Differentiation of Fish Species in Taiwan Strait by PCR-RFLP and Lab

Food Control 44 (2014) 26e34

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Food Control

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Differentiation of ﬁsh species in Taiwan Strait by PCR-RFLP and lab-on-a-chip system

Shuangya Chen a,*, Yongxiang Zhang b, Hong Li c, Jiahe Wang a, Weiling Chen a, Yu Zhou a, Shan Zhou d a Xiamen Entry-Exit Inspection and Quarantine Technology Center, No. 2165, Jiangang Road, Haicang District, Xiamen 361026, China b Xinglin Entry-Exit Inspection and Quarantine Bureau, Xiamen 361022, China c China National Accreditation Service for Conformity Assessment, Beijing 100088, China d Agilent Technologies Co. Ltd., Beijing 100102, China article info abstract

Article history: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-a- Received 9 January 2014 chip system were used to identify 62 commercial fish species in Taiwan Strait. The fish species include 10 Received in revised form groupers, 12 bream species, 9 Sciaenidae species, 5 puffer species and 26 other fish species. A fragment of 3 March 2014 464 bp length of mitochondrial cytoehrome b gene was amplified by PCR and the products were digested Accepted 6 March 2014 with restriction enzymes DdeI, HaeIII and NlaIII, individually. The fragments generated after digestion Available online 27 March 2014 were further resolved on the DNA chip. The results demonstrated that PCR-RFLP analysis and lab-on-a- chip system provided a fast, easy, automated and reliable analysis approach and it will be useful for the Keywords: fi Fish species identification control of the adulteration of food with sh tissue content in Taiwan Strait. Ó Taiwan Strait 2014 Elsevier Ltd. All rights reserved. PCR-RFLP Lab-on-a-chip

1. Introduction large sampling volume. On the contrary, the new lab-on-a-chip system has advantages in simple and convenient manipulation In recent years, more and more cases of adulteration including and speed. Moreover, the lab-on-a-chip system is more secure due mislabeling, fraud and substitution with cheaper fish arose in the to eliminating the gel staining step and it has been used for dif- fish market. Hence, techniques that enable authentication of ferentiation of white fish (Dooley, Sage, Clarke, Brown, & Garrett, commercial fishery products are highly requested to guarantee 2005). accurate labeling and fraudulent substitution. Taiwan Strait is a 180-km-wide, 360-km-long, and 60-m-deep Species identification of fish is typically based on morphological channel separating Mainland China and Taiwan, and links the East characteristics. However, most of the external features allowing China Sea with the South China Sea. As an important fishing morphological identification of whole fish are not apparent after resource, there are more than 700 ocean fish species. Among them processing. DNA and protein based methods are also used for fish over one hundred are important commercial species and most of identification. Protein method has less advantage on processed them belong to Order Perciformes. Due to big variety in quality and sample in which protein may be already denatured or degraded. price, differentiate fish with similar morphology but different in DNA based methods tend to be more favorite and reliable due to commercial value is an important issue. their high specificity and sensitivity, strong stability and easy The purpose of the present work was to discriminate fish species application. In particular, because of ease-of-use, low cost and in Taiwan Strait using PCR-RFLP approach and lab-on-a-chip sys- repeatable, PCR-RFLP has become widely used for fish species tem, and offer a rapid, ease-of-use, safe and effective analysis identification, such as cod fish (Akasaki, Yanagimoto, Yamakami, method for label certification. Tomonaga, & Sato, 2006), mackerel (Aranishi, 2005), tuna (Lin & Hwang, 2007) and shark (Mendonca et al., 2009). 2. Material and methods The shortcomings of traditional electrophoresis include complicate preparation of the gel, long electrophoresis time and 2.1. Samples

* Corresponding author. Tel.: þ86 592 3269929; fax: þ86 592 3269921. A total of 62 species (Table 1) were purchased from seafood E-mail address: [email protected] (S. Chen). markets of Xiamen, China, and taxonomically identiﬁed by an http://dx.doi.org/10.1016/j.foodcont.2014.03.019 0956-7135/Ó 2014 Elsevier Ltd. All rights reserved. S. Chen et al. / Food Control 44 (2014) 26e34 27

Table 1 Fish species collected for PCR-RFLP differentiation study.

