doi:10.1046/j.1365-2052.2003.01071.x SHORT COMMUNICATION Detection and characterization of SNPs useful for identity control and parentage testing in major European dairy breeds F. A. O. Werner*, G. Durstewitz*, F. A. Habermann*, G. Thaller*, W. Kra ¨ mer*, S. Kollers*, J. Buitkamp † , M. Georges ‡ , G. Brem § , J. Mosner ¶ and R. Fries* *Department fu ¨ r Tierwissenschaften, Lehrstuhl fu ¨ r Tierzucht der Technischen Universita ¨ t Mu ¨ nchen, Alte Akademie 12, Freising- Weihenstephan, Germany. † Institut fu ¨ r Tierzucht, Bayerische Landesanstalt fu ¨ r Landwirtschaft, Prof. Du ¨ rrwa ¨ chter-Platz 1, Poing, Germany. ‡ Department of Genetics, Faculty of Veterinary Medicine, University of Lie ` ge, Lie ` ge, Belgium. § Agrobiogen GmbH, Hilgertshausen-Tandern, Germany. ¶ GAG BioScience GmbH, Hochschulring, Bremen, Germany Summary We propose the use of single nucleotide polymorphisms (SNPs) instead of polymorphic microsatellite markers for individual identification and parentage control in cattle. To this end, we present an initial set of 37 SNP markers together with a gender-specific SNP for identity control and parentage testing in the Holstein, Fleckvieh and Braunvieh breeds. To obtain suitable SNPs, a total of 91.13 kb of random genomic DNA was screened yielding 531 SNPs. These, and 43 previously identified SNPs, were subjected to the following selection criteria: (1) the frequency of the minor allele must be larger than 0.1 in at least two of the three examined breeds, and (2) markers should not be linked closely. Allele frequencies were estimated by analysing sequencing traces of pooled DNA or by genotyping individual DNA samples. The selected SNP loci were physically mapped by radiation hybrid mapping or by fluorescence in situ hybridization, and tested against the neutral mutation hypothesis. The presented marker set theoretically allows probabilities of identity less than 10 )13 for individual verification and exclusion powers exceeding 99.99% for parentage testing. Keywords digital DNA signatures, individual identification, parentage control, single nucleotide polymorphism. Individual identification and parentage control are essential for consumer protection and efficient management of ani- mal populations. Today, highly polymorphic microsatellite markers are well established in cattle and used successfully for these purposes (Glowatzki-Mullis et al. 1995; Heyen et al. 1997). However, single nucleotide polymorphisms (SNPs) promise considerable advantages over microsatellite markers: (1) lower mutation rates, (2) more robust in laboratory handling and data interpretation (Krawczak 1999), (3) suitability for standardized representation of genotyping results as a digital DNA signature (Fries & Durstewitz 2001), and (4) suitability for various genotyping techniques and high potential for automation (Kruglyak 1997). One disadvantage is that any SNP has a lower information content, compared with a highly polymorphic microsatellite. But this disadvantage can be compensated for by a higher number of markers. Recently, a set of SNP markers for animal identification and paternity testing in US Beef cattle was presented by Heaton et al. (2002). Here we report on the development of a similar set of SNP markers for use in the major European dairy and dual-purpose breeds: Holstein, Fleckvieh and Braunvieh. Two hundred and three bovine bacterial artificial chro- mosome (BAC) clones from the BAC library Bovine II, no. 754 (Buitkamp et al. 2000) (RZPD, Berlin, Germany) were chosen at random. The BAC ends were sequenced directly using vector-specific primers. Repetitive and coding sequences were identified by BLAST searches. To obtain polymerase chain reaction (PCR) fragments in the range of 500 to 800 bp, primers were designed with a T m of 60 °C and a 3¢-GC clamp using the Primer3 program (Rozen & Skaletsky 2000). To identify SNPs and to estimate their respective allele frequencies, we selected unrelated bulls belonging to the Bos taurus taurus breeds German Holstein (n ¼ 35), German Fleckvieh (n ¼ 33), German Braunvieh (n ¼ 32), Kerry Address for correspondence Ruedi Fries Alte Akademie 12 D-85354 Freising-Weihenstephan Deutschland/Germany. E-mail: Ruedi.Fries@tz.agrar.tu-muenchen.de Accepted for publication 1 October 2003 Ó 2004 International Society for Animal Genetics, Animal Genetics, 35, 44–49 44