Animal Biology 68 (2018) 353–365 brill.com/ab Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa (Pleuronectiformes: Cynoglossidae) Li Gong1,2, Wei Shi3,4, Min Yang3,4 and Xiaoyu Kong3,4,∗ 1 National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, 316022, Zhoushan, China 2 National Engineering Research Center for Facilitated Marine Aquaculture, Marine Science and Technology College, Zhejiang Ocean University, 316022, Zhoushan, China 3 Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 510000, Guangzhou, China 4 South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, 510000, Guangzhou, China Submitted: October 22, 2017. Final revision received: March 16, 2018. Accepted: April 29, 2018 Abstract The eukaryotic ribosomal DNA (rDNA) cluster consists of multiple copies of three genes (18S, 5.8S, and 28S rDNA) and two internal transcribed spacers (ITS1 and ITS2). In recent years, an increasing number of rDNA sequence polymorphisms have been identified in numerous species. In the present study, we provide 33 complete ITS (ITS1-5.8S-ITS2) sequences from two Symphurus plagiusa in- dividuals. To the best of our knowledge, these sequences are the first detailed information on ITS sequences in Pleuronectiformes. Here, two divergent types (Type A and B) of the ITS1-5.8S-ITS2 rDNA sequence were found, which mainly differ in sequence length, GC content, nucleotide diversity (π), secondary structure and minimum free energy. The ITS1-5.8S-ITS2 rDNA sequence of Type B was speculated to be a putative pseudogene according to pseudogene identification criteria. Cluster analysis showed that sequences from the same type clustered into one group and two major groups were formed. The high degree of ITS1-5.8S-ITS2 sequence polymorphism at the intra-specific level indicated that the S. plagiusa genome has evolved in a non-concerted evolutionary manner. These re- sults not only provide useful data for ribosomal pseudogene identification, but also further contribute to the study of rDNA evolution in teleostean genomes. Keywords Blackcheek tonguefish; multigene family; non-concerted evolution; pseudogene; ribosomal RNA gene ∗ ) Corresponding author; e-mail: [email protected] © Koninklijke Brill NV, Leiden, 2018 DOI 10.1163/15707563-17000134 Downloaded from Brill.com09/26/2021 07:03:23AM via free access 354 L. Gong et al. / Animal Biology 68 (2018) 353–365 Introduction Ribosomal RNA gene (rDNA) plays a pivotal role in protein synthesis in eukary- otes and changes to these genes can profoundly affect overall function and growth of the organism (Weider et al., 2005). rDNA is composed of several to thousands of copies of tandemly repeated transcription units within the genome, with each unit consisting of three rRNA genes (18S, 5.8S and 28S) and two internal transcribed spacers (ITS1 and ITS2) (Long and Dawid, 1980; Reed & Phillips, 2000). The molecular evolution of rDNA differs fundamentally from that of single-copy genes. Individual rDNA copies do not evolve independently but instead appear to evolve in a concerted fashion, with the result that copies are similar within a species and different between species. Although the exact mechanisms determining concerted evolution remain unclear, two fundamental mechanisms – unequal crossing over and gene conversion – are thought to drive the homogenization process (Szostak & Wu, 1980; Mullins & Fultz, 1991). Nevertheless, it is important to point out that although gene homogenization appears to be common, intra-genomic variation has also been observed. Since the first cases were reported in the amphibian Xenopus laevis (Wegnez et al., 1972; Peterson et al., 1980) and in the teleost fish Misgur- nus fossilis (Mashkova et al., 1981), where two types of 5S rRNA transcripts were identified, an increasing number of studies have reported a high degree of intra- individual rDNA sequence polymorphism in many taxa (Zhuo et al., 1995; Hugall et al., 1999; Harpke & Peterson, 2006; Freire et al., 2010; Hoy & Rodriguez, 2013; Li et al., 2013). More recently, several different types of 18S rDNA and ITS1 se- quences have been found in fish species (Sajdak & Phillips, 1997; Krieger et al., 2006; Xu et al., 2009). These strikingly multiple variants suggest that the rate of ho- mogenization may be too slow to prevent significant levels of intra-genomic rDNA polymorphism. Therefore, it is considerably necessary to investigate the degree of this rDNA variation to accurately reflect polymorphism, especially since it cannot be assumed that only one type sequence exists in any given species. Among the rDNA polymorphism, pseudogenes are one of the predominant con- tributors and an increasing number of ribosomal pseudogenes have been described in various species (Márquez et al., 2003; Harpke and Peterson, 2008a; Zuriaga et al., 2015; Gong et al., 2016a). Identifying ribosomal pseudogenes is difficult due to the continuous changes between functional copies and