# Insights into the chloroplast genome diversity of the genus Isatis in China

## TL;DR

This study analyzes chloroplast genomes of seven Isatis species in China to resolve taxonomic confusion and identify useful genetic markers.

## Contribution

The study provides new chloroplast genome data and identifies the rpl32–trnL spacer as a novel marker for species identification in Isatis.

## Key findings

- Chloroplast genomes of seven Isatis species were sequenced and analyzed for structural and sequence variation.
- The rpl32–trnL intergenic spacer was found to be the most polymorphic region and a promising molecular marker.
- Phylogenomic analysis confirmed the monophyly of Isatis and revealed misidentifications in public databases.

## Abstract

The genus Isatis contains medicinally important but taxonomically controversial species in China. Reliable genomic resources are urgently needed for accurate species identification and phylogenetic clarification.

We assembled and characterized the complete chloroplast genomes of seven Isatis species. The genomes (153,260–153,872 bp) exhibited the typical quadripartite structure. Sequence variation was heterogeneous: the small single-copy (SSC) region was the most polymorphic (12.3 single-nucleotide polymorphisms (SNPs)/kb), followed by the large single-copy (LSC, 8.9 SNPs/kb) and inverted repeat (IR, 1.7 SNPs/kb) regions. Noncoding sequences showed 3.2-fold greater polymorphism than coding sequences. The rpl32–trnL intergenic spacer was identified as a promising molecular marker due to its high nucleotide diversity (π = 0.0582). While most protein-coding genes were under strong purifying selection, ycf1 and ycf2 exhibited elevated substitution rates. Phylogenomic analyses strongly supported the monophyly of the tribe Isatideae and the genus Isatis (100% bootstrap support (BS)), validating the recent inclusion of I. gymnocarpa and I. multicaulis. We detected significant misidentification in public databases for key taxa (I. costata, I. tinctoria, I. indigotica). The rpl32–trnL spacer effectively distinguished most species but revealed the non-monophyly of I. costata, with one lineage being indistinguishable from I. tinctoria. The phylogenetic separation of the morphologically similar I. minima and I. violascens suggests potential convergent evolution.

Our study provides essential chloroplast genomic resources for Isatis. The findings not only clarify long-standing taxonomic controversies and validate recent reclassifications but also highlight the utility of the rpl32–trnL spacer as a powerful marker for species discrimination and evolutionary studies within this genus.

The online version contains supplementary material available at 10.1186/s12870-026-08240-3.