Identifying SNP markers associated with distinctness, uniformity, and stability testing in Egyptian fenugreek genotypes

Distinctness, uniformity, and stability (DUS) test is the legal requirement in crop breeding to grant the intellectual property right for new varieties by evaluating their morphological characteristics across environments. On the other hand, molecular markers accurately identify genetic variations and validate the purity of the cultivars. Therefore, genomic DUS can improve the efficiency of traditional DUS testing. In this study, 112 Egyptian fenugreek genotypes were grown in Egypt at two locations: Wadi El-Natrun (Wadi), El-Beheira Governorate, with salty and sandy soil, and Giza, Giza governorate, with loamy clay soil. Twelve traits were measured, of which four showed a high correlation above 0.94 over the two locations. We observed significant genotype-by-location interactions (GxL) for seed yield, as it was superior in Wadi, with few overlapping genotypes with Giza. We attribute this superiority in Wadi to the maternal habitat, as most genotypes grew in governorates with newly reclaimed salty and sandy soil. As a first step toward genomic DUS, we performed an association study, and out of 38,142 SNPs, we identified 39 SNPs demonstrating conditional neutrality and four showing pleiotropic effects. Forty additional SNPs overlapped between both locations, each showing a similar impact on the associated trait. Our findings highlight the importance of GxL in validating the effect of each SNP to make better decisions about its suitability in the marker-assisted breeding program and demonstrate its potential use in registering new plant varieties.