Sequence analysis of the ribosomal DNA internal transcribed spacers and 5.8S ribosomal RNA gene in representatives of the clam family veneridae (Mollusca: Bivalvia).
KEY WORDS: Veneridae, rDNA, internal transcribed spacer, 5.8S rRNA gene, phylogenetic analysis, species identification
The internal transcribed spacers (ITSs) of nuclear ribosomal DNA (rDNA) is one of the most extensively sequenced molecular markers, and the region is a component of a rDNA cistron, which consists of 18S, ITS1, 5.8S, ITS2 and 28S. ITSs exist in several hundred copies in most eukaryotes. They are located in one or several loci and distributed in one or several chromosomes. The nuclear rDNA copies within a genome can be highly homogeneous because of concerted evolution of intra and interchromosomal loci. ITS1 and ITS2 are noncoding regions located in rDNA between 18S and 5.8S rRNA genes, and between 5.8S and 28S rRNA genes, respectively (Insua et al. 2003, Jansen et al. 2006, Won & Renner 2005).
Because ITSs sequences show more divergence than their flanking coding regions and are easily amplified, they are routinely used to distinguish related species and to infer phylogenetic relationships from populations to families and even higher taxonomic levels (Coleman & Vacquier 2002). In bivalve mollusks, a variety of methods, such as PCR amplification alone, PCR amplification followed by restriction analysis or sequencing was used to differentiate related species (Ding et al. 2004, Fernandez et al. 2001, Insua et al. 2003, Kenchington et al. 2002, Lopez-pinon et al. 2002, Yu et al. 2000) and exploring the phylogenetic relationship (He et al. 2005, King et al. 1999, Vidigal et al. 2000, Vidigal et al. 2004, Yu et al. 2001) among bivalve species.
The family Veneridae is a group of bivalve mollusks prevalent in seawater all over the world. There are more than 500 species, many of which are commercially valuable and ecologically crucial because of their dominance in benthic communities. A study of the phylogenesis of Veneridae was carried out using partial sequence of the mitochondrial 16S rRNA gene (Canapa et al. 2003).
The purpose of this study is to amplify and sequence the ITS1 and ITS2 regions of 6 Veneridae species, Meretrix meretrix, Cyclina sinensis, Mercenaria mercenaria, Protothaca jedoensis, Dosinia corrugata and Ruditapes philippinarum, to provide the basic characteristics of these sequences and to assess the similarity among species. The results from this study will provide useful information in species identification and phylogenetic analysis of Veneridae clams.
MATERIALS AND METHODS
Sample Collection and DNA Extraction
Specimens of Meretrix meretrix and Cyclina sinensis were collected from Dalian, Liaoning province, China, and Mercenaria mercenaria, Protothaca jedoensis, Dosinia corrugata and Ruclitapes philippinarum belonging to five subfamilies of Veneridae, from Lianyungang, Jiangsu province. Total genomic DNA was extracted from approximately 50 mg of adductor muscle following a modified CTAB protocol (Winnepenninckx et al. 1993). Briefly, the tissue was incubated for 15 min at 55[degrees]C in 600 [micro]l CTAB buffer containing 25 [micro]l 10 mg/mL proteinase K, homogenized with a pestle, and incubated for an additional 60 min. After extractions with saturated phenol and then chloroform: isoamyl alcohol (24:1), genomic DNA was ethanol-precipitated, resuspended in 50-[micro]l TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and stored at -20[degrees]C for future use.
