J Appl Genet 48(4), 2007, pp. 371–374

Short communication

Polymorphisms in intron 1 of the porcine POU1F1 gene

Cheng-Yi Song1,BoGao1, Shang-Hui Teng1, Xiao-Yang Wang1, Fei Xie1, Guo-hong Chen1, Zhi-Yue Wang1, Rong-bin Jing1, Jiu-De Mao2

1College of Animal Science & Technology, University, , China 2Division of Biological Sciences, University of Missouri-Columbia, Columbia, MO, USA

Abstract. This study was conducted to detect polymorphisms in intron 1 of porcine POU1F1 (POU domain, class 1, transcription factor 1, Pit1, renamed as POU1F1) by comparative sequencing. Within the intron, 23 sites of variation were identified, including 16 single-nucleotide substitutions, 4 single-nucleotide indels, 2 short (3-bp and 17-bp), and one long (313-bp) indels. Several important regulatory motifs were found within the 313-bp indel by in silico analysis. The 313-bp indel was next genotyped in 11 Chinese native pig breeds and 4 western meat-type pig breeds. The appearance of genotypes varied between breeds: among Chinese native breeds, no AA and AB genotypes were found in Tibetan, Lingao, Min, Rongchang, and Songliao Black pigs, no AA genotype was found in Fenjing and Leping Spotted pigs, whereas in Pietrain and Landrace there were no BB genotypes, and all 19 Duroc pigs were AA homozygotes. The western meat-type pigs had high A allele frequencies and the Chi- nese pigs had more B alleles, except Jianquhai pigs. A positive association of the AA genotype with birth weight was observed in a commercial pig line. This paper demonstrated that the genetic variation in intron 1 of the pig POU1F1 gene was high and these polymorphisms may provide useful makers for QTL analysis.

Keywords: early growth traits, genotyping, intron 1, pig, polymorphism, Pit1, POU1F1.

The POU1F1 transcription factor is expressed in et al. 1998; Yu et al. 2001). Previous research has the pituitary gland, where it regulates pituitary de- revealed its polymorphisms by using the POU1F1 velopment and the expression of the growth hor- POU domain probe, RFLP and PCR-RFLP tech- mone, prolactin and thyrotropin b-subunit genes niques. The MspI polymorphisms were signifi- (Cohen et al. 1996). The POU1F1 protein contains cantly correlated with pig birth weight in the Iowa 2 domains, termed POU-specific and POU-homeo, State University pig resource families (Yu et al. which are both necessary for high-affinity DNA 1995). The associations of POU1F1 haplotypes binding to genes encoding growth hormone and and the markers around POU1F1 (10–20 cM prolactin (Sturm et al. 1988). Mutations within the apart) with early weights and growth rate were POU1F1 gene are associated with growth hor- confirmed by Yu et al. (1999). We demonstrated mone, thyroid-stimulating hormone beta subunit, a significant genotype effect of POU1F1 on 45, and prolactin deficiency and hypopituitarism in 70, and 180-day body weight and average daily humans (Hendriks-Stegeman et al. 2001). It is weight gain (ADG) in the finishing period in proved that dwarfism in mice and humans can be a crossbred pig population as well (Song et al. associated with a low or absent POU1F1 gene ac- 2005). However, most of them were within the last tivity. 3 exons, and no polymorphism near the 5’ flanking The porcine POU1F1 gene is located on chro- region was reported. Actually, such polymorphisms mosome 13, and it has 6 exons and 5 introns. are generally associated with the sequence elements Its cDNA sequence and partial genomic sequence and may completely abolish the inducibility of the have been characterized in several labs (Chung promoter or decrease its activity significantly.

Received: May 25, 2007. Revised: July 8, 2007. Accepted: August 18, 2007. Correspondence: C.Y. Song, College of Animal Science & Technology, Yangzhou University, 12 Wenhui East Road, Yangzhou, Jiangsu Province, 225009, P.R, China; e-mail: [email protected] 372 C.Y. Song et al.

