V/cm on 12% acrylamide gels. The DNA bands on the gel were stained by 0.2% AgNO3 for 20 min and then 3% Na2CO3 for about 5 min (Qu et al., 2005). Genotypes were recorded according to band patterns. The PCR products of each homozygote were purified using the DNA Fragment Quick Purification/Recover Kit (Takara Biotechnology Co. Ltd., Dalian, China), ligated to the pMD 18-T vector, and transformed into DH5-α Escherichia coli for PCR product cloning (Sambrook et al., 1989). Sequencing was performed on an ABI 377 Sequencer (Applied Biosystems, Foster City, CA) to identify the mutation site.
Statistical Analysis A χ2 test was adopted to analyze the Hardy-Weinberg equilibrium of genotypes of chicken PTH. Individual records for egg weight, eggshell weight, egg shape index, and eggshell breaking strength were the average of 3 or more eggs. Means of eggshell quality traits and blood biochemical indices were calculated for each genotype. Genotypic effects were analyzed by 1-way ANOVA us-
Table 1. Polymerase chain reaction primers for parathyroid hormone Primer
Sequence1
Exon 1
F: 5′-TAAAC CAATT CAGTA GTC-3′ R: 5′-TAAAG CAGTG ATAAA ACC-3′ F: 5′-CATCGTTCCCCAGACTTTTTATC-3′ R: 5′-GCAGATAAAAATGACTTCTAC-3′ F: 5′-CTTGGACAGTTATAATGCTCTTG-3′ R: 5′-AGCCATTATGAGTCCTTTCCTT-3′
Exon 2 Exon 3 1F
= forward; R = reverse.
Annealing temperature (°C)
Amplicon length (bp)
56
110
58
150
57
280
Downloaded from http://ps.oxfordjournals.org/ by guest on June 3, 2015
Houdan hens, an indigenous breed of chicken in France, were used in the experiment. At 20 wk of age, 300 hens were selected at random from a population of 5,000 and transferred to a windowless laying house. Hens were raised in the medium tier of a 3-tier cage system with 1 hen in each cage. They were fed a commercial corn-soybean-based diet with 2,600 kcal/kg of ME and 16.2% CP. Hens had free access to feed and water. The house was automatically ventilated to maintain an ambient temperature between 20 and 28°C with a photoperiod of 16L:8D at a light intensity of 15 lx.
cording to the protocol of RIA Kit (Northern Institute of Biotechnology, Beijing, China).
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RAPID COMMUNICATION
Genotypic Effects of PTH on Eggshell Traits
Figure 1. Band patterns for the polymorphic loci, exon 3 of chicken parathyroid hormone. Lanes 4, 5, and 9 = genotype AA; lanes 2 and 10 = genotype GG; lanes 1, 3, 6, 7, 8, and 11 = genotype AG.
ing the GLM procedure of the SAS Institute (2001) with genotype (G) as a fixed effect, according to the following model:
At 275 to 279 d of age, 148 genotyped hens that individually produced 3 or more eggs were used to analyze the genotypic effects of PTH on eggshell traits. Effects of the genotypes on eggshell traits at 40 wk of age were shown in Table 2. Egg weight, eggshell weight, as well as egg shape index did not differ (P > 0.05) among genotypes. Eggshell percentage and breaking strength for genotypes AG and GG were greater (P < 0.05) than AA, respectively. Except for egg shape index and egg size, eggshell thickness and density are also important traits with high correlation with eggshell strength. The genotypic effects of PHT on them need further study.
Genotypic Effects of PTH on Blood Biochemical Indices
Y = μ + G + e,
RESULTS AND DISCUSSION Polymorphisms in Chicken PTH Of the 3 sets of primers used to amplify the exons, exon 3 was polymorphic and 3 genotypes (AA, AG, and GG) were identified (Figure 1). Sequencing revealed a nucleotide transition, A2205G (GenBank accession no. NC_006092). The SNP did not cause amino acid variation in chicken PTH but caused a codon (encoding the 28th amino acid of the mature PTH) for lysine changing from AAA to AAG. In the experiment, 284 out of 300 individuals were genotyped successfully. The frequencies for genotypes AA, AG, and GG were 0.43, 0.49, and 0.08, respectively, and the allelic frequencies for A and G were 0.67 and 0.33, respectively. The χ2 test showed that the distribution of genotypes was in Hardy-Weinberg equilibrium (χ2 = 2.002 < χ20.05(2) = 5.99).
