Rapid communication: microsatellite DNA markers (BFRO010 and BFRO011) for grayling.

Polymorphism. Two polymorphic microsatellite loci, BFRO010 and BFRO011, were identified in genomic DNA of grayling (Thymallus thymallus). Source and Description. The template DNA was isolated from a library of size-selected (200 to 800 bp) Sau3AI genomic restriction fragments from grayling. The DNA fragments were cloned into pBluescript II SK+ (Stratagene, La Jolla, CA) vector, previously restricted with BamHI, and propagated in Epicurian Coli Competent Cells (Stratagene). Screening of the library was performed with the Chemilluminiscence QuickLight Genome Mapping Probe Kit (FMC BioProducts, Rockland, ME) applying (CA)n and (GA)n as probes (FMC BioProducts). The DNA sequences (GenBank Accession No. AF130409 and AF130410 for loci BFRO010 and BFRO011, respectively) were obtained with an ABI Prism 310 Genetic Analyser using a dRhodamine Terminator Cycle Sequencing Kit (PE Biosystems, Warrington, U.K.). Primers were designed to amplify regions containing repetitive motifs, which were (AC)17 for BFRO010 and (GT)16 for BFROO11. Primer Sequences. Locus BFRO010: Fam-5′-GGACGGAGCCAGCATCAC-3′ and 5′-GATTTCATAATCAGGTCAATAGTCAT-3. Locus BFRO011: Hex-5′CATGGTTGATTGTGGGGGGA-3′ and 5′-AACATCCTTACGCCCTAGCA-3′. Method of Detection. Polymerase chain reaction was carried out in a 10L reaction containing 50 ng of genomic DNA, .25 M each primer, .2 mM dNTP, .5 U of Taq polymerase (PE Biosystems), 1.5 mM MgCl2, 20 mM Tris-HCl, and 50 mM KCl. Thermal cycling reaction was performed in an MJ Research PTC-100 Thermal Cycler under the following conditions: 3 min at 94°C followed by 30 cycles of 94°C for 1 min, 55°C for 20 s, and 72°C for 5 s. The PCR products were mixed with formamide and GENESCAN-350 (TAMRA) Size Standard (PE Biosystems) and genotyped with an ABI