PCR Amplification and Sequencing
ITS1 region was amplified using ITS1-F (5'- GGTGAACCTGCGGATGGA-3') and ITS1-R (5'- GCTGGCTGCGCTCTTCAT-3') as primers, which anneal to the 3' end of 18S ribosomal RNA gene and the 5.8S ribosomal RNA gene, respectively. ITS2 region was amplified using ITS2-F (5'- ATGAAGAGCGCAGCCAGC-3') and ITS2-R (5'- GGCTCTTCCCGCTTCACTC-3') as primers, which anneal to the 5.8S ribosomal RNA gene and the 5' end of 28S ribosomal RNA gene, respectively. Approximate locations of primers are shown in Figure 1. Two pairs of primer were designed based on sequence information obtained from GenBank (AY498751, AF202106, AF131019, AY 198756, AF 120559) using PrimerSelect software (DNAStar package version 5.01). PCRs were set up in a 50-[micro]L volume reaction mixture composed of 2 [micro]L genomic DNA (50 ng/[micro]L), 5.0 [micro]L 10x buffer (20 mM [Mg.sup.2+]), 1.0 [micro]L dNTP (10 mM each), 1.0 [micro]L each primer (25 [micro]M), 0.4 [micro]L AmpliTag DNA polymerase (5U/[micro]L BBI) and dd[H.sub.2]O to 50 [micro]L. Amplifications were carried out in a BIO-RAD thermal cycler (iCycler), using the reaction settings as follows: initial denaturation at 94[degrees]C for 4 min, followed by 35 cycles of denaturing for 40 s at 94[degrees]C, annealing for 30 s at 55[degrees]C, extension for 60 s at 72[degrees]C and a final extension for 7 min at 72[degrees]C. All amplified fragments were selected by 2% w/v agarose gels electrophoresis. The ITS1 and ITS2 regions were PCR amplified from at least 10 individuals from each of six species, but only two PCR products were selected and delivered to Shanghai Sangon Biological Engineering & Technology and Service Co. Ltd. (Shanghai, China) for sequence. The sequence was conducted in both forward and reverse directions using the amplification primers by ABI prism 377 automatic DNA sequencer.
[FIGURE 1 OMITTED]
The forward and reverse sequences were assembled using SeqManII software in DNAStar Package version 5.01 to obtain ITS1 and ITS2 sequences, and each of the different sequences was registered as an ITS1 or ITS2 haplotype in GenBank. Each newly determined sequence was checked against exiting haplotypes using DNAstar and then the sequence was registered as a new haplotype. Because ITS1-R and ITS2-F primers anneal to the same region in 5.8S rDNA (Fig. 1), ITS1 and ITS2 can be assembled into an ITS, and the complete sequence of 5.8S ribosomal RNA gene can be obtained. The boundaries of the coding and spacer regions were determined by comparison with the sequence information of Arctica islandica (GenBank accession No. AF 202106). The sequences were edited and analyzed using the program EditSeq. ITS sequences containing the 5.8S ribosomal RNA gene across six species were aligned using program MegAlign of DNAStar package using the Clustal W method. The trees were produced by Neighbor-Joining (NJ) and Maximum parsimony (MP) methods using MEGA software Version 3.1 (Kumar et al. 2004).
The ITS1 and ITS2 regions were PCR amplified and sequenced among six species, the ITS1 and ITS2 were assembled into a single ITS sequence. For M. meretrix, M. mercenaria, D. corrugata and R. philippinarum, the identical ITS sequences were detected between 2 individuals within a species and named haplotypes WD1, YLI, BL1, FL1, respectively. For other species, C. sinensis and P. jedoensis, the ITS sequence were different between 2 individuals within a species and were named haplotypes QD1, QD2 for C. sinensis, and JL1, JL2 for P. jedoensis. Figure 2 showed the alignment of the ITS sequences including 5.8S rDNA, spanning 18S and 28S rDNA among eight haplotypes from six Veneridae species, and Table 1 displayed the length and GC contents of ITS1, 5.8S ribosomal DNA, and ITS2, and GenBank accession number.
[FIGURE 2 OMITTED]
Sequence Analysis of the First Internal Transcribed Spacer
The length of ITS1 sequence ranged from 522 bp to 900 bp, and GC contents from 57.66% to 65.62% in six species (Table 1). As seen in Table 1 interspecific ITS1 sequences showed remarkable divergence and obvious length polymorphism. The GC contents in ITS1 were higher than AT contents in all species. M. meretrix had the longest ITS1 sequence (900 bp) with 61.67% GC contents, and two trinucleotide microsatellites [(AGT).sub.n] were observed (Fig. 2). As well, a repeat sequence [(AGT).sub.n] GAAAAA(G)GCGAAGGAGCCGCTGGCCCTTC was found occurring twice in ITS1 region in this species. D. corrugata had the shortest ITS1 sequence (522 bp) with 57.66% GC contents. P. jedoensis, M. mercenaria, R. philippinarum and C. sinensis had intermediate ITS1 size, 864 bp, 679 bp, 632 bp and 585 bp, respectively. A dinucleotide microsatellite [(TA).sub.n] was observed in the ITS1 region of C. sinensis (Fig. 2).
