Figure 2.

Mutation in FveGA20ox4 Underlies the R Locus.

(A) Map-based cloning of the R locus (in gray) in linkage group 2 (LGII) (Sargent et al., 2004). The R locus was reduced to 334 kb using microsatellite markers (in black) and to 87.5 kb by recombinant analysis of 1350 individuals.

(B) R locus reduced to 59.11 kb by bulk segregant analysis-WGS. This region includes eight genes. Of the 11 SNPs/InDels linked to the runnerless phenotype identified, one deletion (DEL, blue triangle) is in a predicted GA20ox (gene09034) and 10 SNPs (green diamonds) are either in intergenic regions or in predicted genes (Supplemental Table 2).

(C) Identification of the r mutation responsible for the runnerless trait by screening a large number of recombinant lines (1350 individuals) from the Ilaria F₂ population to identify runnerless ([r] phenotype) lines recombining between the two markers (EMFn134 and UFFxa09F09) flanking the r mutation. The nine recombinant runnerless lines identified were grouped into two bulks according to the position of the breaking point: left to marker UFFxa09F09 (Bulk1 with seven F₂ lines) and right to marker EMFn134 (Bulk2 with two F₂ lines). WGS of these bulks and of Sicile (runnering [R] phenotype) (25× to 35× genome coverage) allowed the identification of all polymorphisms (23 SNPs/InDels) in the chromosomal region carrying r. Comparison with genome sequences of ‘Pawtuckawai’ ([R] phenotype) and ‘Baron Solemacher’ ([r] phenotype) available at https://www.rosaceae.org/ further allowed the reduction of the candidate polymorphisms to 11 SNPs/InDels. The deletion (DEL) in gene FveGA20ox4 was the most likely candidate due to the function of the protein.

(D) Unequivocal identification of DEL in FveGA20ox4 as the causal mutation. Recombinant lines were screened to identify one runnering line ([R] phenotype) named alpha5-58, which was heterozygous at the FveGA20ox4 locus and recombined just after the DEL in FveGA20ox4. After selfing, 26 F₃ lines were obtained, seven of which displayed a runnerless [r] phenotype. All seven lines were homozygous for the DEL in FveGA20ox4, recombined after this mutation, and were homozygous wild type thereafter. The 10 SNPs carried by the chromosomal segment after the DEL in FveGA20ox4 were therefore excluded as candidate polymorphisms. The recessive DEL in FveGA20ox4 (ΔFvega20ox4) was confirmed as the causal mutation for the runnerless [r] phenotype.

(E) DEL of nine nucleotides occurs in exon 2 of GA20ox (gene09034).

(F) A shorter protein with a deletion of three amino acids (Cys²⁶⁸Val²⁶⁹Lys²⁷⁰) is produced in the r mutant (ΔFvega20ox4_r) in comparison with FveGA20ox4_WT.