J. Anim. Sci. 2004. 82:2229-2233
© 2004 American Society of Animal Science
Associations of polymorphisms in the Pit-1 gene with growth and carcass traits in Angus beef cattle1,2
Q. Zhao3,
M. E. Davis and
H. C. Hines
Department of Animal Sciences, The Ohio State University, Columbus 43210
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Abstract
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The Pit-1 gene was studied as a candidate for genetic markers of growth and carcass traits. Angus beef cattle that were divergently selected for high– or low–blood serum IGF-I concentration were used in this study. The single-strand conformation polymorphism method was used to identify polymorphism in the Pit-1 gene including regions from intron 2 to exon 6. Two polymorphisms, Pit1I3H (HinfI) and Pit1I3NL (NlaIII), were detected in intron 3 of the Pit-1 gene. One polymorphism, Pit1I4N (BstNI), was found in intron 4, and a single nucleotide polymorphism, Pit1I5, was found in intron 5. The previously reported polymorphism in exon 6, Pit1E6H (HinfI), was also studied in 416 Angus beef cattle. Associations of the polymorphisms with growth traits, carcass traits, and IGF-I concentration were analyzed using a general linear model procedure. No significant associations were observed between these polymorphisms and growth and carcass traits.
Key Words: Beef Cattle • Carcass Traits • Growth Traits • Pit-1 • Polymorphism
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Introduction
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Growth and carcass traits, which are under the control of multiple genes, are economically important traits in livestock. Selection of animals with higher growth rate and better carcass composition is of great significance to breeders and consumers. Current technologies enable scientists to improve on the accuracy and efficiency of traditional selection methods by applying genetic markers through marker-assisted selection. Therefore, genetic polymorphisms (marker loci) that are significantly associated with certain traits of interest are very useful.
In this study, Pit-1 was examined as a genetic marker candidate gene. It is a pituitary-specific transcription factor that is responsible for pituitary development and hormone expression in mammals (Cohen et al., 1997
). It was shown to control transcription of the growth hormone, prolactin (Nelson et al., 1988; Mangalam et al., 1989), the thyroid-stimulation hormone, ß-subunit (Simmons et al., 1990; Steinfelder et al., 1991), the GHRH receptor genes (Lin et al., 1992), and the Pit-1 gene itself (Rhodes et al., 1993).
Mutations in the Pit-1 gene lead to the absence of growth hormone and to pituitary hypoplasia in mice (Li et al., 1990) and to congenital hypothyroidism, dwarfism, and prolactin deficiency in humans (Pfaffle et al., 1992). In pigs, Pit-1 was found to be related to birth weight (Yu et al., 1996), weaning weight, and ADG (Yu et al., 1995). Also in pigs, associations were discovered with backfat, as well as lean-to-fat ratio (Yu et al., 1995; Stancekova et al., 1999). In cattle, Pit-1 was found to be associated with body composition and milk yields (Renaville et al., 1997a). The current study was designed to screen the Pit-1 gene for polymorphisms and to analyze the association of these polymorphisms with growth and carcass traits in Angus beef cattle.
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Materials and Methods
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Experimental Animals
Angus beef cattle, which were divergently selected for blood serum insulin-like growth factor I concentration, were used as the experimental animals. Selection began in 1989 at the Eastern Ohio Resource Development Center (EORDC), using 100 spring-calving (50 high-line and 50 low-line) and in 1990 using 100 fall-calving (50 high-line and 50 low-line) purebred Angus cows with unknown IGF-I concentrations. Cows from the base population were randomly assigned to the selection lines. Details of the method of selection, management, data collection, and response to selection were described by Davis and Simmen (1997). Four hundred sixteen Angus cattle born in 1995, 1996, and 1997 were genotyped for the Pit1E6H polymorphism, which was described by Woollard et al. (1994). Because the polymorphisms in introns 3 and 4 (Pit1I3H, Pit1I3NL, and Pit1I4N) showed a tight linkage with the Pit1E6H polymorphism, only 98 Angus cattle born in 1995, 1996, and 1997 were genotyped for these three polymorphisms. For the intron 5 polymorphism, Pit1I5, 185 Angus cattle born in 1995 and 1996 were genotyped. Frequency of allele O was extremely low (0.045), and no animals with OO genotype were observed.
