Fine mapping of the Chilli veinal mottle virus resistance 4 (cvr4) gene in pepper (Capsicum annuum L.)

The single recessive Chilli veinal mottle virus resistance locus, cvr4, was fine-mapped in pepper through bulked segregant RNA sequencing combined with gene silencing analysis. Chilli veinal mottle virus (ChiVMV) is a widespread pathogen affecting the production of peppers (Capsicum annuum L.) in Asia and Africa. Few loci conferring resistance to ChiVMV have been identified, severely limiting the development of resistant cultivars. To identify ChiVMV resistance genes, we constructed an F_2:3 segregating population derived from a cross between the ChiVMV-resistant cultivar 'CV9' and the susceptible cultivar 'Jeju'. The inheritance study of F_2:3 populations showed a 1:3 ratio of resistant to susceptible individuals, demonstrating the existence of a single recessive ChiVMV resistance gene in CV9; we named this gene cvr4. To map the cvr4 locus, we employed bulked segregant analysis by RNA sequencing (BSR-seq) of pools from resistant and susceptible F_2:3 individuals. We mapped cvr4 to the telomeric region of pepper chromosome 11. To narrow down the cvr4 locus, we developed additional molecular markers in the cvr4 target region, leading to a 2-Mb region of chromosome 11 showing complete co-segregation with the ChiVMV resistance phenotype. Using the polymorphisms identified during BSR-seq, we defined a list of 15 candidate genes for cvr4, which we tested through virus-induced gene silencing analysis for ChiVMV resistance. Of these, the silencing of several genes (DEM.v1.00021323, DEM.v1.00021336, and DEM.v1.00021337) restricted virus spread. Although DEM.v1.00021323 transcript levels were similar between the resistant and susceptible bulks, its alternative spliced isoforms differed in abundance, suggesting that the splicing variants of DEM.v1.00021323 might affect viral infection. These findings may facilitate the breeding of ChiVMV-resistant cultivars in pepper.