Order Family Species Order Family Species

Perciformes Serranidae Epinephelus akaara Perciformes Priacanthidae Priacanthus tayenus Epinephelus moara Lutjanidae Lutjanus malabaricus Epinephelus fuscoguttatus Stromateidae Pampus argenteus Epinephelus coioides Formionidae Formio niger Epinephelus awoara Carangidae Trachinotus blochii Epinephelus quoyanus Decapterus maruadsi Promicrops lanceolatus Selaroides leptolepis Plectropomus leopardus Centrolophidae Psenopsis anomala Plectropomus maculatus Nemipteridae Nemipterus marginatus Cromileptes altivelis Scolopsis vosmeri Lateolabrax japonicus Labridae Cheilinus undulatus Siniperci chuatsi Siganidae Signus fuscescens Sciaenidae Larimichthys crocea Leiognathidae Leiognathus equulus Nibea albiﬂora Echeneidae Echeneis naucrates Otolithoides biauritus Gerreidae Gerres ﬁlamentosus Chrysochir aureus Kyphosidae Kyphosus bigibbus Sciaenops ocelcatus Microcanthus strigatus Larimichthys polyactis Tetraodontiformes Tetraodontidae Takifugu oblongus Collichthys niveatus Takifugu xanthopterus Collichthys lucidus Lagocephalus gloveri Johnius grypotus Lagocephalus wheeleri Coryphaenidae Hapalogenys mucronatus Lagocephalus lunaris Hapalogenys nigripinnis Torpediniformes Narkidae Narke japonica Plectorhinchus cinctus Rajiformes Rhynchobatidae Rhynchobatus djiddensis Parapristipoma trilineatum Rajiformes Rajidae Raja porosa Sparidae Acanthopagrus latus Rajiformes Dasyatidae Dasyatis zugei Acanthopagrus schlegel Orectolobiformes Orectolobidae Chiloscyllium plagiosum Parargyrops edita Carcharhiniformes Triakidae Mustelus griseus Pagrosomus major Scorpaeniformes Scorpaenidae Scorpaenopsis neglecta Rhabdosargus globiceps Scorpaeniformes Scorpaenidae Sebastiscus marmoratus Oplegnathidae Oplegnathus fasciatus Mugiliformes Polynemidae Elentheronema tetradactylum

ichthyologist at Third Institute of Oceanography, State Oceanic 2.4. RFLP analysis Administration, P. R. China. Three individuals per species were tested. Fish muscle samples from live or frozen specimens of each PCR products were digested with three restriction enzymes species were obtained freshly and immediately processed by cut- DdeI, HaeIII and NlaIII (New England Biolabs, Inc.). Restriction re- ting small muscle portions (1e2 g) and subsequently put them actions were carried out in 5 mL final volume containing 2.5 mLof at 20 C until use. PCR product, 0.5 mL of restriction enzyme, and 0.5 mL of restriction enzyme buffer supplied by the manufactures and incubated at 37 C 2.2. DNA extraction for 2 h. Reactions were terminated by incubation at 65 C for 20 min. PCR products (5 mL) were mixed with 1 mL 60 mmol/L EDTA Total genomic DNA was extracted from fish muscle. Fresh and to achieve a final concentration of 10 mmol/L EDTA. 1 mL of reaction frozen pieces of samples (100e400 mg) were homogenized in mix was loaded on to Agilent DNA 1000 chips and analyzed on the 2100 Bioanalyzer. RFLP profile was detected and compared with the liquid N2. DNA was extracted using Qiagen DNeasy Blood & Tissue Kit (Spin-Column Protocol) and eluted in ultrapure water. Con- positions of size markers. centrations (ng/mL) of DNA were assessed at 260 nm using a Thermo nanodrop 2000C spectrophotometer. 3. Results

