Fig. 2: Whole-genome sequencing and genome variant analysis (WGS-GVA) and fine mapping reveal nonsynonymous SNPs in MePOLD1 that segregate with resistance.

a TME204-WT and F1 progeny, TME419-WT, 60444-WT and TME204, TME419 and 60444 plants regenerated from tissue culture (FEC) were tested for resistance and susceptibility. TME204 WT, F1-3, F1-7, F1-8, and TME419-WT plants had CMD2 resistance while all other plants were susceptible to ACMV infections. The resistance phenotype is indicated on the left bar (Red – Resistant; Blue – Susceptible). A haplotype 1-restricted G to C transversion in the TME204 MePOLD1 gene at location 9,081,215 bp causes a heterozygous V528L mutation in MePOLD1. Two large (n ≈ 1000) F₁ mapping populations derived from NASE14×TME204-LCR were used to fine-map CMD2 (b–e). b An in silico bulk segregant approach was performed using the field phenotyping and genotyping by sequencing (GBS) data (Fig. 1c). The tricube-smoothed allele frequency enrichment (ΔSNP-index) across the TME204-hap1 assembly. In (c) and (d) the red line denotes the 95% confidence interval. The highlighted region on Chr12 defines the significantly linked CMD2 region. c Enlargement of the CMD2 locus mapping results. Each point represents a SNP and its corresponding ΔSNP-index. The dashed lines indicate the borders of the mapped locus between ~5–13 Mb. The previously reported associated marker from Rabbi et al. is indicated by black arrow⁹. d Examining the GBS SNP data from individual recombinants within the locus improves the mapping resolution to ~300 kb. Genotypes are extended downstream until the next SNP called. Two non-recombinant homozygous resistant and susceptible lines are added as a control (top and bottom). Based on the location of the mapped locus, and the previously identified GWAS marker, KASP markers (M1-8) were developed for fine mapping (positions denoted by dot-dash lines in (c) and (d). e A second fine-mapping population was phenotyped in the greenhouse using a virus-induced gene silencing-based infection assay. Recombinants within the region place CMD2 in the 190Kb interval between markers M3 and M7. Lines P1581 and P1561 are non-recombinant susceptible and resistant controls, respectively. In (c) and (e) the genotype at each SNP or marker is indicated by the colour (Allele 1, Red, linked to Resistance; Allele 2, Blue, linked to Susceptibility). The resistance phenotype is indicated on the left bar as above. f Genomic rearrangements within the fine-mapped CMD2 locus introduce new gene candidates.