Development, validation and high-throughput analysis of sequence markers in nonmodel species

Area of Science:

• Evolutionary Biology
• Genomics
• Bioinformatics

Background:

• Nuclear genome DNA sequences are crucial for phylogeography and phylogenetics.
• Identifying suitable markers is challenging for non-model species, especially those with large, repetitive genomes.
• Existing methods often require sequenced genomes, limiting their application.

Purpose of the Study:

• To present a cost-effective framework for designing, validating, and sequencing multiple genetic markers in non-model species without sequenced genomes.
• To demonstrate the utility of this framework in closely related newt species (urodeles).
• To facilitate historical inferences in population genetics and phylogenetics.

Main Methods:

• Developed a broadly applicable framework for marker design and high-throughput sequencing.
• Utilized 3' untranslated transcript regions (3'UTR) to generate over 80 markers (approx. 600 bp each).
• Applied standard bioinformatic tools for data processing to obtain phase-resolved sequences, without requiring barcoded PCR primers.

Main Results:

• Successfully designed and validated over 80 nuclear markers from 3'UTR regions.
• Demonstrated effective multiplexing and sequencing of these markers in newt species.
• The cost of library preparation was independent of the number of markers investigated.

Conclusions:

• The presented framework is a cost-effective and broadly applicable solution for marker development in non-model species.
• This approach enables robust phylogeographic and phylogenetic analyses, even in species with large genomes and no prior genomic data.
• The method is expected to benefit researchers studying genetic structure, species boundaries, and evolutionary history.