The rice nuclear genome continuously integrates, shuffles, and eliminates the chloroplast genome to cause chloroplast-nuclear DNA flux

Abstract

Plastid DNA fragments are often found in the plant nuclear genome, and DNA transfer from plastids to the nucleus is ongoing. However, successful gene transfer is rare. What happens to compensate for this? To address this question, we analyzed nuclear-localized plastid DNA (nupDNA) fragments throughout the rice (Oryza sativa ssp japonica) genome, with respect to their age, size, structure, and integration sites on chromosomes. The divergence of nupDNA sequences from the sequence of the present plastid genome strongly suggests that plastid DNA has been transferred repeatedly to the nucleus in rice. Age distribution profiles of the nupDNA population, together with the size and structural characteristics of each fragment, revealed that once plastid DNAs are integrated into the nuclear genome, they are rapidly fragmented and vigorously shuffled, and surprisingly, 80% of them are eliminated from the nuclear genome within a million years. Large nupDNA fragments preferentially localize to the pericentromeric region of the chromosomes, where integration and elimination frequencies are markedly higher. These data indicate that the plant nuclear genome is in equilibrium between frequent integration and rapid elimination of the chloroplast genome and that the pericentromeric regions play a significant role in facilitating the chloroplast-nuclear DNA flux.

Figure 1.

Frequency of the Appearance of nupDNA Fragments throughout the Rice Chloroplast Genome. The rice chloroplast genome was divided into 100-bp segments. The numbers of nupDNA fragments corresponding to individual segments are shown by histograms. The rice chloroplast genome is a double-stranded circular DNA molecule of 134.5 kb, which contains two copies of an identical 20.8-kb inverted repeat (IRa and IRb) separated by a large single-copy region (LSC; 80.6 kb) and a small single-copy region (SSC; 12.4 kb) (Sugiura, 1992), as is schematically illustrated in Figure 3. The circular map is linearized at the junction between IRa and the large single-copy region. The yellow boxes indicate the regions whose copies are found in the rice mitochondrial genome. The red line indicates the expected number of nupDNA fragments if they originated from throughout the chloroplast genome with equal frequency.

Figure 2.

Locations of nupDNA Fragments on the Rice Genetic Map. The vertical axis represents the genetic map (cM) of each chromosome, and the horizontal bar shows the sum of the nupDNA fragments (kb) located at each locus. nupDNA fragments larger than 10 kb are denoted A to L in order of decreasing size and are indicated by heavy lines. Fragments B and H are located within the same locus. A scale bar of 10 kb is shown in the box. Arrowheads indicate centromeres.

Figure 3.

Schematic Structures of nupDNA Fragments. nupDNA fragments larger than 10 kb are denoted A to L in order of decreasing size. As shown in the box, the rice chloroplast genome is a circular DNA molecule of 135 kb, which contains two copies of an identical IR (IRa and IRb) separated by large (LSC) and small single-copy (SSC) regions (Sugiura, 1992). The entire chloroplast genome was divided into 5-kb segments (arrows), denoted 1 to 23. The corresponding segments found in nupDNA fragments are represented by numbered arrows. The IRs and their segments are shown in red. Asterisks indicate gaps where the genomic sequence was not available when this analysis was performed.

Figure 4.

nupDNA Fragments Are Derived from Ancestral Chloroplast Genomes. (A) Schematic illustration of the IR regions of nupDNA fragments F, A, and G, compared with that of the present chloroplast genome. LSC, large single-copy region; SSC, small single-copy region. (B) The predicted structures of the ancient chloroplast genomes from which nupDNA fragments F, A, and G are derived. Cp indicates the present chloroplast genome. Thick lines of the F, A, and G genomes represent the DNA fragments found in the present nupDNAs, and gray lines represent the parts predicted to be lost or not transferred during evolution.

Figure 5.

Age Distribution and Localization of nupDNA Fragments Larger than 200 bp. The combined lengths of these fragments account for 84% of the total nupDNA. (A) Total amount of nupDNAs generated in the respective periods. (B) Total numbers of nupDNA fragments generated in the respective periods, classified into four groups based on size. (C) Age distribution of nupDNAs occurring within 5 cM of the centromere. (D) Age distribution of nupDNAs occurring more than 5 cM from the centromere. (E) Size distribution of young nupDNA fragments (<1 Myr) in the pericentromeric regions (dark-gray bars) and in the rest (light-gray bars). Size classes: I, 200 to 400 bp; II, 400 to 800 bp; III, 800 to 1600 bp; IV, 1600 to 3200 bp; V, >3200 bp. (F) Integration frequency of young nupDNA fragments (<1 Myr) in the pericentromeric regions. The red line indicates the expected number of integration sites if they were equally distributed throughout the nuclear genome (see Methods). Letters represent the giant nupDNA fragments (Figure 2).

Figure 6.

DNA Flux from Plastid to Nucleus. The nuclear genome continually engulfs the plastid DNA and eliminates it by genome shuffling. This evolutionary process mainly proceeds at unique loci of the nuclear genome, such as the pericentromeric regions. CP, chloroplast.