Materials and Methods
Animals
Angus beef cattle, which were divergently selected for blood serum IGF-I concentration, were used as the experimental animals. Selection began in1989 at the Eastern Ohio Resource Development Center (EORDC), using 100 spring-calving (50 high line and 50 low line) and 100 fall-calving (50 high line and 50 low line) purebred Angus cows with unknown IGF-I levels. Each year, four bull calves with the highest and four bull calves with the lowest IGF-I concentration were selected for breeding within the selection lines (Davis and Simmen, 1997). A total of 185 (cattle born in spring, 1995 through spring, 1996), 426 (cattle born in spring, 1995 througn fall, 1997) and 80 (cattle born in spring, 1995) cattle were genotyped for the intron 5 SSCP, exon 6 HinfI, and intron 2 MspI polymorphisms, respectively.
Methods
Genomic DNA was isolated from blood samples collected during the IGF-I selection experiment. Polymerase chain reaction (PCR) was used to amplify the regions of interest. For the intron 5 variation, a fragment of 360 bp was amplified with primers (forward 5'-CCTCTGTCCATGGGATTTTC-3' and reverse 5'-AAATGTCCCCCAGAACTCAG-3'). The PCR was performed in a 30 ml reaction volume containing 10 pmol of forward primer and the same amount of reverse primer, 200 mM dNTP's, 1x reaction buffer, which contained 1.5 mM MgCl2, 1 unit of Taq-DNA polymerase, and 100 ng of genomic DNA as template. Conditions were 97oC for 2 min, followed by 35 cycles of 95 oC for 45 seconds, 63 oC for 1 min, and 72oC for 90 seconds. After 35 cycles, reactions were finished by an extension of 5 min at 72 oC, and finally bulked at 4 oC in a DNA Thermal Cycler.
For the exon 6 HinfI polymorphism, the primers were designed and the PCR conditions were followed as previously described (Woollard et al., 1994). The PCR reaction mixture was the same as the one for the intron 5 variation described above. For the intron 2 MspI polymorphism, a 3,000 bp fragment was amplified with primers (forward 5'- CTGTTCCTTTCTGTCATTAT-3' and reverse 5'-AGGAAAACCATGACTCAA-3'). The PCR reaction mixture was again the same as the one for intron 5 described above. Conditions were 97oC for 2 min, followed by 35 cycles of 95 oC for 45 seconds, 52 oC for 1 min, and 68 oC for 90 seconds. After 35 cycles, reactions were finished by an extension of 5 min at 72 oC and finally bulked at 4 oC.
When amplification of the 360 bp intron 5 fragment was achieved, SSCP was performed to screen for mutations within the amplified segment. The samples were loaded on 8% polyacrylamide gels and run in 1x TBE buffer at 200 to 250 volts for 16 to 20 hours at a constant temperature of 10°C. Gels were stained with 0.01% Ethidium Bromide for 10 min and viewed under UV light. A total of 185 Angus cattle were genotyped for this marker.
To genotype animals for the two RFLPs, the corresponding PCR products were digested with restriction enzymes HinfI and MspI, respectively, and the digested DNA was run on an agarose gel. The gels were viewed under UV light. A total of 416 and 80 animals, respectively, were genotyped for these two polymorphisms.