The study of the distribution of some known regu- electrophoresis and visualized by ethidium bro- latory motifs in the human genome revealed that mide staining. The PCR amplification products the first introns within most genes play a particu- were purified with a DNA fragment purification larly important regulatory role in transcription kit (TaKaRa, Japan) and then were sequenced by control (Majewski and Ott 2002). The objective of a primer-walking approach. Two different sizes of the current study was to identify the porcine intron were obtained from wild (3262 bp) and do- POU1F1 intron 1 sequence and to examine nucle- mestic (3574 bp) pigs and the sequences were de- otide sequence variation. posited in the GenBank database under accession A domestic pig (Landrace) and a wild pig ge- nos. AY948544 and DQ133895. The MegAlign nome were used for comparative sequencing. Ear Program of DNASTAR software (DNASTAR, tissue samples were collected and DNA was ex- Inc. www.dnastar.com) was used for comparative tracted according to standard procedures. A pair of sequence analysis. Within the first intron, 23 sites primers (P1: TTT TAC TTC GGC TGA CAC of variation were identified, including 16 sin- CTT TAT; P2: GGT TTC CAT AAT GAC AGG gle-nucleotide substitutions, 4 single-nucleotide AAG GG) spanning exons 1 and 2 were designed indels, 2 short indels (3-bp and 17-bp), and one for comparative sequencing according to the re- long (313-bp) indel. The 3-bp nucleotide variation leased gene sequence (GenBank accession no. was an indel of GTC, while the 17-bp segment mu- AF016251). Genomic DNA (300 ng) was ampli- tation was an indel of 17 Ts. fied in 50 mL of reaction solution containing To test the potential functional importance of m the 313-bp indel, the insertion sequences were 200 M dNTPs, 10 pM primer, 1.5 mM MgCl2, in silico 2U LA Taq polymerase (TaKaRa, Japan), analysed and by the use of NSITE pro- and 1 × LA PCR buffer. The PCR was carried out gram (Softberry, Inc, USA, www.softberry.com). according to the following protocol: The DNA Motifs of 4 crucial transcription factors (including template was denatured at 94oC for 4 min, then SP1, NF-k B, AP-2 and Nmp4), a nerve growth 30 cycles of 94oC for 40s, 60oC for 90s, 72oC for factor-induced early-response gene (NGFI-C) 240s, and at last 10 min of elongation at 72oC. and 3 early growth response genes (Egr-1, Egr-2 PCR products were resolved by 0.6% agarose gel and Egr-3) were found.

Table 1. Genotype and allele frequencies of the 313-bp indel in various pig breeds

Breed Sample location N Genotypes Alleles AA AB BB A B Meishan Breeding Centre of Taihu Pig, Jiangsu 35 0.06 0.29 0.65 0.06 0.94 Province Erhualian Suzhou Breeding Centre of Taihu Pig, Jiangsu 33 0.12 0.21 0.67 0.23 0.77 Province Fenjing Suzhou Breeding Centre of Taihu Pig, Jiangsu 18 0 0.28 0.72 0.14 0.86 Province Huai Pig breeding farm of Anhui Provincial Academy 32 0.22 0.41 0.37 0.43 0.57 of Agricultural Science Jianquhai Jianyan pig breeding farm, Jiangsu Province 46 0.48 0.43 0.09 0.70 0.30 Leping Leping city livestock breeding farm, Jiangxi 31 0 0.52 0.48 0.29 0.71 Spotted Province Tibetan Gongbujiangda county, Tibet autonomous region 36 0 0 1.00 0 1.00 Lingao Lingao county, Hainan Province 15 0 0 1.00 0 1.00 Rongchang Rongchang pig breeding farm, Chongqing munic- 25 0 0 1.00 0 1.00 ipality Songliao Pig breeding farm of Jiling Provincial Academy 19 0 0 1.00 0 1.00 Black of Agricultural Science Min Lanxi county pig breeding farm, Heilongjian 14 0 0 1.00 0 1.00 Province Yorkshire Pig breeding farm of Anhui Provincial Academy 19 0.37 0.58 0.05 0.66 0.34 of Agricultural Science, originally from UK Landrace Pig breeding farm of Anhui Provincial Academy 21 0.76 0.24 0 0.88 0.12 of Agricultural Science, originally from Denmark Pietrain Changshu pig breeding farm, Jiangsu Province, 15 0.67 0.33 0 0.83 0.17 originally from Belgium Duroc Changshu pig breeding farm, Jiangsu Province, 19 1.00 0 0 1.00 0 originally from USA Polymorphisms in intron 1 of pig POU1F1 gene 373

