Received 16 October 2005; accepted in principle 14 December 2005; accepted for publication 17 January 2006

Received 16 October 2005; accepted in principle 14 December 2005; accepted for publication 17 January 2006 A new strategy for species identification ...
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Received 16 October 2005; accepted in principle 14 December 2005; accepted for publication 17 January 2006

A new strategy for species identification of planktonic larvae: PCR-RFLP analysis of the internal transcribed spacer region of ribosomal DNA detected by agarose gel electrophoresis or DHPLC SHI WANG1, ZHENMIN BAO1*, LINGLING ZHANG1, NING LI1, AIBIN ZHAN1, WENBO GUO2, XIAOLONG WANG1 and JINGJIE HU1 1

Laboratory of Marine Genetics and Breeding (MGB), Division of Life Science and Technology,

Ocean University of China, Qingdao 266003, People’s Republic of China 2

Key Laboratory of Mariculture (KLM), Division of Life Science and Technology, Ocean

University of China, Qingdao 266003, People’s Republic of China

Key words:

species identification, PCR-RFLP, internal transcribed spacer, planktonic larvae,

DHPLC Correspondence: Zhenmin Bao, Laboratory of Marine Genetics and Breeding (MGB), Division of Life Science and Technology, Ocean University of China, Qingdao 266003, People’s Republic

of

China;

Tel:

(+86)-532-82031960;

Fax:

(+86)-532-82031960;

[email protected].

Communicating Editor - KJ Flynn © The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

1

E-mail:

Abstract Planktonic survey is important for understanding the dynamics and structure of populations or communities. However, the small size of early planktonic larvae, usually less than 500µm, make it difficult or impossible to discriminate closely related taxa based on morphological characters. We developed a new strategy for species identification of planktonic larvae, namely, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) region detected by agarose gel electrophoresis or denaturing high-performance liquid chromatography (DHPLC). A total of 4 restriction enzymes were selected for PCR-RFLP analysis. Based on any one of 4 restriction maps, 12 commercial shellfish species could be differentiated from each other in agarose gel analysis. But more accurate results were obtained in DHPLC analysis. As an example of larval identification, hybrid larvae were successfully identified by their showing diagnostic peaks of both parents in DHPLC analysis. These results suggest that this strategy can meet the demands for plankton survey and studies of hybridization.

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Introduction Many marine invertebrates pass through a planktonic larval phase during their early life-history. In sessile and benthic organisms having little mobility, the planktonic larval phase represents the only opportunity to acquire new habitats and to cross with other populations (Hosoi et al. 2004). The resultant variability in recruitment is potentially important for the dynamics and structure of populations and communities (Caley et al. 1996). However, many of the youngest larval stages, particularly of bivalves, are very similar in appearance during their early development (Garland and Zimmer 2002). Ecological studies of early life stages of marine organisms have been hampered by the difficulties to identify larvae and juveniles at species level (Levin 1990). Traditionally, morphological characters were used to identify planktonic larvae (Chanley and Andrews 1971; Nichols and Black 1994). With the application of scanning electromicroscopy and diverse optical imaging systems, more and more morphological characters became applicable for larval identification (Lutz et al. 1982; Pena et al. 1998; Tiwari and Gallager 2003). However, morphological characters are known to be heavily influenced by environmental factors (Seed 1968) and it is difficult to be implemented for the investigation of early stages of larvae in plankton. Instead, a variety of molecular techniques such as immunological techniques (Miller et al. 1991; Demers et al. 1993; Paugam et al. 2000), allozyme techniques (Hu et al. 1992), species-specific DNA probing (Medeiros-Bergen et al. 1995; Bell and Grassle 1998), RAPD technique (Coffroth and Mulawka 1995; Andre et al. 1999), multiplex PCR (Hare et al. 2000) have been developed for the identification of planktonic larvae. However, all of these techniques have the intrinsic disadvantages: variation of the reliability at different stages in immunological analysis (Medeiros-Bergen et al. 1995), difficulty of scoring allozymes in allozyme analysis (Wood et al. 2003), requirement of previous knowledge about the genome of interest in species-specific probing analysis (Andre et al. 1999) and low stability and reproducibility of RAPD and multi-PCR analysis. In this study, we developed a new strategy for species identification of planktonic larvae, that is, PCR-RFLP analysis of the internal transcribed spacer (ITS) region of ribosomal DNA detected by agarose gel electrophoresis or denaturing high-performance liquid chromatography (DHPLC). This strategy mainly bases on the following advantages: (1) PCR-RFLP technique is a simple, rapid and more cost-effective technique in comparison with techniques mentioned above and has been applied to identification of some marine invertebrate larvae (Evans et al. 1998; Toro 1998; Hosoi et al. 2004). (2) The sequence of ITS is conservative within a species, but diverse between species (Insua

