INTRODUCTION
Pyropia species is one of the major seaweeds cultivated in Korea, Japan, and China (Niwa et al. 2004, Hwang et al. 2014). Strain identification at the intraspecific level of Pyropia yezoensis (Ueda) M. S. Hwang & H. G. Choi is important to the Pyropia aquaculture industry for the maintenance of aquaculture strains and for the development of new cultivars. Although various molecular markers have been developed to discriminate P. yezoensis at the inter / intraspecific levels (Niwa et al. 2004, 2005, Hwang et al. 2005, Park et al. 2008, Niwa and Kobiyama 2009), more efficient tools for precise discrimination are still required.
Red algal mitochondrial genomes (mtDNAs) have interesting genes and structural composition (Odintsova and Yurina 2002, Smith et al. 2012, Yang et al. 2015). Such genetic features have provided useful information for interpreting the evolutionary history of Bangiophycean species (Smith et al. 2012). Particularly, the gene composition and structural variations of mtDNAs of Pyropia species have provided useful genetic information at the inter / intraspecific levels (Hwang et al. 2013, 2014, Hughey et al. 2014).
The study of mtDNAs in Pyropia / Porphyra (Bangiales, Rhodophyta) showed that they exhibit diverse variation in genome size and genetic structure, and also that they contain higher number of introns and intronic open reading frames (ORFs) in comparison to other red algae (Hughey et al. 2014, Hwang et al. 2014, Yang et al. 2015). Specifically, structural variation in the exon / intron structure of the large subunit ribosomal RNA gene (rnl) and cytochrome c oxidase subunit 1 gene (cox1) was reported in Pyropia / Porphyra species (Hwang et al. 2013, 2014, Hughey et al. 2014). These genetic variations significantly affected the size of mitochondrial genome of Pyropia / Porphyra species (Hwang et al. 2013, 2014, Hughey et al. 2014); a similar phenomenon was observed in the brown alga, Pylaiella littoralis (Ikuta et al. 2008).
Hwang et al. (2013, 2014) and Kong et al. (2014) examined the complete genome of mtDNA of P. tenera and P. yezoensis, and explored the genetic variation of exon / intron structure and the phylogenetic relationship among the intronic ORFs. Hughey et al. (2014) also reported the presence of intronic ORFs and the size variations of mtDNA of P. perforata. In this study, we examined the exon / intron structure of rnl and cox1 of P. yezoensis at the intraspecific level. The presence / absence of intron patterns were examined among the different strains of P. yezoensis.
MATERIALS AND METHODS
We analyzed 40 strains of P. yezoensis deposited in the Seaweed Research Center (National Institute of Fisheries Science, Mokpo, Korea) (Table 1). Blades of 27 P. yezoensis strains were collected from Korean coastal regions or aquaculture farms, and the conchocelis filaments were induced and cultured in the Provasoli enrichment medium (Provasoli 1968) at 20°C under white fluorescent irradiation of 20 μmol photon m−2 s−1 (14 L : 10 D cycle). Thirteen Japanese P. yezoensis strains were also analyzed (Table 1). The conchocelis filaments were used for the molecular analyses. Total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions with an extension of incubation time by one hour.
To reveal the exon / intron structure of rnl and cox1 regions of P. yezoensis, we designed new primer sets having the binding sites on each exon region. These exon-primed intron-crossing (EPIC) markers showed high efficiency in revealing the exon / intron structure (Palumbi and Baker 1994, Bierne et al. 2000). Three and four primer sets were developed to reveal the genetic structure of rnl and cox1 regions, respectively (Tables 2 & 3, Fig. 1).
The mtDNAs of P. tenera (Kjellm.) N. Kikuchi, M. Miyata, M. S. Hwang & H. G. Choi (NC_021475, Hwang et al. 2013) and P. yezoensis (NC_017837, Kong et al. 2014; KF561997, Hwang et al. 2014) were used as reference sequences for the primer design. We performed long range polymerase chain reaction (LPCR) to amplify the exon / intron structure of rnl and cox1 genes (Hwang et al. 2013). LPCR was carried out in a 20 μL volume containing 10–50 ng of total genomic DNA, utilizing the LA Taq polymerase system (TaKaRa Bio, Shiga, Japan). The amplification conditions were as follows: 2 min at 94°C, followed by 30 cycles at 94°C for 10 s, 60°C for 30 s, and at 68°C for 5 min, with a final extension at 68°C for 7 min. Band patterns of LPCR products were analyzed by agarose gel electrophoresis.
We also determined the sequences of the cox1 in 40 P. yezoensis strains, which has been used as the standard target DNA region for DNA barcoding of red algae (Saunders 2005). To amplify the cox1 region, we designed a new primer, cox1e1R16F (5′-TGCCAAGACAGGTACTGCT-3′) having the binding position from 30738 to 30756 in P. yezoensis (NC_017837), located at the 5′ end of cox1 gene. The primer pair, cox1e1R16F (this study) and cox1e1Fpy1R (Hwang et al. 2013), was used for the amplification and sequencing of cox1 region in P. yezoensis samples. The amplification conditions were as follows: 3 min at 95°C, followed by 40 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 7 min. PCR products were sequenced commerically (Genotech, Daejeon, Korea) and the sequences were assembled in Sequencher 5.4.6 (Gene Codes Corporation, Ann Arbor, MI, USA). Pairwise distances were calculated using the MEGA 6.0 program (Tamura et al. 2013).
