W. Ge, M. E. Davis1, H. C. Hines, and K. M. Irvin
The Ohio State University Department of Animal Sciences
Marker-assisted selection (MAS) may increase rate of genetic gain in farm animal breeding. Identifying useful genetic markers is the first and most critical step in MAS. This study was designed to identify genetic markers in beef cattle. Genomic DNA was isolated from blood samples of purebred Angus beef cattle divergently selected for high or low blood-serum IGF-I concentrations. Fragments of DNA in the IGF-I, GH, and GHR genes were amplified using PCR and examined for polymorphism using SSCP, DGGE, and sequencing methods. Nine single nucleotide polymorphisms were identified: one in the promoter region of the IGF-I gene, three in the promoter region of the GH gene, and one in the promoter region and four in exon 10 of the GHR gene. Rapid genotyping methods were developed for determination of genotypes for these markers. Seven hundred sixty-three calves were genotyped for the IGF-I polymorphism, and 473 calves were genotyped for six other polymorphisms. Statistical analyses are being conducted to test the association of these markers with blood serum IGF-I concentration and growth traits in Angus beef cattle.
Application of genetic markers in animal selection and breeding may dramatically expedite genetic improvement, especially in beef cattle, which have a long generation interval. Kashi et al. (1990) proposed that marker-assisted selection (MAS) could increase rate of genetic gain by 15 to 30% in livestock. Total gain expected with MAS was estimated to range from 44.7 to 99.5%, depending on the model (Edwards and Page, 1994). Accuracy of selection for qualitative traits can also be improved using MAS (Brenneman et al., 1996). By combining MAS with advanced reproductive techniques, generation interval can be shortened from 69 to 45 months in cattle (Bishop et al., 1995). However, few genetic markers have been identified for growth and carcass traits in beef cattle. Mapping quantitative trait loci (QTL) is an effective way to identify useful genetic markers, but studying candidate genes known to influence animal growth and body development is more economical.
Growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor I (IGF-I) are critical regulators of animal growth and body development (Rappaport, 1991; Werner et al., 1994). Polymorphisms (markers) in the regulatory region (e.g., promoter region) and coding region (exons) of these genes may affect the expression of the genes and the functions of the proteins, respectively. Therefore, the objective of this study was to examine the promoter region and some coding regions of the bovine IGF-I, GH, and GHR genes for polymorphisms.
Purebred Angus beef cattle divergently selected for blood serum IGF-I concentrations (Davis and Simmen, 1997) at the Eastern Ohio Resource Development Center (EORDC) were used in this study. Genomic DNA was isolated from the blood samples using the alcohol deposition method. PCR primers were designed for the promoter regions of the three genes, all five exons of the GH gene, and the 10th exon of the GHR gene. Fragments of DNA were amplified from genomic DNA using the PCR method and examined for polymorphisms using single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) methods. PCR primers and conditions, and SSCP and DGGE conditions were described in earlier reports ( Ge et al., 1997; 1999a; 1999b).
DNA sequence analysis was done using the ABI dye terminator cycle sequencing protocol. The single nucleotide polymorphisms (SNP) shown on the sequences were verified by the restriction fragment length polymorphism (RFLP) method. In cases in which the polymorphism was not recognized by any restriction enzyme, new primers were designed to introduce a recognition site for some enzyme. Thus, PCR-RFLP methods were developed for the polymorphisms and used to genotype the calves.
One SSCP polymorphism was identified in the promoter region of the bovine IGF-I gene (Ge et al., 1997). Sequence analysis of this fragment identified a T to C transition (GenBank accession number AF017143). Another primer was designed to introduce a SnaBI recognition site. PCR fragments were digested with the enzyme at 37ºC for two hours and run on Agarose gel. For the 763 calves genotyped, 43.5% were of TT genotype, 41.2% TC genotype, and 15.3% CC genotype (allele frequency, T: 64.1%, C: 35.9%).