2.3. PCR ampliﬁcation 3.1. DNA extraction and PCR ampliﬁcation

Cyt b gene primers (Russell et al., 2000) named L14735 (50- Among all the specimens, majorities (48 fish species) belong to AAAAACCACCGTTGTTATTCAACTA-30) and H15149 (50-GCICCTCAR- Perciformes. Others belong to Tetraodontiformes, Rajiformes, Tor- AATGAYATTTGTCCTCA-30) were synthesized by Takara Co., Ltd., and pediniformes, Scorpaeniformes, Mugiliformes and Orectolobi- used to amplify a fragment of 464 bp. The amplification reactions formes. Most of Perciformes samples came from Serranidae, were carried out in 25 mL final reaction volume containing 12.5 mLof Sciaenidae, Sparidae and Coryphaenidae. 2 PCR mix (Guangzhou Dongsheng Biotech Co., Ltd.), 1 mL of each All of the DNA extracts from 62 fish species produced 464 bp primer (10 mM), 1 mL of template DNA (2.5e25.0 ng/mL) and 9.5 mL PCR fragment respectively, indicated that the DNA were success- sterile distilled water. The amplification conditions according to fully extracted and the template DNA could be used for PCR Agilent DNA Fish ID Ensemble Protocol (Agilent Technologies Inc.) on amplification. BIO-RAD PTC-200 DNA Engine Thermal Cycler were: initial denaturation at 95 C for 5 min, 40 cycles of amplification (denaturation at 3.2. RFLP analysis of fish species 95 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 30 s), and final extension at 72 C for 7 min. The PCR products were 3.2.1. RFLP analysis of groupers in family Serranidae detected and compared with the positions of size markers using Groupers are valuable fish species and in Taiwan Strait Agilent DNA 1000 Kit on the Agilent 2100 Bioanalyzer. mostly point to fishes belongs to genera Epinephelus, Promicrops, 28 S. Chen et al. / Food Control 44 (2014) 26e34

Plectropomus and Cromileptes. Since there are huge price differ- In present study, we collected 12 Serranidae species, including ences exist in different grouper species, meats of cheaper grouper six Epinephelus species, two Plectropomus species, Promicrops lan- are occasionally used to adulterate expensive ones, such as Cro- ceolatus, C. altivelis, L. japonicus and S. Chuatsi, and used a PCR-RFLP mileptes altivelis and Plectropomus leopardus. Sometimes, Lateo- method to discriminate them. labrax japonicus and Siniperci chuatsi are also used to adulterate The restriction patterns of 12 fish species were shown in Fig. 1 groupers. and mean fragment sizes were shown in Table 2. Combined three Previous publication showed that PCR approaches using 16S restriction patterns, all the species except Epinephelus akaara and rRNA gene had been developed for grouper identification (Epi- Epinephelus moara showed obvious differences in the patterns, nephelus and Mycteroperca species) from substitute species such as suggesting the PCR-RFLP method is useful for differentiating Nile perch (Lates niloticus) and wreck fish (Polyprion americanus) L. japonicus and S. Chuatsi from grouper species, as well as differ- (Trotta et al., 2005). While some highly consumed expensive entiating C. altivelis and P. leopardus from other cheaper groupers. grouper species in China market, like P. leopardus and C. altivelis, However, E. akaara and E. moara had same patterns, indicated these were not belong to Epinephelus or Mycteroperca. Therefore this two grouper could not be distinguished by three enzymes. method was not appropriate in China market. Furthermore, TaqI, MseI, EcoRI, BamHI and XhoI enzymes could not

Fig. 1. PCR-RFLP proﬁles of Serranidae species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). M, Molecular mass marker; 1, E. akaara;2,E. moara;3,E. fuscoguttatus;4,E. coioides;5,E. awoara;6,E. quoyanus;7,E. lanceolatus;8,P. leopardus;9,P. maculatus;10,C. altivelis;11,L. japonicus;12,S. chuatsi. S. Chen et al. / Food Control 44 (2014) 26e34 29