Carcass traits of approximately 146 (among 416 animals genotyped) and 71 (among 185 animals genotyped) Angus bulls that were not saved for breeding were analyzed for effects of Pit1E6H and Pit1I5, respectively. Age at slaughter ranged from 11 to 15 mo. Bulls born in the years 1995 and 1996 were slaughtered at the Herman Falter Packing Co., Columbus, OH. Bulls born in 1997 were slaughtered at the Mahan Packing Co., Bristolville, OH.
Genotype Determination
Genomic DNA was isolated from blood of Angus cattle. The polymerase chain reaction was used to amplify the DNA fragments from genomic DNA. Primer pairs were selected so that the regions from intron 2 to exon 6, including all introns and exons in between, of the Pit-1 gene were amplified and screened for polymorphisms. The four primer pairs, Pit1I3, Pit1I4, Pit1I53, and Pit1E6 (Table 1), were used to amplify introns 3, 4, and 5, and exon 6, in which all the polymorphisms were detected in this study. Other primers with no polymorphism detected in their amplification regions are not listed in this table. Primer pairs were designed based on either the DNA sequence or the cDNA sequence of the bovine Pit-1 gene from GenBank. The PCR was performed in a 30-µL reaction mixture containing 10 pmol of forward primer and the same amount of reverse primer, 200 µM dNTP (deoxyribonucleotide triphosphate), and 1x reaction buffer, which contained 1.5 mM MgCl2, 1 unit of Taq-DNA polymerase, and 100 ng of genomic DNA as template. For the primer pair Pit1I3, PCR conditions were 97°C for 2 min, followed by 35 cycles of 95°C for 45 s, 60°C for 1 min, and 72°C for 90 s. After 35 cycles, reactions were finished by an extension of 5 min at 72°C. The conditions for the Pit1I4 primer pair were 97°C for 2 min, followed by 35 cycles of 95°C for 45 s, 63°C for 1 min, and 70°C for 1 min. After 35 cycles, reactions were finished by an extension of 5 min at 72°C. For the primer pair Pit1I53, the PCR conditions were 97°C for 2 min, followed by 35 cycles of 95°C for 45 s, 63°C for 1 min, and 72°C for 1 min. After 35 cycles, reactions were finished by an extension of 5 min at 72°C. The PCR conditions for the primer Pit1E6 were described by Woollard et al. (1994).
Single-strand conformation polymorphism (SSCP) method was used to screen for mutations within the amplified region. Because the optimal size of DNA for SSCP is under 400 base pairs, the long DNA fragments amplified by primer pairs Pit1I3 and Pit1I4 were digested with HinfI to decrease the size of the DNA fragments before performing SSCP. The reaction mixture, which included 10 µL of digested PCR product, 10 µL of ddH2O, and 12 µL of loading dye, was denatured at 95°C for 5 min, and placed in ice for 10 min. The samples were then loaded on 10% nondenaturing polyacrylamide gels, with 10% urea or 10% formamide to improve the resolution of the DNA bands on the gel (Yip et al., 1999). The samples were run in 1x TBE (Tris-EDTA) buffer at 200 V for 16 h at a constant temperature of 12°C. Gels were then stained with 0.01% ethidium bromide for 10 min and viewed under UV light. After a polymorphism was detected, the PCR products of the homozygous genotypes were sent to the Plant-Microbe Genomics Facility at Ohio State University for sequencing and analyzed for nucleotide changes. One SNP—Pit1I5, with two alleles—was detected in the fragment amplified with primer pair Pit1I53 by SSCP. Because no restriction site exists for this SNP, SSCP was used to genotype the animals. Two polymorphisms in intron 3, amplified with primer pair Pit1I3, and one in intron 4, amplified with primer pair Pit1I4, were also found. These polymorphisms, Pit1I3H, Pit1I3NL, and Pit1I4N, all have two alleles and can be genotyped using restriction enzymes HinfI, NlaIII, and BstNI, respectively. Designations of alleles are shown in Table 2.
