Xiaoling Jiang; Lifan Zhang; Ningying Xu^*; Jipeng Zhai; Zunzhou Lv

College of Animal Science, Zhejiang University, Hangzhou, Zhejiang 310029, P.R. China

ABSTRACT

Glycoprotein hormones in the pituitary gland affect a myriad of biological processes such as development, growth, metabolic control and gametogenesis. The quantitative trait loci (QTL) near their common glycoprotein alpha(A) subunit gene (CGA) have been reported inconsistently. The aim of this study was to dissect potential genetic factors for these unstable results and validate the association or linkage relationship of CGA gene with growth and carcass traits. By resequencing all the exons and part of the introns of the porcine CGA gene, 22 polymorphisms in total were identified in this study. Five single nucleotide polymorphism (SNP) markers were chosen and evaluated in six pure-bred pig breeds (n = 228). Breed-specific haplotypes were found and a map of the porcine CGA polymorphisms' evolution history was inferred. A resource family (n = 365) with different genetic backgrounds from those used in other papers was used to perform an association study. The resource family was created based on crosses of Pietrain and Jinhua pigs (Central China type pigs). Results indicated that a low correlation between haplotype blocks may abolish each other's effects. Moreover, a significant association of SNP C-925T with growth rate and back-fat thickness in this study confirmed the existence of previously reported QTL. SNP C-925T and SNP A+15599G could be useful linkage markers and SNP C-925T may also be a candidate causative SNP for the corresponding traits. Further investigation for variants within of the CGA promoter region and their association with growth rate and back-fat traits is suggested.

Keywords: Common glycoprotein alpha(A) subunit gene, haplotype, SNP, tetra-primer ARMS, swine

Introduction

The glycoprotein hormones form a family consisting of the luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyrotrophin (TSH) in the anterior pituitary. The general biological roles of LH and FSH are the stimulation of testicular and ovarian functions via the regulation of gametogenesis and steroid hormone synthesis in the gonads, whereas TSH stimulates the thyroid gland to produce and release the thyroid hormones, 3,3',5,5'-tetraiodothyronine (T4) and 3,3',5-triiodothyronine (T3), which affect a myriad of biological processes such as skeletal maturation, linear growth, lipid synthesis and lipolysis, and carbohydrate metabolism (Yen, 2001). The thyroid hormones also potentiate the effects of many other hormones such as insulin, the growth hormone (GH), glucocorticoids and glucagon, and has been frequently called a permissive hormone (Bolander, 2004).

Each of the glycoprotein hormones is a heterodimer, formed by the non-covalent association of an alpha(A) subunit that is common in all the members of the family with distinct β subunits that confer hormone specificity (Bolander, 2004). The common alpha(A) subunit of these hormones, encoded by the unique, single-copy gene, CGA (also named as GSUalpha(A)), is important to receptor binding and signal transduction (Szkudlinski et al., 1996). No inactivating mutations of the CGA gene have been detected in humans and mice yet (Kendall et al., 1995; Huhtaniemi & Alevizaki, 2007), for its widespread consequences in the form of hypogonadism, hypothyroidism and so on. Mice with the CGA gene knockout were viable, but exhibited severe growth insufficiency and infertility (Kendall et al., 1995).

The porcine CGA gene is located on the p arm of Sus scrofa chromosome 1 (SSC 1) and consists of four exons and three introns (Kato et al., 1991). A microsatellite marker in the intron 1 (named as PGHAS or ALPHA) has been identified (Moran, 1993) and used in genetic studies. Considering the extensive effects of the glycoprotein hormones, especially TSH, their common CGA gene shows to be a promising candidate gene for economic important traits. However, comparing with the large number of Quantitative trait loci (QTL) that have been mapped on SSC 1, there are only a limited number of QTL adjacent to the CGA gene, and they have only been reported in some studies (Malek et al., 2001; Beeckmann et al., 2003; Evans et al., 2003; Liu et al., 2007) and were undetectable in others (Rohrer & Keele, 1998; Bidanel et al., 2001; De Koning et al., 2001; Beeckmann et al., 2003; Rohrer et al., 2006; Liu et al., 2007; Edwards et al., 2008).

