Phylogenetic Assessment of Senna tora and Senna obtusifolia Using Chloroplast Genome and 45S nrDNA Sequences

Background/Objectives: Senna tora and Senna obtusifolia are closely related herbaceous plant species with highly similar morphology that serve as important sources of herbal medicine. These two species have long been subject to taxonomic ambiguity. In this study, we aimed to clarify their phylogenetic relationship using chloroplast genome sequences and 45S nuclear ribosomal DNA (nrDNA) sequences. Methods: We assembled and analyzed complete chloroplast genomes and 45S nrDNA sequences of three Senna accessions (K276760, IT104602, and K276758) and used these datasets for phylogenetic analysis together with those of previously reported Senna species. We then performed comparative analyses to identify informative polymorphisms between S. tora and S. obtusifolia. Results: The chloroplast genomes of K276760 and IT104602 were identical in length (162,218 bp), whereas the K276758 genome was slightly larger (162,347 bp). All three chloroplast genomes exhibited quadripartite structure typical of angiosperm chloroplast genomes and contained 112 unique genes. The 45S nrDNA sequences of all three accessions were 5852 bp long and consisted of 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 26S rRNA. Phylogenetic analysis based on both chloroplast genome and 45S nrDNA sequences resolved S. tora and S. obtusifolia into separate, well-supported clades. K276760 and IT104602 were assigned to the S. obtusifolia clade, whereas K276758 showed greater affinity towards S. tora. We identified single-nucleotide polymorphisms (SNPs) and insertion/deletion (InDel) polymorphisms, and developed three candidate InDel markers to distinguish S. tora from S. obtusifolia. Conclusions: The congruent chloroplast genome and 45S nrDNA phylogenies provide strong molecular evidence for species-level differences between S. tora and S. obtusifolia. The sequence datasets and candidate InDel markers developed in this study will be useful for species identification and future taxonomic and evolutionary studies within Senna.