Statistically, the difference of all blood biochemical indices at 40 wk of age among genotypes was not significant (P > 0.05; Table 3); however, the total serum calcium for genotype GG was higher than genotype AA and AG by 11.8 and 10.1%, respectively; the serum phosphate for genotype GG was greater than genotypes AA and AG by 16.7 and 12%, respectively. But, all genotypes shared the same calcium:phosphate ratio and demonstrated the biofunction of PTH in the maintenance of serum calcium-phosphate homeostasis. For serum PTH, genotype GG was approximately 30% higher than genotype AA. In this study, the eggshell percentage and breaking strength for genotype GG as well as AG were significantly higher than AA, indicating that allele G was favorable for eggshell quality. In chickens, injection of 200 pg of pCEP4-PTH gene plasmid significantly (P < 0.05) elevated the levels of serum calcium and PTH and thus significantly (P < 0.05) improved the eggshell quality (Zhang et al., 2009). Therefore, the relatively higher serum calcium and PTH level may explain the better eggshell quality in chickens with allele G. Although synonymous mutations, also called silence SNP, do not change the amino acid sequences and the biofunctions of proteins, they may be associated with
Table 2. Means ± SEM of parathyroid hormone (PTH) genotypes on eggshell traits at 40 wk of age in hens PTH genotype1 Trait
AA (n = 56)
AG (n = 74)
GG (n = 18)
Egg weight (g) Shell weight (g) Egg shape index Eggshell percentage (%) Eggshell strength (kg/cm2)
44.7 4.71 1.32 10.5 3.28
44.7 4.94 1.32 11.1 3.71
44.3 5.03 1.32 11.3 3.69
a,bMeans 1A
± ± ± ± ±
0.4 0.08 0.01 0.1a 0.09a
± ± ± ± ±
0.4 0.06 0.01 0.1b 0.07b
in a row with different superscripts differed at P < 0.05. total of 148 out of 300 hens were successfully tested for the eggshell traits.
± ± ± ± ±
1.0 0.13 0.01 0.3b 0.17b
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where Y was the dependent variable, μ the population mean, and e the random error. Significant differences among means of different genotypes were calculated using Duncan’s multiple range test, and the significance was determined at P < 0.05.
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Jiang et al. Table 3. Means ± SEM of parathyroid hormone (PTH) genotypes on serum biochemical indices at 40 wk of age in hens PTH genotype1 Item
AA (n = 47)
Serum calcium (mg/dL) Serum phosphate (mg/dL) Calcium:posphate Serum PTH (pg/mL) 1A
6.8 2.4 2.8 25.1
± ± ± ±
0.2 0.1 0.1 3.1
AG (n = 59) 6.9 2.5 2.8 29.8
± ± ± ±
0.1 0.1 0.1 3.0
GG (n = 17) 7.6 2.8 2.8 32.6
± ± ± ±
0.2 0.2 0.1 5.1
total of 123 out of 148 hens were successfully assayed for the serum biochemical indices.
ACKNOWLEDGMENTS This work was supported by grants from China S&T Pillar Program (2008BADB2B06, 2008BADA9B06) and the National High Technology Research and Development Program (“863” Program) of China (2008A101009).
REFERENCES Brommage, R., E. H. Charlotte, J. L. Cynthia, W. S. Melanie, M. H. Janet, and P. J. Christopher. 1999. Daily treatment with human recombinant parathyroid hormone-(1–34), LY333334, for 1 year increases bone mass in ovariectomized monkeys. J. Clin. Endocrinol. Metab. 84:3757–3763. Capon, F., M. H. Allen, and M. Ameen. 2004. A synonymous SNP of the corneodesmosin gene leads to increased mRNA stability and demonstrates association with psoriasis across diverse ethnic groups. Hum. Mol. Genet. 13:2361–2368.
