Novel Gene‐Copy‐Specific KASP Markers for Efficient Marker‐Assisted Development of Low Vicine Faba Bean (<scp><i>Vicia faba</i></scp> L.)

ABSTRACT Vicine and convicine (v‐c) pose significant challenges to the global use of faba beans, causing favism in susceptible individuals. Developing low v‐c faba beans is crucial to enhance their utilization and safety, but progress has been hindered by insufficient genomic tools for marker‐assisted selection. This challenge is linked to the complex nature of the responsible locus, which makes marker development difficult. While a two‐base pair insertion/deletion in VC1 correlates with v‐c phenotype, converting this variant into a reliable marker remains challenging due to multiple VC1 copies. Here, we developed a set of five robust, breeder‐friendly selection markers targeting VC1 and a diverging homologue, RIBA2 , for v‐c breeding. The marker SNP08 was designed based on the 2 bp inactivating insertion in VC1 . To overcome the bias caused by unexpressed homologous VC1 copies, we employed a target‐specific preamplification strategy, incorporating a preliminary PCR step to amplify specifically the VC1 copy harbouring the mutation associated with v‐c. This was followed by genotyping using a Kompetitive allele‐specific PCR (KASP) assay, resulting in a robust VC1 ‐copy‐specific KASP marker capable of predicting v‐c phenotypes with 100% accuracy. Additionally, we developed four simple, easy‐to‐use KASP markers based on single nucleotide polymorphisms (SNPs) within the single‐copy neighbouring VC1 homologue, RIBA2 . Subsequent validation in a diverse panel showed consistent co‐segregation with v‐c phenotypes. The results demonstrated high accuracy of the markers in predicting v‐c phenotypes, highlighting their suitability for marker‐assisted development of low v‐c faba beans. These genomic tools are valuable for faba bean breeding and will facilitate the development of high‐quality faba bean protein.