Premium
Characterization of hypervariable microsatellite loci in the threespine stickleback Gasterosteus aculeatus
Author(s) -
RICO C.,
ZADWORNY D.,
KUHNLEIN U.,
FITZGERALD G. J.
Publication year - 1993
Publication title -
molecular ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.619
H-Index - 225
eISSN - 1365-294X
pISSN - 0962-1083
DOI - 10.1111/j.1365-294x.1993.tb00017.x
Subject(s) - gasterosteus , library science , biology , fish <actinopterygii> , fishery , computer science
Single locus minisatellite and microsatellite DNA fingerprinting have become increasingly important to studies of relatedness in wild animals (Pembertonet al. 1991, and references therein). Here we describe the characterisation of polymorphic single-locus microsatellites for a fish species the threespine stickleback Gasterosteus aculeatus L. and briefly discuss their potential applicability in studies of the evolutionary ecology of this fish. A total of five repetitive GT/AC clones were sequenced. Using the polymerase chain reaction (PCR) we directly amplified microsatellite loci using the flanking sequences as primers. The primer sequences are shown in Table 1. The number of repeats per locus ranged from 20 to 38. All clones had perfect repeats of the dinucleotide with the exception of one clone which had a transversion of a G to a C near the middle of the repetitive sequence. PCR reactions were performed in 10 ml using the thermostable Taq polymerase under the recommended conditions of the supplier (Pharmacia). About 200 ng of genomic DNA were used per reaction (template DNA was prepared as described in Ricoet al. 1992). Final buffer concentrations were 5 0 m ~ KCI, 10-mM Tris-HCI (pH 8.3 at 21”C), 1 .5-m~ MgCl,, 0.01 % gelatine, 5% deionized formamide, and 100 pmol of each primer. DNA was denatured at 94°C prior to the addition of 0.5 units of Taq polymerase and a mixture of four deoxynucleotides. The final concentration of each iiucleotide was 125 pmol and 4.5 pmol of [CX-~~SI~ATP were included. Subsequently, the temperature was cycled between 92°C for 60 s, 58°C for 60 s and 72°C for 120 s for 35 cycles using a DNA Thermal Cycler (Perkin Elmer Cetus Corp.). The reaction products were resolved by electrophoresis in an 8% denaturing polyacrylamide sequencing gel (Sambrook et al. 1989) where single base length differences could be resolved. A sequencing ladder was used as a molecular weight marker. Using genomic DNA from nine randomly collected, and therefore presumably unrelated individuals, we esti-