Comparative chloroplast genomes place Rhododendron ambiguum near R. concinnum but leave genus relationships unresolved

A comparative chloroplast-genome study placed the newly assembled Rhododendron ambiguum chloroplast genome close to that of R. concinnum in Bayesian phylogenetic analyses, but broader relationships among the sampled Rhododendron species remained unresolved. The study characterized the R. ambiguum genome and compared complete chloroplast genomes from 18 Rhododendron species, using three Vaccinium species as outgroups.

Fresh R. ambiguum leaves were collected at an elevation of 3,291 meters in Gongga Mountain National Nature Reserve in Sichuan, China, and a corresponding voucher specimen was deposited at the West China Subalpine Botanical Garden in Dujiangyan under voucher number 2019-GG-015. DNA was extracted with a modified CTAB method and sequenced on an Illumina HiSeq X Ten platform. The project generated 8 gigabases of paired-end reads, corresponding to an estimated sequencing depth of about 18-fold, which the authors compared with the approximately 650-megabase size reported for most Rhododendron genomes.

The researchers assembled the reads with MIRA and NOVOplasty, using the published chloroplast genomes of R. × pulchrum and R. calophytum as references. Annotation was performed with CpGAVAS and supplemented by manual curation and BLAST gene verification. The completed R. ambiguum chloroplast genome was deposited in GenBank under accession OR455462. The assembly was circular and quadripartite, spanning 207,478 base pairs: two inverted repeats of 47,249 base pairs each, a large single-copy region of 110,367 base pairs and a small single-copy region of 2,613 base pairs.

The R. ambiguum genome contained 110 unique genes, comprising 77 protein-coding genes, 29 transfer RNA genes and four ribosomal RNA genes. Sixteen genes were duplicated in the inverted repeats, and overall GC content was 35.8%. Fourteen genes contained introns: 10 protein-coding genes and four transfer RNA genes. The ycf3 gene contained two introns, while the annotated-gene table also marked rps12 as trans-spliced. These features followed the typical organization reported for many terrestrial plant chloroplast genomes, although the assembled sequence was larger than the 120,000–160,000-base-pair range cited for a typical angiosperm chloroplast genome.

Comparisons across the 18 Rhododendron genomes revealed extensive variation in gene order and in the boundaries of the large single-copy, small single-copy and inverted-repeat regions. Eleven species had genomes near 200,000 base pairs and relatively small small-single-copy regions. R. simsii, R. latoucheae and R. × pulchrum had shorter total genomes, although R. simsii possessed a 69,783-base-pair small-single-copy region. R. molle and R. huadingense had the longest large single-copy regions and shortest inverted repeats, while R. kawakamii and R. datiandingense differed markedly in regional lengths and gene arrangement. Across all 18 genomes, total length ranged from 146,941 base pairs in R. × pulchrum to 230,777 base pairs in R. kawakamii. The authors cautioned that mapping or annotation differences could also contribute to unusual structural patterns.

Sliding-window analysis of 11 species with similar genome structures, using 600-base-pair windows and 200-base-pair steps, identified four highly variable non-coding regions with nucleotide-diversity values of at least 0.095: trnI-rpoB, ndhE-psaC, rpl32-ndhF and rrn16S-trnI. The first two were located in the large single-copy region, rpl32-ndhF in the small single-copy region and rrn16S-trnI in an inverted repeat. Non-coding regions were more variable than coding regions in this dataset. The R. ambiguum genome also contained 76 simple sequence repeats, including 69 mononucleotide, six dinucleotide and one trinucleotide repeat. Fifty-nine were in intergenic regions, and none was detected in the small single-copy region.

Codon analysis across the 18 genomes identified a general preference for codons ending in A or U. Relative synonymous codon-usage values ranged from 0.317 for CGC to 2.17 for UUA, with UUA preferentially used for leucine. Selection-pressure estimates were calculated for 53 shared protein-coding genes. Among 42 genes for which an applicable nonsynonymous-to-synonymous substitution ratio, or Ka/Ks ratio, was obtained, 38 had ratios below 1 and the mean was 0.496. Four genes—cemA, rps4, rpl16 and rpl14—had estimated ratios above 1. These were interpreted by the authors as signs of positive selection, rather than as direct measurements of adaptive function.

Bayesian inference based on shared protein-coding genes divided the sampled Rhododendron species into three broad clades, with node values reported as approximately 1 for nearly all nodes. Analyses of both shared genes and complete chloroplast genomes placed R. ambiguum near R. concinnum. However, the two datasets produced inconsistent topologies, and relationships among Rhododendron subgenera remained unresolved. The authors associated much of this uncertainty with the placement of subgenus Tsutsusi and noted that natural hybridization, introgression and intermediate morphologies complicate taxonomic identification within the genus.

The study provides a complete chloroplast-genome resource for R. ambiguum and identifies variable regions that may serve as candidate markers in future phylogenetic analysis. Its comparative scope was limited to 18 Rhododendron chloroplast genomes, with V. bracteatum, V. macrocarpon and V. uliginosum added as outgroups, and the phylogenetic results did not establish a genus-wide topology. Genome mapping and annotation may influence some apparent structural differences, while the Ka/Ks estimates do not directly demonstrate the causes of selection. The assembled sequence and associated datasets are available through the NCBI Bioproject and GenBank accession OR455462.

Subject of Research: Comparative chloroplast genomics and phylogeny of Rhododendron species

Article Title: The complete chloroplast genome of Rhododendron ambiguum and comparative genomics of related species

Article References: Ma, W. B., Ou, Y., Dayananda, B., Ji, H. J., & Yu, T. (2024). The complete chloroplast genome of Rhododendron ambiguum and comparative genomics of related species. Comparative Cytogenetics, 18, 143-159. https://doi.org/10.3897/compcytogen.18.119929

Image Credits: AI Generated

DOI: 10.3897/compcytogen.18.119929

Keywords: Rhododendron ambiguum, chloroplast genome, comparative genomics, phylogenetics, plant molecular biology, Rhododendron, Bayesian inference, DNA barcoding, codon usage, selection pressure, complete, chloroplast