Chamary, J. V., and L. D. Hurst. 2005. Evidence for selection on synonymous mutations affecting stability of mRNA secondary structure in mammals. Genome Biol. 6:R75. Chamary, J. V., J. L. Parmley, and L. D. Hurst. 2006. Hearing silence: Non-neutral evolution at synonymous sites in mammals. Nat. Rev. Genet. 7:98–108. Comeron, J. M. 2004. Selective and mutational patterns associated with gene expression in humans: Influences on synonymous composition and intron presence. Genetics 167:1293–1304. De Ferrari, G. V., A. Papassotiropoulos, and T. Biechele. 2007. Common genetic variation within the low-density lipoprotein receptor-related protein 6 and late-onset Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 104:9434–9439. Dhawale, A. 2008. Abnormal eggs cause subnormal profits. World Poult. 24:20–23. Duan, J. 2003. Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Hum. Mol. Genet. 12:205–216. Duan, J., and M. A. Antezana. 2003. Mammalian mutation pressure, synonymous codon choice, and mRNA degradation. J. Mol. Evol. 57:694–701. Eastin, W. C., Jr., and E. S. Paziani. 1978. On the control of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19:493–504. Goswami, R., T. Mohapatra, N. Gupta, R. Rani, N. Tomar, A. Dikshit, and R. K. Sharma. 2004. Parathyroid hormone gene polymorphism and sporadic idiopathic hypoparathyroidism. J. Clin. Endocrinol. Metab. 89:4840–4845. Hamilton, R. N. G., K. G. Hollands, P. W. Voisey, and A. A. Grunder. 1979. Relationship between egg shell quality and shell breakage and factors that affect shell breakage in the field—A review. World’s Poult. Sci. J. 35:177–190. Hosoi, T., M. Miyao, S. Inoue, S. Hoshino, M. Shiraki, H. Orimo, and Y. Ouchi. 1999. Association study of parathyroid hormone gene polymorphism and bone mineral density in Japanese postmenopausal women. Calcif. Tissue Int. 64:205–208. Karlsson, O., and C. Lilja. 2008. Eggshell structure, mode of development and growth rate in birds. Zoology 111:494–502. Khosla, S., M. Demay, M. Pines, S. Hurwitz, J. T. Potts Jr., and H. M. Kronenberg. 1988. Nucleotide sequence of cloned cDNAs encoding chicken preproparathyroid hormone. J. Bone Miner. Res. 3:689–698. Lavner, Y., and D. Kotlar. 2005. Codon bias as a factor in regulating expression via translation rate in the human genome. Gene 345:127–138. Laws, S. M., E. Hone, S. Gandy, and R. N. Martins. 2003. Expanding the association between the APOE gene and the risk of Alzheimer’s disease: Possible roles for APOE promoter polymorphisms and alterations in APOE transcription. J. Neurochem. 84:1215–1236. Messens, W., K. Griispeerdt, K. De Reu, B. De Ketelaere, K. Mertens, F. Bamelis, B. Kemp, J. De Baerdemaeker, E. Decuypere, and L. Herman. 2007. Eggshell penetration of various types of hens’ eggs by Salmonella enterica serovar Enteritidis. J. Food Prot. 70:623–628. Murray, T. M., G. R. Leticia, D. Paola, and F. R. Bringhurst. 2005. Parathyroid hormone secretion and action: Evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl-terminal ligands. Endocr. Rev. 26:78–113.