ITS1 sequences of six species were aligned. The percentages of sequence divergence and identity were showed in Table 2. Interspecific ITS1 sequence divergences were very high, because of the presence of transition, transversion and indels (insertions/ deletions). The percentages of interspecific sequence divergence were from 27.2% (M. mercenaria and P. jedoensis) to 102.6% (M. meretrix and R. philippinarum). P. jedoensis exhibited 0.1% intraspecific sequence divergence between two individuals. For other species, M. meretrix, C. sinensis, M. mercenaria, D. corrugata and R. philippinarum, the ITS1 sequence was identical between two individuals within a species.
We found one relatively conserved motif in the ITS1 region across the six species of Veneridae (Fig. 2), with a size of 54 bp showed 18 polymorphic sites, 12 of which were transition and six were transversion.
Sequence Analysis of the Second Internal Transcribed Spacer
The length of ITS2 sequences ranged from 281 bp to 412 bp and GC contents from 65.21% to 67.87% (Table 1). The interindividual variation for ITS2 was detected in both P. jedoensis and C. sinensis. In contrast, M. meretrix, M. mercenaria, D. corrugata and R. philippinarum had the same ITS2 sequences in two individuals. The GC contents of ITS2 sequences were higher than AT contents in all species. M. meretrix had the longest ITS2 sequence (412 bp) with 65.29% GC content, P. jedoensis had the shortest ITS2 sequence (281 bp to 282 bp) with 67.38% to 67.62% GC contents; C. sinensis, M. mercenaria, D. corrugata and R. philippinarum had intermediate ITS2 sequence (Table 1). A quadrunucleotide microsatellite [(AGCG).sub.n] in M. meretrix and three dinucleotied microsatellites [(AG).sub.n] in M. meretrix, R. philippinarum and D. corrugata were observed (Fig. 2)
ITS2 regions from 8 haplotypes in six species were aligned. Percentages of sequence divergence and identity were listed in Table 3. ITS2 showed remarkable interspecific sequence divergences and obvious length polymorphism but to less compared with ITS1. Percentages of interspecific sequence divergence were from 21.6% (P. jedoensis and M. mercenaria) to 80.7% (M. meretrix and C. sinensis). Intraspecific sequence divergences in ITS2 region between two individuals were detected in C. sinensis (0.5%) and P. jedoensis (1.1%). For other species, M. meretrix, M. mercenaria, D. corrugata and R. philippinarum, the ITS2 sequence was identical between two individuals within a species.
We also detected two relatively conserved motifs in ITS2 region across six species (Fig. 2). The first relatively conserved motif with a size of 31 bp displayed five transition or transversion polymorphic sites, and the second with a size of 24 bp showed three transition or transversion polymorphic sites.
Sequence Analysis of the 5.8S Ribosomal RNA Gene
Through assembling ITS1 and ITS2 sequences, we obtained complete sequences of the 5.8S ribosomal RNA gene of six Veneridae species. The length of the 5.8S ribosomal RNA gene was 157 bp in all species (Fig. 2), and the GC contents ranged from 57.96% to 58.60% depending on species. The sequence divergences ranged from 0.0% to 6.0% across six species (Table 4). The 5.8S rRNA genes contained 10 polymorphic sites in these species, four of which were transition and six were transversion. Moreover, P. jedoensis, M. mercenaria, D. corrugata and R. philippinarum showed identical 5.8S rDNA sequence.
Phylogenetic Analysis in Terms of ITS2 Containing Partial 5.8S and 28S Ribosomal RNA Gene Sequences
Using ITS2 spanning 5.8S and 28S rDNA sequences as a molecular marker, the phylogenetic tree of Veneridae clam was constructed using Arctica islandica (GenBank accession number AF202106) as an outgroup species with Neighbor-Joining (NJ) and Maximum parsimony (MP) methods (Fig. 3).
