Development of InDel markers and construction of DNA fingerprints of wax gourd (Benincasa hispida) based on whole genome re-sequencing

• By utilizing whole-genome re-sequencing, identified insertion-deletion sites in 55 wax gourd germplasm resources and clarified the distribution of InDel sites in the whole genome of wax gourd. • A set of 33 polymorphic InDel marker primer pairs were developed, including gene-linked markers, which are suitable for constructing DNA fingerprints of commercial wax gourd varieties. • A subset of 12 core InDel markers was optimized to simplify the DNA fingerprinting process, enabling the differentiation of 84 commercial wax gourd varieties and enhancing the efficiency of hybrid purity verification and variety identification in commercial breeding. The development of new wax gourd varieties is accelerating. However, the limited genetic data on wax gourd populations and the lack of advanced molecular marker identification techniques have posed significant challenges to the commercial breeding of wax gourd. In this study, we performed whole-genome re-sequencing of 55 wax gourd germplasm resources to identify insertion-deletion (InDel) markers. A total of 964,087 InDels were identified across the 55 germplasm resources, yielding an average density of approximately 1098 InDels per Mb. From these, 33 InDel markers were selected through polymerase chain reaction (PCR) amplification technique. The average values for the major allele frequency, gene diversity index ( Hs ), expected heterozygosity ( He ), and polymorphism information content ( PIC ) of 33 InDel markers were 0.641, 0.444, 0.351, and 0.344, respectively. Based on the in-depth study and analysis of 33 InDel markers, 12 core InDel markers were screened in accordance with the principle of minimizing the number of primers, and the DNA fingerprint of 84 commercial varieties of wax gourd was successfully constructed. Subsequently, the heterozygous genotypes of the markers in the fingerprint were verified, and the results showed that among the 37 commercial varieties of wax gourd, each variety had 1 to 7 pairs of InDel markers that could be used for seed purity identification. This study developed InDel markers for wax gourd variety identification and seed purity verification, and constructed DNA fingerprints, which provides a basis for wax gourd genetic diversity, variety identification, property rights disputes and molecular breeding.