Mitochondrial and nuclear DNA sequence variation of presumed Crassostrea gigas and Crassostrea angulata specimens: a new oyster species in Hong Kong?

Aquaculture 228 (2003) 15 – 25 www.elsevier.com/locate/aqua-online

Mitochondrial and nuclear DNA sequence variation of presumed Crassostrea gigas and Crassostrea angulata specimens: a new oyster species in Hong Kong? Pierre Boudry *, Serge Heurtebise, Sylvie Lape`gue Laboratoire de Ge´ne´tique et Pathologie, Station IFREMER, Ronce-les-bains, Mus-du-Loup, 17390 La Tremblade, France Received 4 April 2003; received in revised form 17 June 2003; accepted 19 June 2003

Abstract In several cases, oyster taxa have been misidentified owing to their high morphological plasticity, uncertain geographical range and undocumented introductions. Recently though, molecular techniques have been efficiently applied to discriminate between oysters and to quantify genetic divergence within and among species. In the present paper, we report mitochondrial (16S and COI) and nuclear (28S) DNA sequences of presumed Crassostrea gigas and Crassostrea angulata, two taxa of aquacultural importance. Mitochondrial DNA sequences are compared with previously published sequences and PCR-RFLP haplotypes. Within C. gigas, divergence was less than 0.5% for COI, and less than 0.2% for 16S. Within C. angulata, divergence was less than 1.1% for COI and 0.2% for 16S. Our results also confirm the close genetic relationship between C. gigas and C. angulata and further document their level of divergence: 2 – 3% for COI and 0.5 – 1% for 16S. However, the initially presumed C. gigas oysters farmed in Hong Kong (Pearl River delta), presented DNA sequences strongly divergent from both C. gigas and C. angulata: 13 – 14% for COI, 3 – 4% for 16S and 1.2 – 1.6% for 28S. The closest related species are C. gigas and Crassostrea nippona with a divergence of 12 – 13% for COI, 3 – 4% for 16S and 1.2 – 1.6% for 28S. Comparisons with existing DNA sequence data available in the nucleotide sequence databases shows that this is either a new species or that it corresponds to a species for which no DNA sequence is yet available. Further studies are required to document morphological characteristics and geographical range of this putative new species. D 2003 Elsevier B.V. All rights reserved. Keywords: Crassostrea; 16S gene; Cytochrome oxidase I gene; 28S gene; Taxonomy; Oysters

* Corresponding author. Tel.: +33-5-46-36-76-18; fax: +33-5-46-36-37-51. E-mail address: [email protected] (P. Boudry). 0044-8486/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0044-8486(03)00443-5

