"Less is more": a whole-genome-sequencing-enabled workflow to identify minimal nuclear marker sets for resolving closely related plant taxa
Standard DNA barcodes, typically derived from plastid regions and nuclear ribosomal ITS, often fail to discriminate closely related plant species, resolving only to species-group or genus levels, while the extant nuclear conserved gene sets and kmer-based methods each have their limitations. Here we present a workflow that exploits whole-genome sequence data to develop minimal multi-locus nuclear marker sets that retain the species-level resolution of genome-scale datasets. Building upon whole-genome data generated from multiple individuals across two taxonomically challenging genera—Salix (willows) and Potamogeton (pondweeds)—we compare two strategies for marker discovery: i) mining universally conserved gene sets widely used in plant phylogenetics (Angiosperms353 and BUSCO orthologs), and ii) targeting taxon-specific genomic regions exhibiting elevated species-diagnostic variation. Both approaches yielded minimal marker sets of four to twelve nuclear regions (approximately 1000 bp each) capable of resolving species. However, the taxon-specific strategy proved superior. A four-region panel resolved all Salix species as monophyletic with the exception of two highly polyploid taxa (S. myrsinifolia and S. phylicifolia), where misidentification, imperfect taxonomy, or frequent hybridization are suspected. In Potamogeton, a separate four-region panel resolved all species as monophyletic. Critically, these minimal marker sets balance species-diagnostic variation against conserved primer-binding sites, enabling development of simple, cost-effective high-throughput assays suitable for biodiversity assessment, ecological research, and conservation applications. The successful application across two distantly related, taxonomically complex genera demonstrates the general utility of this workflow for developing nuclear barcoding markers in any plant lineage.
Standard DNA barcodes, typically derived from plastid regions and nuclear ribosomal ITS, often fail to discriminate closely related plant species, resolving only to species-group or genus levels, while the extant nuclear conserved gene sets and kmer-based methods each have their limitations. Here we present a workflow that exploits whole-genome sequence data to develop minimal multi-locus nuclear marker sets that retain the species-level resolution of genome-scale datasets. Building upon whole-genome data generated from multiple individuals across two taxonomically challenging genera—Salix (willows) and Potamogeton (pondweeds)—we compare two strategies for marker discovery: i) mining universally conserved gene sets widely used in plant phylogenetics (Angiosperms353 and BUSCO orthologs), and ii) targeting taxon-specific genomic regions exhibiting elevated species-diagnostic variation. Both approaches yielded minimal marker sets of four to twelve nuclear regions (approximately 1000 bp each) capable of resolving species. However, the taxon-specific strategy proved superior. A four-region panel resolved all Salix species as monophyletic with the exception of two highly polyploid taxa (S. myrsinifolia and S. phylicifolia), where misidentification, imperfect taxonomy, or frequent hybridization are suspected. In Potamogeton, a separate four-region panel resolved all species as monophyletic. Critically, these minimal marker sets balance species-diagnostic variation against conserved primer-binding sites, enabling development of simple, cost-effective high-throughput assays suitable for biodiversity assessment, ecological research, and conservation applications. The successful application across two distantly related, taxonomically complex genera demonstrates the general utility of this workflow for developing nuclear barcoding markers in any plant lineage.
Royal Botanic Garden Edinburgh