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et al. 2003). Therefore ITS is an ideal marker for PCR-RFLP analysis and has been used for species identification (Guillamon et al. 1998; chambers et al. 1999; Fernandez et al. 2001; Lopez-Pinon et al. 2002). (3) DHPLC is a recently developed technique. Because of rapidity, automation, high sensitivity and specificity, DHPLC is a good choice for analysis of PCR products, restriction fragments, and other DNA fragments under non-denaturing mode (Devaney and Marino 2001). Previous studies were mostly targeted to identify specified species but not identify multiple species simultaneously. Moreover, only a few studies concerned about identification of hybrid larvae based on morphological characters (Nichols and Black 1994) or molecular techniques (Wood et al. 2003). Establishment of methods for identification of hybrid larvae would facilitate studies of hybridization, and larval dispersal and gene flow (Wood et al. 2003). In this study, identification standards were first set up for 12 commercial shellfish species based on the new strategy. All 12 species can be differentiated from each other based on any one of 4 restriction maps in agarose gel analysis, but more accurate results were obtained in DHPLC analysis. As an example of larval identification, hybrid larvae were successfully identified by the presence of diagnostic peaks attributed to both parents in DHPLC analysis. These results suggest that this strategy can meet the demands for plankton survey and studies of hybridization. Method Sampling of adults, spawning and cross Twelve commercial shellfish species used in this study are shown in Table I. Adult individuals of 12 species were sampled from different origins (local markets in Qingdao, aquacultural hatcheries in Penglai and Weihai, Shandong Province, China). The muscle tissues were preserved at -20ºC. Fifteen individuals of each species from at least two origins were used in PCR-RFLP analysis. Artificial hybridization between C. farreri and A. irradians was carried out in laboratory. The parental scallops were induced to spawn using thermal and chemical stimuli. After fertilization, hybrid larvae were reared at 20ºC. Samples of larvae were taken at the trochophore larval stage (about 20 hours after fertilization) and stored in ethanol at 4ºC. DNA Extraction

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The genomic DNA of adult shellfish was extracted from frozen muscle tissues with phenol/chloroform extraction as described by Sambrook et al. (Sambrook et al., 1989). The larval DNA extraction method described as follows. Larvae were transferred from ethanol to a cavity slide and left until the ethanol evaporated completely. Then the larvae were rinsed and agitated with sterile pure water. In total, 30 larvae were individually isolated through micro-operation and then transferred on a mounted needle to a 0.2mL PCR tube containing 10µL of STE solution (100mM NaCl; 10mM Tris-Cl [pH8.0]; 1mM EDTA [pH8.0]; 0.5mg/mL proteinase K). The tubes were kept at 56ºC for 30 min to break the cells and expose the DNA, and then 95ºC for 5 min to inactivate proteinase K. PCR Amplification, Cloning and Sequencing PCR amplifications were set up in a 20µL volume composed of 100ng adult genomic DNA or 10µL larval DNA solution, 0.2µM each primer, 2mM MgCl2, 0.2mM each dNTP, 1×PCR reaction buffer,

and

1U

Taq

polymerase

(Promega).

A

pair

of

primers

(forward:

GTTTCTGTAGGTGAACCTG; reverse: CTCGTCTGATCTGAGGTCGGA) were used according to the primers designed for Mytilus mussels (Heath et al., 1995). These anneal at the 3'end of the 18S rRNA gene and the 5'end of the 28S rRNA gene, amplifying the ITS-1, 5.8S gene, and ITS-2. Thermal cycling used a PTC-100 cycler (MJ research, USA). All cycling began with an initial denaturation at 94ºC for 3 minutes, followed by 30 cycles of 94ºC for 1 min, 54ºC for 1 min, and 72ºC for 1min 30s, and a final extension at 72ºC for 10 minutes. PCR product of each species was ligated into pMD18-T (Takara Inc., Dalian, China) and subsequently transformed into E.coli DH5α cells. Recombinant clones were screened for inserts of correct size. Positive clones were sequenced using a 3730 automatic sequencer (ABI Inc., USA). The nucleotide sequences of some species have been deposited in the GenBank database under accession numbers AY690599-AY690600, AY695798-AY695803. Restriction Enzyme Selection and Digestion Restriction enzymes were selected by comparing multiple restriction maps of 12 species provided by the BioEdit program (Hall 1999) which uses the REBASE database (Roberts and Macelis 2001). Digestions were performed in 20µL volumes, containing 10µL PCR product, 3U restriction