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1. Introduction Molecular DNA markers have become essential tools for the identification of marine species today. In oysters, due to their large level of phenotypic plasticity, morphology is often of limited value for unambiguous identification of specimens and for taxonomy as a whole. Additionally, long distance larval dispersal, combined with numerous intentional or accidental introductions, has often led to uncertainty as to the geographic range of many oyster species. Since hybridisation between related taxa has been demonstrated under laboratory conditions, this might also represent a complicating factor in the study of oyster populations. In this context, oyster systematics, the identification of oysters species and the knowledge of their geographical range, have greatly benefited from the development of molecular DNA markers in recent years. In some cases, different oyster taxa initially described in separate geographical areas have since been grouped as a single species following molecular DNA studies. For example, Anderson and Adlard (1994) proposed that Saccostrea commercialis and Saccostrea glomerata should be regarded as synonymous taxa on the basis of rDNA internal transcribed spacer data. More recently, Kenchington et al. (2002) suggested that Ostrea edulis and Ostrea angasi are conspecific. Conversely, DNA tools can also be used to correct misidentification of species and to confirm or revise their geographical range. For example, O’Foighil et al. (1999) confirmed the transoceanic range (New Zealand and Chile) of Ostrea chilensis using mitochondrial COI sequence data and proposed that dispersal by rafting was the most likely explanation for this distribution. Similarly, the mangrove oyster Crassostrea gasar was shown to be present not only along the coasts of Western Africa but also along the Atlantic coasts of South America (Lape`gue et al., 2002). In other cases, the distinction between oyster taxa was confirmed and their genetic divergence quantified on the basis of sequence data. For example, the genetic differentiation between Crassostrea gigas, Crassostrea ariakensis and Crassostrea sikamea, three Asian cupped oysters known to hybridise under laboratory conditions (Gaffney and Allen, 1993), was confirmed (Banks et al., 1993; O’Foighil et al., 1995) and markers were used to genetically identify pure stocks of C. sikamea introduced into the USA (Gaffney et al., 1998). Similarly, markers were also used to identify wild specimens sampled in the Ariake Sea (Japan), proving that C. sikamea was still present in this area (Hedgecock et al., 1999). However, many species have been described for which no molecular studies have yet been made to confirm their status. China is the country with the largest C. gigas production (in 1997: Mainland China: 2.3  106 metric tonnes, Taiwan: 24  103 metric tonnes, Hong Kong: 66 metric tonnes, according to FAO, 1999). However, many other different taxa have been reported along Chinese coasts and species identification is often uncertain. In Northern China, Crassostrea talienwhanensis, Crassostrea plicatula and C. gigas are considered as sibling species (Liu and Dai, 1998). In the East and South China, at least nine species have been described: C. ariakensis (Wakiya, 1929), Crassostrea belcheri (Sowerby, 1871), C. gigas (Thu¨nberg, 1793), Crassostrea inequivalvis (Sowerby, 1871), Crassostrea lugubris (Sowerby, 1871), Crassostrea nippona (Seki, 1934), Crassostrea paulucciae (Crosse, 1869), Crassostrea rivularis (Gould, 1861) and Crassostrea vitrefacta (Sowerby, 1871)

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(Bernard et al., 1993). In Taiwan, populations initially presumed to be C. gigas were shown to be Crassostrea angulata, a closely related taxon (O’Foighil et al., 1998), on the basis of COI PCR-RFLP (Boudry et al., 1998) and microsatellite markers (Huvet et al., 2000). On the basis of shell morphology, authors (e.g. Mok 1974a,b; Morton and Wong, 1975; Cheung and Wong, 1992; Chan et al., 1999) concluded that C. gigas was one of the oyster species present in Hong Kong. However, analysis of COI PCR-RFLP patterns of the presumed C. gigas specimens showed that they only present a new haplotype (‘‘G’’), suggesting that a ‘‘significant name change’’ would be necessary (Lam et al., in press). However, the genetic divergence between this haplotype and other Crassostrea species remained to be determined. In the present paper, we studied 16S, COI and 28S DNA sequence variation of oysters presumed to be C. gigas or C. angulata from various origins. Sequence data were compared with previously published sequences for other oyster species. The results demonstrate that specimens collected in Hong Kong are neither C. gigas nor C. angulata and might be classified as a new species.

2. Materials and methods 2.1. Studied material and corresponding PCR-RFLP haplotypes Presumed C. gigas and C. angulata specimens were collected from European (France, Spain, Portugal, United Kingdom) and Asian (China) populations. These populations had previously been studied using PCR-RFLP methods, leading to the definition of PCRRFLP COI haplotypes, as described in Boudry et al. (1998) and Lam et al. (in press). For each of the most frequent haplotypes (A, B, C, D, E, and G) two individuals were sequenced. Table 1 details the geographic origin of the studied samples, the taxa they belong to and the PCR-RFLP haplotype they exhibit. Moreover, two specimens of the Asian oysters C. belcheri and Crassostrea iredalei were studied with the 16S fragment to complete the phylogenetic analysis.

Table 1 Geographical origin, presumed identification before DNA studies and after PCR-RFLP analysisa of the studied samples Origin

Presumed taxa (before DNA studies)

Results after PCR-RFLP analysisa Haplotype

Taxa

Rio Mira (Portugal) Cadiz (Spain) Bangor (United Kingdom) Gravelines (France) Bangor (United Kingdom) Pearl River delta, Hong Kong (China)

C. C. C. C. C. C.

A B C D E G

C. C. C. C. C. ?

a

angulata angulata gigas gigas gigas gigas

Results from Boudry et al. (1998) and Lam et al. (in press).

