Complete nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species

Abstract

Background: The recent determination of complete chloroplast (cp) genomic sequences of various plant species has enabled numerous comparative analyses as well as advances in plant and genome evolutionary studies. In angiosperms, the complete cp genome sequences of about 70 species have been determined, whereas those of only three gymnosperm species, Cycas taitungensis, Pinus thunbergii, and Pinus koraiensis have been established. The lack of information regarding the gene content and genomic structure of gymnosperm cp genomes may severely hamper further progress of plant and cp genome evolutionary studies. To address this need, we report here the complete nucleotide sequence of the cp genome of Cryptomeria japonica, the first in the Cupressaceae sensu lato of gymnosperms, and provide a comparative analysis of their gene content and genomic structure that illustrates the unique genomic features of gymnosperms.

Results: The C. japonica cp genome is 131,810 bp in length, with 112 single copy genes and two duplicated (trnI-CAU, trnQ-UUG) genes that give a total of 116 genes. Compared to other land plant cp genomes, the C. japonica cp has lost one of the relevant large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperms, such as Cycas and Gingko, and additionally has completely lost its trnR-CCG, partially lost its trnT-GGU, and shows diversification of accD. The genomic structure of the C. japonica cp genome also differs significantly from those of other plant species. For example, we estimate that a minimum of 15 inversions would be required to transform the gene organization of the Pinus thunbergii cp genome into that of C. japonica. In the C. japonica cp genome, direct repeat and inverted repeat sequences are observed at the inversion and translocation endpoints, and these sequences may be associated with the genomic rearrangements.

Conclusion: The observed differences in genomic structure between C. japonica and other land plants, including pines, strongly support the theory that the large IRs stabilize the cp genome. Furthermore, the deleted large IR and the numerous genomic rearrangements that have occurred in the C. japonica cp genome provide new insights into both the evolutionary lineage of coniferous species in gymnosperm and the evolution of the cp genome.

Figure 1

Gene organization of the C. japonica chloroplast genome (see Table 1). Genes shown outside the circle are transcribed clockwise, while those located inside are transcribed counter-clockwise. Intron-containing genes are indicated by asterisks. Red boxes, ribosomal RNA genes; black boxes, transfer RNA genes; light orange boxes, large subunit of ribosomal protein genes; dark orange boxes, small subunit of ribosomal protein genes; dark purple boxes, DNA dependent RNA polymerase genes; dark green boxes, rbcL gene; yellowish-green boxes, subunits of photosystem I genes; green boxes, subunits of photosystem II genes; light blue boxes, subunits of cytochrome genes; dark blue boxes, subunits of ATP synthase genes; light yellow boxes, ORF genes; dark yellow boxes, subunits of NADH dehydrogenase genes; light purple boxes, chlorophyll biosynthesis genes. The pseudogene is indicated by ψ (pseudo-).

Figure 2

Amino acid sequences of the rps16 genes from five plant cp genomes, including C. japonica. The histogram below the sequences represents the degree of similarity. Peaks indicate positions of high similarity, and valleys positions of low similarity. Numbers at the C-terminal ends indicate the length of the amino acid sequences in each species.

Figure 3

Nucleotide sequences of the trnT-GGU genes of six land plant cp genomes, including C. japonica. The trnT-GGU gene is missing from the C. taitungensis genome and is too short to form a secondary structure in C. japonica. The bold characters show the anti-codon GGU. The position of the trnT-GGU gene in each cp genome is shown above each sequence. The secondary structure of the trnT-GGU gene is described at the top.

Figure 4

Alignment of amino acid sequences of the accD gene in five land plant cp genomes. The histogram indicates the degree of similarity (see Figure 2). The number on the right indicates the length of the accD reading frame in each cp genome. The amino acid length of the accD gene product in C. japonica is approximately twice that of the other five plant cp genomes.