To dissect potential genetic factors for these inconsistent results and validate the association or linkage relationship of the CGA gene with the growth and carcass traits, a resource family with different genetic backgrounds from those used in other studies was utilized. The resource family was created based on crosses of Pietrain and Jinhua pig (Central China type pigs). First, DNA variations of the sequence of the CGA gene were identified by resequencing all exons and part of the introns. Single nucleotide polymorphisms (SNP) were chosen to develop new SNP markers for association analyses. Prior to the association analyses, the SNP markers were evaluated in six pure-bred pigs including pure-bred Pietrain and Jinhua pig breeds to construct breed-specific haplotypes and investigate their evolution history. Both porcine CGA haplotypes and single SNP were analyzed in the association study to examine the association of variants with growth and carcass traits.

Materials and Methods

Genomic DNA samples were obtained from 622 pigs: 228 belonged to six pure-bred breeds (57 Yorkshire, 30 Duroc, 27 Landrace, 24 Pietrain, 49 Jinhua pigs, 41 Jiaxing Black pigs) and the others to a three-generation resource family from crosses of Pietrain and Jinhua pigs. The resource population was created by first mating three Pietrain boars and three sows to two Jinhua pig sows and three boars, respectively. Seven litters of F₁ individuals were produced. From six of the seven F₁ litters, six boars and 23 sows were chosen to produce 39 litters of F₂ animals. Besides, two of the F₁ boars were also mated back to the two founder Jinhua sows, and three of the F₁ sows were mated back to the Pietrain boars. DNA samples were extracted from whole blood or ear samples using the traditional phenol and chloroform method (Sambrook et al., 2001). The concentrations of DNA samples were measured by spectrophotometer and each was diluted to 10 ng/µL, exactly.

To scan the porcine CGA gene for DNA sequence variations, the direct sequencing method was employed using DNA from 4 F₁ boars, 1 Pietrain and 1 Jinhua pig. The porcine CGA sequence was obtained from the Ensembl database (http://www.ensembl.org/Sus_scrofa/Info/Index) using the Sscrofa9 assembly. Upstream sequence of the gene was also obtained and aligned to the sequence published by Kato et al. (1991) to target the 5'-untranslated exon 1. Figure 1 describes the gene structure and the polymorphisms identified by alignment (shown as solid lines). Five sets of primers were designed to amplify all exons and part of the introns. The amplicons were sequenced in both directions. Sequence traces were assembled with the Seqman program (DNAStar, USA) and analyzed to search for SNP.

The tetra-primer amplification refractory mutation system (ARMS) PCR procedure (Ye et al., 2001) was utilized here for SNP genotyping. This method employs four primers to amplify fragments representing each of the two allelic forms and a larger amplicon containing the SNP as control. Primers were designed by using the online computer program made accessible by Ye et al. (2001) (http://cedar.genetics.soton.ac.uk/public_html/primer1.html) for five SNP loci: C-925T, C+14547T, C+15065T, A+15599G and T+15944A. Genotyping PCR was performed in a total volume of 10 µL containing 20 ng of template DNA, appropriate concentration of each primer (see Table 1), 200 µM dNTPs, 1 µL 10 x buffer and 0.4 U of Taq polymerase (TaKaRa, Dalian, China). The PCR profile was the following: an initial step of denaturation of 3 min step at 94 ºC; then 35 cycles of 30 seconds at 94 ºC, 1 min at respective annealing temperature and 1 min at 72 ºC; and the final extension step was for 10 min at 72 ºC. The PCR products were separated by standard electrophoresis on 2% agarose gels.

