Species Identification of Processed Products of Tuna and Bonito in Taiwan by Using PCR Technique and Phylogenetic Relationships Based on Mitochondrial Cyt b Sequences

Autor: Wen-Feng Lin, 林文風
Rok vydání: 2007
Druh dokumentu: 學位論文 ; thesis
Popis: 95
Tuna and bonito are both popular seafood in the world. In Taiwan, there are 5 Thunnus species defined as tuna, including T. thynnus (bluefin tuna), T. alalunga (albacore), T. albacares (yellowfin tuna), T. obesus (bigeye tuna) and T. tonggol (longtail tuna). In addition, 5 fish species among 3 genera are defined as bonito, including Euthynnus pelamis (or Katsuwonus pelamis; skipjack), E. affinis (eastern little tuna), Auxis rochei (frigate mackerel), A. thazard (frigate tuna), and Sarda orientalis (oriental bonito). Cytochrome b gene (Cytb) is a functional gene between tRNAGlu and tRNAThr in mtDNA, and which was usually used to study about phylogeny and evolution in vertebrate. In order to identify the original species of raw materials and processed products, 1,141 bp complete Cyt b sequences from 5 tuna species, 5 bonito species and other 4 related Scombridae species including T. maccoyii, Somber japonicus, Scomberomorus commerson and Acanthocybium solandri, were established by PCR (polymerase chain reaction) technique in this study. Six pairs of primer were successfully used to reconstruct the Cyt b sequences in 14 species, and the newly determined nucleotides of 14 species are available from DDBJ/EMBL/GenBank databases under accession numbers EF141171 to EF141185. First, the PCR-RFLP (PCR-restriction fragment length polymorphism) technique was developed to identify the species of T. thynnus, T. alalunga, T. obesus, T. albacares, E. pelamis, E. affinis, A. thazard and S. orientalis in products of canned tuna. To increase the efficiency and sensitivity of DNA extraction, a recent developed method of binding magnetic beads was applied in this study. Two sets of primer were designed to amplify 126 bp and 146 bp of partial Cyt b, and 5 restriction enzymes including Bsp1286 I, Hinc II, Rsa I, Sca I and Mbo II were determined to analyze the short length fragments. The developed PCR-RFLP method was applied to identify species of 18 commercial canned tuna successfully. Attempts are made to establish one step multiplex PCR assay for distinguishing 5 species of raw and cooked bonito including E. pelamis, E. affinis, A. rochei, A. thazard and S. orientalis. The 5 sets of species-specific primer were designed to amplify different Cyt b fragments in each species individually. The amplified lengths of fragments were respectively 143 bp for A. rochei, 236 bp for E. pelamis, 318 bp for A. thazard, 398 bp for E. affinis and 506 bp for S. orientalis, which could be obviously differentiated from each other on DNA electrophoresis. The 5 sets of species-specific primer were mixed and applied to simultaneously detect bonito species. All of 12 commercial fish raw and 5 out of 8 cooked bonito fillets were successfully identified species by the multiplex PCR assay. Furthermore, the species-specific PCR method was developed to identify the species of dried bonito product (katsuobushi) produced from 5 bonito species. Other 5 pairs of species-specific primer were designed to amplify short length fragments among bonito species, respectively. The developed species-specific PCR method was successfully applied to authenticate species of commercial dried bonito products. Hence, these methods really provided a useful and academic technique to identify the sources of tuna and bonito processed products. Finally, the complete 1,141 bp Cyt b sequences were also used to establish the phylogenetic relationships of family Scombridae. The phylogenetic trees were built by 3 methods including neighbor-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML). Cyt b sequences from 14 species in this study combined with 6 related Scombridae species and 2 billfish species as outgroup were evaluated by 3 different models. The result shows that the phylogenetic relationships in MP tree and ML tree based on complete Cyt b sequences are quite the same with traditional morphological taxonomy in family Scombridae.
Databáze: Networked Digital Library of Theses & Dissertations
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