Figure 5

Percentage identity plots and gene order surrounding the clpP gene. Gene identities between C. japonica and C. taitungensis (A), and between C. taitungensis and six other plants including C. japonica (B-G) are shown by MultiPipMaker. The directions of arrows indicate the transcribed sense/antisense strands. The colored boxes show the group of genes and the relevant coding region of each gene. Mutual comparisons of the clpP gene between C. japonica and C. taitungensis (A, B), show the first and third exon are completely absence in the C. japonica cp genome. For the gene order surrounding the clpP gene, the clpP to rpl20 gene order is extremely conserved to be co-transcribed in the cp genomes of all the land plants of this study (see Figure 7 for the detailed gene order). On the other hand, the clpP gene in the C. japonica cp genome is found halfway between the psbJ and accD gene, and is clearly not co-transcribed with the rps12 and rpl20 genes.

Figure 6

Alignment of the ycf68 regions of seven land plant cp genomes. Sequences of the ycf68 region of P. thunbergii and O. sativa were obtained from databases of each complete cp genome sequence, and relevant regions of the other plants were obtained by alignment with that of P. thunbergii or O. sativa. Codons highlighted in red represent stop codons.

Figure 7

Gene order and cp genomic architecture of the seven land plant species, including C. japonica. Each colored gene segment shows the same gene order region among the seven land plants cp genomes. Gray, blue and orange boxes for each gene order show the relevant regions of LSC (Large Single Copy), SSC (Small Single Copy) and IR (Inverted Repeat) regions in the E. globulus (A), O. sativa (B), A. capillus (C), M. polymorpha (D), and C. taitungensis (E) cp genomes, respectively. In the P. thunbergii (F) and C. japonica (G) cp genomes, gray, blue and orange boxes show the relevant regions of the SSC and IR regions of the C. taitungensis cp genome. Red boxes in the P. thunbergii cp genome show the defined IR in the P. thunbergii cp genome. In the C. japonica cp genome, the black and white arrows show duplicated genes; trnI-CAU (black arrows), trnQ-UUG (white arrows).

Figure 8

Harr plot analyses comparing the cp genome of C. japonica with those of C. taitungensis and P. thunbergii. Each dotplot shows the positions where 45 out of 50 nucleotides match in the two sequences. The plot analysis was carried out using Pipmaker software. Sequences along the Y-axis are set from the top to the bottom, and along the X-axis are from left to right. Relative lengths of sequences are shown to the side and below the boxes. The colored gene segments along the X- and Y-axes correspond with common gene units of the seven cp genomes (shown in Figure 7). At the expected endpoint of inversion or translocation mutation, the gene name is attached based on the X-axis (C. japonica cp genome). The pseudogene is indicated by ψ (pseudo-). The representative inversion and translocation are represented by gene segment I (the trnT-UGU to trnQ-UUG of C. japonica cp genome) and gene segment II (thr trnV-GAC to pseudo-clpP of C. japonica cp genome). The detailed comparisons of the gene segment I and II are shown in the following Figure 9 and Figure 10.

Figure 9

Expected inversion event in the C. japonica cp genome. The expected inversion corresponds with the gene segment I in Figure 8. Genes are represented by boxes extending above or below the base-line depending on the direction of transcription. The colored boxes indicate the same gene units among the seven cp genomes, including C. japonica. The tRNA anti-codon is abbreviated in the six plant cp genomes excluding C. japonica. The character highlighted in red represents the duplicated trnQ-UUG in the C. japonica cp genome. The pseudogene is indicated by ψ (pseudo-).

Figure 10

Expected inversion or translocation endpoints and dispersed repetitive sequences of the C. japonica cp genome. The expected inversion corresponds with gene segment II in Figure 8. Genes are represented by boxes extending above or below the base-line depending on the direction of transcription. The colored boxes indicate the same gene units among the seven cp genomes including C. japonica. The number above the gene segments of the C. japonica cp genome correspond with positions of each repetitive sequence, and the character (similarity, length, repeat type, location, and sequence) of each repetitive sequence is shown in additional file 3. The trnL-CAA gene sequence of the C. japonica cp genome is shown with its secondary structure. The trnL-CAA with ψ (pseudo-) is incomplete in length to form its secondary structure.

Figure 11

A three-step model for genome rearrangement with the clpP pseudogene in C. japonica cp genome. (A) the hypothesized ancestral cp genome of C. japonica; (B), (C) the hypothesized genome rearrangement; (D) the present form of C. japonica. The number (IV) in the figure indicate the formed repetitive sequence (see Figure 10 and additional file 3), and its transition position during genome rearrangement.