Isolamento e caracterização de marcadores moleculares de Hoplias intermedius (Günther, 1864): genética e forense

Abstract

The conservation of Brazilian ichthyofauna is facing a growing threat, which highlights the importance of new tools for the enrichment of conservation measures. Thus, Forensic Genetics and the design of molecular markers, emerge to improve illegal fishing control activities and species detection on poorly preserved samples. The choice of the target species of this study, Hoplias intermedius (Günther, 1864), responds to the environmental impact of the basins where species is distributed, its increasing importance as a fishing resource and the great taxonomic complexity of the genus. The objectives of our study include the sequencing and assembly of the whole mitochondrial genome, design of mitochondrial species-specific primers for H. intermedius and its validation as a mitochondrial molecular marker for its applications in forensic aims. DNA was extracted and a genomic library was constructed by using new generation sequencing. Fragments were obtained with Nextera DNA Sample Preparation kit and MiSeq Reagent kit V3-600 paired-end. The mtDNA assembly was made with CLC Workbench software v8.5.1 obtaining coverage of 304.45% and its annotation was performed on the MITOfish platform, showing a typical mitogenome arrangement of vertebrates (13 protein coding genes, two ribosomal RNA genes, 22 RNA transporter genes and one control region). MEGA6 software was used for the alignment of mitochondrial sequences of H. intermedius and other mtDNA with 99% of identity. The detection of informative sequence regions for the target species was validated by SPIDER R-package. Sensitivity, specificity and efficiency tests were performed by using samples collected in the field, from scientific collections and from fishmongers. PCR reactions were standardized for each primer combination using 5U/ul Taq Polymerase, 2mM dNTPs, and specific combinations of buffer solutions IB, IC, IIC and 10X IVB, 2M KCl (Phoneutria) and DMSO. The annealing temperature range varied among 59°, 62° and 64° C and the number of cycles between 30 and 35. Results showed that the mtDNA of H. intermedius is a circular DNA molecule of 16629 bp, with 43.97% GC content. The ATG start codon was found in 13 non-coding protein genes encoded in the (+) strand, and as expected the ND6 gene encoded in the (-) strand. Primer pairs Hin5F-Hin3R and Hin5F-Hin4R were highly specific for H. intermedius, both design for the 16S rRNA region. Amplicon sequences were obtained by using bidirectional sequencing, and subsequently constructed an NJ tree for each marker. Our results demonstrate that molecular markers for H. intermedius pursue a huge potential to identify the target species, and its application includes integrative taxonomy for Hoplias species and wildlife forensic identification of poor DNA quality samples.