Statistical Analysis
Associations of the animal genotypes with growth traits and IGF-I concentrations were determined by analysis of variance of quantitative traits, which included birth weight; weaning weight; preweaning gain; on-test weight; weight at d 28 and 56 of the 140-d postweaning test; off-test weight; weight gain during the 20-d period between weaning and the beginning of the postweaning test (GAIN20); postweaning gain; serum IGF-I concentration on d 28, 42, and 56 of the 140-d postweaning test; and mean serum IGF-I concentration over the three measurements on d 28, 42, and 56 for a given calf using GLM procedures in SAS (SAS Inst. Inc., Cary, NC.). A Bonferroni correction was used to adjust significance levels of each test, because a total of 20 growth and carcass traits were tested for polymorphisms Pit1E6H and Pit1I5 and 13 growth traits for polymorphisms Pit1I3H, Pit1I3NL, and Pit1I4N. Fixed effects of genotype, year, season of birth (spring vs. fall), age of dam (2, 3, 4, 5 to 9, 
10 yr), sex (bull vs. heifer), and IGF-I selection line (high vs. low) were included as independent variables in the linear model. Weaning age of calf was treated as a covariate in the model when analyzing weaning weight, preweaning gain, and GAIN20. On-test age of calf was used as a covariate in the model when analyzing on-test weight; weight at d 28 and 56; off-test weight; postweaning gain; IGF-I on d 28, 42, and 56; and mean IGF-I concentration. Calf age was not included in the analysis of birth weight. Data were also analyzed separately within the high- and low-IGF-I selection lines using the same model, except that the selection line was omitted from the model.
Carcass traits analyzed included backfat thickness (cm); LM area (cm2); kidney, pelvic, and heart fat (%); hot carcass weight (kg); marbling score (1 = devoid, 2 = practically devoid, 3 = traces, 4 = slight, 5 = small, 6 = modest, 7 = moderate, 8 = slightly abundant, 9 = moderately abundant, and 10 = abundant); quality grade (6 = Standard–, 7 = Standard, 8 = Standard+, 9 = Select–, 10 = Select, 11 = Select+, 12 = Choice–, 13 = Choice, and 14 = Choice+); and yield grade (1 to 5). The model used to analyze carcass traits was the same as the one for growth traits, except that sex was deleted from the model and slaughter age was used as a covariate instead of on-test age.
Differences in genotypic frequencies between the high- and low-IGF-I selection lines were analyzed using a 
2 test.
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Results and Discussion
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Two polymorphisms, Pit1I3H (HinfI) and Pit1I3NL (NlaIII), were detected in intron 3 of the Pit-1 gene. One polymorphism, (BstNI), was found in intron 4. Each of these three genetic variations had three genotypes. One polymorphism, Pit1I5, was found in intron 5. However, for the 185 animals genotyped for this polymorphism, only genotypes OP and PP were detected with across-line frequencies of 0.09 and 0.91(Table 2
), respectively; no OO individuals were observed. For the Pit1I3H polymorphism, genotypes CC, CD, and DD were detected with across-line frequencies of 0.12, 0.49, and 0.39, respectively. For the Pit1I3NL polymorphism, across-line genotypic frequencies of 0.12, 0.47, and 0.41 were determined for GG, GH, and HH, respectively. The Pit1I4N polymorphism in intron 4 was found to have genotypes EE, EF, and FF with across-line frequencies of 0.12, 0.47, and 0.41, respectively. Across-line genotypic frequencies of 0.11, 0.44, and 0.45 were detected for AA, AB, and BB, respectively, for the previously reported polymorphism in exon 6 (Woollard et al., 1994), Pit1E6H (HinfI). All of the new polymorphisms detected in the current study were in the intronic regions, indicating that the intronic regions of the Pit-1 gene are much more variable than the exons. Thus far, only one exonic polymorphism has been detected in cattle (Woollard et al., 1994).
Genotypic frequencies for the various polymorphisms were not found to be significantly different between the high- and low-IGF-I selection lines based on a 2 test (genotypic frequencies by line are summarized in Table 2). This finding indicates that selection for serum IGF-I concentration did not have a significant effect on the frequencies of these polymorphisms.