angulata angulata gigas gigas gigas

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DNA extraction of ethanol-preserved gill fragments was performed either by a Chelexbased method, as described in Estoup et al. (1996), or by a phenol/chloroform method, as described by Moore (1993). 2.2. DNA sequencing We amplified the 16S mitochondrial fragment (16SrDNA: the large subunit rRNAcoding gene) with primers described by Banks et al. (1993), according to the protocol detailed in Boudry et al. (1998). A partial COI (Cytochrome Oxidase C subunit I) fragment was amplified according to the primers and conditions detailed in Folmer et al. (1994). We amplified the 28S ribosomal fragment (28SrDNA: the large subunit rRNA-coding gene) with primers described by Littlewood (1994). The PCR products were purified with a High Pure PCR Product Purification Kit (Boehringer-Mannheim, Germany). The sequencing reaction, consisting of a first step of denaturation (2 min, 92 jC) and 30 cycles (30 s, 95 jC, denaturating; 30 s, 50 jC, annealing; 1 min, 72 jC, elongation), was performed with the Sequitherm EXCELk II DNA sequencing kit-LC (Epicentre Technologies). The fragments were separated on a LiCorR 4200 automated DNA sequencer. All the novel sequences were submitted to the EMBL nucleotide sequence database. 2.3. DNA sequence analysis The COI sequences of mitochondrial haplotypes A, B, C, D, E, and G ; together with some sequences already obtained for C. gigas, C. angulata, Crassostrea virginica, C. sikamea, C. ariakensis (Accessions AF152565, AF152567, AF152566, AF152568 and AF152569, respectively; O’Foighil et al., 1998), C. nippona (Accession AF300616; Lee et al., 2000), C. belcheri and C. iredalei (Accessions AY038077, AY038078) were aligned with the software CLUSTALW (Thompson et al., 1994). The same procedure was performed for the 16S sequences of individuals corresponding to COI haplotypes A, B, C, D, E, and G and the specimens of the species C. belcheri and C. iredalei. These sequences where aligned together with some already obtained for C. gigas (Accession AF280611), C. virginica (Accession AF092285), C. gasar, Crassostrea rhizophorae (Accessions AJ312937 and AJ312938, respectively; Lape`gue et al., 2002), C. nippona, and C. ariakensis (Accessions AY007426 and AY007427, respectively; Lee et al., 2000). The 28S sequences obtained from two specimens from Hong Kong were compared with previously published sequences (Littlewood, 1994; O’Foighil and Taylor, 2000) for C. gigas (Accessions AF137051 and Z29546, respectively named C. gigas 1 and C. gigas 2), C. ariakensis (Accessions AF137052 and Z29548, respectively named C. ariakensis 1 and C. ariakensis 2), C. virginica (Accession AF137050), C. rhizophorae (Accession AF137049), and C. belcheri (Accession Z29545). Pairwise sequence divergences between species were estimated by the DNADIST program from PHYLIP (Felsenstein, 1989) according to Kimura’s two-parameter model (Kimura, 1980). Phylogenic analyses were computed using the program FITCH. Bootstrap analysis with 100 replicates was performed by the SEQBOOT and CONSENSE programs.

Table 2 Pairwise sequence divergences, for the mt COI DNA (above the diagonal) and 16S rDNA (below the diagonal) fragments

C. gigas Haplotype C Haplotype D Haplotype E Haplotype A Haplotype B C. nippona Haplotype G C. sikamea C. belcheri C. iredalei C. ariakensis C. virginica C. rhizophorae C. gasar

0 0 0 0.002 0.007 0.005 0.044 0.029 na 0.052 0.051 0.057 0.166 0.182 0.184

0 0.002 0.007 0.005 0.044 0.029 na 0.052 0.051 0.062 0.150 0.163 0.172

0.002 0.002 0.002 0.007 0.005 0.044 0.029 na 0.052 0.051 0.062 0.150 0.163 0.172

0.004 0.004 0.005 0.010 0.007 0.048 0.032 na 0.055 0.053 0.065 0.153 0.166 0.175

0.022 0.022 0.020 0.026 0.002 0.041 0.027 na 0.044 0.048 0.059 0.150 0.163 0.172

0.028 0.028 0.026 0.032 0.005 0.045 0.029 na 0.047 0.051 0.062 0.154 0.167 0.176

0.161 0.165 0.163 0.168 0.169 0.172 0.041 na 0.064 0.072 0.089 0.160 0.221 0.203

0.132 0.132 0.130 0.132 0.132 0.138 0.125 na 0.041 0.040 0.043 0.186 0.173 0.172

0.100 0.101 0.100 0.101 0.095 0.093 0.165 0.146 na na na na na na

0.192 0.190 0.188 0.195 0.185 0.183 0.195 0.191 0.186 0.041 0.059 0.159 0.178 0.172

