Long-Read Assembly and a Within-Genus Structural Survey Reveal an Exceptionally Long Inverted Repeat in the...

Background/Objectives: Expanded inverted repeats (IRs) occur in Rhododendron plastomes, but their extent and evolutionary context remain incompletely resolved because repeat-rich genomes can be difficult to assemble from short reads. Methods: We assembled and curated the plastome of Rhododendron spinuliferum using publicly available PacBio HiFi reads and evaluated whether the same junction architecture was present using Illumina PE400 reads from a different individual. Results: Bandage-guided graph resolution and junction-spanning reads supported a 209,482 bp quadripartite plastome comprising a 108,321 bp large single-copy (LSC) region, a 2629 bp small single-copy (SSC) region, and two 49,266 bp IRs. All four inferred junctions were supported by HiFi reads and were also detected in the independent Illumina dataset, and short-read mapping covered every reference position without zero-depth sites. In a broad within-genus survey of 26 structurally resolved Rhododendron plastomes, R. spinuliferum had the longest validated conventional IR in the curated dataset. Twenty-four plastomes shared a 19-gene expanded-IR core and retained ndhF in the SSC, whereas R. vialii and R. datiandingense represented extreme SSC contraction and short-IR architecture, respectively. Ancestral-state reconstruction identified the expanded-IR core as the most likely crown state. After one IR copy was removed, repeat density was not correlated with IR length, and comparison with R. delavayi showed that the additional IR sequence in R. spinuliferum was distributed predominantly across intergenic regions. Conclusions: These results provide a validated plastome resource and clarify the structural evolution of expanded IRs in the sampled Rhododendron plastomes.