Integrating image-based phenotyping and GWAS to map tolerance to Spittlebug nymphs in interspecific Urochloa grasses

Urochloa grasses are among the most widely used forage grasses across the tropics. Spittlebugs (Hemiptera: Cercopidae) are major pests of tropical Urochloa (syn. Brachiaria) pastures, severely reducing forage productivity and quality. Understanding the genetic basis of host-plant resistance is essential for developing durable resistant cultivars. Here, we combined high-throughput image-based phenotyping and genome-wide association studies (GWAS) to dissect the genetic architecture of tolerance to Aeneolamia varia nymphs in 339 interspecific F hybrids derived from crosses between resistant sexual and susceptible apomictic Urochloa parents. Digital image analysis using both unsupervised (DQU) and supervised (DTR) quantification pipelines enabled precise estimation of plant damage, yielding moderate to high broad-sense heritability estimates (H{superscript 2} = 0.49-0.66). In contrast, insect survival (NTS) exhibited low to moderate correlations with all damage traits and lower heritability estimates (H{superscript 2} = 0.42). Using 57,051 high-quality SNPs aligned to the genome of the hybrid cultivar Basilisk, GWAS models identified 18 quantitative trait loci (QTL) for plant damage traits, but none for insect survival (antibiosis). Six robust QTL on chromosomes 1, 6, 7, 27, 29, and 36 were consistently detected across models and phenotyping methods, explaining up to 21.5% of phenotypic variance. Candidate gene analysis revealed proteins involved in hormone signalling, oxidative stress response, and cell wall modification, suggesting multifaceted tolerance mechanisms. These results provide a foundational set of molecular markers associated with spittlebug tolerance in Urochloa, useful for marker-assisted and genomic selection in our forage breeding programme.