First environmental DNA (eDNA) record of central Amazon in a floodplain lake: extraction method selection and validation / Primeiro registro de DNA ambiental (eDNA) da Amazônia central em um lago de várzea: seleção e validação de método de extração

Danniel Rocha Bevilaqua, Sabrina Araújo de Melo, Carlos Edwar de Carvalho Freitas, Ana Caroline Viana da Silva, Jacqueline da Silva Batista

Abstract


In recent years, environmental DNA (eDNA) has emerged as an effective method for detecting aquatic organisms. The current study tested a method of DNA extraction from water samples from Central Amazonian floodplain lake. Water collections were performed in a floodplain lake on Paciência Island, on the Solimões River (Amazon Basin, Brazil) during the high water period. Seven commercial DNA extraction methods/kits were tested with samples collected from Lake Cacau. After quantification, eDNA samples were amplified with 12S and 16S mitochondrial gene universal primers. The DNeasy Blood & Tissue kit (Qiagen), with modifications developed during the current study (adapt 03), gave the best overall result showing expected gene amplification, and intense agarose gel banding. eDNA concentrations ranged from 0.00 to 22.90 ng/µL for the other methods, with either lower intensity of the amplicon size to target gene correspondence when compared to that obtained with the DNeasy Blood & Tissue kit adapt 03 method, or there was an absence of amplification.The concentration of eDNA extracted from the lake sample was 2.68ng/µL. Prochilodus nigricans and Potamorhina altamazonica genomic DNA (gDNA) samples were extracted for use as a positive controls. The 12S and 16S genes from eDNA were amplified and sequenced with next generation Sequencing (NGS). Using a metabarcode approach, we used the 12S (12S-V5) and 16S (L2513/H2714) minibarcode universal primers to detect fish species DNA traces present in water samples from the Cacau lake. The NGS produced 18 and 492,454 12S and 16S minibarcode readings, respectively, for three taxonomic groups (Actinopterygii, Mammalia and Squamata). Adding the two genes, a total of 492,472.00 readings, with 15 readings with 12S and 4,431.082 readings with 16S for fish. Fish (Actinopterygii) represented 83.33% and 0.60% (three Orders, five families and five genus identified according to BLASTN), mammals had 11.11% and 99.40% and reptiles with 5.56% and 0.00% for the total readings of the 12S and 16S respectively, with more than 97% similarity to NCBI-available reference sequences. Taxonomic classification at the level of species was not very resolute when comparing OTU's with sequences published in NCBI.This study sets a milestone in the methodology of DNA extraction from floodplain lake, and shows the method can be used to monitor Amazon basin ichthyofauna, as it was possible to detect the presence of fish species DNA in collected water samples.


Keywords


Monitoring, Environmental DNA, DNA extraction

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DOI: https://doi.org/10.34117/bjdv6n11-254

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