SHORT COMMUNICATION doi:10.1111/j.1365-2052.2004.01156.x Ovine alpha-amylase genes: isolation, linkage mapping and association analysis with milk traits
J. H. Calvo*, S. Marcos*, A. E. Beattie†, C. Gonzalez*, J. J. Jurado* and M. Serrano* *Departamento de Mejora Gene´ tica Animal, INIA, 28040 Madrid, Spain. †AgResearch Molecular Biology Unit, Department of Biochemistry and Centre for Gene Research, University of Ottago, Dunedin, New Zealand
Summary On the basis of comparisons between cattle and sheep genome mapping information the ovine a-amylase gene was examined as a possible genetic marker for milk traits in sheep. The objective of the present study was to isolate, map and determine whether this gene is a candidate gene for milk traits. DNA fragments (832 and 2360 bp) corresponding to two different AMY genes were isolated, and one SNP in intron 3 and one GTG deletion in exon 3 of the 2360 bp DNA fragment were found. The 2360 bp ovine AMY DNA fragment was located on chromosome 1 by linkage mapping using the International Mapping Flock. No association was found between estimated breeding values for milk yield, protein and fat contents and AMY genotypes in a daughter design comprising 13 Manchega families with an average of 29 daughters (12–62) per sire.
Keywords amylase gene, association, linkage mapping, sheep.
a-Amylase belongs to a family of enzymes that hydrolyses in Table 1. Genomic DNA (100 ng) was amplified in a final the a1-4 bonds in glucose polymers. In humans, a-amylases volume of 25 ll containing 5 pmol of each primer, 200 lM are encoded by a multigene family, clustered within a dNTPs, 2.0 mM MgCl2,50mM KCl, 10 mM Tris-HCl, 0.1% 250 kb region, containing five active genes, including two Triton X-100 and 0.75 U Taq polymerase (EcoTaq Plus, pancreatic genes (AMY2A and AMY2B) and three salivary Ecogen, Madrid, Spain). After denaturation at 94 C for genes (AMY1A, AMY1B and AMY1C) (Gumucio et al. 2 min, 30 amplification cycles were performed comprising 1988). a-Amylase genes have been mapped to bovine denaturation at 94 C for 1 min, annealing temperature chromosome 3 (Laurent et al. 1999), human chromosome according with the primer pair for 1 min, extension at 1 (Dracopoli & Meisler 1990), and caprine chromosome 13 72 C for the time considering the length of the expected (Schibler et al. 1998). fragment (1 min for every kb), followed by a further 5 min Quantitative trait loci (QTL) for some milk traits have extension at 72 C. The PCR products were cloned in been reported on bovine chromosome 3 (BTA3) close to the pMOSBlue (Amersham Pharmacia, Little Chalfont, UK) and AMY cluster (Lipkin et al. 1998; Zhang et al. 1998). sequenced using an ABI PRISM 3700 (Applied Biosystem, Therefore, we examined the ovine AMY genes as potential Madrid, Spain) and standard protocols. genetic markers for milk traits in sheep. Specific sheep primers were designed for each DNA frag- A comparative walking strategy using DNA sequence ment (Table 1, PCR 4, 5), and direct sequencing of PCR from bovine (EMBL: AF054834) and human genomic DNA products from six different animals (two animals of (EMBL: NT_030567) was used to obtain the partial genomic Manchega, one animal from each of the Awassi, Assaf and DNA sequences of 832 bp (Table 1, PCR 2, 3) and 2360 bp Rasa Aragonesa sheep breeds and one Mouflon) was per- (Table 1, PCR 1) fragments corresponding to two different formed using an ABI PRISM 3700 automatic sequencer ovine AMY genes. Primer sequences, the annealing tem- (Applied Biosystem) and standard protocols. peratures and the size amplified of the fragments are shown The exons were identified in the 832 and 2360 bp sheep AMY DNA fragments by homology with known AMY Address for correspondence sequences of cattle, swine, rat and humans, using BLAST
Unidad de Tecnologia en Produccion Animal, Centro de Investigacion y (National Center for Biotechnology Information: http:// Tecnologia Agroalimentaria de Aragon, Apartado 727, 50080 www.ncbi.nlm.nih.gov/BLAST/), DNASIS (Hitachi Software Zaragoza, Spain. Engineering, Ltd, 1991), CLUSTALW (http://www.ebi.ac.uk/ E-mail: [email protected] clustalw/) and GENEVIEWER (http://www.itba.mi.cnr.it/ Accepted for publication 3 May 2004 webgene/) software. The isolated 832 bp AMY DNA