Thirteen Camellia chloroplast genome sequences determined by high-throughput sequencing: genome structure and phylogenetic relationships

Abstract

Background: Camellia is an economically and phylogenetically important genus in the family Theaceae. Owing to numerous hybridization and polyploidization, it is taxonomically and phylogenetically ranked as one of the most challengingly difficult taxa in plants. Sequence comparisons of chloroplast (cp) genomes are of great interest to provide a robust evidence for taxonomic studies, species identification and understanding mechanisms that underlie the evolution of the Camellia species.

Results: The eight complete cp genomes and five draft cp genome sequences of Camellia species were determined using Illumina sequencing technology via a combined strategy of de novo and reference-guided assembly. The Camellia cp genomes exhibited typical circular structure that was rather conserved in genomic structure and the synteny of gene order. Differences of repeat sequences, simple sequence repeats, indels and substitutions were further examined among five complete cp genomes, representing a wide phylogenetic diversity in the genus. A total of fifteen molecular markers were identified with more than 1.5% sequence divergence that may be useful for further phylogenetic analysis and species identification of Camellia. Our results showed that, rather than functional constrains, it is the regional constraints that strongly affect sequence evolution of the cp genomes. In a substantial improvement over prior studies, evolutionary relationships of the section Thea were determined on basis of phylogenomic analyses of cp genome sequences.

Conclusions: Despite a high degree of conservation between the Camellia cp genomes, sequence variation among species could still be detected, representing a wide phylogenetic diversity in the genus. Furthermore, phylogenomic analysis was conducted using 18 complete cp genomes and 5 draft cp genome sequences of Camellia species. Our results support Chang's taxonomical treatment that C. pubicosta may be classified into sect. Thea, and indicate that taxonomical value of the number of ovaries should be reconsidered when classifying the Camellia species. The availability of these cp genomes provides valuable genetic information for accurately identifying species, clarifying taxonomy and reconstructing the phylogeny of the genus Camellia.

Figure 1

Gene map of the Camellia chloroplast genomes. Genes shown outside the outer circle are transcribed clockwise and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. Dashed area in the inner circle indicates the GC content of the chloroplast genome.

Figure 2

The comparison of the LSC, IR and SSC border regions among the eighteen Camellia chloroplast genomes.

Figure 3

Visualization alignment of chloroplast genome sequences. VISTA-based identity plots showing sequence identity between the eighteen sequenced Camellia chloroplast genomes and nine other representative flowering plants, with Camellia sinensis var. assamica as a reference. Thick black lines show the inverted repeats (IRs) in the chloroplast genomes. Genome regions are color-coded as protein coding, rRNA coding, tRNA coding or conserved noncoding sequences (CNS).

Figure 4

Analyses of repeated sequences in the five Camellia chloroplast genomes. A Number of the three repeat types; B Frequency of the direct repeats by length; C Frequency of the reverse repeats by length; D Frequency of palindromic repeats by length; E Location of repeats; F Summary of shared repeats among the five Camellia chloroplast genomes. IGS, intergenic spacer.

Figure 5

The distribution of simple sequence repeats (SSRs) in the five Camellia chloroplast genomes. M: Mononucleotide; T: Tetranucleotide; H: Hexanucleotide.

Figure 6

The distribution of indel types in the five Camellia chloroplast genomes. The pairwise comparisons were performed to identify indels among the five Camellia chloroplast genomes. A includes ASSA vs. OLEI/PUBI/PETE/RETI and OLEI vs. PUBI. B includes OLEI vs. PETE/RETI, PUBI vs. PETE/RETI and PETE vs. RETI.

Figure 7

Percentages of variable characters in homologous regions across the five Camellia chloroplast genomes. A Coding regions; B Non-coding regions.

Figure 8

Levels of evolutionary divergences among different regions of the five Camellia chloroplast genomes.

Figure 9

Phylogenetic relationships of the thirteen species of section Thea and the eighteen species of Camellia constructed by maximum likelihood (A, C) and maximum parsimony (B, D) with C. reticulata and Coffea arabica as outgroup. The A and C ML trees have a -InL = 124830.0859 and -InL = 290325.4563. The B MP tree has a length of 1,129 with a consistency index of 0.890 and a retention index of 0.766. The D MP tree has a length of 14,892 with a consistency index of 0.983 and a retention index of 0.796. Numbers above node are bootstrap support values (>50%).

Figure 10

Distribution of indels within introns and coding sequences of the five Camellia chloroplast genomes. The phylogenetic tree was a subtree of Figure 10 using C. arabic as outgroup. The insertions are indicated as ‘+’ and deletions are marked as ‘-’ on the branch. The genes are designated as ‘*’. Synapomorphy and homoplasy are shown by black and gray bars, respectively.