[FIGURE 3 OMITTED]
The topology of the tree by MP method was similar to that by NJ method. Tree topologies showed that six Veneridae species were in the same group and forming a clade. In Veneridae, P. jedoensis was first grouped with M. mercenaria with a high bootstrap value of 94% in MP tree and 99% in NJ tree and then formed a clade with D.corrugata with a lower bootstrap value of 66% in MP tree and 80% in NJ tree; in contrast, M. meretrix, C. sinensis and R. philippinarum formed a monophyletic group. This indicated that P. jedoensis and M. mercenaria has a closer relationship than other species.
In contrast to ITS2, the construction of phylogeny using ITS1 was not applicable because of some disadvantages, such as ITS1 length variation, the presence of tandem repeated sequences, and large number of indels. ITS1 sequences analysis, using MP and NJ methods, gave poor resolution in genus level. Therefore ITS1 was not used for phylogenetic analysis.
The current study provided information about the nucleotide sequences of ITS1 and ITS2 regions, complete sequence of the 5.8S ribosomal RNA gene in six Veneridae clams (M. meretrix, C. sinensis, M. mercenaria, P. jedoensis, D. corrugata and R. philippinarum). Their characteristics and variation were demonstrated through PCR amplification and sequencing. The length of ITS1 in the family Veneridae were longer than that in four Pectinidae scallop with size 209 bp to 276 bp for ITS1 and 270 bp to 294 bp for ITS2 (Insua et al. 2003), respectively. It should be noted that the length range in ITS1 reported in the current study was among the largest observed in bivalves. The size of ITS is species-dependent and the difference could be significant among species. The largest ITS1, such as that in Ladybird beetle Exocomus quadripustulatus, was as long as 2572 bp (Von der Schulenburg et al. 2001), and the shortest one only 70 bp to 80 bp in Acropora species (Odorico & Miller 1997). Depending on species, the length of ITS2 can be twice or more that of ITS1 (Dahlgren et al. 2000, Fernandez et al. 2001, Yu et al. 2000), both situation, the length of ITS1 similar to ITS2 or larger than ITS2 were also reported (Chen et al. 2002, Coleman & Vacquier 2002). The GC contents ranged from 57.66% to 65.62% for ITS1 and from 65.21% to 67.87% for ITS2 in Veneridae tested in the current study. These species had higher GC contents than other bivalve species. For instance, the GC contents in Pectinidae scallop were 43% to 49% for ITS1 or 44% to 49% for ITS2 (Insua et al. 2003) and 51.9% to 55.5% for ITS2 in Pearl Oyster (He et al. 2005). A frequent characteristic of spacers is a balanced GC content between ITS1 and ITS2, and this also occurs in the Veneridae spacers, this fact could indicate the coevolution between the two spacers at the level of base composition.
Both ITS1 and ITS2 regions in family Veneridae exhibit extensive sequence variation and obvious length polymorphisms, as is similar to Crustacea Eriocheir formoca (Chu et al. 2001) and other bivalves (Ding et al. 2004). In this study, the construction of topology in Veneridae clam using ITS1 sequence information is not applicable because of the high length variation, the presence of tandem repeated sequences and large number of indels in ITS1. However, it may be useful in phylogentic analysis at lower taxonomic levels, such as subspecies, breeds or population levels. In contrast, the interspecific ITS2 sequence similarity in these species were higher than that of ITS1, and considering the length of ITS2 were shorter than that of ITS1, providing advantage and convenience in designing primers and sequencing, ITS2 is an ideal candidate for the study of genetic structure in Veneridae. The further study on the efficiency for combing the method in the current study with other techniques, such as PCR-RFLP, is promising.
In this study, a relatively conserved motif in ITS1 region and two relatively conserved motifs in ITS2 region were found in these species. This indicated that these motifs might be involved in certain nucleotide acid-related functions, such as in rRNA processing (Insua et al. 2003). Beside, two trinucleotide microsatelillites [(AGT).sub.n] in M. meretrix and a dinucleotide microsatelillite [(TA).sub.n] in C. sinensis were found locating in ITS1 region, and a quadrunucleotide microsatellite [(AGCG).sub.n] and three dinucleotied microsatellites [(AG).sub.n] was found locating ITS2 region. These microsatellites may be served as good markers in future studies. In the bivalve species, a dincleotide microsatellite [(GT).sub.n] and a trinucleotide microsatellite [(TAC).sub.n] were found in ITS sequence of Lasmigona (King et al., 1999), but so far there has been no report from Veneridae.
