Dentex dentex is one of the most commercially caught Sparidae species in the Mediterranean Sea and Atlantic Ocean. It is very appreciated in European markets and consequently more subjected to species substitution frauds [1]. The currently mitochondrial (mt) DNA sequences used for fish species identification in prepared and processed products are cytochrome b-CYTB, cytochrome c oxidase I-COI, 16S and 12S genes. Recent researches showed that the study of the whole mtDNA allows to identify new, effective and specie-specific barcode markers [2]. In particular, NAD5 gene has high discrimination capacity for Sparidae species. However, the use of all these genes needs amplification and a sequencing process [2,3]. Therefore, a valuable species identification requires many laboratory steps and is time consuming. The aim of this research was to analyze and compare the whole mtDNA sequence of Sparidae species to find a barcoding marker useful to identify the sparid species D. dentex, avoiding the sequencing step. Thirteen Sparidae complete mitogenomes were compared in this study. They were aligned by UGENE software. Hamming Distance algorithm was used to evaluate in percent the genetic dissimilarity among species and genes. Overall mean p-genetic distance analyses were conducted using the Maximum Composite Likelihood model. The nucleotide sequence variability was determined by aligning gene-by-gene sequences of Sparidae species using MEGA 6.0. Primers were designed by eye after multiple alignment of the Sparidae complete mtDNA sequences using BioEdit Sequence Alignment Editor. Primers efficiency for D. dentex identification was tested using PCR reaction. Results of Hamming Distance, nucleotide sequence variability and p-genetic distance analysis showed the potentiality of NAD2 gene as barcode marker for sparids. The PCR reaction confirmed the discrimination capacity of NAD2 gene. In particular, the amplification of the selected NAD2 fragment was possible only for the species D. dentex. In conclusion, NAD2 gene showed high interspecific nucleotide dissimilarity to provide unambiguous results for D. dentex species authentication without sequencing, reducing time, costs and efficiency. In fact, species identification results can be obtained in a few hours of lab work. Therefore, competent national authorities responsible for monitoring and enforcing could improve and make full use of DNA-testing methods in order to deter operators from false labelling of seafood. In agreement with Regulation (EU) 1379/2013, this study contributes to the molecular traceability of fishery products. [1] Katavic et al. Growth performance of pink dentex as compared to four other sparids reared in marine cages in Croatia. Short Communication. Aquaculture International, 8:455–461, 2000. [2] Ceruso et al. Frauds and fish species authentication: study of the complete mitochondrial genome of some Sparidae to provide specific barcode markers, Food Control, accepted for publication, 2019. [3] Armani et al. DNA and Mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market. Food Control, 50: 589-596, 2015.

Comparative mitogenomic analysis of Sparids and evaluation of a new potential DNA barcoding marker for Dentex dentex.

Celestina Mascolo;Marina Ceruso
;
Giorgio Smaldone;Tiziana Pepe
2019

Abstract

Dentex dentex is one of the most commercially caught Sparidae species in the Mediterranean Sea and Atlantic Ocean. It is very appreciated in European markets and consequently more subjected to species substitution frauds [1]. The currently mitochondrial (mt) DNA sequences used for fish species identification in prepared and processed products are cytochrome b-CYTB, cytochrome c oxidase I-COI, 16S and 12S genes. Recent researches showed that the study of the whole mtDNA allows to identify new, effective and specie-specific barcode markers [2]. In particular, NAD5 gene has high discrimination capacity for Sparidae species. However, the use of all these genes needs amplification and a sequencing process [2,3]. Therefore, a valuable species identification requires many laboratory steps and is time consuming. The aim of this research was to analyze and compare the whole mtDNA sequence of Sparidae species to find a barcoding marker useful to identify the sparid species D. dentex, avoiding the sequencing step. Thirteen Sparidae complete mitogenomes were compared in this study. They were aligned by UGENE software. Hamming Distance algorithm was used to evaluate in percent the genetic dissimilarity among species and genes. Overall mean p-genetic distance analyses were conducted using the Maximum Composite Likelihood model. The nucleotide sequence variability was determined by aligning gene-by-gene sequences of Sparidae species using MEGA 6.0. Primers were designed by eye after multiple alignment of the Sparidae complete mtDNA sequences using BioEdit Sequence Alignment Editor. Primers efficiency for D. dentex identification was tested using PCR reaction. Results of Hamming Distance, nucleotide sequence variability and p-genetic distance analysis showed the potentiality of NAD2 gene as barcode marker for sparids. The PCR reaction confirmed the discrimination capacity of NAD2 gene. In particular, the amplification of the selected NAD2 fragment was possible only for the species D. dentex. In conclusion, NAD2 gene showed high interspecific nucleotide dissimilarity to provide unambiguous results for D. dentex species authentication without sequencing, reducing time, costs and efficiency. In fact, species identification results can be obtained in a few hours of lab work. Therefore, competent national authorities responsible for monitoring and enforcing could improve and make full use of DNA-testing methods in order to deter operators from false labelling of seafood. In agreement with Regulation (EU) 1379/2013, this study contributes to the molecular traceability of fishery products. [1] Katavic et al. Growth performance of pink dentex as compared to four other sparids reared in marine cages in Croatia. Short Communication. Aquaculture International, 8:455–461, 2000. [2] Ceruso et al. Frauds and fish species authentication: study of the complete mitochondrial genome of some Sparidae to provide specific barcode markers, Food Control, accepted for publication, 2019. [3] Armani et al. DNA and Mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market. Food Control, 50: 589-596, 2015.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/763017
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