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enzyme, and 2µL buffer supplied by the manufacturer. The reaction was incubated at 65ºC (MseI and TaqI) or 37ºC (HaeIII and MboI) for 8 hours and then stopped by inactivating the restriction enzyme at 80ºC for 20min. Agarose Gel Electrophoresis Before DHPLC analysis, PCR and RFLP products of all samples were separated on 3% agarose gels and were visualized under UV light after staining with ethidium bromide. Then the fragment lengths of PCR and RFLP products were compared to the expected ′in silico′ fragment lengths. DHPLC Analysis DHPLC were performed on a 3500HT WAVE DNA fragment analysis system (Transgenomic Inc., USA). The PCR products or restriction fragments were loaded on a C18 reverse-phase column based on alkylated polystyrene-divenylbenzene particles (DnaSepTM column). The temperature of separation column was fixed at 50ºC which is the temperature for size separation. Size separation analysis was carried out with an acetonitrile gradient formed by mixing buffers A (0.1M triethylammonium acetate, 0.025% acetonitrile) and B (0.1M triethylammonium acetate, 25% acetonitrile). The optimized gradient was 0.0 min in 71% buffer A–29% buffer B, 0.5 min in 66% buffer A–34% buffer B, 5.1 min in 43% buffer A–57% buffer B, 9.7 min in 37% buffer A–63% buffer B, 14.2 min in 35% buffer A–65% buffer B, and 18.8 min in 34% buffer A–66% buffer B. Flow-rate was 0.9 mL/min and DNA was detected with a UV detector at 260 nm. PCR and RFLP products of 12 sequenced clones were used in DHPLC analysis for setting up standard maps. RFLP products of hybrid larvae with two enzymes (MseI and TaqI) were detected by DHPLC and then compared with the standard maps of two parents. Results PCR Amplification The ITS fragments could be amplified successfully from all adult individuals of 12 species and 97% hybrid larvae in this study. PCR products of 12 species detected by agarose gel electrophoresis

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and DHPLC are shown in Fig. 1. In agarose gel analysis, some species could be differentiated from each other by the ITS lengths. But when length difference between two species is less than 20bp, it is difficult to accurately differentiate them (e.g. A1-2, B1-2 and C1-2; G1-2 and H1-2; I1-2 and J1-2 in Fig. 1 I). In DHPLC analysis, all 12 species could be well differentiated from each other based on the retention times, which suggests that DHPLC is superior to agarose gel electrophoresis in differentiating multiple species based on the ITS lengths. Restriction Enzyme Selection and Digestion In total, 4 restriction enzymes (MseI, TaqI, HaeIII and MboI) were selected for PCR-RFLP analysis. Sizes of restriction fragments expected for the selected enzymes are shown in Table II. Because signals of fragments less than 50bp are usually weak and not always perceptible in DHPLC analysis, they were not included in Table II. PCR-RFLP results of 12 species with 4 restriction enzymes are shown in Fig. 2 (detected by agarose gel electrophoresis) and Fig. 3 (detected by DHPLC). Restriction fragment lengths of all individuals were identical to the expected and exceptional individuals were not found. In agarose gel analysis, 12 species could be differentiated from each other based on the species-specific bands with any one of the 4 enzymes. Restriction fragments less than 100bp could not always be identified in agarose gel analysis. Moreover, when differences of several fragment lengths in a species were less than 20bp, these restriction fragments were difficult to separate on agarose gel (e.g. G1-2, J1-2, K1-2 and L1-2 in Fig. 2 III; F1-2 in Fig. 2 IV). Considering the resolution of all species-specific bands in agarose gel analysis, MseI and MboI that have less cut sites are thought to be better than TaqI and HaeIII in multiple species identification. In DHPLC analysis, 12 species could also be differentiated from each other very well based on any one of the 4 restriction maps. Restriction fragments that had little length difference in a species could be readily identified in DHPLC analysis (e.g. J1-2, K1-2 and L1-2 in Fig. 2 III; F1-2 in Fig. 2 IV). Based on this advantage of DHPLC, TaqI and HaeIII that have more cut sites may be more efficient than MseI and MboI in multiple species identification when DHPLC is used. In DHPLC analysis, identification of a hybrid larva with MseI and TaqI is shown in Fig. 4. The restriction maps of the hybrid larva exhibited diagnostic peaks of both parents. So the larva could be definitely identified as the hybrid offspring of C. farreri and A. irradians.