The nucleotide characterizations of these polymorphisms were revealed by sequencing. The Pit1I3H polymorphism was shown to have an AAT deletion in allele C. The Pit1I3NL polymorphism had a G-to-C transition in allele G (Genbank accession No. AY183916). For the Pit1I4N polymorphism, a G-to-T transversion was observed in allele E (Genbank accession No. AY183917). For Pit1I5, the O allele had a single nucleotide transversion from G to A.
Among 98 animals genotyped, polymorphisms for Pit1I3H, Pit1I3NL, Pit1I4N, and Pit1E6H indicated the possible existence of linkage disequilibrium in these cattle. All but two of the animals with BB genotypes also had DD, FF, and HH genotypes. Genotype AA was always observed in animals with genotype CC, EE, and GG. Therefore, we propose the existence of two linkage groups, B-D-F-H and A-C-E-G. Although no definite linkage conclusions can be reached from the heterozygous genotypes, the fact that the great majority of heterozygous genotypes occurred together supports our proposal of linkage disequilibrium. Because these 98 animals were randomly selected from different seasons, sexes, and IGF-I selection lines, it was believed that this linkage may exist in the entire population of cattle under divergent selection for IGF-I at EORDC. Therefore, results from the association study of growth and carcass traits for the Pit1E6H polymorphism may also represent the relationships of polymorphisms Pit1I3H, Pit1I3NL, and Pit1I4N with growth and carcass traits in the Angus herd.
After the Bonferroni correction, no significant (P > 0.05) associations with IGF-I concentration, growth, or carcass traits were observed for the polymorphisms Pit1E6 (Table 3). Results for the association study of the polymorphisms Pit1I3H, Pit1I3NL, and Pit1I4N with IGF-I concentration and growth traits associations were not significant (P > 0.05) as well. For the Pit1I5 polymorphism, no significant association was observed. Because few OP and no OO animals were found in the 185 animals genotyped, there was not sufficient power in analyzing this polymorphism. No significant results were found for these polymorphisms when data were analyzed separately within the high- and low-IGF-I selection lines. Because there are many correlated traits analyzed in this study, Bonferroni correction makes this analysis very conservative.
The polymorphism Pit1E6 in exon 6 was detected by Woollard et al. (1994). Previous studies of this polymorphism in Italian Holstein-Friesian bulls revealed that allele A had a positive effect on milk yield traits, body depth, angularity, and rear leg set (Renaville et al., 1997a). The same authors also found a relationship of allele B with higher body weight at 7 mo of age in double-muscled Belgian Blue bulls (Renaville et al., 1997b). Allele A was shown to have more desirable daily milk yield and milk composition in Polish Black-and-White cows (Zwierzchowski et al., 2002). However, Di Stasio et al. (2002) found no association of the genotypes with meat production traits in Piemontese cattle. In addition, Zwierzchowski et al. (2001) found no relationship of this marker with growth and carcass traits in beef cattle. The present results also did not show significant association of this polymorphism with growth and carcass traits in Angus beef cattle. Therefore, the effects of this genetic marker have varied from study to study, which could be due to different statistical models used, different numbers of animals genotyped, or genetic composition of the breeds studied. More animals need to be studied to better understand the effect of this marker on production traits in cattle.
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Implications
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The polymorphisms for Pit-1, a pituitary transcription factor gene, identified in the current study do not seem to affect growth and carcass traits in fall- and spring-calving Angus cattle divergently selected for high– or low–blood serum IGF-I concentration. However, the HinfI polymorphism in exon 6 was reported in other studies to be associated with body composition and milk yields in dairy cattle and early-age body weight in beef cattle. Therefore, this genetic marker seems to have different effects in different populations.
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Footnotes
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1 Salaries and research support provided by state and federal funds appropriated to the Ohio Agric. Res. and Dev. Center, Ohio State Univ. 
2 The laboratory assistance of J. Riggenbach is appreciated.
3 Correspondence: Children’s Research Institute, Columbus Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205 (phone: 614-722-3070; fax: 614-722-2774; e-mail: zhao.62{at}osu.edu).
Received for publication January 13, 2004.
Accepted for publication April 28, 2004.
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Abstract
Introduction
