Plastid RNA Editing in Glycyrrhiza uralensis: Landscape Characterization and Comparative Assessment of RNA‑seq Library Strategies for Detection

Background: Plastid RNA editing is widespread in angiosperms, yet remains underexplored in the medicinal nonmodel species Glycyrrhiza uralensis. This study aimed to: (i) comprehensively identify plastid RNA editing sites in G. uralensis; and (ii) compare the detection performance of three library construction strategies: total RNA sequencing, rRNA-depleted RNA sequencing, and mRNA sequencing. Methods: Three individuals were resequenced for plastome assembly with GetOrganelle and annotation by PGA. Strandspecific RNAseq libraries were mapped to samplematched plastomes using HISAT2. Variants were called by REDItools2 under uniform thresholds. Candidates were visually verified in IGV and read origins confirmed by BLAST; artefacts were removed via strandspecific filtering. Results: After stringent filtering, 38 high-confidence RNA editing sites were identified across 19 genes. Total RNA seq performed best, detecting 37/38 sites consistently, whereas rRNA depleted libraries detected fewer genuine sites and produced numerous rRNA linked, noncanonical, noncoding strand dominant artefacts. Despite very low plastid mapping, mRNA seq recovered a large fraction of bona fide sites under stringent, strand aware filtering. Conclusions: This study establishes a set of 38 high-confidence plastid RNA editing sites in G. uralensis and reveals the potential adaptive implications of editing in ndh-related genes. Methodologically, total RNA-seq is recommended in de novo RNA editing identification for optimal sensitivity and low false-positive rates, and datasets from mRNA-seq can be reutilized in reliable retrieval of RNA editing sites, provided that stringent strand-specific filtering is applied.