0.181 0.177 0.175 0.182 0.172 0.172 0.172 0.168 0.191 0.196 0.065 0.173 0.178 0.173

0.170 0.168 0.170 0.168 0.168 0.174 0.160 0.151 0.176 0.190 0.186 0.176 0.191 0.173

0.260 0.257 0.254 0.262 0.251 0.256 0.259 0.267 0.266 0.279 0.270 0.298 0.038 0.131

na na na na na na na na na na na na na

na na na na na na na na na na na na na na

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C. Haplotype Haplotype Haplotype Haplotype Haplotype C. Haplotype C. C. C. C. C. C. C. gigas C D E A B nippona G sikamea belcheri iredalei ariakensis viginica rhizophorae gasar

0.107

na: Not available.

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3. Results PCR-amplified fragments of 710 base-pairs from the COI mitochondrial gene were obtained for individuals previously characterized using the PCR-RFLP method (Boudry et al., 1998; Lam et al., in press) as haplotypes A, B, C, D, E and G. These DNA fragments were sequenced and registered as Accessions AJ553901, AJ553902, AJ553903, AJ553904, AJ553905, AJ553906, respectively. The two sequences obtained for each pair of individuals were identical for each haplotype. The distance analysis computed after their alignment with the other Crassostrea COI sequences is presented in Table 2 (above the diagonal). The same analyses were performed for the 16S rDNA fragment. The sequences of the specimens corresponding to the COI haplotypes A, B, C, D, E, and G were registered as Accessions AJ553907, AJ553908, AJ553909, AJ553910, AJ553911, AJ553912, respectively. The two sequences obtained for each pair of individuals were identical for each haplotype. Additionally, we also sequenced the 16S rDNA fragment of C. belcheri and C. iredalei (Accessions AJ553913 and AJ553914). The differences between these sequences were compared with previously published 16S sequence data from other Crassostrea species. Pairwise divergences are presented in Table 2 (below the diagonal).

Fig. 1. Phylogenetic trees obtained from sequence divergence of the COI (A) and 16S (B) mitochondrial DNA fragment according to Kimura’s model (Kimura, 1980) for presumed C. gigas and C. angulata specimens. Angul1 to Angul4 correspond to C. angulata haplotypes described by O’Foighil et al. (1998). Numbers on the branches indicate bootstrap values. Asterisks indicate the sequences obtained in the present study.

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These mitochondrial DNA sequences group haplotypes C, D and E, previously associated with C. gigas (Boudry et al., 1998) on the basis of the PCR-RFLP analysis, within a C. gigas cluster (Fig. 1). The C. gigas COI sequence previously published by O’Foighil et al. (1998) corresponds to haplotype C, which is the most common haplotype in C. gigas (Huvet et al., 2000). Within this C. gigas group (Table 2), divergence is less than 0.5% for the COI fragment, and less than 0.2% for the 16S fragment. The divergence between haplotypes A and B, previously associated with C. angulata (Boudry et al., 1998) is 0.5% for the COI fragment (Table 2). The four C. angulata sequences previously published by O’Foighil et al. (1998) (among which ‘‘angul2’’ corresponds to Accession AF152567), are also found to be very close (divergence < 1.1%). The 16S sequences of the individuals previously exhibiting COI haplotypes C and D are identical to the C. gigas sequence (Accession AF280611) and are close to Haplotype E (divergence = 0.2%). In the same way, the two C. angulata sequences, corresponding to COI haplotypes A and B, are very similar (divergence = 0.2%). The divergence between these two groups (C, D and E versus A and B) is relatively low (2 –3% for COI and 0.5 –1% for 16S). However, their distinction is clear as illustrated in Fig. 1. Sequences corresponding to haplotype G, i.e. specimens from Hong Kong initially thought to be C. gigas, are clearly divergent from both C. gigas and C. angulata clusters (Fig. 1), showing a respective divergence of 13% and 13– 14% for COI, and 3% and 3 –

