Abstract
Human ribosomal protein (RP) gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR). In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis), 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.
Highlights
Because introns are a good source to find DNA polymorphisms in the eukaryotic genome, exon-primed introncrossing (EPIC) polymerase chain reaction (PCR) assays have been developed for genetic analyses (Lessa 1992; Palumbi and Baker 1994; Corte-Real et al 1994; Chow and Hazama 1998; Chow 1998; Quattro and Jones 1999; Hassan et al 2002; Jarman et al 2002; Chow and Nakadate 2004; Nakadate and Chow 2008; Pinho et al 2010; Jennings and Etter 2011; Chow et al 2015)
The intron/exon structures of human ribosomal protein (RP) genes have been reported by Kenmochi et al (1998) and Yoshihama et al (2002), and the nucleotide sequences are available in the GenBank database
A total of 12 primer pairs expected to amplify RP gene introns of the Pacific bluefin tuna with lengths of 500–1000 bp were selected for ease of sizing, and PCR amplification was attempted using template DNA of the Japanese pilchard (Sardinops melanostictus), the Japanese puffer (Takifugu rubripes), starry flounder (Platichthys stellatus), the Pacific bluefin tuna (T. orientalis), broadbanded thornyhead (Sebastolobus macrochir), and the Japanese eel (Anguilla japonica), all derived from the tissue collection in our laboratory
Summary
Because introns are a good source to find DNA polymorphisms in the eukaryotic genome, exon-primed introncrossing (EPIC) polymerase chain reaction (PCR) assays have been developed for genetic analyses (Lessa 1992; Palumbi and Baker 1994; Corte-Real et al 1994; Chow and Hazama 1998; Chow 1998; Quattro and Jones 1999; Hassan et al 2002; Jarman et al 2002; Chow and Nakadate 2004; Nakadate and Chow 2008; Pinho et al 2010; Jennings and Etter 2011; Chow et al 2015). Keywords Universal primers Á Ribosomal protein genes Á Intron Á Single copy nuclear loci Á Teleostei
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