Although interindividual sequence divergences in ITS1 and ITS2 regions were detected in C. sinensis (0.3% for ITS2) and P. jedoensis (0.1% for ITS1 and 0.6% for ITS2). Because of the presence of polymerase and sequencing error, the sequences cannot be thought as different only if when sequence divergence more than 0.9% (Kong et al. 2002), C. sinensis and P. jedoensis showed no intraspecific variations in ITS1 and ITS2. The 5.8S ribosomal RNA genes were highly conserved across these species studied in the current study, and had a length of 157 bp, which was reported in other bivalves, such as Pectinidae scallop species (A. opercularis, M. varia, H. distortus, P. maximus) (Insua et al. 2003) and Arctica islandica (Dahlgren et al. 2000).
The spacer regions, ITS1 and ITS2, of the rDNA are widely and routinely used in analysis of species relationships by using a phylogenetic reconstruction method in various organisms. It was successfully applied in analysis of phylogenetic relationship among the Biomphalaria species and among Pearl Oysters, and the conclusions from phylogenetic tree were well in agreement with those from analysis based on morphological systematics and other molecular techniques, such as polymerase chain reaction and restriction fragment length polymorphism analysis (He et al. 2005, Vidigal et al. 2000, Vidigal et al. 2004). Our study demonstrated that ITS1 provided weak phylogenetic signal in discrimination of Veneridae, whereas, ITS2 regions were effective in identifying phylogenetic relationships among Veneridae species. In this study, the tree obtained by ITS2 sequence analysis revealed that P. jedoensis and M. mercenaria, belonging to subfamilies Chioninae, has a close relationship than other species, belonging to subfamilies Meretricinae, Tapetinae and Cyclininae. Phylogenetic analysis of ITS2 sequences through both methods generated trees with similar topologies that were very concurrent with the morphological taxonomy proposed by Keen. Therefore ITS2 sequence characteristics are an efficient tool in reconstruction of evolutionary relationship among these organisms, and they can be applied in establishment of species relationship, or reevaluation of the traditional taxonomy.
The authors thank Dr. Liu FJ in The Institute of Environmental and Human Health, Texas Tech University, USA, for advice on this paper, and Mr. Wang HZ in Zhewang Agricultural Technology Service Center of Ganyu County, Lianyungang, Jiangsu province, China, for providing the M. mercenaria samples. This Work was jointly supported by National Key Technologies R & D Program in the 10th five-year plan of China (2004BA526B0403), Jiangsu Key Laboratory of Marine Biotechnology (2006HS001), Natural Science Foundation Program (Z2004024) and Start-up Package Program for attracting talented people (KK03035) from HuaiHai Institute of Technology (HHIT).
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CHENG HAN-LIANG, (1) * MENG XUE-PING, (1) JI HONG-JU, (2) DONG ZHI-GUO (1) AND CHEN SHU-YIN (2)
(1) HuaiHai Institute of Technology, Jiangsu Key Laboratory of Marine Biotechnology, Lianyungang 222005, P.R. China; (2) Institute of Marine Fisheries, Jiangsu Province, Nantong 226007, P. R. China
* Corresponding author. E-mail: CHL3139@163.com
