Ovine alpha-amylase genes: isolation, linkage mapping and association analysis with milk traits

Summary On the basis of comparisons between cattle and sheep genome mapping information theovine a-amylase gene was examined as a possible genetic marker for milk traits in sheep. Theobjective of the present study was to isolate, map and determine whether this gene is acandidate gene for milk traits. DNA fragments (832 and 2360 bp) corresponding to twodifferent AMY genes were isolated, and one SNP in intron 3 and one GTG deletion in exon 3of the 2360 bp DNA fragment were found. The 2360 bp ovine AMY DNA fragment waslocated on chromosome 1 by linkage mapping using the International Mapping Flock. Noassociation was found between estimated breeding values for milk yield, protein and fatcontents and AMY genotypes in a daughter design comprising 13 Manchega families withan average of 29 daughters (12–62) per sire.Keywords amylase gene, association, linkage mapping, sheep.a-Amylase belongs to a family of enzymes that hydrolysesthe a1-4 bonds in glucose polymers. In humans, a-amylasesare encoded by a multigene family, clustered within a250 kb region, containing five active genes, including twopancreatic genes (AMY2A and AMY2B) and three salivarygenes (AMY1A, AMY1B and AMY1C) (Gumucio et al.1988). a-Amylase genes have been mapped to bovinechromosome 3 (Laurent et al. 1999), human chromosome1 (Dracopoli & Meisler 1990), and caprine chromosome 13(Schibler et al. 1998).Quantitative trait loci (QTL) for some milk traits havebeen reported on bovine chromosome 3 (BTA3) close to theAMY cluster (Lipkin et al. 1998; Zhang et al. 1998).Therefore, we examined the ovine AMY genes as potentialgenetic markers for milk traits in sheep.A comparative walking strategy using DNA sequencefrom bovine (EMBL: AF054834) and human genomic DNA(EMBL: NT_030567) was used to obtain the partial genomicDNA sequences of 832 bp (Table 1, PCR 2, 3) and 2360 bp(Table 1, PCR 1) fragments corresponding to two differentovine AMY genes. Primer sequences, the annealing tem-peratures and the size amplified of the fragments are shownin Table 1. Genomic DNA (100 ng) was amplified in a finalvolume of 25 ll containing 5 pmol of each primer, 200 l