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target traits via affecting the stability of mRNA and translation efficiency (Laws et al., 2003; Capon et al., 2004; Chamary et al., 2006; De Ferrari et al., 2007). Synonymous mutations alter the primary structure of mRNA, which causes the change in mRNA secondary structure and thus affects the stability of mRNA. When A or U in the third nucleotide of a codon changes into G or C, the incidence of mRNA being degraded by enzymes lowered and the mRNA was more stable (Duan, 2003; Duan and Antezana, 2003; Chamary and Hurst, 2005). On the other hand, codon usage bias may affect the translation efficiency (Comeron, 2004; Lavner and Kotlar, 2005). In humans, analysis of 1224 lysine from 78 genes showed that the usage frequencies of codons AAG and AAA were 69.6 and 30.4%, respectively (Shi et al., 2000); the same phenomenon was also seen in Phalaenopsis hainanensis (Pang et al., 2005). In chicken PTH, we found 10 codons encoding lysine, in which the usage frequencies of codons AAG and AAA were 70 and 30%, respectively. Therefore, the synonymous mutation A2205G probably increases the stability of mRNA and translation efficiency in chicken PTH. In conclusion, the synonymous mutation A2205G in PTH affected the eggshell percentage and breaking strength in this study, and it may be associated with the variation of serum calcium and PTH level. Therefore, the SNP has the potential for utilization in a MAS program for eggshell quality in chicken.
RAPID COMMUNICATION Ogawa, H., T. Takahashi, T. Yasuoka, T. Kuwayama, K. Tanaka, and M. Kawashima. 2000. Parathyroid hormone receptor binding property in the shell gland of oviduct of the guineafowl during an oviposition cycle. Poult. Sci. 79:575–579. Pang, G. X., G. C. Wang, S. N. Hu, and C. K. Tseng. 2005. Acquirement and analysis of expressed sequence tags of filamentous sporophyte of P. haitanensis. Oceanol. Limnol. Sin. 36:452– 458. Potts, J. T. 2005. Parathyroid hormone: Past and present. J. Endocrinol. 187:311–325. Qu, L. J., X. Y. Li, G. Q. Wu, and N. Yang. 2005. Efficient and sensitive method of DNA silver staining in polyacrylamide gel. Electrophoresis 26:99–101. Roberts, J. R. 2004. Factors affecting egg internal quality and egg shell quality in laying hens. Jpn. Poult. Sci. 41:161–177. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. SAS Institute. 2001. Version 8.2. SAS Institute Inc., Cary, NC.
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Shi, X. F., J. F. Huang, C. R. Liang, S. Q. Niu, J. Xie, and C. Q. Liu. 2000. Synonymous codon usage bias and binding ability of codon and anticodon in human genome. Chin. Sci. Bull. 45:2520–2525. Sunthornthepvarakul, T., S. Churesigaew, and S. Ngowngarmratana. 1999. A novel mutation of the signal peptide of the preproparathyroid hormone gene associated with autosomal recessive familial isolated hypoparathyroidism. J. Clin. Endocrinol. Metab. 84:3792–3796. Tenne, M., F. E. McGuigan, H. Ahlborg, P. Gerdhem, and K. A. Kesson. 2010. Variation in the PTH gene, hip fracture, and femoral neck geometry in elderly women. Calcif. Tissue Int. 86:359– 366. van de Velde, J. P., N. Loveridge, and J. P. Vermeiden. 1984. Parathyroid hormone responses to calcium stress during eggshell calcification. Endocrinology 115:1901–1904. Zhang, S. Y., L. Yu, Y. J. Wang, X. H. Lin, and Y. F Wang.. 2009. Effect of pCEP4-PTH gene plasmid on eggshell quality and serum hormone levels in layer. J. Zhejiang Univ. 35:201–208.
