Lycophytes and ferns represent one of the earliest-diverging lineages of vascular plants, with the Lycopodiaceae family constituting the basal clade among lycophytes. In this research, we successfully assembled and annotated the complete Lycopodium japonicum Thunb. (L. japonicum) mitochondrial genome (mitogenome) utilizing PacBio HiFi sequencing data, resulting in a single circular molecule with a size of 454,458 bp. 64 unique genes were annotated altogether, including 34 protein-coding genes, 27 tRNAs and 3 rRNAs. It also contains 32 group II introns, all of which undergo cis-splicing. We identified 195 simple sequence repeats, 1,948 dispersed repeats, and 92 tandem repeats in the L. japonicum mitogenome. Collinear analysis indicated that the mitogenomes of Lycopodiaceae are remarkably conserved compared to those of other vascular plants. We totally identified 326 RNA editing sites in 31 unique protein-coding genes with 299 sites converting cytosine to uracil and 27 sites the reverse. Notably, the L. japonicum mitogenome has small amounts foreign DNA from plastid or nuclear origin, accounting for only 2.81% of the mitogenome. The maximum likelihood phylogenetic analysis based on 23 diverse land plant mitogenomes and plastid genomes supports the basal position of lycophytes within vascular plants and they form a sister clade to all other vascular lineages, which is consistent with the PPG I classification system. As the first reported mitogenome of Lycopodioideae subfamily, this study enriches our understanding of Lycopodium mitogenomes, and sets the stage for future research on mitochondrial diversity and evolution within the lycophytes and ferns.

Malus Mill., a genus of temperate perennial trees with great agricultural and ecological value, has diversified through hybridization, polyploidy and environmental adaptation. Limited genomic resources for wild Malus species have hindered the understanding of their evolutionary history and genetic diversity. We sequenced and assembled 30 high-quality Malus genomes, representing 20 diploids and 10 polyploids across major evolutionary lineages and geographical regions. Phylogenomic analyses revealed ancient gene duplications and conversions, while six newly defined genome types, including an ancestral type shared by polyploid species, facilitated the detection of strong signals for extensive introgressions. The graph-based pan-genome captured shared and species-specific structural variations, facilitating the development of a molecular marker for apple scab resistance. Our pipeline for analyzing selective sweep identified a mutation in MdMYB5 having reduced cold and disease resistance during domestication. This study advances Malus genomics, uncovering genetic diversity and evolutionary insights while enhancing breeding for desirable traits.

Plant mitochondrial genomes (mitogenomes) exhibit fluid genome architectures, which could lead to the rapid erosion of genome synteny over a short evolutionary time scale. Among the species-rich orchid family, the leafy Cymbidium lancifolium and leafless Cymbidium macrorhizon are sister species with remarkable differences in morphology and nutritional physiology. Although our understanding of the evolution of mitochondria is incomplete, these sister taxa are ideal for examining this subject. In this study, the complete mitogenomes of C. lancifolium and C. macrorhizon, totaling 704,244 bp and 650,751 bp, respectively, were assembled. In the 2 mitogenomes, 38 protein-coding genes, 18 cis- and 6 trans-spliced introns, and approximately 611 Kb of homologous sequences are identical; overall, they have 99.4% genome-wide similarity. Slight variations in the mitogenomes of C. lancifolium and C. macrorhizon in repeat content (21.0 Kb and 21.6 Kb, respectively) and mitochondrial DNA of plastid origin (MIPT; 38.2 Kb and 37.5 Kb, respectively) were observed. The mitogenome architectures of C. lancifolium and C. macrorhizon are complex and comprise 23 and 22 mini-circular chromosomes, respectively. Pairwise comparisons indicate that the two mitogenomes are largely syntenic, and the disparity in chromosome numbers is likely due to repeat-mediated rearrangements among different chromosomes. Notably, approximately 93.2 Kb C. lancifolium mitochondrial sequences lack any homology in the C. macrorhizon mitogenome, indicating frequent DNA gains and losses, which accounts mainly for the size variation. Our findings provide unique insights into mitogenome evolution in leafy and leafless plants of sister species and shed light on mitogenome dynamics during the transition from mixotrophy to mycoheterotrophy.

Posttranscriptional modifications, including intron splicing and RNA editing, are common processes during regulation of gene expression in plant organelle genomes. However, the intermediate products of intron-splicing, and the interplay between intron-splicing and RNA-editing were not well studied. Most organelle transcriptome analyses were based on the Illumina short reads which were unable to capture the full spectrum of transcript intermediates within an organelle. To fully investigate the intermediates during intron splicing and the underlying relationships with RNA editing, we used PacBio DNA-seq and Iso-seq, together with Illumina short reads genome and transcriptome sequencing data to assemble the chloroplast and mitochondrial genomes of Nymphaea ‘Joey Tomocik’ and analyze their posttranscriptional features. With the direct evidence from Iso-seq, multiple intermediates partially or fully intron-spliced were observed, and we also found that both cis- and trans-splicing introns were spliced randomly. Moreover, by using rRNA-depleted and non-Oligo(dT)-enrichment strand-specific RNA-seq data and combining direct SNP-calling and transcript-mapping methods, we identified 98 and 865 RNA-editing sites in the plastome and mitogenome of N. ‘Joey Tomocik’, respectively. The target codon preference, the tendency of increasing protein hydrophobicity, and the bias distribution of editing sites are similar in both organelles, suggesting their common evolutionary origin and shared editing machinery. The distribution of RNA editing sites also implies that the RNA editing sites in the intron and exon regions may splice synchronously, except those exonic sites adjacent to intron which could only be edited after being intron-spliced. Our study provides solid evidence for the multiple intermediates co-existing during intron-splicing and their interplay with RNA editing in organelle genomes of a basal angiosperm.