Genotype and allele frequencies of the five SNPs in six pig pure-breds were calculated using PopGen32 software (Yeh et al., 1999). Then, haplotypes were constructed by program PHASE (Stephens & Donnelly, 2003) and visualized in Haploview software (Barrett et al., 2005). Linkage disequilibrium between SNPs was estimated with D' and r², and the linkage blocks were defined using the four gamete rule (Wang et al., 2002).

Haplotypes of the resource family were inferred by program, PedPhase 3.0 (Li & Li, 2009). Polymorphism C+14547T was omitted from further analyses because of its serious amplification failure. Afterwards, associations between the haplotypes or genotypes of the CGA gene and the investigated traits were assessed using the SAS MIXED procedure (SAS Institute Inc.). The model used was:

Y_ijkl = µ + H_i + S_j + L_k + β * X_ijkl + e_ijkl

Where Y_ijkl = phenotypic value of traits; µ = population mean; H_i = fixed effect of haplotype or genotype; S_j = fixed effect of sex; L_k = random effect of litter; β = regression coefficient; X_ijkl = live weight at slaughter as a covariate for carcass traits; and e_ijkl = random residual error. Multiple comparisons of least square means were adjusted with Bonferroni correction.

Results

The complete CGA gene was located on SSC 1 from position 58,190,063 to 58,206,411. A schematic representation of the porcine CGA gene and the identified SNPs are shown in Figure 1. Eleven potential polymorphisms were identified by comparison of the sequence obtained from the Ensembl database with that published by Kato et al. (1991). Fourteen polymorphic loci were detected by direct sequencing in this study, three of which were identical to the ones identified by sequence comparison.

Haplotypes were constructed by the program, PHASE, and the haplotype frequencies of each breed were calculated and presented in Table 3. Haplotype CCCGT existed nearly in all breeds, whereas haplotype CTCGT and TTCGT are only shared in Chinese breeds. Haplotype CCCAT, CCTGT and CCTAT are only shared in Western breeds. CCCGA and TCCGA are common in Jinhua pig, but are also found in Yorkshire and Pietrain breeds. Haplotype CCTGA was detected only in the Pietrain breed.

Linkage disequilibrium between SNPs (D' and r²) was estimated by Haploview software. The linkage blocks defined using the four gamete rule are shown in Figure 3. SNP A+15599G and T+15944A appeared to be in complete linkage disequilibrium (D' = 1), but their correlation coefficient is quite low (r² = 0.043), indicating that one could not substitute the other one and both of them were required for providing the haplotype information. A similar relationship existed between SNP C+14547T and C+15065T. Though values of D' and r² between SNP C-925T and others are low, lack of recombinant gamete types caused them to be correlated into one block. A recombination hot spot appears to exist between SNP C+15065T and SNP A+15599G.

Because of the serious amplification failure of SNP C+14547T in the resource family, this variant was omitted from further analyses. Haplotypes of the resource family were inferred by program PedPhase 3.0, and the results indicated that the founder Jinhua pigs possessed haplotypes C-CGT (i.e. CCCGT/CTCGT) and T-CGT (i.e. TTCGT), while the founder Pietrain had haplotypes C-CAT (i.e. CCCAT) and C-TGA (i.e. CCTGA. Recombination between the two blocks was observed in the pedigree at a rate of approximately 0.06, and resulted in the emergence of another three haplotypes: C-CGA, C-TGT and T-CAT.

Association analysis showed that no traits were associated (P <0.05) with the CGA haplotypes. However, significant association of SNP C-925T with carcass weight, BFT at last rib and ADG, and association of SNP C+15065T with intramuscular water content were observed (P values of genetic effects are presented in Table 4). The haplotype block 1, containing these two SNPs, is significantly associated with ham external fat weight and LEA, besides BFT and ADG. But no significant differences between haplotype block 1 groups were observed in average BFT and BFT at shoulder traits using multiple comparisons adjusted by Bonferroni correction.

SNP A+15599G is significantly associated with ham external fat weight, while SNP T+15944A is significantly associated with intramuscular water content. Significant association of haplotype block 2 with the LM area in addition to the ham external fat weight and intramuscular water content were observed.