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Key words: chicken, parathyroid hormone, single nucleotide polymorphism, eggshell quality 2010 Poultry Science 89:2101–2105 doi:10.3382/ps.2010-00888
INTRODUCTION Broken and cracked eggshells cause significant economic losses to the egg production industry (Hamilton et al., 1979; Dhawale, 2008). Poor shell strength enhances the occurrence of hairline cracks and therefore increases the risks of bacterial contamination of eggs (Messens et al., 2007). The successful development of avian embryos is dependent upon a robust eggshell for mechanical protection, for protection from infection, for prevention of excessive water loss from egg, and as a primary source of calcium for the embryonic skeleton (Karlsson and Lilja, 2008). Therefore, eggshell quality is important in chicken breeding. Eggshell quality is affected by many factors involving genetics, disease, nutrition, and environment (Roberts, 2004). The formation of eggshell requires calcium from blood, bones, and the gastrointestinal tract (Eastin and Paziani, 1978). Egg laying and shell calcification
©2010 Poultry Science Association Inc. Received May 10, 2010. Accepted July 17, 2010. 1 Corresponding author: [email protected]
impose severe extra demands on calcium homeostasis, and parathyroid hormone (PTH) is involved in this homeostasis (Potts, 2005). Parathyroid hormone, a protein hormone released by the parathyroid gland, controls the level of blood calcium and phosphate (Brommage et al., 1999; Murray et al., 2005) and maintains the calcium homeostasis by regulating the calcium liberation from the bone and resorption from the kidney (Ogawa et al., 2000; Tenne et al., 2010). In avians, PTH acts either directly or indirectly on the oviduct, limiting eggshell calcification (van de Velde, 1984). During the period of eggshell calcification, PTH bioactivity is elevated; after formation of the shell, plasma PTH decreases to a low level (van de Velde et al., 1984), indicating that changes in bioactive PTH play an important role in eggshell calcification during eggshell formation. Nucleotide variations in the PTH gene and the genetic effects on hypoparathyroidism and bone mineral density have been reported in humans (Hosoi et al., 1999; Sunthornthepvarakul et al., 1999; Goswami et al., 2004; Tenne et al., 2010). Chicken PTH has been cloned and the analysis of nucleotide sequence showed that the gene contains 3 exons and the encoding region consists of 357 nucleotides, which predicts a mature
2101
Downloaded from http://ps.oxfordjournals.org/ by guest on June 3, 2015
ABSTRACT Parathyroid hormone (PTH), released by the parathyroid gland of animals, plays an important role in regulating the metabolism of calcium and phosphate. As a candidate gene for eggshell quality traits, the SNP was screened and its genetic effects on eggshell qualities and levels of serum calcium, phosphate, and PTH were analyzed in this study. Three hundred Houdan hens, an indigenous breed of chicken in France, were used for genotyping and data recording. Of the 3 sets of primers used to amplify the exons, exon 3 was polymorphic and 3 genotypes were identified. Sequencing revealed a nucleotide transition, A2205G (GenBank accession no. NC_006092), which was a synonymous mutation and caused a codon for lysine to change from AAA to AAG. Eggshell percentage and break-
2102
Jiang et al.
chicken PTH of 88 amino acids in contrast to the 84 amino acids of the mammalian hormones (Khosla et al., 1988). However, the nucleotide variation in chicken PTH has not been reported. In the present study, the SNP was identified and the genetic effects of SNP on eggshell quality were evaluated.
MATERIALS AND METHODS Populations and Management
Measurements Traits related to eggshell were individually recorded from 275 to 279 d of age. During that period, only hens that produced 3 or more eggs were tested for the traits including egg weight, egg shape index, eggshell weight, and eggshell breaking strength. All of these traits were tested 24 h after laying. Eggshell weight was measured after washing the interior egg membrane and after drying overnight at 80°C; egg shape index was represented as egg length:breadth; the eggshell percentage was expressed as (100 × eggshell weight/egg weight); eggshell breaking strength was measured using Nabel DET6000 (Nabel Co. Ltd., Kyoto, Japan) by applying increased pressure to the broad pole of the shell. At 20 wk of age, 1 mL of blood was collected individually for DNA isolation. At 40 wk of age, 5 mL of blood was collected by wing vein puncture for each hen whose eggshell traits were recorded. Blood biochemical indices included the concentrations of total serum calcium, phosphate, and PTH. Total serum calcium and phosphate levels were done by standard methods using a Hitachi 917 Multianalyser (Hitachi Co. Ltd., Tokyo, Japan). The serum PTH was assayed using RIA ac-
Genotyping for PTH Genomic DNA was isolated from blood samples by the phenol-chloroform method. The 3 exons of PTH were amplified by PCR. The PCR primers (Table 1) were designed according to the sequence of PTH (GenBank accession no. NC_006092) using the online primer design procedure Primer 5.0 (http://www.premierbiosoft. com). The 20-μL PCR volume included 50 ng of DNA templates, 0.20 mM deoxynucleoside triphosphate, 2.5 mM MgCl2, 0.20 mM primers, and 0.5 U of Taq DNA polymerase. The PCR protocol was 94°C for 5 min followed by 35 cycles of 94°C for 45 s, 57°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 10 min. The PCR products were genotyped by the method of single-stranded conformational polymorphism. Two microliters of PCR product of each individual was mixed with 5 μL of denaturing buffer (98% formamide, 0.09% xylene cyanole FF, and 0.09% bromophenol blue) and denatured at 94°C for 5 min, followed by a rapid chill on ice for 10 min. The denatured PCR products were electrophoresed for 14 h at 8 V/cm on 12% acrylamide gels. The DNA bands on the gel were stained by 0.2% AgNO3 for 20 min and then 3% Na2CO3 for about 5 min (Qu et al., 2005). Genotypes were recorded according to band patterns. The PCR products of each homozygote were purified using the DNA Fragment Quick Purification/Recover Kit (Takara Biotechnology Co. Ltd., Dalian, China), ligated to the pMD 18-T vector, and transformed into DH5-α Escherichia coli for PCR product cloning (Sambrook et al., 1989). Sequencing was performed on an ABI 377 Sequencer (Applied Biosystems, Foster City, CA) to identify the mutation site.