A DGGE polymorphism with six genotypes was identified in the promoter region of the GH gene. Sequence analysis later revealed three SNPs in a 60 bp region. All three SNPs were C to T transitions (GenBank accession number AF118837). One of the SNPs can be recognized by restriction enzyme MaeIII, and 249 calves were genotyped for this marker (genotype frequencies, CC: 93.6%, CT: 6.4%, TT:0%). MspI and XmnI recognition sites were introduced by PCR primers to determine the other two SNPs at the same time. Only three haplotypes (linked alleles) were found among the 473 calves: C..C, T..C, and T..T. Thus, six genotypes were identified: C/C..C/C (9.7%), C/T..C/C (13.8%), C/T..C/T (25.6%), T/T..C/C (8.3%), T/T..C/T (26.9%), and T/T..T/T (15.7%).
An A to G transition was determined by sequencing the DNA fragment following the detection of a DGGE polymorphism in the promoter region of the GHR gene (Ge et al., 1999a; GenBank accession number AF126288). Restriction enzyme NsiI recognizes this transition and was used to determine the genotype of this SNP for 473 calves, of which 35.3% were of GG genotype, 50.1% AG genotype, and 14.6% AA genotype.
Four SNPs were discovered by sequence analyses after a DGGE polymorphism was detected in a 450 base pair fragment of exon 10 of the GHR gene (Ge et al., 1999b; GenBank accession number AF140284). They were C-T, A-G, C-T, and A-G transitions. The second variation changed the codon from ACC (coding for Thr) to GCC (coding for Ala), and the fourth one changed the codon from AGC (coding for Ser) to GGC (coding for Gly). The first and third SNPs do not change the amino acid encoding. The first, third, and fourth SNPs were recognized by MaeII, NlaIII, and AluI restriction enzymes. A NarI recognition site was introduced to determine the genotype for the second polymorphism. Genotypes of the markers other than the first one were determined for 473 calves. Genotypic frequencies were: 34.7% GG, 51.6% AG, and 13.7% AA for the second marker; 34.0% CC, 49.5% CT, and 16.5% TT for the third marker; and, 4.0% GG, 35.5%AG, and 60.5% AA for the fourth marker.
Statistical analyses are being conducted to test the relationships of these markers with growth traits and blood serum IGF-I concentrations, including birth weight, weaning weight, on-test weight, off-test weight, postweaning gain, and IGF-I concentrations on day 28, 42, and 56 of the 140-day postweaning test.
Bishop, M. D., Hawkins, G. A., and Keener, C. L. 1995. Use of DNA markers in animal selection. Theriogenology 43:61.
Brenneman, R. A., Davis, S. K., Sanders, J. O., Burns, B. M., Wheeler, T. C., Turner, J. W., and Taylor, J. F. 1996. The polled locus maps to BTA1 in a Bos indicus x Bos taurus cross. J. Hered. 87:156.
Davis, M. E., and Simmen, R. C. M. 1997. Genetic parameter estimates for serum insulin-like growth factor I concentration and performance traits in Angus beef cattle. J. Anim. Sci. 75:317.
Edwards, M. D., and Page, N. J. 1994. Evaluation of marker-assisted selection through computer simulation. Theor. Appl. Genet. 88:376.
Ge, W., Davis, M. E., and Hines, H. C. 1997. Two SSCP alleles identified at the 5’-flanking region of the bovine IGF1 gene. Anim. Genet. 28:155.
Ge, W., Davis, M. E., Hines, H. C., and Irvin, K. M. 1999a. Two-allelic DGGE polymorphism detected in the promoter region of the bovine GHR gene. Anim. Genet. 30:71.
Ge, W., Davis, M. E., Hines, H. C., and Irvin, K. M. 1999b. Polymorphism in exon 10 of the bovine GHR gene detected by PCR-DGGE. Anim. Genet. 30:167.
Kashi, Y., Hallerman, E., and Soller, M. 1990. Marker-assisted selection of candidate bulls for progeny testing program. Anim. Prod. 51:63.
Rappaport, R. 1991. A touch of growth. Horm. Res. 36:166.
Werner, H., Adamo, M., Roberts Jr., C. T., and LeRoith, D. 1994. Molecular and cellular aspects of insulin-like growth factor action. Vitam. Horm. 48:1.
1 For more information, contact at: The Ohio State University, 221 Plumb Hall, 2027 Coffey Road, Columbus, OH 43210; (614) 292-4984, Fax (614) 292-7116; email:davis.28@osu.edu