Wheat chloroplast pangenome reveals frequent intramolecular recombination in the inverted repeat regions

In eukaryotes, the mutation rate of the chloroplasts is lower than that of the nuclear genomes. Advances in Next-Generation Sequencing (NGS) and Third-Generation Sequencing (TGS) technologies, together with improvements in genome assembly algorithms, have substantially propelled research in chloroplast genomics. Although nearly 9,000 chloroplast genomes have been released, chloroplast population genetics for specific species remains unexplored. The chloroplast genome possesses a quadripartite structure consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeats (IRs). A longstanding question is why the maternally inherited chloroplast genome does not appear to suffer from the Muller’s ratchet effect. It has been hypothesized that intramolecular recombination within the chloroplast genome may counteract this effect; however, direct evidence for such recombination remains lacking. We conducted chloroplast population data analysis in hexaploid wheat and its ancestral relatives. One the basis of the pan-genome constructed and phylogeny analysis of chloroplast genomes of all samples, we calculated the chloroplast diversity of hexaploid wheat (π = 0.0001) is the lowest among the three ploidy types tested. Additionally, we found that during the formation of hexaploid wheat, only the chloroplasts from tetraploid wheat were inherited. Moreover, Aegilops tauschii contributed solely as the paternal provider of nuclear genome material. In the chloroplast genome assembly, we assembled IRa (inverted repeat A) and IRb (inverted repeat B), revealing multiple insertion/deletion sequence differences between them. Importantly, we discovered that recombination occurs between the IR regions of the chloroplast genome. Frequent recombination results in two structural configurations existing in nearly equal proportions within a single sample. This phenomenon has led to an increase in the nucleotide diversity of the chloroplast IR region within the wheat population, which was originally low among species in Poaceae. This study demonstrates the feasibility of assembling chloroplast genomes using low-depth whole-genome sequencing (WGS) and confirms that the chloroplast genome of hexaploid wheat originates from tetraploid wheat rather than Aegilops tauschii. Furthermore, we provide evidence of frequent intra-molecular recombination in the chloroplast IR regions, leading to the coexistence of two equimolar inversion isomers. Despite strong purifying selection, recombination increases genetic diversity within the IR regions, facilitating adaptation and maintaining the functional stability of essential genes. Our findings highlight the role of recombination in balancing genetic stability and flexibility in chloroplast genome evolution, offering new insights into nuclear-cytoplasmic interactions and polyploid adaptation.