Reconfiguration of the plastid genome in Lamprocapnos spectabilis: IR boundary shifting, inversion, and intraspecific variation

Abstract

We generated a complete plastid genome (plastome) sequence for Lamprocapnos spectabilis, providing the first complete plastome from the subfamily Fumarioideae (Papaveraceae). The Lamprocapnos plastome shows large differences in size, structure, gene content, and substitution rates compared with two sequenced Papaveraceae plastomes. We propose a model that explains the major rearrangements observed, involving at least six inverted repeat (IR) boundary shifts and five inversions, generating a number of gene duplications and relocations, as well as a two-fold expansion of the IR and miniaturized small single-copy region. A reduction in the substitution rates for genes transferred from the single-copy regions to the IR was observed. Accelerated substitution rates of plastid accD and clpP were detected in the Lamprocapnos plastome. The accelerated substitution rate for the accD gene was correlated with a large insertion of amino acid repeat (AAR) motifs in the middle region, but the forces driving the higher substitution rate of the clpP gene are unclear. We found a variable number of AARs in Lamprocapnos accD and ycf1 genes within individuals, and the repeats were associated with coiled-coil regions. In addition, comparative analysis of three Papaveraceae plastomes revealed loss of rps15 in Papaver, and functional replacement to the nucleus was identified.

Figure 1

Circular gene map of the Lamprocapnos spectabilis plastome. Thick lines on the inner circle indicate the inverted repeats (IR_A and IR_B, 51,309 bp), which separate the genome into small (SSC, 1,645 bp) and large (LSC, 86,358) single-copy regions. Genes on the inside and outside of the map are transcribed in clockwise and counterclockwise directions, respectively. The ring of bar graphs on the inner circle indicates the GC content in dark grey. Asterisks indicate genes transferred from single-copy regions to the IR and φ denotes a pseudogene.

Figure 2

All complete angiosperm plastomes from the NCBI Genome database, accessed on January 1, 2018. (A) Size and GC content (red) of the Lamprocapnos spectabilis plastome relative to 1,936 angiosperm plastomes from the NCBI Genome database. (B) Boxplot distribution of the sizes of the total genomes (red), large single-copy (LSC, green), small single-copy (SSC, blue) and inverted repeat (IR, purple) among 1,857 angiosperms containing two IRs. The numbers on the boxes indicate the median genome size, LSC, SSC and IR. Arrows with a closed circle indicate the positions of L. spectabilis.

Figure 3

Analysis of plastome arrangements in Lamprocapnos. (A) PCR strategy for detecting inversions and translocations. Arrowheads with red lines on the inside or outside of the circle indicate the position and orientation of the PCR primers used to confirm the structure of the plastid genome. (B) Assay results using primers designed to amplify 11 regions. Lane M contains the SolGent^TM 1 kb plus DNA ladder, and lanes (1–11) correspond to the numbers of the 11 PCR amplicons in (A) or (C). The full-length electrophoretic gel is presented in Supplementary Fig. 12. (C) Linear plastome map of Lamprocapnos with the 11 PCR amplicon sets. The genes above and below the horizontal line correspond to the genes in Fig. 1. (D) Graph showing the base per base depth of the sequencing coverage across the Lamprocapnos plastome with one IR region. The red dot-dashed line indicates the coverage of the plastome with two IRs.

Figure 4

Structural alignments of Papaveraceae plastomes using Mauve. The colored blocks represent collinear sequence blocks shared by all plastomes. Blocks drawn below the horizontal line indicate sequences found in an inverted orientation. Individual genes and strandedness are represented below the Euptelea genome block. Only one copy of the inverted repeat (IR) is shown for each plastome and pink boxes below each plastome block indicate its IR.

Figure 5

Model of plastome rearrangement in Lamprocapnos. (A) The ancestral plastome architecture of Papaveraceae (top), the hypothetical intermediate states (a to f), and the current Lamprocapnos plastome (bottom) are shown. Ancestral angiosperm genome structure is represented by Nicotiana tabacum, which is also conserved with gene order identical to three related species (Papaver, Coreanomecon, and Euptelea). The genes above and below the horizontal line are transcribed in rightward and leftward directions, respectively. Gray shading highlights inverted repeat (IR) regions with IR boundary shifts. The colored arrows correspond to the IR boundary shifts (dark green, the first IR contraction; light green, the subsequent IR contraction; dark blue, the first IR expansion; light blue, the subsequent IR expansion). The red boxes indicate the inferred inversion regions. The blue box indicates the rps2-atpA operon regions. (B) The hypothetical model for IR expansion and contraction is illustrated. IR expansion is generated with a double-strand break (DBS) event in IR_B, followed by strand invasion, expansion, and recombination in IR_A. IR contraction can likely occur via a similar mechanism. IR expansion and contraction can also occur from different directions.

Figure 6

Length variation in plastid accD and ycf1 of Lamprocapnos. (A) Schematic diagram of the genomic regions surrounding plastid accD. Boxes inside the accD gene (gray) indicate the conserved domain (acetyl-CoA carboxylase beta subunit; pink). The nucleotide and amino acid sequences of the plastid Lamprocapnos accD gene are shown in detail. Red boxes indicate the conserved domain of the acetyl-CoA carboxylase beta subunit. Blue boxes indicate amino acid repeat (AAR) motifs. (B) Amino acid sequences of plastid accD copies from six Lamprocapnos individuals. The dark and light blue boxes correspond to each AAR motif in (A). An asterisk indicates an amino acid sequence mismatch of the AAR motif. (C) Schematic diagram of genomic regions surrounding the plastid ycf1. Boxes inside the ycf1 gene (gray) indicate the conserved domain (pink) and three hotspot regions (red). Each amino acid sequence of the two hotspot regions of ycf1 copies from six Lamprocapnos individuals. Purple and orange boxes indicate amino acid repeat (AAR) motifs.

Figure 7

Variation in sequence divergence among Lamprocapnos. (A) Sequence divergence among Coreanomecon, Lamprocapnos, and Papaver plastid genes or functional groups of genes. (B) Boxplot distribution of the relative rates of d_N and d_S values for SC-to-IR genes from Lamprocapnos.