Discussion

The porcine CGA gene is composed of four exons and three introns spanning 16,349 bp of DNA sequence. The ATG translation starting site of this gene is in exon 2. Except for the two well-reported microsatellites, a total of 22 potential sequence variations were identified. Twelve of them were transition mutations, five were transversion mutations, and two were indel mutations. The remaining three sequence variations (TT+14423G, TAATG+15607AATGT, GA+15723AG) cannot be put under any of the three classes, and that might have been caused by a sequencing error from the Kato's CGA DNA sequence. No coding mutation was detected in this study.

Based on the haplotype frequencies among six pig breeds, haplotype CCCGT appeared to be the ancestral type. Then, a mutation of C to T at locus C+14547T occurred and segregated in Chinese pigs, while a mutation of G to A at locus A+15599G and a mutation of C to T at locus C+15065T occurred in Western breeds (Figure 4). The recombination existing between SNP C+15065T and A+15599G generated the new haplotype, CCTAT in Western breeds.

Association analysis in the resource family showed that allele T from the Jinhua pig at locus C-925T was associated with lower carcass weight and higher back-fat thickness at the last rib, and the heterozygotes showed significantly faster growth rates than the homozygotes (Table 5). However, when incorporating the SNP C+15065T, i.e. using block 1 as genetic factor, the association possibility decreased. This suggested that only SNP C-925T, which locates in the promoter region, may be the candidate causative SNP or in linkage at a high r² with the causative SNP which is responsible for these traits' variations. Our results confirmed the existence of QTLs adjacent to the CGA region for carcass composition, growth rate and back-fat thickness which have been reported previously (Malek et al., 2001; Beeckmann et al., 2003; Liu et al., 2007). Variations in the promoter region of CGA gene, but not the other gene components might be the candidates for these QTLs and further investigation is worthwhile.

Allele A at locus A+15599G, which is present in Western breeds, was significantly associated with lower ham external fat weight. A QTL for ham external fat weight on SSC1 in the European wild boar and Pietrain resource family has been reported by Beeckmann et al. (2003), and the CGA SNP A+15599G could be in linkage with the causative SNP for this QTL. Interestingly, haplotype block 1 also showed significant association with ham external fat weight, but combining the haplotype block 1 and SNP A+15599G decreased the association possibility. This result may be due to the low correlation between these markers.

The A allele of T+15944A and the T allele of SNP C+15065T originated from the same haplotype C-TGA. Thus, it was not surprising that they had similar results in the association analysis. However, after being adjusted with the Bonferroni correction, no significant differences in intramuscular water content existed following comparisons of their least square means. Animals with block 2 diplotype, --GA/--AT, had a lower intramuscular water content. To date, no QTL for intramuscular water content adjacent to the CGA region has been reported.

Thyrotropin (TSH), one of the CGA gene composing hormones, regulates the synthesis and release of thyroid hormones. The thyroid hormone could stimulate lipolysis directly and indirectly as a result of the potentiation of the effects of GH, glucocorticoid and glucagon. Many of the effects of GH are mediated by a group of hormones called insulin-like growth factors (IGF's). GH and IGF's secretion will decline when thyroid hormone levels are low (Porterfield & White, 2007). It's known that IGF's and their binding proteins are also associated with growth and fatness traits (Makgahlela et al., 2009; Switonski et al., 2010). Effects of the CGA gene on the same characters might be mediated partly by IGFs pathways. Interactions between variations of CGA and IGF's were strongly suggested to exist.

Conclusion

Several novel, non-coding polymorphisms were found in the porcine CGA gene in this study. Different single nucleotide polymorphisms emerged in specific pig breed populations. At least two linkage disequilibrium blocks were detected in the CGA gene. It is suggested that the two microsatellites in this area may act as the recombination hot spots. Association of SNP C-925T with growth rate and back-fat thickness in this study confirmed the existence of previously reported QTL and provides evidence for the roles of SNP C-925T and SNP A+15599G in affecting specific quantitative traits. Further investigation on the CGA promoter region with the growth rate and back-fat traits is recommended.

