The First Complete Chloroplast Genome of Spider Flower (Cleome houtteana) Providing a Genetic Resource for Understanding Cleomaceae Evolution

Abstract

In the present study, the sequencing and analysis of the complete chloroplast genome of Cleome houtteana and its comparison with related species in the Cleomaceae family were carried out. The genome spans 157,714 base pairs (bp) and follows the typical chloroplast structure, consisting of a large single-copy (LSC) region (87,506 bp), a small single-copy (SSC) region (18,598 bp), and two inverted repeats (IRs) (25,805 bp each). We identified a total of 129 genes, including 84 protein-coding genes, 8 ribosomal RNA (rRNA) genes, and 37 transfer RNA (tRNA) genes. Our analysis of simple sequence repeats (SSRs) and repetitive elements revealed 91 SSRs, with a high number of A/T-rich mononucleotide repeats, which are common in chloroplast genomes. We also observed forward, palindromic, and tandem repeats, which are known to play roles in genome stability and evolution. When comparing C. houtteana with its relatives, we identified several highly variable regions, including ycf1, ycf2, and trnH-psbA, marking them as propitious molecular markers for the identification of species as well as phylogenetic studies. We examined the inverted repeat (IR) boundaries and found minor shifts in comparison to the other species, particularly in the ycf1 gene region, which is a known hotspot for evolutionary changes. Additionally, our analysis of selective pressures (Ka/Ks ratios) showed that most genes are under strong purifying selection, preserving their essential functions. A sliding window analysis of nucleotide diversity (Pi) identified several regions with high variability, such as trnH-psbA, ycf1, ndhI-ndhG, and trnL-ndhF, highlighting their potential for use in evolutionary and population studies. Finally, our phylogenetic analysis, using complete chloroplast genomes from species within Cleomaceae, Brassicaceae, and Capparaceae, confirmed that C. houtteana belongs within the Cleomaceae family. It showed a close evolutionary relationship with Tarenaya hassleriana and Sieruela rutidosperma, supporting previous taxonomic classifications. The findings from the current research offer invaluable insights regarding genomic structure, evolutionary adaptations, and phylogenetic relationships of C. houtteana, providing a foundation for future research on species evolution, taxonomy, and conservation within the Cleomaceae family.

Keywords: SSRs; Ycf1 gene; chloroplast genome; inverted repeat boundaries; nucleotide diversity; phylogenetic analysis; repetitive elements; selective pressure.

Figure 1

Genome map of the C. houtteana plastome. The IR regions are shown in dark colors, dividing the chloroplast genome into large (LSC) and small (SSC) single-copy regions. Genes inside the circle are transcribed clockwise, while those outside are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. The inner ring represents GC content (light green) and AT content (dark green), with a legend added for clarity. A genome length scale (in kb) is included around the outer circle to provide a size reference. The circular chloroplast genome map was generated using Chloroplot (https://irscope.shinyapps.io/Chloroplot/, accessed on 10 November 2024).

Figure 2

Repetitive sequences in C. houtteana and eight related plastomes: (A) total number of repetitive sequences. (B) lengthwise frequency of forward repeats in plastomes; (C) lengthwise frequency of palindromic repeats; (D) lengthwise frequency of reverse repeats; (E) lengthwise frequency of tandem repeats.

Figure 3

Analysis of the simple sequence repeats (SSRs) in C. houtteana and eight related plastomes: (A) total number of SSRs in genomes; (B) frequency of the simple sequence repeat motif in the chloroplast genome of C. houtteana and eleven related plastomes; (C) summary of genes lost across C. houtteana and related species plastomes. The blue color shows the missing genes, the green color shows single genes, and the red color shows the genes duplicated in plastomes.

Figure 4

(A) Heatmap showing pairwise sequence distance of 70 genes from C. houtteana and related plastomes. (B) pairwise ratios of non-synonymous rates (Ka) to synonymous rates (Ks) in C. houtteana. This heatmap illustrates the Ks/Ks ratios for 77 protein-coding genes across nine species from the Cleomaceae family. (C) Sliding window analysis of nucleotide variability among C. houtteana and related plastomes (window length: 600 bp; step size: 100 bp).

Figure 5

Distances between adjacent genes and junctions of the small single-copy (SSC), large single-copy (LSC), and two inverted repeat (IR) regions among C. houtteana and related plastomes. Boxes above and below the primary line indicate the adjacent border genes. The figure is not scaled in terms of sequence length and only shows relative changes at or near the IR/SC borders.

Figure 6

Synteny plot of C. houtteana plastome with eleven related species’ plastomes. The synteny plot shows normal links with a chocolate color, inverted links with a lime-green color, and gene features with a sky-blue color.

Figure 7

Visual alignment of C. houtteana and eight related plastomes from the Cleomaceae family. VISTA-based identity plot showing sequence identity among these species, using C. houtteana as a reference. The vertical scale indicates percent identity, ranging from 50 to 100%. The horizontal axis indicates the coordinates within the chloroplast genome. Arrows indicate the annotated genes and their transcription direction.

Figure 8

Phylogenetic trees were constructed from the whole-plastome dataset among 25 members of the order Brassicales, representing 12 different genera, using different methods such as Bayesian inference (BI) and Maximum Likelihood (ML). Numbers above the branches are the posterior probabilities of BI and bootstrap values of ML.