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Disscussion Sample collection and PCR amplification In order to decrease the risk of sampling from one genetically distinct subpopulation, adult individuals of 12 species were sampled from different origins. For the same purpose, this strategy was also adopted in other studies (Fernandez et al. 2001; Lopez-Pinon et al. 2002). The ITS fragments were amplified successfully from all adult individuals of 12 species even though these species belong to different classes. The primers used in this study seem to be widely applicable to shellfish species. The proportion (97%) of successful amplification for the hybrid larvae is higher than those reported in other studies (Corte-Real et al. 1994; Evans et al. 1998; Hosoi et al. 2004), which suggests that the larval DNA extraction method used in this study is more effective. Feasibility of PCR-RFLP analysis of ITS sequences for species identification PCR-RFLP analysis of ITS sequences for sea shellfish identification has been reported previously (Fernandez et al. 2001; Lopez-Pinon et al. 2002). In this study, for all adult individuals of 12 species, the results of PCR-RFLP analysis based on 4 restriction enzymes were identical to the expected. Exceptional individuals were not found. PCR-RFLP analysis in this study is thus relatively stable. Even though individuals that contain mutated restriction sites really exist, results of PCR-RFLP analysis based on 4 restriction enzymes can be integrated to make the correct taxonomic assessment. If extremely precise conclusions are needed in some conditions, it can be accomplished through sequencing PCR products followed by a detailed comparison of individual sequences. It should be mentioned that PCR-RFLP analysis of ITS sequences for species identification may not be suitable for all sea shellfish species. Yu et al. (Yu et al., 2000) demonstrated that the ITS-1 sequences of Tridacna crocea exhibited very high polymorphism, which caused the variation of restriction maps in different geographically separated populations. Thus it is necessary to investigate the variation of ITS sequences from different geographically separated populations before application of this technique. Advantages of application of ITS sequences to species identification

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The gene sequences applying to PCR-RFLP analysis commonly have two origins: mitochondria (16s rDNA, COI, cytB et al.) and nuclear genome (ITS, GLU-5 et al.). Different from mitochondria genes that are maternally inherited, nuclear genes contain genetic information of both parents and thus can be applied to identification of hybrids. Hosoi et al. (Hosoi et al., 2004) developed PCR-RFLP analysis based on the large subunit (LSU) rRNA gene D1/D2/D3 region for identification of bivalve larvae of multiple species collected from Maizuru Bay. Because of the high conservatism of rRNA genes, only 1 enzyme (HaeIII) of the over 100 enzymes that would theoretically cleave at one or more locations could provide species-specific restriction patterns for all analyzed species. The difficulty of screening more enzymes for simultaneous identification of multiple species limits the application of this method. In this study, based on PCR-RFLP analysis of ITS sequences, each of 4 enzymes could produce species-specific restriction patterns for all analyzed species. Moreover, compared with LSU- and SSU-rDNA, ITS region evolves more rapidly and is more useful in comparing closely related species (Shao et al. 2004). Therefore, ITS sequence appears as an ideal marker for multiple species identification when compared with other nuclear markers. Application of DHPLC system in species identification DHPLC is based on the principle of liquid chromatography. The DNA fragments mixed with TEAA buffer were separated in DNAsep catridge. Separated DNA fragments are detected by ultraviolet detector. Corresponding signals are input into computer and subsequently converted into corresponding peak signals. The longer the DNA fragment is, the later the corresponding peak appears. The DHPLC system has the advantages of high precision, high sensitivity, automatization, good repeatability, rapidity (Xiao and Oefner 2001; Frueh and Noyer-Weidner 2003). It is very suitable for species identification of a small quantity of samples or samples owning complex DNA makeup. Specially, with its high resolving power, it allows the separation of fragments that differ by as little as 1% in chain length up to about 200 base pair (Devaney et al. 2000). The advantages of DHPLC in identification of hybrid larvae are well demonstrated in this study. The hybrid larvae have the following characteristics: (1) The hybrid larvae can not be distinguished from other scallop larvae using only morphological characters. (2) The hybrid larvae sampled are very small (less than 100µm). (3) The genome of the hybrid larvae is heterozygous. Identification of hybrid larvae is equal to identification of two species at the same time, which is more difficult than common larval

