Polymorphism. A polymorphic locus, characterized by multiple dinucleotide repeats (TG- and AG-microsatellites), was identified in genomic DNA of marble trout (Salmo marmoratus Cuvier 1817) and brown trout (Salmo trutta Linnaeus 1758) by PCR and designated as BFRO 001.
Source and Description. The template DNA was isolated from a library of size selected (100 to 600 bp) Sau3AI genomic restriction fragments from marble trout cloned into pBluescript II SK+ (Stratagene) vector, previously restricted with BamHI, and propagated in Epicurian Coli Competent Cells (Stratagene). Screening of the library was performed with the Chemilluminescence Quick-LightTM Genome Mapping Probe Kit (FMC) applying (CA)n and (GA)n as probes. The DNA sequence (GenBank Accession No. U90327) was obtained with the conventional dideoxy method using DIG Taq DNA Sequencing Kit (Boehringer Mannheim). Primers were designed to amplify a region containing a cluster of repetitive motifs: 5'-(TG)13(AG)4(TG)2CAT-GTGCGAC(T G)12-3'.
Method of Detection. Polymerase chain reaction was carried out using primers SM 10/9 F: 5'-TTT GGA ATG ATA TGG ATA TGG -3' (CA/CT strand) and 5'-digoxigenin labeled SM 10/9 R: 5'-CTT ACA GCC ACC TTT ATG CG-3'. Each reaction (10 µL final volume) contained 50 ng of genomic DNA, .75 µM each primer, .2 mM dNTP, .5 U of Taq polymerase (Gibco), 1.5 mM MgCl2, 20 mM Tris-HCl, and 50 mM KCl. Thermal cycling reaction was performed in a Perkin Elmer 2400 Cycler under the following conditions: 3 min at 94oC followed by 29 cycles of 94oC for 45 s and 60oC for 25 s. The PCR products were run on a direct blotting electrophoresis device GATC 1500 (Gesellshaft für Analyse-Technik und Consulting mbH) using 4% denaturating polyacrylamide gel and detected by color reaction using alkaline phosphatase conjugated anti-digoxigenine antibodies.
Description of Polymorphism. In the test material, 12 alleles were detected in 57 marble trout, originating from two geographically remote wild populations, and in 24 brown trout, originating from three different river basins. The observed allele sizes were as follows: 202, 204, 206, 212, 216, 218, 220, 224, 228, 232, 236, and 238 bp. The alleles 478, 480, and 488 were characteristic for marble trout. The observed frequencies of these three alleles differed between the two sampling locations and were .18202, .07204, and .75212 and .19202, .78204, and .03212, respectively (chi2 = 63.9, P < .001). The other alleles were evenly distributed among the brown trout populations except for the alleles 218, 228, and 232, which were restricted only to a certain geographic location. The results from 16 specimens, indicating all the alleles observed, are shown in Figure 1.
Comments. The PCR with SM 10/9 F and SM 10/9 R primers failed to amplify genomic DNA from rainbow trout (Oncorhynchus mykiss) and grayling (Thymallus thymallus).
Key Words: Fishes, Brown Trout, Microsatellites, DNA, Polymorphism
© 1997, by the American Society of Animal Science. All rights reserved.
J. Anim. Sci. 1997. 75:1983 Back to Table of Contents