Acknowledgement

Reference

Barrett, J.C., Fry, B., Maller, J. & Daly, M.J., 2005. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263-265.        [ Links ]

Beeckmann, P., Schroffel, J., Moser, G., Bartenschlager, H., Reiner, G. & Geldermann, H., 2003. Linkage and QTL mapping for Sus scrofa chromosome 1. J. Anim. Breed. Genet. 120, 1-10.        [ Links ]

Bidanel, J.P., Milan, D., Iannuccelli, N., Amigues, Y., Boscher, M.Y., Bourgeois, F., Caritez, J.C., Gruand, J., Le Roy, P., Lagant, H., Quintanilla, R., Renard, C., Gellin, J., Ollivier, L. & Chevalet, C., 2001. Detection of quantitative trait loci for growth and fatness in pigs. Genet. Sel. Evol. 33, 289-309.        [ Links ]

Bolander, F.F., 2004. Molecular Endocrinology. 3^rd ed. Elsevier Ltd. Oxford, UK. pp. 31.        [ Links ]

Cesar, A.S.M., Silveira, A.C.P., Freitas, P.F.A., Guimaraes, E.C., Batista, D.F.A., Torido, L.C., Meirelles, F.V. & Antunes, R.C., 2010. Influence of Chinese breeds on pork quality of commercial pig lines. Genet. Mol. Res. 9, 727-733.        [ Links ]

De Koning, D.J., Rattink, A.P., Harlizius, B., Groenen, M.A.M., Brascamp, E.W. & Van Arendonk, J.A.M., 2001. Detection and characterization of quantitative trait loci for growth and reproduction traits in pigs. Livest. Prod. Sci. 72, 185-198.        [ Links ]

Edwards, D.B., Ernst, C.W., Raney, N.E., Doumit, M.E., Hoge, M.D. & Bates, R.O., 2008. Quantitative trait locus mapping in an F-2 Duroc x Pietrain resource population: II. Carcass and meat quality traits. J. Anim. Sci. 86, 254-266.        [ Links ]

Evans, G.J., Giuffra, E., Sanchez, A., Kerje, S., Davalos, G., Vidal, O., Illan, S., Noguera, J.L., Varona, L., Velander, I., Southwood, O.I., de Koning, D.J., Haley, C.S., Plastow, G.S. & Andersson, L., 2003. Identification of quantitative trait loci for production traits in commercial pig populations. Genetics 164, 621-627.        [ Links ]

Huhtaniemi, L. & Alevizaki, M., 2007. Mutations along the hypothalamic-pituitary-gonadal axis affecting male reproduction. Reprod. Biomed. Online 15, 622-632.        [ Links ]

Kato, Y., Ezashi, T., Hirai, T. & Kato, T., 1991. The gene for the common alpha-subunit of porcine pituitary glycoprotein hormone. J. Mol. Endocrinol. 7, 27-34.        [ Links ]

Kendall, S.K., Samuelson, L.C., Saunders, T.L., Wood, R.I. & Camper, S.A., 1995. Targeted disruption of the pituitary glycoprotein hormone alpha-subunit produces hypogonadal and hypothyroid mice. Gene. Dev. 9, 2007-2019.        [ Links ]

Li, S.J., Yang, S.H., Zhao, S.H., Fan, B., Yu, M., Wang, H.S., Li, M.H., Liu, B., Xiong, T.A. & Li, K., 2004. Genetic diversity analyses of 10 indigenous Chinese pig populations based on 20 microsatellites. J. Anim. Sci. 82, 368-374.        [ Links ]

Li, X. & Li, J., 2009. An almost linear time algorithm for a general haplotype solution on tree pedigrees with no recombination and its extensions. J. Bioin. Comput. Biol. 7, 521-545.        [ Links ]