RESULTS
PCR amplification of cox1 gene generated a 549 bp in all 40 samples, excluding the primer binding sites. Analysis of cox1 sequences revealed 4 haplotypes among the 27 Korean P. yezoensis strains (C1–C4) (Table 1). On the other hand, 13 strains of Japanese P. yezoensis shared the same haplotype (C5). The pairwise distance among the haplotypes ranged from 0.2 to 0.9% (1–5 bp).
We found four exons and three introns in the rnl in all of the sampled P. yezoensis strains, also reported in previous studies (Hwang et al. 2013, 2014); and five exons and four introns were revealed in the cox1 (Fig. 1). The results revealed five genotypes (R111, R110, R101, R011, and R010) in the rnl intron and six genotypes (C1101, C0111, C0101, C0100, C0001, and C0000) in the cox1 intron based on the existence of introns (Tables 1 & 3). Combined structural variations of rnl and cox1 exhibited 12 genotypes among the 40 P. yezoensis strains (Table 1). Korean P. yezoensis exhibited 12 genotypes (H1–H12) among 27 strains. The genotype 5 (H5) was dominant, and broadly distributed in nine Korean strains. All of the Japanese P. yezoensis strains had the same gene structure (H12) (Table 1).
DISCUSSION
The presence of introns and intronic ORFs is the main reason for variation in the size of the mitochondrial genome of Pyropia species. Hwang et al. (2013, 2014) was the first to report this intron structure from P. tenera and P. yezoensis in red algae. In this study, we examined the genetic features of intron structure of rnl and cox1 of P. yezoensis at the intraspecific level, and found 12 genetic types from 40 culture strains from Korea and Japan (Tables 1 & 3).
The sequences of mitochondrial cox1 region have been recommended as DNA barcode markers to identify animal and algal species, and the cox1 region exhibited high efficiency in describing species boundaries (Hebert et al. 2003, Saunders 2005). In this study, five haplotypes of the cox1 gene were revealed for 40 P. yezoensis strains (Table 1). Korean P. yezoensis exhibited 4 haplotypes (haplotypes C1–C4) for 27 strains with 0.2–0.9% pairwise genetic distances. All the 13 Japanese P. yezoensis strains had the same cox1 sequence (haplotype C5), and showed 0.2–0.7% pairwise distances from the Korean haplotypes.
Some of the P. yezoensis strains yielded the same cox1 sequence, but different introns (Table 1). The most variable haplotype C3 of cox1 was sub-divided into nine genotypes (H1, H4, H5, H6, H7, H8, H9, H10, and H12), using the combined rnl and cox1. The genotype C1 was sub-divided into three genotypes (H1, H3, and H12). Therefore, the gene structure of the rnl and cox1 regions exhibited higher genetic resolution to discriminate P. yezoensis strains in comparison to the cox1 sequence variations in these samples.
Besides cox1 sequences, several genetic markers discriminating the Pyropia / Porphyra strains at the inter / intra-specific level, such as SSU ribosomal DNA (rDNA), ITS1, rbcL, and the RuBisCO spacer have been studied (Brodie et al. 1998, Lindstrom and Fredericq 2003, Hwang et al. 2005, Nelson et al. 2006, Niwa et al. 2009, Sutherland et al. 2011, Kucera and Saunders 2012, Mols-Mortensen et al. 2014, Guillemin et al. 2016). Nuclear SSU rDNA and rbcL sequences were shown to discriminate the Korean P. yezoensis strains from the Japanese P. yezoensis strains (Hwang et al. 2005). In this study, cox1 sequence analysis divided the 27 Korean P. yezoensis strains into four types; the presence / absence of introns in rnl and cox1 genes divided the 27 Korean P. yezoensis strains into 12 types; and the combination of both markers divided the Korean P. yezoensis strains into 14 types. These results implied that the combination of both markers was more useful in comparison to a single marker.
On the other hand, only one genotype was found in all the Japanese P. yezoensis strains, which was in contrast to the Korean strains. All Japanese P. yezoensis strains were aquaculture strains, except JP-Tu1. Niwa et al. (2008, 2009) reported the presence of three genotypes in 13 Japanese aquaculture strains of P. yezoensis; 11 strains had the same type, and each of the remaining two strains had a unique type based on ITS1 region analysis. The Japanese materials used in this study had only one genotype, which is the same type as that of P. yezoensis f. narawaensis reported by Niwa et al. (2008, 2009).
Theoretically, the presence / absence of introns can produce eight types in the rnl and 16 types in the cox1, and 128 types from the combination of rnl and cox1 of P. yezoensis. Additional genotypes of intron could be found across global samplings of P. yezoensis strains, including other Pyropia species in Korea, Japan, and China. In this study we found high intraspecific genetic variation in the rnl and cox1 genes, and showed that these two genes can better discriminate strains of P. yezoensis. These molecular markers can be useful to maintain the diversity of aquaculture strains and for the development of new cultivars.
