Complete Chloroplast Genome Sequence of Hibiscus cannabinus and Comparative Analysis of the Malvaceae Family

Abstract

Kenaf (Hibiscus cannabinus) is one of the most fast-growing bast in the world and belongs to the family Malvaceae. However, the systematic classification and chloroplast (cp) genome of kenaf has not been reported to date. In this study, we sequenced the cp genome of kenaf and conducted phylogenetic and comparative analyses in the family of Malvaceae. The sizes of H. cannabinus cp genomes were 162,903 bp in length, containing 113 unique genes (79 protein-coding genes, four rRNA genes, and 30 tRNA genes). Phylogenetic analysis indicated that the cp genome sequence of H. cannabinus has closer relationships with Talipariti hamabo and Abelmoschus esculentus than with Hibiscus syriacus, which disagrees with the taxonomical relationship. Further analysis obtained a new version of the cp genome annotation of H. syriacus and found that the orientation variation of small single copy (SSC) region exists widely in the family of Malvaceae. The highly variable ycf1 and the highly conserved gene rrn32 were identified among the family of Malvaceae. In particular, the explanation for two different SSC orientations in the cp genomes associated with phylogenetic analysis is discussed. These results provide insights into the systematic classification of the Hibiscus genus in the Malvaceae family.

Keywords: Hibiscus cannabinus; LSC; Malvaceae; SSC; cp; gene orientation.

FIGURE 1

Gene map of the H. cannabinus chloroplast genome. The genes inside and outside of the outer circle are transcribed in the clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are shown in different colors. The inner circle represents different regions of the cp genome. IRA, inverted repeat region A; IRB, invert repeat region B; LSC, large single-copy region; SSC, small single-copy region. The line-chart in gray shows GC content along the genome.

FIGURE 2

Simple Sequences Repeats (SSR) Analysis in H. cannabinus. The distributions of mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide repeats were shown.

FIGURE 3

Evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 0.92533808 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al., 2004) and are in units of the number of base substitutions per site. The analysis involved 40 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were 226151 positions in total in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).

FIGURE 4

Sequence identity plot comparing the chloroplast genome of Hibiscus cannabinus. Hibiscus syriacus, Gossypium hirsutum, and Gossypium raimondii. The vertical scale indicates the percentage of identity, ranging from 50 to 100%. The horizontal axis indicates the coordinates within the cp genome. Genome regions are color-coded as protein-coding, rRNA, tRNA, intron, and conserved non-coding sequences (CNS).

FIGURE 5

The pattern of nucleotide substitutions between Hibiscus cannabinus and Gossypium raimondii chloroplast genomes. The patterns were divided into six types as indicated by the six non-stand-specific base-substitution types (i.e., numbers of considered G to A and C to T sites for each respective set of associated variation types). The cp of Gossypium raimondii was used as a reference.

FIGURE 6

Chloroplast genome comparative analysis between H. cannabinus and G. raimondii. Sliding window plots of nucleotide diversity (π) across the complete cp genomes of Hibiscus cannabinus and Gossypium raimondii (window length: 600 bp, step size: 100 bp). Y-axes: nucleotide diversity (π) of each window; X-axes: the position of the midpoint of a window.

FIGURE 7

Comparative analysis of chloroplast genomic DNA sequences of Hibiscus cannabinus and Hibiscus syriacus. (A) The number of repeated sequences in Hibiscus cannabinus. (B) The number of repeated sequences Hibiscus syriacus. (C) Sliding window plots of nucleotide diversity (π) across the complete cp genomes of Hibiscus cannabinus and Gossypium raimondii (window length: 600 bp, step size: 100 bp). Y-axes: nucleotide diversity (π) of each window; X-axes: the position of the midpoint of a window.