Development of a Liquid-Phased Probe Array for Upland Cotton and Its Application in Cultivar Identification

Single-nucleotide polymorphism (SNP) genotyping arrays are important tools for crop genetic research. Addressing the current issues of insufficient accuracy in upland cotton cultivar identification and difficulties in distinguishing closely related germplasm and hybrids, developing an SNP array enabling rapid and accurate cotton cultivar identification and applicable to molecular breeding is a key demand in cotton cultivar identification and genetic breeding. This study aims to develop a low-cost and high-precision SNP array for upland cotton (<i>Gossypium hirsutum</i> L.) based on Genotyping by Target Sequencing (GBTS) technology. The array will integrate high accuracy in cultivar identification with applicability to molecular breeding, and this study further aims to clarify its application in cultivar identification. The Cotton 13K SNP array contains 13,571 high-quality SNP loci, including 8658 polymorphic sites derived from resequencing data and 4913 functional loci linked to key agronomic traits. All these loci are relatively evenly distributed across the genome. Genotyping 219 upland cotton cultivars/lines accurately clustered them into four genetic subgroups (K = 4), which closely matched their breeding institutions and geographical origins. Analysis of 44 experimental cotton materials (including sister lines and backcross materials) established a genetic similarity threshold of ≥90% for effectively distinguishing closely related germplasm. Comparative analysis of 38 F₁ hybrids and conventional cotton cultivars demonstrated that the average heterozygosity (Het) of hybrids (16.01%) was significantly higher than that of conventional cultivars (5.52%, <i>p</i> < 0.001). A preliminary threshold of Het ≥ 10% was identified for accurate discrimination of cotton hybrids. In conclusion, the Cotton 13K SNP array is a robust tool for population genetic analysis, discrimination of closely related cultivars, and hybrid identification. It also facilitates key molecular breeding steps, including parental evaluation, backcross monitoring, and marker-assisted selection (MAS). Its integration into breeding pipelines is expected to accelerate the development of new cotton varieties.