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identification. Successful identification of hybrid larvae suggests that DHPLC is more competitive than other detecting means in application of plankton survey and studies of hybridization. Although all 12 species could be differentiated from each other in agarose gel or DHPLC analysis, the two detecting means should be chose according to different conditions in practice. In some instances (e.g. large numbers of samples need be processed), agarose gel is an effective detection means for PCR-RFLP analysis because of decreasing the cost of time and money. When more accurate results are needed, DHPLC becomes a necessary means for PCR-RFLP analysis because of its high precision and high sensitivity. Identification of other shellfish species for plankton survey Considering the request of identification of other shellfish species in plankton survey, we compared the ITS sequences of other shellfish species in GenBank database. Some of them can be differentiated from each other merely by the ITS length, whereas others that have similar ITS lengths can also be distinguished from each other by PCR-RFLP analysis used in this study. Therefore, this new strategy can meet the demands for plankton surveys. Acknowledgements The authors thank Prof. Guanpin Yang and Prof. Quanqi Zhang for reviewing this manuscript. This work is supported by grants from Hi-Tech Research and Development Program of China (2003AA603022 and 2005AA603220), National Natural Science Foundation of China (30300268), Outstanding Scientists Research Foundation of Shandong Province (01BS10). References Andre, C., Lindegarth, M., Jonsson, P. R. et al. (1999) Species identification of bivalve larvae using random amplified polymorphic DNA (RAPD): differentiation between Cerastoderma edule and C. lamarcki. J. Mar. Biol. Ass. UK., 79, 563-565. Bell, J. L. and Grassle, J. P. (1998) A DNA probe for identification of larvae of the commercial surclam (Spisula solidissima). Mol. Mar. Biol. Biotechnol., 7, 127-137. Caley, M. J., Carr, M. H., Hixon, M. A. et al. (1996) Recruitment and the local dynamics of open

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marine populations. Annu. Rev. Ecol. Syst., 27, 477-500. Chambers, S. M., Sawyer, N. A. and Cairney, J. W. G. (1999) Molecular identification of co-occurring Cortinarius and Dermocybe species from southeastern Australian sclerophyll forests. Mycorrhiza, 9, 85-90. Chanley, P. E. and Andrews, J. D. (1971) Aids for identification of bivalve larvae of Virginia. Malacologia, 11, 45-119. Coffroth, M. A. and Mulawks, J. M. (1995) Identification of marine invertebrate by means of PCR-RAPD species-specific markers. Limnol. Oceanogr., 40, 181-189. Corte-Real, H. B. S. M., Holland, P. W. H. and Dixon, D. R. (1994) Inheritance of a nuclear DNA polymorphism assayed in single bivalve larvae. Mar. Biol., 120, 415-420. Demers, A., Lagadeug, Y., Dodson, J. J. et al. (1993) Immunofluorescence identification of early life history stages of scallops (Pectinidae). Mar. Ecol. Prog. Ser., 97, 83-89. Devaney, J. M., Girard, J. E. and Marino, M. A. (2000) DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography. Anal. Chem., 72, 858-864. Devaney, J. M. and Marino, M. A. (2001) Purification methods for preparing polymerase chain reaction products for capillary electrophoresis analysis. Methods Mol. Biol., 162, 43-49. Evans, B. S., White, R. W. G. and Ward, R. D. (1998) Genetic identification of asteroid larvae from Tasmania, Australia, by PCR-RFLP. Mol. Ecol., 7, 1077-1082. Fernandez, A., Garcia, T., Asensio, L. et al. (2001) PCR-RFLP analysis of the internal transcribed spacer (ITS) region for identification of 3 clam species. J. Food Sci., 66, 657-661. Frueh, F. W. and Noyer-Weidner, M. (2003) The use of denaturing high-performance liquid chromatography (DHPLC) for the analysis of genetic variations: impact for diagnostics and pharmacogenetics. Clin. Chem. Lab. Med., 41, 452-461. Garland, E. D. and Zimmer, C. A. (2002) Techniques for the identification of bivalve larvae. Mar. Ecol. Prog. Ser., 225, 299-310. Guillamon, J. M., Sabate, J., Barrio, E. et al. (1998) Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region. Arch. Microbiol., 169, 387-392. Hall, T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor program for Windows 95/98NT. Nucl. Acids Symp. Ser., 41, 95-98. Hare, M. P., Palumbi, S. R. and Butman, C. A. (2000) Single-step species identification of bivalve larvae using multiplex polymerase chain reaction. Mar. Biol., 137, 953-961.

