Mitochondrial gene marker identification for a targeted hornbill eDNA assay

Abstract

The effectiveness of traditional hornbill monitoring through visual and acoustic surveys is often hampered by their cryptic behavior and habitat preference in dense rainforests. The challenges in hornbill detection have led to insufficient population data, posing a significant challenge to hornbill conservation efforts. Environmental DNA (eDNA) offers a complementary approach that enables non-invasive detection of species from trace genetic material shed into the surrounding environments. Targeted eDNA assay using real-time PCR (qPCR) allows the detection and quantification of low amount of target DNA in complex environmental samples. The design of primers defines the region of target DNA to be amplified from the complex DNA sequences present in environmental samples. Gene marker selection is critical to ensure assay specificity by identifying regions that are conserved enough within target species but variable enough between target and non-target species, used for design of primers. This study identifies the mitochondrial gene markers for the development of a targeted eDNA assay, using Anthracoceros albirostris as a proof-of-concept study. We compiled mitochondrial DNA (mtDNA) sequences of target and non-target, closely related species from the GenBank nucleotide database. Due to the lack of hornbills’ sequence data, we performed Sanger sequencing to generate mtDNA sequences targeting cytochrome c oxidase subunit 1 (COI), NADH dehydrogenase subunit 2 (ND2), and displacement loop (d-loop) genes using feather samples from four hornbill individuals. Analysis of sequence alignment revealed a high level of sequence similarity (>90%) among the three mtDNA genes. Despite the high similarity, specific regions within ND2 genes exhibited a sufficient level of sequence variation. This study presents the preliminary development of targeted hornbill eDNA assay, utilizing ND2 as gene marker for primer design. It serves as a foundational step towards the broader application of molecular tools to protect the iconic hornbills in Borneo.