In silico phylogenetic analysis reveals nuclear ribosomal DNA as the most effective marker for DNA barcoding of Cucumis L. species

Cucumis L. species represent a globally important crop genus with high economic value and genetic diversity, yet their wild relatives remain understudied and potentially threatened by habitat loss and genetic erosion. This knowledge gap hinders effective conservation strategies and limits the utilization of wild genetic resources for crop improvement. A deeper understanding of Cucumis diversity and evolution is especially critical for conserving wild relatives of cultivated species, which may harbor valuable traits for climate resilience and disease resistance. This study investigated the effectiveness of 3 DNA barcode markers (matK, rbcL and nrDNA) for phylogenetic analysis across 38 Cucumis species, addressing a key methodological gap in Cucurbitaceae systematics with direct applications in germplasm conservation and crop improvement programmes. Sequences were retrieved from NCBI and analyzed using MEGA 11, DnaSP v6 and PopArt. The hypothesis that nuclear ribosomal DNA (nrDNA) would outperform plastid markers (matK and then rbcL) in resolving species relationships was validated. The nrDNA displayed the highest genetic distance and clearest clade separation of intraspecific variation, making it particularly valuable for: (i) accurate identification of wild Cucumis germplasm in conservation efforts, (ii) precise selection of genetic resources for breeding programmes and (iii) resolution of taxonomic uncertainties in agricultural and ecological studies. matK provided moderate resolution, while rbcL performed poorly at the intraspecific level. These findings provide a robust molecular toolkit for biodiversity conservation and sustainable agricultural development, enabling more effective utilization and protection of Cucumis genetic resources worldwide.