Statistical Analysis A χ2 test was adopted to analyze the Hardy-Weinberg equilibrium of genotypes of chicken PTH. Individual records for egg weight, eggshell weight, egg shape index, and eggshell breaking strength were the average of 3 or more eggs. Means of eggshell quality traits and blood biochemical indices were calculated for each genotype. Genotypic effects were analyzed by 1-way ANOVA us-
Table 1. Polymerase chain reaction primers for parathyroid hormone Primer
Sequence1
Exon 1
F: 5′-TAAAC CAATT CAGTA GTC-3′ R: 5′-TAAAG CAGTG ATAAA ACC-3′ F: 5′-CATCGTTCCCCAGACTTTTTATC-3′ R: 5′-GCAGATAAAAATGACTTCTAC-3′ F: 5′-CTTGGACAGTTATAATGCTCTTG-3′ R: 5′-AGCCATTATGAGTCCTTTCCTT-3′
Exon 2 Exon 3 1F
= forward; R = reverse.
Annealing temperature (°C)
Amplicon length (bp)
56
110
58
150
57
280
Downloaded from http://ps.oxfordjournals.org/ by guest on June 3, 2015
Houdan hens, an indigenous breed of chicken in France, were used in the experiment. At 20 wk of age, 300 hens were selected at random from a population of 5,000 and transferred to a windowless laying house. Hens were raised in the medium tier of a 3-tier cage system with 1 hen in each cage. They were fed a commercial corn-soybean-based diet with 2,600 kcal/kg of ME and 16.2% CP. Hens had free access to feed and water. The house was automatically ventilated to maintain an ambient temperature between 20 and 28°C with a photoperiod of 16L:8D at a light intensity of 15 lx.
cording to the protocol of RIA Kit (Northern Institute of Biotechnology, Beijing, China).
2103
RAPID COMMUNICATION
Genotypic Effects of PTH on Eggshell Traits
Figure 1. Band patterns for the polymorphic loci, exon 3 of chicken parathyroid hormone. Lanes 4, 5, and 9 = genotype AA; lanes 2 and 10 = genotype GG; lanes 1, 3, 6, 7, 8, and 11 = genotype AG.
ing the GLM procedure of the SAS Institute (2001) with genotype (G) as a fixed effect, according to the following model:
At 275 to 279 d of age, 148 genotyped hens that individually produced 3 or more eggs were used to analyze the genotypic effects of PTH on eggshell traits. Effects of the genotypes on eggshell traits at 40 wk of age were shown in Table 2. Egg weight, eggshell weight, as well as egg shape index did not differ (P > 0.05) among genotypes. Eggshell percentage and breaking strength for genotypes AG and GG were greater (P < 0.05) than AA, respectively. Except for egg shape index and egg size, eggshell thickness and density are also important traits with high correlation with eggshell strength. The genotypic effects of PHT on them need further study.