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Heath, D. D., Rawson, P. D. and Hilbish, T. J. (1995) PCR-based nuclear markers identify alien blue mussel (Mytilus spp.) genotypes on the west coast of Canada. Can. J. Fish. Aquat. Sci., 52, 2621-2627. Hosoi, M., Hosoi-Tanabe, S., Sawada, H. et al. (2004) Sequence and polymerase chain reaction-restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: simple and widely applicable technique for multiple species identification of bivalve larva. Fisheries Sci., 70, 629-637. Hu, Y. P., Lutz, R. A. and Vrijenhoek, R. C. (1992) Electrophoretic identification and genetic analysis of bivalve larvae. Mar. Biol., 113, 227-230. Insua, A., Lopez-Pinon, M. J., Freire, R. et al. (2003) Sequence analysis of the ribosomal DNA internal transcribed spacer region in some scallop species (Mollusca: Bivalvia: Pectinidae). Genome, 46, 595-604. Levin, L. (1990) A review of method for labeling and tracking marine invertebrate larvae. Ophelia, 32, 115-144. Lopez-Pinon, M. J., Insua, A. and Mendez, J. (2002) Identification of four scallop species using PCR and restriction analysis of the ribosomal DNA internal transcribed spacer region. Mar. Biotechnol., 4, 495-502. Lutz, R., Goodsell, J. and Castagnan, M. (1982) Preliminary observations on the usefulness of hinge structure for identification of bivalve larvae. J. Shellfish Res., 2, 65-70. Medeiros-Bergen, D. E., Olson, R. R., Conroy, J. A. et al. (1995) Distribution of holothurian larvae determined with species-specific genetic probes. Limnol. Oceanogr., 40, 1225-1235. Miller, K. M., Jones, P. and Roughgraden, J. (1991) Monoclonal antibodies as species-specific probes in oceanographic research: examples with intertidal barnacle larvae. Mol. Mar. Biol. Biotechnol., 1, 35-47. Nichols, S. J. and Black, M. G. (1994) Identification of larvae: the zebra mussel (Dreissena polymorpha), quagga mussel (Dreissena rosteriformis bugensis), and Asian clam (Corbicula fluminea). Can. J. Zool., 72, 406-417. Paugam, A., Pennec, M. L. and Genevieve, A. F. (2000) Immunological recognition of marine bivalve larvae from plankton samples. J. Shellfish Res., 19, 325-331. Pena, J. B., Rios, C., Pena, S. et al. (1998) Ultrastructural morphogenesis of pectinid spat from the western Mediterranean: a way to differentiate seven genera. J. Shellfish Res., 17, 123-130. Roberts, R. and Macelis, D. (2001) REBASE-restriction enzymes and methylases. Nucleic Acids

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Res., 29, 268-269. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning a Laboratory Manval, 2nd edn. Cold Spring Harbor Laboratory Press, New York. Seed, R. (1968) Factors influencing shell shape in the mussel Mytilus edulis. J. Mar. Biol. Ass. UK., 48, 561-584. Shao, P., Chen, Y., Zhou, H. et al. (2004) Genetic variability in Gymnodiniaceae ITS regions: implications for species identification and phylogenetic analysis. Mar. Biol., 144, 215-224. Tiwari, S. and Gallager, S. (2003) Machine learning and multiscale methods in the identification of bivalve larvae. Proceedings of the Ninth IEEE International Conference on Computer Vision, 2, 1-8. Toro, J. E. (1998) Molecular identification of four species of mussels from southern Chile by PCR-based nuclear markers: the potential use in studies involving planktonic surveys. J. Shellfish Res., 17, 1203-1205. Wood, A. R., Beaumont, A. R., Skibinski, D. O. F. et al. (2003) Analysis of a nuclear-DNA marker for species identification of adults and larvae in the Mytilus edulis complex. J. Moll. Stu., 69, 61-66. Xiao, W. and Oefner, P. J. (2001) Denaturing high-performance liquid chromatography: a review. Hum. Mutat., 17, 439-474. Yu, E. T., Juinio-Menez, M. A. and Monje, V. D. (2000) Sequence Variation in the Ribosomal DNA internal transcribed spacer of Tridacna crocea. Mar. Biotechnol., 2, 511-516.