Table 2 Table 3 PCR-RFLP fragment sizes of 12 Serranidae species. PCR-RFLP fragment sizes of 12 species belong to Coryphaenidae, Sparidae, Opleg- nathidae, Priacanthidae and Lutjanidae. Sample Fragment size/bp Sample Fragment size/bp DdeI HaeIII NlaIII DdeI HaeIII NlaIII E. akaara 447 119, 169, 177 180, 285 E. moara 446 119, 170, 178 181, 287 H. mucronatus 95, 123, 255, 261 182, 287 92, 106, 127, 168 E. fuscoguttatus 194, 269 178, 288 182, 284 H. nigripinnis 122, 327 129, 342 95, 105, 297 E. coioides 447 180, 295 182, 286 P. cinctus 89, 120, 215 129, 333 184, 279 E. awoara 462 117, 174 183, 280 P. trilineatum 456 47, 134, 293 485 E. quoyanus 457 38, 182, 251 495 A. latus 65, 392 463 184, 285 E. lanceolatus 451 48, 133, 160 181, 286 A. schlegel 462 463 88, 110, 270 P. leopardus 469 39, 103, 127, 189 90, 101, 152 P. edita 169, 298 130, 149, 191 94, 107, 291 P. maculatus 115, 340 38, 102, 128, 187 88, 98, 244 P. major 166, 256 129, 148, 191 91, 105, 287 C. altivelis 122, 330 59, 75, 103, 233 479 R. globiceps 396 128, 332 82, 89, 285 L. japonicus 89, 117, 259 114, 321 446 O. fasciatus 200, 245 131, 335 102, 146, 186 S. chuatsi 447 138, 147, 188 108, 373 P. tayenus 150, 289 119, 130, 151 92, 105, 256 L. malabaricus 458 126, 150, 176 364

Fig. 2. PCR-RFLP profiles of 12 fish species belong to Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). M, Molecular mass marker; 1, H. mucronatus;2,H. nigripinnis;3,P. cinctus;4,P. trilineatum;5,A. latus;6,A. schlegel;7,P. edita;8,P. major;9,R. globiceps;10, O. fasciatus;11,P. tayenus;12,L. malabaricus. 30 S. Chen et al. / Food Control 44 (2014) 26e34 differentiate E. akaara and E. Moara either (data not shown), processed into filets. 12 common fish species (Hapalogenys implied that other gene or other method such as DNA sequence mucronatus, Hapalogenys nigripinnis, Plectorhinchus cinctus, Para- analysis may be chosen to discriminate E. akaara and E. moara. pristipoma trilineatum, Acanthopagrus latus, Acanthopagrus schlegel, Parargyrops edita, Pagrosomus major, Rhabdosargus globiceps, 3.2.2. RFLP analysis of 12 morphologically similar species in family Oplegnathus fasciatus, Priacanthus tayenus and Lutjanus malabar- Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and icus) in the Xiamen fish market coming from five different fish Lutjanidae families were chosen to compare with the PCR-RFLP files. Some fish species from the family Coryphaenidae, Sparidae, PCR-RFLP patterns generated with enzymes DdeI, HaeIII and Oplegnathidae, Priacanthidae and Lutjanidae have a joint name NlaIII were shown in Fig. 2 and mean fragment sizes were shown in “bream” in China and have similar morphological characteristics, Table 3. The great difference of the patterns showed that these which result in false identification, especially when the fish were morphologically similar species could be discriminated with three

Fig. 3. PCR-RFLP proﬁles of Sciaenidae species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, L. crocea;2,N. albiﬂora;3,O. biauritus;4,C. aureus;5,S. ocelcatus;6,L. polyactis;7,C. niveatus;8,C. lucidus;9,J. grypotus.) S. Chen et al. / Food Control 44 (2014) 26e34 31