Since the 313-bp indel contains these potential found in Fenjing and Leping Spotted pigs, regulatory elements and has a possible biological whereas in the studied sample of Pietrain and function, it was further genotyped in 4 western Landrace there were no BB genotypes, and all 19 meat-type pig breeds and 11 Chinese native Duroc pigs were AA homozygotes. The western breeds. The number and location of pig samples meat-type pigs had a high A allele frequency and selected from each breed, and the recorded fre- the Chinese native pigs had more B alleles, except quencies of alleles and genotypes are given in Ta- Jianquhai pigs. ble 1. A total of 370 individuals of a non-inbred com- P2 and P3 (ATA GGT TGG GAT GAG AAG mercial line (Sujiang) were used for correlation AAT) primers were used to genotype the new analysis between the 313-bp indel and early 313-bp indel. The PCR was performed in a vol- growth traits by using the general linear model. ume of 20 mL, containing 100 ng of template The genotype frequencies of AA, AB and BB were DNA, 0.25 mM primers, 50 mM KCl, 10 mM 0.65, 0.28 and 0.064, respectively. The AA geno- Tris–HCl (pH 8.0), 1.5 mM MgCl , 2.5 mM type was associated with higher birth weight 2 P dNTPs, and1UofTaqpolymerase. The reaction ( < 0.05). was carried out as initial denaturation for 180 s Several studies identified polymorphisms of POU1F1 at 95oC, 30 cycles of denaturation for 30 s at 95oC, the gene and their associations with annealing for 60 s at 60oC, and elongation for 180 quantitative traits (Renaville et al. 1997; Moody sat72oC, followed by a 10-min final extension at et al. 1996; Brunsch et al. 2002). The current paper 72oC. Then the PCR products were separated by presents the first study to examine intron 1 in de- electrophoresis in 0.8% agarose gel and stained tail and identify its DNA variations. Agarose gel with ethidium bromide. Two alleles, allele A electrophoresis and DNA sequencing revealed (1926 bp) and allele B (2239 bp, inserted 313-bp a large variation in size, mainly caused by fragment) and 3 genotypes, AA(1926 bp), a 313-bp indel near the end of intron 1, and 22 AB(1926 bp and 2239) and BB(2239 bp) were ob- other mutations. in silico served (Figure 1). The analysis revealed regulatory mo- tifs, such as SP1, AP2 and early growth response genes, in the 313-bp indel. The deletion of these motifs can affect the transcription of POU1F1, which may influence complex traits, like birth weight. So the 313-bp indel has a potential biolog- ical function. Genotyping the 313-bp indel in different breeds revealed that western meat-type pigs have a high A allele frequency and most Chinese fat-type pigs have more B alleles, which suggested that the 313-bp indel was possibly associated with high values of growth traits. The positive associa- tion of the 313-bp variant with pig birth weight confirmed this possible function as well. This re- sult was in accordance with a previous report, Figure 1. PCR results: the 313-bp indel in intron 1 of the which indicated that the POU1F1 locus was asso- porcine POU1F1 gene. Lane1=ABgenotype (1926 bp ciated with higher birth weight (Yu et al. 1995). and 2239 bp); lane 2 = BB genotype (1926-bp); lane 3 = Results of the present study suggest that research AA genotype (2239 bp); lane 4 = GenerulerTM 100-bp DNA Ladder Plus marker (Sangon, ) on the function of the 313-bp indel should be con- tinued, with special emphasis on analysis of inter- The distribution of the 313-bp polymorphism action with transcription factors. genotypes and allele frequency in the meat-type Additionally, the POU1F1b transcript, which pigs and Chinese pigs are summarized in Table 1. is one of the POU1F1 isoforms, can be spliced by No deviation from the Hardy-Weinberg equilib- using an alternative donor site of intron 1 and addi- rium was detected. The appearance of genotypes tional 26 amino acids are then inserted in front varied between breeds: no AA and AB genotypes of exon 2 in rats and humans, but the isoform was were found in Tibetan, Lingao, Min, Rongchang, identified in pigs as well (Yu et al. 2001). By se- and Songliao Black pigs, no AA genotype was quencing the first intron, we demonstrated that the 374 C.Y. Song et al.