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Table and Figure Legends

Table I *, each specific name is designated a capital letter for being conveniently referred to in the Fig. 1-3. Fig. 1. PCR products of 12 species detected by agarose gel electrophoresis (I) and DHPLC (II). (M1: 1kb DNA ladder; M2 or M: 100bp DNA ladder; other symbols are the same as those in Table 1) Fig. 2. PCR-RFLP results of 12 species with MseI (I), TaqI (II), HaeIII (III), MboI (IV) detected by agarose gel electrophoresis. (M: 100bp DNA ladder; other symbols are the same as those in Table 1) Fig. 3. PCR-RFLP results of 12 species with MseI (I), TaqI (II), HaeIII (III), MboI (IV) detected by DHPLC. (M: 100bp DNA ladder; other symbols are the same as those in Table 1) Fig. 4. Identification of a hybrid larva with MseI (I), TaqI (II) detected by DHPLC. (M: 100bp DNA ladder; A: C. farreri; B: A. irradians; C: hybrid larva; a: specific peak of C. farreri; b: specific peak of A. irradians)

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Tables Table I Twelve commercial shellfish species investigated in present study Class

Subclass

Order

Family

Genus and species

Lamellibranchia

Pteriomorphia

Pterioida

Pectinidae

Chlamys farreri (A*) Chlamys nobilis (B) Patinopecten yessoensis (C) Argopecten irradians (D)

Heterodonta

Ostridae

Crassostrea gigas (J)

Arcoida

Arcidae

Scapharca sativa (F)

Mytiloida

Mytilidae

Mytilus edulis (H)

Veneroida

Solenidae

Sinonovacula constricta (G)

Veneridae

Ruditapes philippinarum (I) Ruditapes variegata (K) Meretrix meretrix (L)

Gastropoda

Prosobranchia

Archaeogastropoda

15

Haliotidae

Haliotis discus hannai (E)

Table II. Size of restriction fragments expected to be produced by the selected enzymes Specific name

ITS length

MseI

TaqI

HaeIII

MboI

C. farreri (A)

741bp

203 168 151 143 76

325 164 95 58 52

526 117 98

425 171 84

C. nobilis (B)

731bp

380 193 158

351 322 58

526 117 88

418 193 84

P. yessoensis (C)

734bp

234 193 98 82 66 61

312 221 143 58

529 205

431 187 56

A. irradians (D)

769bp

395 204 143

345 250 93 58

343 244 182

333 249 100 51

807bp

413 394

384 110 109 93 58

342 202 145 99

423 214 106

S. sativa (F)

883bp

409 317 94 63

365 160 156 78 66 58

798 85

230 219 204 194

S. constricta (G)

928bp

491 437

237 158 148 94 88 78 58 53

219 172 167 147 135 59

370 310 165

M. edulis (H)

948bp

458 197 169 70 54

268 153 152 117 83 76 58

597 235 116

369 247 83 79 69

1215bp

724 491

329 253 173 166 111 58 58

581 126 110 103 68 68 64

529 342 278 57

324 321 169 127 95 83

222 199 144 117 97 86 80 77 62

74

58

234 209 204 171 149 107 67 55

548 353 88 84 74

442 272 267 107 76 62 58

410 152 147 131 83 77 61 51 50

527 438 218 88

513 276 161 119 103 93 81 61 58

370 252 157 133 109 103 86 77 57

55

57

H. discus hannai (E)

R. philippinarum (I) C. gigas (J)

1225bp

R. variegata (K)

1307bp

819 488

M. meretrix (L)

1520bp

993 404 83

16

565 504 247 155

Figures

Fig. 1

(I)

(II)

17

Fig. 2

(I)

(II)

(III)

(IV)

18

Fig. 3

I

II

III

IV

19

Fig. 4

I

II

20

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