Table 4 has not yet reached the level of species variation, which is different PCR-RFLP fragment sizes of 9 Sciaenidae species. from the morphological conclusion. The small difference in COI Sample Fragment size/bp gene sequences between two species also suggested that DNA fi DdeI HaeIII NlaIII method using COI gene would be insuf cient for discriminate them. With the present result, other fast-evolving genes such as NADH L. crocea 121, 332 131, 342 80, 92, 107, 219 dehydrogenase (ND) gene or internal transcribed spacer (ITS) N. albiflora 125, 318 127, 159, 174 102, 365 O. biauritus 116,317 73,111,125,143 90,96,119,164 sequence may be better for the discrimination of L. polyactis, C. aureus 468 124, 133, 180 105, 370 C. niveatus and C. lucidus. S. ocelcatus 445 75, 117, 130, 146 91, 105, 285 L. polyactis 440 128, 338 90, 100, 287 3.2.4. RFLP analysis of 5 puffer species C. niveatus 441 126, 336 88, 98, 286 fi C. lucidus 446 130, 335 91, 103, 285 The meat of puffer sh is delicious, nutritious and has high J. grypotus 166, 302 476 187, 283 economic value. So that puffer fish are known as “the king of fish”. However, deaths by puffer fish toxin which has high toxicity, had been reported all over the world every year. There are more than enzymes. Among them, the patterns of P. edita and P. major were forty puffer fish species in China, only seven or eight species very similar, the only difference between them was that PCR among them are safe to eat. In order to confirm the puffer species product of P. edita was digested to two fragments of around 169 bp of the food and protect the health of consumers, simple and and 298 bp by DdeI, while PCR product of P. major was digested to effective identification method is needed to differentiate puffer two fragments of around 166 bp and 256 bp. fish species. Due to geography and temperature reasons, the distribution of 3.2.3. RFLP analysis of 9 Sciaenidae species puffer fish species is limited. In Taiwan Strait the most common Large yellow croaker (Larimichthys crocea) is an economically puffer species are low toxic Takifugu oblongus, Takifugu xanthopte- important marine fish in China. Due to its high price, other cheaper rus, Lagocephalus gloveri, Lagocephalus wheeleri and toxic Lagoce- fish species such as Nibea albiflora were found to be dyed with stain phalus lunaris. In present study, PCR-RFLP method and lab-on-a- and labeled as yellow croaker by some vendors, especially in those chip system were applied to discriminate these five puffer species. frozen products. Thus, it is necessary to identify L. crocea from Isoelectric focusing electrophoresis (IEF), sodium dodecyl sul- morphologically similar species. fate e polyacrylamide gel electrophoresis (SDS-PAGE) and two- Some researches on genetic analysis of L. crocea have been re- dimensional electrophoresis (2DE) techniques using protein mol- ported (Cui, Liu, Li, You, & Chu, 2009; Wu, Liu, Cai, Ye, & Wang, ecules were applied to identify puffer fish species (Chen & Hwang, 2011). However, the study on identification method of this spe- 2002; Chen, Shiau, Noguchi, Wei, & Hwang, 2003; Chen, Shiau, Wei, cies is very limited so far. Here, we collected 9 Sciaenidae species & Hwang, 2004). PCR-RFLP was also reported using mitochondrial (L. crocea, N. albiflora, Otolithoides biauritus, Chrysochir aureus, 16S rRNA gene to differentiate nine puffer fish species collected Sciaenops ocelcatus, Larimichthys polyactis, Collichthys niveatus, from the coastal area of Okinawa Islands in Japan (Ishizaki et al., Collichthys lucidus and Johnius grypotus) from Xiamen market and 2006). A PCR method was also established for detecting puffer intended to use PCR-RFLP method to discriminate large yellow fish, while this method can not differentiate puffer fish species croaker from other croakers. (Chen et al., 2010). PCR-RFLP patterns generated with enzymes were shown in PCR-RFLP patterns generated with enzymes DdeI, HaeIII and Fig. 3 and mean fragment sizes were shown in Table 4. The result NlaIII were shown in Fig. 4 and mean fragment sizes were shown in indicated that L. crocea could be easily discriminated from other Table 5. eight fish species with three enzymes. While L. polyactis, C. niveatus Results showed that patterns generated with enzymes DdeI and C. lucidus had the same RFLP patterns, indicating that they were different between Takifugu and Lagocephalus. T. oblongus and could not be discriminated by this method. T. xanthopterus both generated only one band using DdeI enzyme, Liu, Chen, Dai, and Zhuang (2010) compared 75 COI gene se- suggesting that no DdeI site existed in the PCR amplicon. While all quences (650 bp) of 30 species of Sciaenidae and found that the three Lagocephalus species generated two bands bigger than genetic differentiation (0.004) between C. niveatus and C. lucidus 100 bp.