Liu, G., Jennen, D.G.J., Tholen, E., Juengst, H., Kleinwachter, T., Holker,, M., Tesfaye D., Un, G., Schreinemachers, H.J., Murani, E., Ponsuksili, S., Kim, J.J., Schellander, K. & Wimmers, K., 2007. A genome scan reveals QTL for growth, fatness, leanness and meat quality in a Duroc-Pietrain resource population. Anim. Genet. 38, 241-252.        [ Links ]

Makgahlela, M.L., Fan, B., Du, Z.Q. & Rothschild, M.F., 2009. Investigation of effects of three candidate genes on leg action and fat deposition traits in pigs. S. Afr. J. Anim. Sci. 39, 127-130.        [ Links ]

Malek, M., Dekkers, J.C.M., Lee, H.K., Baas, T.J. & Rothschild, M.F., 2001. A molecular genome scan analysis to identify chromosomal regions influencing economic traits in the pig. I. Growth and body composition. Mamm. Genome 12, 630-636.        [ Links ]

Moran, C., 1993. Microsatellite repeats in pig (Sus domestica) and chicken (Gallus domesticus) genomes. J. Hered. 84, 274-280.        [ Links ]

Pierce, J.G. & Parsons, T.F., 1981. Glycoprotein hormones - structure and function. Annu. Rev. Biochem. 50, 465-495.        [ Links ]

Porterfield, S.P. & White, B.A., 2007. Endocrine Physiology. 3^rd ed. Mosby Inc, St Louis, USA. pp 124-152.        [ Links ]

Rohrer, G.A. & Keele, J.W., 1998. Identification of quantitative trait loci affecting carcass composition in swine: I. Fat deposition traits. J. Anim. Sci. 76, 2247-2254.        [ Links ]

Rohrer, G.A., Thallman, R.M., Shackelford, S., Wheeler, T. & Koohmaraie, M., 2006. A genome scan for loci affecting pork quality in a Duroc-Landrace F-2 population. Anim. Genet. 37, 17-27.        [ Links ]

Sambrook, J, Fristsch, E.F. & Maniatis, T., 2001. Molecular cloning: A laboratory manual. 3^rd ed. Cold Spring Harbor Laboratory, New York, USA.        [ Links ]

Stephens, M. & Donnelly, P., 2003. A comparison of Bayesian methods for haplotype reconstruction from population genotype data. Am. J. Hum. Genet. 73, 1162-1169.        [ Links ]

Switonski, M., Stachowiak, M., Cieslak, J., Bartz, M. & Grzes, M., 2010. Genetics of fat tissue accumulation in pig: a comparative approach. J. Appl. Genet. 51, 153-168.        [ Links ]

Szkudlinski, M.W., Grossmann, M. & Weintraub, B.D., 1996. Structure-function studies of human TSH - New advances in design of glycoprotein hormone analogs. Trends Endocrin. Met. 7, 277-286.        [ Links ]

Wang, N., Akey, J.M., Zhang, K., Chakraborty, R. & Jin, L., 2002. Distribution of recombination crossovers and the origin of haplotype blocks: The interplay of population history, recombination, and mutation. Am. J. Hum. Genet. 71, 1227-1234.        [ Links ]

Ye, S., Dhillon S., Ke, X.Y., Collins, A.R. & Day, I.N.M., 2001. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res. 29, art. no.-e88.        [ Links ]

Yeh, F.C., Yang, R.C. & Boyle T., 1999. POPGENE Version 1.31, Microsoft window-based freeware for population genetic analysis. University of Alberta, Canada.        [ Links ]

Yen, P.M., 2001. Physiological and molecular basis of thyroid hormone action. Physiol. Rev. 81, 1097-142.        [ Links ]

Copyright resides with the authors in terms of the Creative Commons Attribution 2.5 South African Licence.
See: http://creativecommons.org/licenses/by/2.5/za
Condition of use: The user may copy, distribute, transmit and adapt the work, but must recognise the authors and the South African Journal of Animal Science
* Corresponding author: nyxu@zju.edu.cn