pseudogenes; nevertheless, pseudogenes still possess characteristic features. The primary criteria for ribosomal pseudogene identification are truncated sequences combined with more indels (in- sertions and deletions), higher nucleotide diversity (π), lower GC content, a less stable secondary structure, and a lower minimum free energy (Bailey et al., 2003; Xu et al., 2009; Li et al., 2013). In the 18S rDNA of the stone flounder Kareius bi- coloratus, a pseudogene had a 15-22 bp deletion compared to the functional gene, and the nucleotide diversity (π) was also higher (0.0052 versus 0.0012) (Xu et al., 2009). In the 5.8S rDNA of the medicinal fungus Ophiocordyceps sinensis,the pseudogene was found to have a lower GC content (36.13% to 38.71%) than the Downloaded from Brill.com09/26/2021 07:03:23AM via free access L. Gong et al. / Animal Biology 68 (2018) 353–365 355 functional gene (49.68% to 50.32%), as well as lower secondary structure stability (−34.82 to −25.91 kcal mol−1 versus −41.50 to −34.48 kcal mol−1) (Li et al., 2013). In addition, variations in the conserved rRNA genes – especially indels – also are useful and swift indicators of the functionality of the connective ITS re- gion. One well-known case is applying the conserved 5.8S motif and its secondary structure for the pseudogenic ITS1 and ITS2 identification in three angiosperm taxa (Araceae, Quercus and Mammillaria) (Harpke & Peterson, 2008b). To date, rDNA sequence polymorphisms have been documented in three flat- fishes, including identification of 18S-ITS1 rDNA sequence polymorphisms in the stone flounder Kareius bicoloratus (Xu et al., 2009), 18S rDNA sequence poly- morphisms in Cynoglossus lineolatus (Gong et al., 2016a) and ITS2 sequence polymorphisms in the Zanzibar tonguesole Cynoglossus zanzibarensis (Gong et al., 2016b). However, to our knowledge, there have been no studies of the ITS region (ITS1-5.8S-ITS2) in teleosts. In the present study, we investigated polymorphic rDNA sequences in the blackcheek tonguefish, Symphurus plagiusa (Pleuronecti- formes: Cynoglossidae), a small demersal flatfish widely distributed in the Western Atlantic. The results revealed remarkable intra-genomic variation in the ITS re- gion, even including the very highly conserved 5.8S rDNA. Sequence alignments identified two types of ITS1, 5.8S and ITS2 – types A and B – coexisting in the S. plagiusa genome. Further comparisons of the sequence length, indel events, GC content, secondary structure, and minimum free energy between these two sequence types suggested that the Type A genes were functional and the Type B genes were putative pseudogenes. The results presented here may provide useful data for the study of the ITS region in flatfishes and may further contribute to our understand- ing of the evolutionary mode of rDNA sequences in teleostean fishes. Materials and methods Sampling, DNA extraction, PCR amplification, cloning and sequencing Two S. plagiusa individuals were collected from New York and Campoton, respec- tively, and were immediately stored in 95% alcohol. Total genomic DNA was ex- tracted from muscle tissue as per the instructions of a marine animal genomic DNA extraction kit (Tiangen Biotech Co., China). Based on a previous study (Kumar et al., 2013), a forward primer Z-18S-1720 (5-TCGCTACTACCGATTGGATGG TTTA-3) and a reverse primer F-28S-100 (5-GCTCTTCCCTCTTCACTCG-3) were designed for the amplification of 18S (partial)-ITS1-5.8S-ITS2-28S (partial) rDNA. In previous studies, the use of dimethyl sulfoxide (DMSO) was found to en- hance amplification diversity during PCR (Buckler et al., 1997; Gong et al., 2016b). Accordingly, 8% (generally no more than 10%) DMSO was added to the reaction system for the highest amplification efficiency. PCR was carried out in a 25 μl reaction volume containing 2.0 mM MgCl2, 0.4 mM of each dNTP, 0.5 μMof each primer, 1.0 U of Taq polymerase (TaKaRa, Beijing, China), 2.5 μlof10× Downloaded from Brill.com09/26/2021 07:03:23AM via free access 356 L. Gong et al. / Animal Biology 68 (2018) 353–365 Taq buffer, and approximately 50 ng of DNA template. PCR cycling conditions in- cluded an initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, an annealing at 50°C for 1.5 min, and elongation at 72°C for 1 min. The PCR reaction was completed by a final extension at 72°C for 10 min. PCR products were purified using a TaKaRa Agarose Gel DNA Purification Kit (TaKaRa) and were then inserted into the pMD19-T vector (TaKaRa). Sequencing was performed on an ABI genetic analyser (Applied Biosystems, Beijing, China). Data analyses Sequenced fragments were assembled using CodonCode Aligner (vers. 3, Codon- Code Corporation, Dedham, MA, USA). The boundaries of the coding genes and ITS region were determined using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov). Sequence alignments were performed using ClustalX 2.0 (Larkin et al., 2007) and were checked manually using BioEdit v7 (Hall, 1999).