78-bp sequence at the 3´ end of the intron, encod- Kondrashov FA, Koonin EV, 2003. Evolution of alter- ing the additional 26 amino acids, were conserved native splicing: deletions, insertions and origin of functional parts of proteins from intron se- in humans and rats. Both the 313-bp and the 17-bp quences. Trends Genet 19: 115–119. repeat indel were close to the 3´ end of the first Majewski J, Ott J, 2002. Distribution and characteriza- intron and may affect splicing activity and RNA tion of regulatory elements in the human genome. level since some evidence showed that functional Genome Res 12: 1827–1836. parts of proteins originate from intron sequences Moody DE, Pomp D, Newman S, MacNeil M, 1996. due to deletions or insertions (Kondrashov et al. Characterization of DNA polymorphisms in three 2003). More investigations of the possible rela- populations of Hereford cattle and their associations tionship between these indels and alternative with growth and maternal EPD in line 1 Herefords. J Anim Sci 74: 1784–1793. splicing would also be of great interest. Renaville R, Gengler N, Vrech E, Prandi A, Massart S, Corradini C, et al. 1997. PIT1 gene polymorphism, Acknowledgements. This work was funded by both milk yield and conformation traits for Italian Hol- the Jiangsu Natural Science Foundation and stein-Friesian bulls. J Dairy Sci 80: 3431–3438. Yangzhou University Natural Science Fund, through Song CY, Gao B, Teng Y, Wang XY, et al. 2005. MspI grants nos. BK2006068 and NK0513129. polymorphisms in the 3rd intron of the swine POU1F1 gene and their associations with growth REFERENCES performance. J Appl Genet 46: 285–289. Sturm RA, Herr W, 1988. The POU domain is a bipar- Brunsch C, Sternstein I, Reinecke P, Bieniek J, 2002. tite DNA-binding structure. Nature 336: 60–604. Analysis of associations of PIT1 genotypes with Yu TP, Sun HS, Wahls S, Sanchez-Serrano I, growth, meat quality and carcass composition traits Rothschild MF, Tuggle CK, 2001. Cloning of the in pigs. J Appl Genet 43: 85–91. full-length pig PIT1 (POU1F1) cDNA and a novel PIT1 Chung HO, Kato T, Tomizawa K, Kato Y, 1998. Mo- alternative transcript, and functional studies lecular cloning of Pit-1 cDNA from porcine ante- of their encoded proteins. Anim Biotechnol 12: rior pituitary and its involvement in pituitary 1–19. stimulation by growth hormone releasing. Exp Clin Yu TP, Tuggle CK, Schmitz CB, Rothschild MF, 1995. Endocrinol Diabetes 106: 203–210. Association of PIT1 polymorphisms with growth Cohen LE, Wondisford FE, Radovick S, 1996. Role of and carcass traits in pigs. J Anim Sci 73: Pit-1 in the gene expression of growth hormone, 1282–1288. prolactin and thyrotropin. Endocrinol Metab Clin Yu TP, Wang L, Tuggle CK, Rothschild MF, 1999. North Am 25: 523–540. Mapping genes for fatness and growth on pig chro- Hendriks-Stegeman BI, Augustijn KD, Bakker B, et al. mosome 13: a search in the region close to the pig PIT1 2001. Combined pituitary hormone deficiency gene. J Anim Breed Genet 116: 269–280. caused by compound heterozygosity for two novel mutations in the POU domain of the Pit1/POU1F1 gene. J Clin Endocrinol Metab 86: 1545–1550.