Introduction

Because introns are a good source to find DNA polymorphisms in the eukaryotic genome, exon-primed intron-crossing (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). Polymorphisms detected in introns have been used for genetic population analysis (Palumbi and Baker 1994; Chow and Takeyama 2000; Williams et al. 2002; Nohara et al. 2003; Touriya et al. 2003; Nakadate et al. 2005; Berrebi et al. 2005; Atarhouch et al. 2007; Chow et al. 2007; Yanagimoto et al. 2012), investigations of interspecific hybridization (Daguin et al. 2001; Pacheco et al. 2002; Yao et al. 2015), and phylogenetic analysis (DeBry and Seshadri 2001; Berrebi et al. 2005; Creer 2007; Near and Cheng 2008; He et al. 2008; Pinho et al. 2010).

The universal specificity of primers may depend on the conservation of exon sequences whereas the efficiency of isolating a single copy of the target nuclear genome may be a function of the number of duplicate gene copies, paralogs, and/or pseudogenes. Ribosomal proteins (RPs), which play fundamental roles in protein synthesis, are the most highly conserved proteins among eukaryotes, and each protein is typically encoded by a single gene (Wool 1979; Wool et al. 1995; Kenmochi et al. 1998; Yoshihama et al. 2002). Therefore, RP genes may be particularly suitable for designing universal primers. In spite of this potential, the number of studies reporting universal primers for the amplification of RP gene introns of aquatic animals is small (Chow and Hazama 1998; Pinho et al. 2010; Chow et al. 2015). Here we report the development of universal primer pairs to amplify 128 intron regions of 41 RP genes in fishes, which may be potentially applicable to distantly related fish species.

Materials and methods

The intron/exon structures of human 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 (http://www.ncbi.nlm.nih.gov/genbank/). Reference cDNA data of Danio rerio, Ictalurus punctatus, Salmo salar, Takifugu rubripes, and Paralichthys olivaceus, corresponding to orthologs of the human RP genes were obtained from the GenBank database. These fish species are taxonomically distant one another and offer abundant data set of ribosomal protein gene. The Orders to which these fish species belong were Cypriniformes, Siluriformes, Salmoniformes, Tetraodontiformes, and Pleuronectiformes, respectively, and we anticipated that primers designed using conserved sequences among these distant fish species may have a universal specificity. The draft genome of the Pacific bluefin tuna, Thunnus orientalis (accession nos. BADN01000001–BADN01133062 in the DNA Data Bank of Japan) (Nakamura et al. 2013) was also utilized. The Pacific bluefin tuna belongs to the Order Perciformes. Multiple sequence alignments were performed using ClustalX version 1.83 (Thompson et al. 1997), and conserved exon regions were visually inspected. Rules for primer design included; primer length of 20 nucleotides, less than two degenerate bases, a moderate GC content (approximately 50 %), no degenerated bases within last two positions, and avoiding presence of the primer secondary structure produced by inter- and intra-molecular interactions. Furthermore, more than 20 bp distances was placed between the splicing site and 3′-end of at least one of a primer in each pair, which may be used to authenticate amplicons by nucleotide sequence analysis. All these procedures were performed by eyes.

Because intron size cannot be predicted and there may be considerable variations even between closely related taxa, draft genome sequences of the Pacific bluefin tuna were used to select primer pairs for PCR testing. 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. These fishes belong to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes, respectively, and used to evaluate universal specificity of the primer pairs selected. PCR amplification was carried out in a 12 µL final volume containing 1 µL of template DNA (10–50 ng/µL), 1.2 µL of 10 × reaction buffer (containing 20 mM MgCl2), 1 mM of each deoxynucleotide triphosphate, 0.4 µM of each primer, and 0.5 U of EX Taq polymerase (Takara Bio, Inc., Shiga, Japan). The same reaction conditions were used for all primer pairs, in which the reaction mixtures were preheated to 94 °C for 4 min, followed by 35 amplification cycles (denaturing at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 50 s), with a final extension at 72 °C for 7 min. PCR products were electrophoresed on 1.5 % agarose gels (UltraPure Agarose; Invitrogen Corporation, Carlsbad, CA, USA) and stained with ethidium bromide. The gel images visualized on transilluminator were captured by CCD camera.

Results and discussion

Based on the relatively high degree of conservation of the exon sequences among distant reference fish species and human, a total of 128 primer pairs for the amplification of 41 RP genes were designed for EPIC PCR. Among the 41 RP genes listed in Table 1, whole or partial sequences of 29 genes (Table 1, dagger) were found in the draft genome sequences of the Pacific bluefin tuna. Twelve primer pairs (Table 1, asterisk) expected to amplify 500–1000 bp fragments in the Pacific bluefin tuna were selected, and the PCR amplification results are shown in Fig. 1 and summarized in Table 2. All amplified fragments were larger than those expected for intronless amplicons when separated using agarose gel electrophoresis. Amplified fragments were observed for all primer pairs in the Japanese puffer (lane 2), starry flounder (lane 3), and the Pacific bluefin tuna (lane 4), whereas no amplification was observed for primer pair 17 in the Japanese eel (lane 6), for primer pair 86 in broadbanded thornyhead (lane 5), and for three primer pairs (17, 68, and 86) in the Japanese pilchard (lane 1). The Orders to which the Japanese eel, broadbanded thornyhead, and the Japanese pilchard belonged were not involved in primer design, which may cause the slight difference in amplification success between the former and later fish groups. The highest score for single fragment amplification was observed in starry flounder (10 of 12 primer pairs) and the lowest was observed in the Pacific bluefin tuna and broadbanded thornyhead (6 of 12 primer pairs), whereas the total score for single fragment amplification was relatively high (62.5 %) (45 of 72 primer pairs). Clear or nearly two-band PCR products were observed for primer pair 26 in Japanese pilchard and Pacific bluefin tuna, for primer pair 38 in Japanese eel, for primer pair 49 in starry flounder and Pacific bluefin tuna, and for primer pair 68 in Pacific bluefin tuna and broadbanded thornyhead. These could be heterozygotes and potential candidates for population genetic analysis. Amplification of three or more fragments was relatively fewer incidence (12 of 72 primer pairs), which may be attributable to paralogs, pseudogenes, and/or non-specific annealing. Elevation of annealing temperature may decrease number of amplified fragments as demonstrated previously (Chow et al. 2015). The sizes of amplified fragments of Pacific bluefin bluefin tuna using nine primer pairs (Table 2, dagger) corresponded to those expected from the genomic data, whereas fragments amplified using three primer pairs (5, 90, and 94) were shorter than expected, indicating that these three primer pairs annealed and amplified non-target regions of the Pacific bluefin tuna genome.

Table 1 Sequences of 128 primer pairs for 41 ribosomal protein (RP) genes
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Fig. 1
figure 1

Agarose gel electrophoresis images of the PCR products amplified using 12 selected primer pairs for ribosomal protein gene (RP) introns. RP genes are shown at the top-left in each gel image and the primer pair number is shown in parenthesis (see also Table 1). The left and right most ends in each gel are the size marker, and the 1st to 6th lanes are the Japanese pilchard, the Japanese puffer, starry flounder, the Pacific bluefin tuna, broadbanded thornyhead, and the Japanese eel, respectively

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Table 2 Number of fragments amplified by 12 primer pairs in six fish species
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Although nucleotide sequence analysis may be necessary to characterize and authenticate the amplicons of interest, the present study offers new sets of nuclear primers that are potentially applicable to wide variety of fish taxa and can be an initial step toward isolating single-copy nuclear DNA sequences. Subsequent polymorphism detection may also provide a more informative database for genetic species identification and population studies.