The development of liquid-phase chip by target sequencing and their application in watermelon molecular breeding

Genotyping by Target Sequencing (GBTS) technology, known for its flexibility, high efficiency, high throughput, and low cost, has been increasingly employed in molecular breeding. However, there is still limited study on the design and development of high-throughput genotyping tools in watermelon. In this study, we identified 112000 high quality SNPs by analyzing the resequencing data of 43 cultivated watermelon accessions. 11921 and 6094 SNPs were selected for developing two sets of watermelon liquid-phase chips with different marker densities, named Watermelon 10K and 5K, respectively. Furthermore, the SNPs and Indels of most mapped gene/QTLs for many agronomic important traits in watermelon were also integrated into the two chips for foreground selection. These chips have been tested using GBTS technology in various applications in watermelon. The genotyping of 76 accessions by Watermelon 5K liquid-phase chip showed an average detection rate of 99.28 % and 81.78 % for cultivated and wild watermelon accessions, respectively. This provided enough markers information for GWAS and two significant QTLs, ssc1.1 and ssc1.2, associated with soluble sugar content were detected. Furthermore, BSA-seq analysis for non-lobed leaf and dwarf traits were validated by liquid-phase chips, and the candidate region was consistent with our previous studies. Additionally, we precisely introduced the Cldw1 and Clbl genes into an elite inbred line WT2 using Watermelon 5K for assisted selection, resulting in the development of three new germplasm with good plant architecture. As a high-throughput genotyping liquid-phase SNP array, the Watermelon 10K and 5K chips will greatly facilitate functional studies and molecular breeding in watermelon.