The PEK locus is responsible for egg-killing
To validate the locus identified by BSA-seq on the top of chromosome B3, we designed KASP markers that targeted the whole 10 Mb region (Supporting information: Table S4). We genotyped the entire BC1 population (BC1-3, n = 66) with five KASP markers. Each KASP marker co-segregated with the HR cell death phenotype, without showing segregation distortion (χ2 test, p> 0.05) (Supporting information: Table S5). The S parent DG1-S1 and susceptible BC1 plants were homozygous (pek- DG1/pek- DG1) while the R donor SF48-O1, the R parent F1_#130 and resistant BC1 plants were heterozygous (PEK -SF48/pek -DG1). Four informative recombinants between M1 and M25 were then genotyped with additional KASP markers, which restricted the locus to the interval 6.11-8.45 Mb between M12 and M19 (Fig. 2c). Validation with KASP markers confirmed that HR was associated with heterozygosity at a single locus on chromosome B3, which we namedPieris -e gg k illing (PEK ) locus.
We then proceeded with fine-mapping the PEK locus through recombinant analysis using a BC1S1population (BC1S1-1, n = 695) that was generated by selfing a resistant BC1-3 plant with heterozygous genotype at the PEKlocus, thus carrying both SF48-O1 allele (PEK -SF48) and DG1-S1 allele (pek -DG1). The whole BC1S1population was genotyped with five KASP markers (M1, M13, M19, M25, M5) spanning the interval. Each marker showed a 1:2:1 allelic segregation ratio (χ2 test, p > 0.05) which was expected given the 3:1 phenotypic segregation ratio between R and S plants (Table 2). The markers were placed on a genetic map of 20.6 cM with a total recombination rate of 2.54 cM/Mb (Fig. 3a). As previously observed in BC1-3, all markers covering the region were associated with HR and marker M13 (6.06 Mb) showed the highest LOD score (Table 2). In total, we could identify 114 recombinants between markers M1 and M5, of which 64 informative recombinants between markers M1 and M19 (Fig. 3b). These 64 plants showed recombination between heterozygous (PEK-SF48/pek- DG1) and homozygous (pek- DG1/pek- DG1) genotypes. Interestingly, all the susceptible BC1S1 plants had homozygous pek- DG1 allele at marker M13. Additional KASP markers were designed between M1 and M19 and four key recombinants (Haplotypes 6 and 7) restricted thePEK locus between M27 and M28. Although we did not find a maker co-segregating with the HR phenotype, fine-mapping restricted thePEK locus to a ~50 kb interval on chromosome B3.