Fig. 4. PCR-RFLP proﬁles of puffer species obtained on microchips. PCR products were digested by restriction enzymes (M, Molecular mass marker; 1, T. oblongus;2,T. xanthopterus; 3, L. gloveri;4,L. wheeleri;5,L. lunaris.). 32 S. Chen et al. / Food Control 44 (2014) 26e34

Table 5 3.2.5. RFLP analysis of 24 other commercial ocean fish species in PCR-RFLP fragment sizes of 5 puffer species. Xiamen market Sample Fragment size/bp It is significant to expand the range of fish species to verify the

DdeI HaeIII NlaIII practicality of this PCR-RFLP method. Twenty four other commercial ocean fish species in Xiamen market were collected. Among the T. oblongus 375 63, 75, 305 467 species, 15 samples belong to Perciformes. PCR-RFLP patterns were T. xanthopterus 372 138, 296 460 L. gloveri 215, 236 130, 299 476 shown in Fig. 5. Other 9 samples belong to other orders and PCR- L. wheeleri 178, 238 119, 132, 183 71, 83, 93, 106, 164 RFLP patterns of these fishes were shown in Fig. 6. Mean frag- L. lunaris 123, 325 45, 128, 255 126, 343 ment sizes of all 24 species were shown in Table 6. The results showed that profiles of each species had specificity and these 24 species could be differentiated via three enzymes. NlaIII profiles had three types, one band for T. oblongus, T. xanthopterus and L. gloveri, several small bands for L. wheeleri, and 4. Discussion two bands of around 126 bp and 343 bp for L. lunaris. For HaeIII profiles, T. xanthopterus and L. gloveri produced similar The cytochrome b (cyt b) gene contains both slowly and rapidly patterns different from other samples. evolving codon positions, as well as more conservation and more According to the above data, none of the puffer species could be reliable regions. Therefore, this gene has been considered one of the identified only by one enzyme. Combined three enzymes patterns, most frequently used gene for phylogenetic studies at the species or these puffer species could be differentiated from each other. genus level (Rasmussen & Morrissey, 2008). In the present study,

Fig. 5. PCR-RFLP proﬁles of 15 Perciformes species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, P. minor;2,F. niger;3,T. blochii;4,D. maruadsi;5,S. leptolepis;6,P. anomala;7,N. marginatus;8,C. undulatus;9,S. fuscescens;10,L. equulus;11,E. naucrates;12,S. vosmeri; 13, G. ﬁlamentosus;14,K. bigibbus;15,M. strigatus.) S. Chen et al. / Food Control 44 (2014) 26e34 33

Fig. 6. PCR-RFLP profiles of 9 fish species in other order obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, N. japonica;2,R. djiddensis;3,R. porosa;4,D. zugei;5,C. Plagiosum;6,M. griseus;7,S. neglecta;8,S. marmoratus;9,E. tetradactylum.) we investigated the PCR-RFLP profiles of fish species in Taiwan Compare with conventional gel electrophoresis, lab-on-a-chip Strait using cyt b gene to discriminate them. The result shows that system made RFLP profiles more accurate, sensitive, rapid and the RFLP profile is specific, effective, rapid and suitable for food easier to perform. DNA fragments greater than 25 bp of chip 1000 inspection. were expected detected using lab-on-a-chip system. However, Although the method described above is useful for identification some small fragments lower than 50 bp were inconsistently purposes, it is not suited for samples containing a mixture of observed. This was probably because they were too close to the different species as profiles become too complex to interpret. The sizing limit (25 bp) or did not fluoresce sufficiently to be detected method is not ideally appropriate for heavily processed products due to low concentration. These small fragments displayed low either, such as canned samples. A smaller (<200 bp) PCR target peak height on the electropherogram and need to be manually would be better suited for canned food. adjusted. 34 S. Chen et al. / Food Control 44 (2014) 26e34

Table 6 Quarantine of the People’s Republic of China (AQSIQ) and a grant PCR-RFLP fragment sizes of 24 ﬁsh species. (No. 3502Z20134055) from Science and Technology Program of Sample Fragment size/bp Xiamen City.

DdeI HaeIII NlaIII

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Multiples PCR method for use in real-time PCR for identification of fish fillets Acknowledgments from grouper (Epinephelus and Mycteroperca species) and common substitute species. Journal of Agricultural and Food Chemistry, 53, 2039e2045. Wu, X., Liu, X., Cai, M., Ye, X., & Wang, Z. (2011). Genetic analysis of farmed and wild This work was supported by grants (No. 2011IK245, 2013IK185) stocks of large yellow croaker Larimichthys crocea by using microsatellite from General Administration of Quality Supervision, Inspection & markers. African Journal of Biotechnology, 10, 5773e5784.