A Large Intergenic Spacer Leads to the Increase in Genome Size and Sequential Gene Movement around IR/SC Boundaries in the Chloroplast Genome of Adiantum malesianum (Pteridaceae)

Abstract

Expansion and contraction (ebb and flow events) of inverted repeat (IR) boundaries occur and are generally considered to be major factors affecting chloroplast (cp) genome size changes. Nonetheless, the Adiantum malesianum cp genome does not seem to follow this pattern. We sequenced, assembled and corrected the A. flabellulatum and A. malesianum cp genomes using the Illumina NovaSeq6000 platform, and we performed a comparative genome analysis of six Adiantum species. The results revealed differences in the IR/SC boundaries of A. malesianum caused by a 6876 bp long rpoB-trnD-GUC intergenic spacer (IGS) in the LSC. This IGS may create topological tension towards the LSC/IRb boundary in the cp genome, resulting in a sequential movement of the LSC genes. Consequently, this leads to changes of the IR/SC boundaries and may even destroy the integrity of trnT-UGU, which is located in IRs. This study provides evidence showing that it is the large rpoB-trnD-GUC IGS that leads to A. malesianum cp genome size change, rather than ebb and flow events. Then, the study provides a model to explain how the rpoB-trnD-GUC IGS in LSC affects A. malesianum IR/SC boundaries. Moreover, this study also provides useful data for dissecting the evolution of cp genomes of Adiantum. In future research, we can expand the sample to Pteridaceae to test whether this phenomenon is universal in Pteridaceae.

Keywords: Adiantum malesianum; chloroplast conformations; genome comparison; inverted repeat; topological tension.

Figure 1

Gene map of A. flabellulatum and A. malesianum cp genomes. The inner circle is A. flabellulatum, while the outer is A. malesianum. Genes on the inside of the circle are transcribed clockwise, while those on the outside are transcribed counter-clockwise. Genes are color-coded based on functions. The dotted arrows indicate the position shift of the same gene in two cp genomes. The thicker black line represents the IRs. Names of the same genes in the two cp genomes are displayed only once.

Figure 2

Analysis of the expansion/contraction of IR/SC boundaries in six Adiantum cp genomes. For each species, genes transcribed on the positive strand are depicted from right to left above the corresponding tracks, while genes on the negative strand are depicted from left to right below the tracks. Arrows indicate the distance from one side of the gene to the LSC/IR boundary. ΦndhB is a partial copy of ndhB in the IRa. ΦtrnT-UGU represents fragmented sequences of trnT-UGU.

Figure 3

The schematic diagram of the location of homologous sequences included in the large rpoB-trnD-GUC IGS of A. malesianum. Gray lines connect different homologous sequences of one specie.

Figure 4

(a) A model to explain the structural change around IR/SC boundaries in the A. malesianum cp genome. It shows that the large rpoB-trnD-GUC IGS causes sequential gene movement in LSC, leading to trnI-CAU being “squeezed” into IRb and replacing part of trnT-UGU; then, the generation of trnI-CAU in IRa leads to the movement of ndhB from IRa into LSC. Adiantum capillus-veneris is used as the reference, because it has the highest level of homology with A. malesianum. Gene ropB is set as the start site of alignment. Black horizontal lines represent gene sequences; “||” represents sequence omission. Black boxes indicate identical genes; colored boxes are genes with location changes at the IR/SC boundaries. Of them, the trnT-UGU gene in A. malesianum is experimentally confirmed as unable to be normally expressed, as it only has a partial fragment remaining. Inset is the sequence alignment of trnT-UGU, trnI-CAU and their intergenic spacer between the two species; gray color indicates an identical site, red dots represent omitted bases present in A. capillus-veneris and “-” represents the gap. The number on the right side of the sequence is the number of bases. (b) Electropherogram showing the amplified DNA fragments corresponding to the four IR/SC boundaries in the A. malesianum cp genome.

Figure 5

(a) Comparative analysis of repeated sequences in the six Adiantum cp genomes. The number of SSRs (above), dispersed repeats (middle) and tandem repeats (below) are presented. (b) Distribution pattern of SSRs, dispersed repeats and tandem repeats in the A. malesianum cp genome. Blue regions are IRs; the red region is the rpoB-trnD-GUC IGS. Dots indicate relative positions of the repeats.