Evaluation of molecular markers associated with tobamoviruses resistance genes in pepper landraces of the Basque Country (northern Spain)

Two local cultivars of pepper (‘Derio’ and ‘Ibarroria’) from the Basque Country are susceptible to tobamoviruses. In 2015, a backcrossing breeding program assisted by molecular markers was started to transfer the L3 and L4 resistance genes to these susceptible local cultivars. Different molecular markers cited in publications were evaluated in the breeding material, but only 3 of 13, and 2 of 7 molecular markers tested for the L3 and L4 genes, respectively, correctly differentiated susceptible and resistant plants. Among the discarded markers, some did not work, others amplified but gave false positives, and some amplified in both positive and negative plants. These molecular markers were described in the literature as co-dominant and thus should be able to discriminate between homozygous and heterozygous plants at the two loci. Marker YB2A19(NK) for the L3 gene functioned as a co-dominant marker. However, when marker 087H3T7 was used for the L4 gene, an amplicon of equal size was obtained in susceptible ‘Derio’ and the heterozygous resistant hybrid ‘Giulio’. After the isolation, purification, sequencing and sequence alignment of the PCR products from ‘Derio’ and ‘Giulio’, three differential SNPs were found. For one of them a restriction enzyme was found that cut at the differential SNP. PCR amplification followed by enzyme digestion allowed distinguishing homozygous and heterozygous resistant plants. The development and use of co-dominant markers was especially helpful in the last generations of marker assisted backcrossing where a co-dominant marker is required for the selection of homozygous resistant plants.