Rapid communication: a polymorphic microsatellite in the promoter region of the bovine calpastatin gene.

Marker Name. CAST. Source and Description of Primers. Primers were designed from a published sequence of the bovine calpastatin promoter region surrounding a cytosine-adenine (CA) repeat 1.4 kb upstream of the transcription start site (Cong et al., 1998), to amplify a product from genomic DNA of approximately 246 bp (primers CSTF5 and CSTR4) that was used for sequencing. A polymorphic 126 to 154 bp amplicon was produced with primers CSTF4 and CSTR4 for microsatellite analysis. Primer Sequences. CSTF4: 5′-GTA AAG CCG CAC AAA ACA CAC CCA GG-3′; CSTF5: 5′-TTC AAC AGC CTC CTG AAA GGC AAT GG-3′; CSTR4: 5′-CCT GGA CCC TCT GGA TGA GGA AGC GG-3′. Method of Detection. The PCR amplification of 50 ng of genomic DNA for cycle sequencing was performed for 35 cycles of 94°C for 30 s, 57°C for 45 s, and 72°C for 45 s, followed by a 5-min final extension at 72°C in an MJ (Watertown, MA) PTC-100 thermocycler. Sequencing was performed as in Geesink et al. (1998). For microsatellite analysis, a total of 25 ng of genomic DNA, 5 pmol of each primer, and .1 U of Taq polymerase were included in an 8-μL reaction containing 1× Taq buffer, 30 μM of dTTP, dGTP, dCTP, and 15 μM dATP, and .1 μCi of [α-32P]ATP. The cycling profile was 1 min at 92°C, with 30 cycles of 30 s at 94°C, 1 min at 55°C, and 1 min at 72°C, followed by a final 5-min extension at 72°C. Description of Polymorphism. The polymorphism was a (CA)n repeat that identified nine alleles in the MARC mapping population (Bishop et al., 1994) with fragment lengths of 126, 132, 136, 142, 146, 148, 150, 152, and