Fig. 2. Phylogenetic trees obtained from sequence divergence of the COI (A) and 16S (B) mitochondrial DNA fragment according to Kimura’s model (Kimura, 1980). C. gigas and C. angulata are represented by haplotype C and A, respectively (i.e. the most common haplotypes in these two taxa). Numbers on the branches indicate bootstrap values. Asterisks indicate the sequences obtained in the present study.

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Table 3 Pairwise sequence divergences for the 28S rRNA fragments

Hong Kong oysters C. gigas 1 C. gigas 2 C. ariakensis 1 C. ariakensis 2 C. belcheri C. virginica C. rhizophorae

C. gigas 1

C. gigas 2

C. ariakensis 1

C. ariakensis 2

C. belcheri

C. virginica

C. rhizophorea

O. edulis

0.0124

0.0162

0.0174

0.0188

0.0265

0.0888

0.0902

0.0799

0.0033

0.0110 0.0144

0.0121 0.0155 0.0011

0.0223 0.0235 0.0257 0.0270

0.0756 0.0796 0.0731 0.0722 0.0734

0.0768 0.0808 0.0743 0.0734 0.0746 0.0033

0.0677 0.0716 0.0665 0.0680 0.0767 0.0821 0.0833

4% for 16S. The phylogenetic relationship of haplotype G with other Crassostrea species is illustrated in Fig. 2. The closest species, aside from C. gigas (haplotype C) and C. angulata (haplotype A) is C. nippona with 12.5% divergence for COI and 4% for 16S. In order to further document the genetic divergence between Hong Kong oysters and other Crassostrea species, PCR-amplified fragments of 1300 base-pairs from the 28S

Fig. 3. Phylogenetic tree obtained from sequence divergence of the 28S nuclear DNA fragment according to Kimura’s model (Kimura, 1980). Numbers on the branches indicate bootstrap values. Asterisk indicates the sequences obtained in the present study.

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rRNA nuclear gene were obtained for two individuals previously characterized as haplotype G. These DNA fragments were sequenced and the unique sequence obtained was submitted as Accession AJ553915. The distances between these sequences were compared to previously published 28S sequence data of other Crassostrea species. They are presented in Table 3. The phylogenetic tree based on these distances is given in Fig. 3. Divergence values between Hong Kong oysters and C. gigas and C. ariakensis sequences are divergent by 1.2 – 1.6% and 1.7– 1.9%, respectively.

4. Discussion DNA-based studies have revealed several previous cases of misidentification of oysters. Direct comparison with existing DNA sequence data available in nucleotide sequence databases and/or with other specimens often allows the correct classification of the studied oysters. This was notably the case for C. gasar populations along the South American Atlantic Coasts (Lape`gue et al., 2002). The present study confirms that the classification of COI PCR-RFLP haplotypes A, B, C, D and E, in presumed C. gigas and C. angulata populations (Boudry et al., 1998; Huvet et al., 2000; Fabioux et al., 2002), corresponds to the sequence data published by O’Foighil et al. (1998). Additionally, the observed divergence between haplotypes A, B, C, D and E, clustering into two groups, confirms (1) the close phylogenetic relationship between these two taxa (O’Foighil et al., 1998) and (2) their genetic distinction (the levels of divergence within each group being lower than that observed between them). However, haplotype G (Lam et al., in press), the only haplotype observed in a population initially identified as C. gigas, is clearly divergent from this species. Furthermore, its nuclear and mitochondrial sequences do not correspond to any available oyster sequence data. Consequently, this poses the question of whether it corresponds to a new species, or to a previously described species, for which no sequence data is available. For example, no sequence data are available for C. talienwhanensis and C. plicatula, two taxa considered as sibling species to C. gigas in northern China on the basis of the RAPD technique (Liu and Dai, 1998). Morphological comparison of Hong Kong oysters with other Crassostrea species might also help to determine if they are present in other areas. However, until now, they have been morphologically classified as C. gigas by most authors (e.g. Mok 1974a,b; Morton and Wong, 1975; Cheung and Wong, 1992; Chan et al., 1999). Oysters are known to be highly plastic in their morphology (see Jozefowicz and O’Foighil, 1998), as this is influenced by environmental growing conditions, so it is uncertain that these oysters could be unambiguously distinguished from C. gigas on the sole basis of morphological characters. Comparison of specimens grown under common conditions would surely help in answering this question. Taking into account the large dispersal of oyster larvae, it is unlikely that the oysters observed in Hong Kong are restricted to this area. Highly fragmented distributions have been reported for several oyster species (e.g. Tiostrea chilensis: New Zealand and Chile; O’Foighil et al., 1999; C. gasar: Western Africa and Southeastern America; Lape`gue et al., 2002), so further studies are needed to determine the range of this putative new species. It should be noted that no specimens of this presumed new species were observed in

