Comparative chloroplast genomes of pinaceae: insights into the mechanism of diversified genomic organizations

Abstract

Pinaceae, the largest family of conifers, has diversified organizations of chloroplast genomes (cpDNAs) with the two typical inverted repeats (IRs) highly reduced. To unravel the mechanism of this genomic diversification, we examined the cpDNA organizations from 53 species of the ten Pinaceous genera, including those of Larix decidua (122,474 bp), Picea morrisonicola (124,168 bp), and Pseudotsuga wilsoniana (122,513 bp), which were firstly elucidated. The results uncovered four distinct cpDNA forms (A-C and P) that are due to rearrangements of two ∼20 and ∼21 kb specific fragments. The C form was documented for the first time and the A form might be the most ancestral one. In addition, only the individuals of Ps. macrocarpa and Ps. wilsoniana were detected to have isomeric cpDNA forms. Three types (types 1-3) of Pinaceae-specific repeats situated nearby the rearranged fragments were found to be syntenic. We hypothesize that type 1 (949 ± 343 bp) and type 3 (608 ± 73 bp) repeats are substrates for homologous recombination (HR), whereas type 2 repeats are likely inactive for HR because of their relatively short sizes (151 ± 30 bp). Conversions among the four distinct forms may be achieved by HR and mediated by type 1 or 3 repeats, thus resulting in increased diversity of cpDNA organizations. We propose that in the Pinaceae cpDNAs, the reduced IRs have lost HR activity, then decreasing the diversity of cpDNA organizations, but the specific repeats that the evolution endowed Pinaceae complement the reduced IRs and increase the diversity of cpDNA organizations.

FIG. 1.—

CpDNA dot-plot analyses of Pinaceae genera. The Cedrus cpDNA is used as the reference. A positive slope denotes that the compared two sequences (horizontal and vertical axes) are matched in the same orientations, whereas a negative one indicates that the two sequences can be aligned but with opposite orientations. Labeled genes are based on their positions in the Cedrus cpDNA.

FIG. 2.—

Experimental verification of cpDNA forms in Pinaceae. (A) Sketches of eight different cpDNA forms. The reduced IR_A and IR_B are denoted by blank arrow boxes. The purple and red curved lines represent the F1 (from trnR-UCU to trnE-UUC) and F2 fragments (from ψrps4 to ψtrnG-GCC), respectively. The arrows denote their relative orientations. Primers (small open arrows) designed to verify different forms are labeled. Combinations of primer pairs are shown in the table below. (B) The primers for PCR verification. Two primer pairs were used to determine a specific cpDNA form. “+” indicates that the primers can produce expected amplicons, whereas “−” indicates that the primers cannot produce expected ones. For example, if the two primer pairs, psbB-R (I) + clpP-R (II) and trnV-F (III) + trnD-R (IV), can simultaneously produce positive fragments, the examined cpDNA may be an “A” form.

FIG. 3.—

Conserved repeats located near the boundaries of the rearranged fragments, F1 (purple arrows) and F2 (red arrows). Genera are arranged on the basis of the phylogenetic frame of Lin et al. (2010). The relative locations and orientations of F1 and F2 for each genus were labeled with “+” or “−” based on the dot-plot analyses in the fig. 1. The type 1, 2, and 3 repeats are abbreviated as T1, T2, and T3 and labeled above the cpDNA of each sampled genus. The sizes of repeats are proportional to each other.

FIG. 4.—

A hypothetical scenario for the formation of four distinct cpDNA forms. The extant and primitive states are highlighted with a yellow and a purple background, respectively. Conversion between two distinct forms can be achieved via hypothesized paths of HR (discontinued arrows). Specific repeats for each HR are shown along the discontinued arrows. Black arrows indicate the evolutionary direction from the primitive to extant states. White arrows along the black ones denote lost events. All arrows are not scaled.