Enrichment Probe Sets Combining Universal and Lineage-Specific Targets Help Resolve Recalcitrant Lineages

Area of Science:

• Evolutionary Biology
• Genomics
• Botany

Background:

• Target capture sequencing (TCS) combined with low-coverage whole genome sequencing (lcWGS), known as Hyb-Seq, integrates natural history collections into biodiversity research.
• Existing probe kits like Angiosperms353 have limitations for resolving challenging plant groups, such as the Malpighiales order.

Purpose of the Study:

• To develop a custom probe set for resolving phylogenetic relationships within the difficult-to-resolve clusioid clade of flowering plants.
• To improve upon the resolution provided by universal probe kits in specific recalcitrant lineages.

Main Methods:

• Designed the Clusioids626 probe set with 39,936 probes targeting 626 nuclear orthologs, including universal and clade-specific targets.
• Applied Hyb-Seq to 70 accessions across all clusioid families and tribes.
• Retrieved plastid coding sequences using a custom target file.

Main Results:

• The Clusioids626 kit recovered a median of ~600 orthologs per sample, with 50.4% read mapping.
• Fully resolved phylogenetic relationships for all clusioid families, establishing a novel clade structure.
• Demonstrated significant incongruence between nuclear and plastid genome-derived phylogenies.

Conclusions:

• The Clusioids626 kit significantly outperforms the Angiosperms353 kit for resolving clusioid phylogeny.
• The custom probe design workflow is adaptable for other lineages needing enhanced phylogenetic resolution.
• Hyb-Seq with custom probe sets is a powerful tool for modern biodiversity genomics.