The cucumber (Cucumis sativus L.) mitochondrial genome: intraspecific structural variation, repetitive architecture,...

Mitochondria are essential organelles with complex mitochondrial genomes (mitogenomes) that challenge conventional assembly and analysis. Compared to plastid and nuclear genomes, plant mitochondrial genomes (mitogenomes) exhibit immense structural complexity and low nucleotide substitution rates. Cucumber (Cucumis sativus L.) is a vital crop and a key model for studying its rare paternal mitogenome inheritance. However, a comprehensive understanding of intraspecific mitogenomic diversity in cucumber has been hindered by the limitations of short-read sequencing, which fails to resolve its complex, repeat-rich architecture.

Here, by leveraging long-read sequencing, we assembled the complete mitogenomes of 12 diverse cucumber accessions. The assemblies revealed massive mitogenomes (1.49-1.61 Mb) with highly reticulate, graph-based structures, moving beyond the simplistic"master-circle" model. We discovered that this profound structural diversity is driven by a small number of large, recombinationally active repeats, which allowed us to classify the mitogenomes into six distinct structural types. Phylogenetic analyses of the plastid (maternal) and mitochondrial (paternal) genomes revealed strikingly discordant evolutionary histories, reflecting their different inheritance modes and evolutionary rates. Analysis of intracellular DNA transfer showed that mitochondrial-to-nuclear transfers (NUMTs) have declined over evolutionary time, whereas plastid-to-nuclear transfers (NUPTs) are more recent and ongoing. Furthermore, the synonymous substitution rate in cucumber mitogenomes was 8-10 times lower than in plastomes, underscoring their extreme sequence conservation.

Our study demonstrates that cucumber mitogenomes are not static but are highly dynamic mosaics shaped by repeat-mediated recombination, resulting in extensive structural variation even within a single species. These findings highlight the power of long-read assembly for studying complex organellar genomes and establish the paternally inherited mitogenome as a valuable resource for tracing paternal lineages and developing novel strategies for crop improvement.