Development of long InDel markers through whole genome resequencing for variety purity detection in cucumber

The purity of commercial cucumber varieties, predominantly F₁ hybrids, is a critical factor in seed quality assessment. While SSR or SNP markers are commonly used, insertion-deletion (InDel) markers offer greater stability and practical advantages, including lower cost, compatibility with agarose gel electrophoresis, and suitability for multiplex PCR. In this study, we developed long InDel (> 30 bp) markers for variety purity detection in cucumber using whole-genome resequencing data from 182 accessions. A total of 326,479 InDel loci were identified, of which 2655 long InDels satisfy the following requirements which have conserved 200 bp flanking sequences, high polymorphism and suitability for primer design. From these, 74 markers with minor allele frequency (MAF) > 0.2 across four cucumber populations were evenly distributed across seven chromosomes and synthesized. All markers successfully amplified target products, and 10 exhibited high heterozygosity and clear bands on agarose gels. These 10 markers were further adapted for multiplex capillary electrophoresis detection by adding different fluorescent groups. DNA fingerprints of 66 commercial cucumber varieties were constructed using these markers. Among them, four markers InD_Cu35, InD_Cu46, InD_Cu61 and InD_Cu66, showed at least one heterozygous genotype across all varieties, making them particularly suitable for purity testing of cucumber hybrid varieties. The practical utility of these markers was confirmed by assessing the purity of a new hybrid variety, Lvlinglong (98.9%). These long InDel markers provide a robust, flexible, and cost-effective tool for routine cucumber variety purity detection and genetic analysis.