Conserved chloroplast genome sequences of the genus Clerodendrum Linn. (Lamiaceae) as a super-barcode

Abstract

Background: The plants of the genus Clerodendrum L. have great potential for development as an ornamental and important herbal resource. There is no significant morphological difference among many species of the genus Clerodendrum, which will lead to confusion among the herbs of this genus and ultimately affect the quality of the herbs. The chloroplast genome will contribute to the development of new markers used for the identification and classification of species.

Methods and results: Here, we obtained the complete chloroplast genome sequences of Clerodendrum chinense (Osbeck) Mabberley and Clerodendrum thomsoniae Balf.f. using the next generation DNA sequencing technology. The chloroplast genomes of the two species all encode a total of 112 unique genes, including 80 protein-coding, 28 tRNA, and four rRNA genes. A total of 44-42 simple sequence repeats, 19-16 tandem repeats and 44-44 scattered repetitive sequences were identified. Phylogenetic analyses showed that the nine Clerodendrum species were classified into two clades and together formed a monophyletic group. Selective pressure analyses of 77 protein-coding genes showed that there was no gene under positive selection in the Clerodendrum branch. Analyses of sequence divergence found two intergenic regions: trnH-GUG-psbA, nhdD-psaC, exhibiting a high degree of variations. Meanwhile, there was no hypervariable region identified in protein coding genes. However, the sequence identities of these two intergenic spacers (IGSs) are greater than 99% among some species, which will result in the two IGSs not being used to distinguish Clerodendrum species. Analysis of the structure at the LSC (Large single copy) /IR (Inverted repeat) and SSC (Small single copy)/IR boundary regions showed dynamic changes. The above results showed that the complete chloroplast genomes can be used as a super-barcode to identify these Clerodendrum species. The study lay the foundation for the understanding of the evolutionary process of the genus Clerodendrum.

Fig 1. Gene map of the C. chinense plastome.

There are six rings on the diagram from the center outwards. The first ring is used to indicate the position of forward (red arcs) and reverse (green arcs) repeats. The second ring is used to indicate tandem repeats (short columns). The third ring is used to indicate microsatellite repeats (short columns). The fourth shaded ring indicates each part and the length of SSC, IRa, IRb, and LSC regions. The optional shaded area stretching from the inner sphere toward the outer circle marks the IR regions The fifth ring indicates the GC content of the chloroplast genome. The outer ring shows the gene names and their optional codon usage bias. The genes are colored based on their functional categories.

Fig 2. Evolutionary tree of family Lamiaceae.

The phylogenetic results included 44 species within family and two out-group species. Mazus pumilus and Phryma leptostachya were set as the outgroups. The species marked with an asterisk are C. Chinense and C. thomsoniae, whose chloroplast genomes were firstly reported. The right of the panel is the name of tribe and subfamily according to the species. Numbers above the branches are the bootstrap support values.

Fig 3. Results of genetic distance analysis of protein coding genes (A) and intergenic regions (B) in Clerodendrum species.

Comparison of the variability of PCG and IGS regions among C. chinense, C. cyrtophyllum, C. lindleyi, C. yunnanense, C. mandarinorum, C. trichotomum, C. thomsoniae and C. japonicum. The X-axis indicates the name of PCG and IGS regions. The top thirty PCG or ICG of K2p distances were showed in panel A and panel B. And the Y-axis shows the range of K2p distances between different pairs of species. The diamond shows the average K2p distance, respectively.

Fig 4. Boundary analysis of the single copy region and inverted repeated region among 8 Clerodendrum species.

The name of each species and its corresponding length of chloroplast genome sequence are described on the left side of each track. Genes shown above the lines are transcribed forward while genes shown below the lines are transcribed reversely. Light blue, orange, and green box represent LSC, IR and SSC region, respectively. The numbers on the box indicate the length of each region. The number near the arrow shows the distance between the start or end coordinates of a given gene and the corresponding junction site. JLB (IRb/LSC), JSB (IRb/SSC), JSA (SSC/IRA) and JLA (IRa/LSC) represent the junction between corresponding regions in the genome respectively.