Synteny assignment of four genes and two microsatellite markers in river buffalo (Bubalus bubalis L.)

Introduction Syntenic comparisons among related species suggest that large chromosomal regions are conserved among mammals ( Womack & Moll 1986 ; Brubacher et al. 1990 ; Copeland & Jenkins 1991 ; Levan et al. 1991 ). Cattle and buffaloes (NAA = 58) belong to family Bovidae which has a high degree of chromosome conservation ( Buckland & Evans 1978 ; Gallagher & Womack 1992 ). Their diploid chromosome numbers are 60 and 50, respectively. The banding homology between cattle and river buffalo chromosomes suggests that each of the five biarmed pairs of the river buffalo originates by centric fusion between two of ten acrocentric pairs of cattle autosomes (R eport of the C ommittee for the Standardization Of Banded Karyotypes Of The River Buffalo 1994 ). Syntenic conservation between cattle and river buffalo has been reported ( De Hondt et al. 1991 , 1997 ; El Nahas et al. 1993 , 1996 , 1999 ; Oraby et al. 1998 ). Materials and methods Forty buffalo/hamster somatic cell hybrids developed from the fusion of buffalo leukocytes and Chinese hamster cell line wg3h ( Echard et al. 1984 ) deficient in hypoxanthine phosphoribosyl transferase ( De Hondt et al. 1991 ) were used. The six investigated markers comprise four coding loci: prolactin (PRL), fibronectin (FN1), inhibin β‐A (INHBA), and lactoglobulin β (LGB) and two specific DNA segments: CSSM14 and CSSM52. The polymerase chain reaction (PCR) primers were kindly provided by Dr J. E. W omack, Department of Pathology, College of Veterinary Medicine, Texas A&M University. They were designed from published bovine sequences ( Table 1 ). Primer sequences and conditions for amplification reaction PCR primer Primer sequence 5′–3′ PCR product size (bp) Annealing temperature (°C) References PRL GGA AAG TGA ACA TGA CTG TCT AG 156 60 Fries et al. (1993) GCC CTC TCT TCT ACA ATG AAC AC FN1 TCC AAC CCA GAG AAA CAA GCG T 278 57 Barendse et al. (1994) GAT CCC AAT CCA AAT CCA AAG T INHBA TTC CAT GTA CTA TGA CGA TGG GCA G 177 67 Neibergs et al. (1993) AAC ATG TCT TCG TGT CAC CAC TGT C LGB TGT GCT GGA CAC CGA CTA CAA AAA G 247 55 Medrano & Aguilar‐Cordova (1990) GCT CCC GGT ATA TGA CCA CCC TCT CSSM14 AAA TGA CCT CTC AAT GGA AGC TTG 145 60 Moore et al. (1994) AAT TCT GGC ACT TAA TAG GAT TCA CSSM52 AAA TGC ACC CGG CTA ATA TGA AAG G 185 60 Moore et al. (1994) GTA TAT ACT GTT GTT TTC TGG CAG Genomic DNA extraction To perform PCR analysis, genomic DNA was extracted from buffalo, hamster cell line, and buffalo‐hamster hybrid cells according to established protocols ( Blin & Stafford 1976 ). Cells were incubated overnight in a shaking water‐bath at 37°C in lysis buffer (10 m m Tris base, 400 m m NaCl, 2 m m sodium EDTA) pH 8.2, 20 % sodium dodecyl sulfate (SDS) and proteinase k (10 mg/ml). Nucleic acids were extracted once with phenol, saturated with Tris‐EDTA (TE) buffer (10 m m Tris, 10 m m NaCl, 1 m m EDTA), followed by extraction in phenol–chloroform–isoamyl alcohol (25 : 24 : 1) until there was no protein at the interface. This was followed by extraction in chloroform–isoamyl alcohol (24 : 1). To each extraction, equal volumes of the solvent were added, followed by thorough mixing and centrifugation for 10 min at 2000 r.p.m. The top layer was carefully transferred to another Falcon tube for the next extraction. To the final aqueous phase, 0.1 volume of 2.5 m Na acetate and 2.5 volume of cold 95 % ethanol were added. The tubes were agitated gently to mix the liquids and a fluffy white ball of DNA was formed. The DNA was picked up with a heat‐sealed Pasteur pipette and washed briefly in 70 % ethanol. The DNA was finally dissolved in an ap