De novo assembly of the first mitochondrial genome in Benincasa reveals structural dynamics and evolutionary insights...

BACKGROUND: Benincasa hispida Cogn. var. chieh-qua How, commonly referred to as the small wax gourd, is a widely cultivated cucurbit vegetable renowned for its considerable nutritional and medicinal benefits. However, the lack of reported mitochondrial genome (mitogenome) information for B. hispida severely restricts our understanding of the organelle evolution within the Cucurbitaceae family. This study bridges this knowledge gap by undertaking the sequencing, assembly, and comprehensive analysis of the complete mitogenome of B. hispida. RESULTS: Using an integrated sequencing approach that combined Illumina and Nanopore technologies, we performed de novo assembly of the mitogenome of B. hispida, followed by comparative analyses with its close relatives. The assembled mitogenome was characterized by a linear structure composed of 18 contigs, with a total length of 431,446 bp and a GC content of 44.9%. Annotation revealed 72 mitochondrial genes, including 40 protein-coding genes (PCGs), 27 tRNA genes, 4 rRNA genes, and 1 pseudogene (rps14). Codon usage analysis showed a preference for codons that ended with A/T, and 488 RNA-editing sites, predominantly C-to-U conversions, were predicted, potentially enhancing the protein hydrophobicity and stability. A notable abundance of repetitive elements was observed, which comprised 106 simple sequence repeats, 20 tandem repeats, and 643 dispersed repeats, collectively accounting for 11.6% of the genome. Furthermore, we identified a significant presence of chloroplast-derived transfer fragments, with 56 homologous fragments that encompassed a length of 50,189 bp (11.6% of the mitogenome). A comparative evolutionary analysis revealed that most PCGs were under purifying selection, where cox1 and cox3 were highly conserved, while nad4 and rpl2 displayed signals indicative of positive selection. A nucleotide diversity analysis across 12 Cucurbitaceae species confirmed general sequence conservation, with rpl5 exhibiting the highest variability. Phylogenetic reconstruction based on 32 conserved mitochondrial PCGs from representative angiosperm species robustly placed B. hispida within the Cucurbitales clade, which revealed its closest relationship with Citrullus lanatus. Collinearity analysis further supported this close affinity, showing extensive homologous blocks between their mitogenomes, alongside structural rearrangements unique to B. hispida. CONCLUSIONS: This study presents the first complete mitogenome resource for B. hispida, elucidating its structural complexity, evolutionary dynamics, and phylogenetic position. Prolific repeats and frequent intracellular gene transfers underscore their crucial roles in shaping the architecture and diversification of cucurbit mitogenomes. This genomic foundation paves the way for future research on molecular breeding, species identification, and evolutionary studies within the genus Benincasa and the broader Cucurbitaceae family.