Rapid communication: Poli-RC35-TUF, a highly polymorphic microsatellite marker in the Japanese flounder, Paralichthys olivaceus.

Source and Description. A partial genomic library (300 to 500 bp) was constructed using DNA obtained from Japanese flounder,Paralichthys olivaceus, digestedwith Sau3AI. The DNA was inserted in pBluescript SK (Toyobo, Japan) digested by BamHI and transformed into competent E. coli cells (strain DH5α). The partial genomic library was screened with a γP-labeled (CA)10 probe. About 800 positive clones were obtained, and part of that was sequenced on an ABI Prism 310 Genetic analyzer (Perkin Elmer Japan Applied Biosystems, Tokyo, Japan) using a BigDyeTerminator cycle sequencing kit (Perkin Elmer). A total of 150 primer pairs flanking the dinucleotide repeats were designed. A marker, named Poli-RC35-TUF, was found to be highly polymorphic. Primer Sequences. Forward: 5′-CAC AAG CCT GCA CAA ATC AT; reverse: 5′-TAT TAC TCT GCC ATC CCC CA. The original sequence has been submitted to the GenBank database (accession no. AB032370). PCR conditions. PCR was performed in a 25L solution containing 12.5 pmol of forward primer and .6 pmol of reverse primer end-labeled with γP-ATP, .6 U of Taq Polymerase (Takara, Japan), 1×PCRbuffer (50mMKCl, 10mMTris-HCl pH8.3, 1.5mMMgCl2), 50 ng of genomic DNA, 200 M of each dNTP, and 1% BSA. Thermal cycling was performed in a GeneAmp PCR System 9600 (Perkin Elmer) and consisted of an initial denaturation at 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 62°C for 1 min, 72°C for 1 min, and a final extension of 3 min at 72°C. The PCR products were separated in a 6% polyacrylamide gel. Allele sizes were determined using an M13 standard sequencing ladder. Mendelian Inheritance. CodominantMendelian inheritance of alleles was observed on 44 individuals in a family of farmed flounder. Two genotypes were generated by the homozygousmale parent and by the heterozygous