A Genomic-Based Workflow for eDNA Assay Development for a Critically Endangered Turtle, Myuchelys georgesi.

Area of Science:

• Conservation Genetics
• Molecular Ecology
• Environmental DNA (eDNA) Analysis

Background:

• Environmental DNA (eDNA) analysis is a key tool for detecting rare and elusive species.
• eDNA assays often target mitochondrial DNA (mtDNA) due to its high copy number and persistence.
• Developing eDNA assays requires reliable mitochondrial reference sequences or de novo sequencing.

Purpose of the Study:

• To design and validate species-specific eDNA primers for the critically endangered Bellinger River turtle (Myuchelys georgesi).
• To utilize a bioinformatically assembled mitochondrial genome (mitogenome) for primer design.
• To confirm the accuracy of the assembled mitogenome against Sanger sequencing data.

Main Methods:

• Bioinformatic assembly of the Bellinger River turtle mitogenome from a reference genome.
• Comparison of the assembled mitogenome with a Sanger-sequenced mitogenome to verify accuracy.
• Design of two 20 bp primers targeting cytochrome oxidase 1 (CO1) and cytochrome B (CytB) genes.
• Validation of primer efficacy through in silico, in vitro, and in situ analyses.

Main Results:

• An accurate mitochondrial genome for Myuchelys georgesi was bioinformatically assembled.
• Two novel eDNA primers targeting CO1 and CytB genes were successfully designed.
• The designed primers demonstrated successful validation across multiple testing stages.

Conclusions:

• Bioinformatically assembled mitogenomes are a viable source for designing effective eDNA primers.
• The developed primers are validated for detecting the critically endangered Bellinger River turtle.
• This approach facilitates conservation efforts for rare species using eDNA technology.