Genotypic Effects of PTH on Blood Biochemical Indices
Y = μ + G + e,
RESULTS AND DISCUSSION Polymorphisms in Chicken PTH Of the 3 sets of primers used to amplify the exons, exon 3 was polymorphic and 3 genotypes (AA, AG, and GG) were identified (Figure 1). Sequencing revealed a nucleotide transition, A2205G (GenBank accession no. NC_006092). The SNP did not cause amino acid variation in chicken PTH but caused a codon (encoding the 28th amino acid of the mature PTH) for lysine changing from AAA to AAG. In the experiment, 284 out of 300 individuals were genotyped successfully. The frequencies for genotypes AA, AG, and GG were 0.43, 0.49, and 0.08, respectively, and the allelic frequencies for A and G were 0.67 and 0.33, respectively. The χ2 test showed that the distribution of genotypes was in Hardy-Weinberg equilibrium (χ2 = 2.002 < χ20.05(2) = 5.99).
Statistically, the difference of all blood biochemical indices at 40 wk of age among genotypes was not significant (P > 0.05; Table 3); however, the total serum calcium for genotype GG was higher than genotype AA and AG by 11.8 and 10.1%, respectively; the serum phosphate for genotype GG was greater than genotypes AA and AG by 16.7 and 12%, respectively. But, all genotypes shared the same calcium:phosphate ratio and demonstrated the biofunction of PTH in the maintenance of serum calcium-phosphate homeostasis. For serum PTH, genotype GG was approximately 30% higher than genotype AA. In this study, the eggshell percentage and breaking strength for genotype GG as well as AG were significantly higher than AA, indicating that allele G was favorable for eggshell quality. In chickens, injection of 200 pg of pCEP4-PTH gene plasmid significantly (P < 0.05) elevated the levels of serum calcium and PTH and thus significantly (P < 0.05) improved the eggshell quality (Zhang et al., 2009). Therefore, the relatively higher serum calcium and PTH level may explain the better eggshell quality in chickens with allele G. Although synonymous mutations, also called silence SNP, do not change the amino acid sequences and the biofunctions of proteins, they may be associated with
Table 2. Means ± SEM of parathyroid hormone (PTH) genotypes on eggshell traits at 40 wk of age in hens PTH genotype1 Trait
AA (n = 56)
AG (n = 74)
GG (n = 18)
Egg weight (g) Shell weight (g) Egg shape index Eggshell percentage (%) Eggshell strength (kg/cm2)
44.7 4.71 1.32 10.5 3.28
44.7 4.94 1.32 11.1 3.71
44.3 5.03 1.32 11.3 3.69
a,bMeans 1A
± ± ± ± ±
0.4 0.08 0.01 0.1a 0.09a
± ± ± ± ±
0.4 0.06 0.01 0.1b 0.07b
in a row with different superscripts differed at P < 0.05. total of 148 out of 300 hens were successfully tested for the eggshell traits.
± ± ± ± ±
1.0 0.13 0.01 0.3b 0.17b
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where Y was the dependent variable, μ the population mean, and e the random error. Significant differences among means of different genotypes were calculated using Duncan’s multiple range test, and the significance was determined at P < 0.05.
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Jiang et al. Table 3. Means ± SEM of parathyroid hormone (PTH) genotypes on serum biochemical indices at 40 wk of age in hens PTH genotype1 Item
AA (n = 47)
Serum calcium (mg/dL) Serum phosphate (mg/dL) Calcium:posphate Serum PTH (pg/mL) 1A
6.8 2.4 2.8 25.1
± ± ± ±
0.2 0.1 0.1 3.1
AG (n = 59) 6.9 2.5 2.8 29.8
± ± ± ±
0.1 0.1 0.1 3.0
GG (n = 17) 7.6 2.8 2.8 32.6
± ± ± ±
0.2 0.2 0.1 5.1
total of 123 out of 148 hens were successfully assayed for the serum biochemical indices.
ACKNOWLEDGMENTS This work was supported by grants from China S&T Pillar Program (2008BADB2B06, 2008BADA9B06) and the National High Technology Research and Development Program (“863” Program) of China (2008A101009).
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