TABLE 1. The length and GC contents of ITS1, 5.8S ribosomal DNA, and ITS2 in eight haplotypes from six species ITSI Length GC Species (Sample size) Haplotype (bp) (%) Meretrix meretrix (2) WD1 900 61.67 Cyclina sinensis (2) QD1 585 61.03 QD2 585 61.03 Mercenaria mercenaria (2) YL1 679 63.03 JL1 864 65.62 Protothaca jedoensis (2) JL2 864 65.51 Dosinia corrugata (2) BL1 522 57.66 Ruditapes philippinarum (2) FL1 632 63.92 5.8S rDNA ITS2 Length GC Length GC Species (Sample size) (bp) (%) (bp) (%) Meretrix meretrix (2) 157 58.60 412 65.29 Cyclina sinensis (2) 157 58.60 386 66.06 157 58.60 388 65.21 Mercenaria mercenaria (2) 157 57.96 361 67.87 157 57.96 282 67.38 Protothaca jedoensis (2) 281 67.62 Dosinia corrugata (2) 157 57.96 294 66.33 Ruditapes philippinarum (2) 157 57.96 379 67.28 Species (Sample size) GenBank accession number Meretrix meretrix (2) DQ132788, DQ191390 Cyclina sinensis (2) DQ132787, DQ191387 DQ132787, DQ191388 Mercenaria mercenaria (2) DQ132789, DQ191391 DQ220290, DQ191389 Protothaca jedoensis (2) DQ220291, DQ132791 Dosinia corrugata (2) DQ346656 Ruditapes philippinarum (2) DQ399404 TABLE 2. Percentage of sequence divergence (below triangle) and identity (above triangle) of ITS1 sequences across seven haplotypes from six species by Clustal W method Species 1 2 3 4 1 Meretrix meretrix *** 31.8 31.8 28.7 2 Cyclina sinensis QD1 91.7 *** 35.4 33.5 3 Mercenaria mercenaria 96.8 46.9 *** 45.4 4 Protothaca jedoensis JL1 88.9 50.4 27.2 *** 5 Protothaca jedoensis JL2 88.4 50.8 27.4 0.1 6 Dosinia corrugata 96.0 43.5 28.2 40.6 7 Ruditapes philippinarum 102.6 63.3 58.4 52.1 Species 5 6 7 1 Meretrix meretrix 28.8 31.0 27.2 2 Cyclina sinensis QD1 33.5 35.6 30.8 3 Mercenaria mercenaria 45.1 48.5 30.2 4 Protothaca jedoensis JL1 99.9 41.8 32.0 5 Protothaca jedoensis JL2 *** 41.8 32.0 6 Dosinia corrugata 40.2 *** 31.2 7 Ruditapes philippinarum 52.5 66.0 *** TABLE 3. Percentage of sequence divergence (below triangle) and identity (above triangle) of ITS2 sequences across eight haplotypes from six species by Clustal W method Haplotypes 1 2 3 4 1 Meretrix meretrix *** 35.8 36.5 34.3 2 Cyclina sinensis QD1 80.7 *** 96.6 40.1 3 Cyclina sinensis QD2 79.7 0.5 *** 40.3 4 Mercenaria mercenaria 61.3 56.4 56.7 *** 5 Protothaca jedoensis JL1 46.8 48.1 47.6 22.6 6 Protothaca jedoensis JL2 47.0 49.2 48.6 21.6 7 Dosinia corrugata 55.2 52.6 53.0 27.0 8 Ruditapes philippinarum 72.8 72.0 71.8 51.4 Haplotypes 5 6 7 8 1 Meretrix meretrix 37.8 39.0 39.7 33.2 2 Cyclina sinensis QD1 39.9 41.8 37.6 39.2 3 Cyclina sinensis QD2 39.6 42.6 37.6 37.9 4 Mercenaria mercenaria 53.0 52.8 62.4 39.2 5 Protothaca jedoensis JL1 *** 97.2 50.9 42.4 6 Protothaca jedoensis JL2 1.1 *** 52.1 42.9 7 Dosinia corrugata 24.5 23.3 *** 44.1 8 Ruditapes philippinarum 47.6 47.1 49.8 *** TABLE 4. Percentage of sequence divergences (below triangle) and base variation (above triangle, transitions/transversions) of 5.8S rDNA across six species by Clustal W method Species 1 2 3 4 1 Meretrix meretrix *** 9 (3/6) 7 (3/4) 7 (3/4) 2 Cyclina sinensis 6.0 *** 4 (2/2) 4 (2/2) 3 Mercenaria mercenaria 4.6 2.6 *** 0(0/0) 4 Protothaca jedoensis 4.6 2.6 0.0 *** 5 Dosinia corrugata 4.6 2.6 0.0 0.0 6 Ruditapes philippinarum 4.6 2.6 0.0 0.0 Species 5 6 1 Meretrix meretrix 7 (3/4) 7 (3/4) 2 Cyclina sinensis 4 (2/2) 4 (2/2) 3 Mercenaria mercenaria 0 (0/0) 0 (0/0) 4 Protothaca jedoensis 0 (0/0) 0 (0/0) 5 Dosinia corrugata *** 0 (0/0) 6 Ruditapes philippinarum 0.0 ***