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Southern Taiwan, which is about 600 km away from Hong Kong. We are aware of the genetic studies developed by the Virginia Institute of Marine Science, USA (Aquaculture Genetics and Breeding Technology Center) on C. ariakensis from Southern China, but none on C. gigas-like populations. Knowing the high morphological similarity between this presumed new species and C. gigas, these studies will be facilitated by the availability of DNA-based markers (Lam et al., in press, this study). 5. Conclusion Many examples of misidentification of oysters have been previously reported. In several cases, comparative analyses of available DNA sequences, morphological comparisons and information about geographic range and/or introductions enabled authors to propose alternative identifications. In the present paper, comparative DNA sequence analysis of presumed C. gigas or C. angulata revealed that specimens farmed in Hong Kong do not belong to this taxon and might be a new species, as no available DNA sequence corresponds to these oysters. Our results once again illustrate that, despite the importance of oyster aquaculture, genetic resources are yet poorly known and that fundamental knowledge, on the taxonomy and systematics of cultured populations, needs to be established. Acknowledgements This research is partly based on a preliminary study carried out together with Dr. K. Lam in La Tremblade, France, and funded by the Research Grants Council of the University of Hong Kong and by IFREMER. This work was also partly supported by the Re´gion PoitouCharentes (Convention 99 RPC-A-201). We thank Drs. K. Lam for providing the samples of oysters from Hong Kong and H. McCombie for help with the English. References Anderson, T.J., Adlard, R.D., 1994. Nucleotide-sequence of a rDNA internal transcribed spacer supports synonymy of Saccostrea commercialis and S. glomerata. J. Molluscan Stud. 60, 196 – 197. Banks, M.A., Hedgecock, D., Waters, C., 1993. Discrimination between closely related Pacific oyster species (Crassostrea) via mitochondrial DNA sequences coding for large subunit rRNA. Mol. Mar. Biol. Biotechnol. 2, 129 – 136. Bernard, F.R., Cai, Y.Y., Morton, B., 1993. Catalogue of the Living Marine Bivalve Molluscs of China. The University of Hong Kong Press, Hong Kong. Boudry, P., Heurtebise, S., Collet, B., Cornette, F., Ge´rard, G., 1998. Differentiation between populations of the Portuguese oyster, Crassostrea angulata (Lamarck) and the Pacific oyster, Crassostrea gigas (Thunberg), revealed by mtDNA RFLP analysis. J. Exp. Mar. Biol. Ecol. 226, 279 – 291. Chan, K.W., Cheung, R.Y.H., Leung, S.F., Wong, M.H., 1999. Depuration of metals from soft tissues of oysters (Crassostrea gigas) transplanted from a contaminated site to clean sites. Environ. Pollut. 105, 299 – 310. Cheung, Y.H., Wong, M.H., 1992. Trace metal contents of the Pacific oyster (Crassostrea gigas) purchased from markets in Hong Kong. Environ. Manage. 16, 753 – 761. Estoup, A., Largiader, C.R., Perrot, E., Chourrout, D., 1996. Rapid one-tube DNA extraction for reliable PCR detection of fish polymorphic markers and transgenes. Mol. Mar. Biol. Biotechnol. 5 (4), 295 – 298.

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