Map and sequence-based chromosome walking towards cloning of the male fertility restoration gene Rf5 linked to R(11) in sunflower

The nuclear fertility restorer gene Rf5 in HA-R9, originating from the wild sunflower species Helianthus annuus, is able to restore the widely used PET1 cytoplasmic male sterility in sunflowers. Previous mapping placed Rf5 at an interval of 5.8 cM on sunflower chromosome 13, distal to a rust resistance gene R(11) at a 1.6 cM genetic distance in an SSR map. In the present study, publicly available SNP markers were further mapped around Rf5 and R(11) using 192 F(2) individuals, reducing the Rf5 interval from 5.8 to 0.8 cM. Additional SNP markers were developed in the target region of the two genes from the whole-genome resequencing of HA-R9, a donor line carrying Rf5 and R(11). Fine mapping using 3517 F(3) individuals placed Rf5 at a 0.00071 cM interval and the gene co-segregated with SNP marker S13_216392091. Similarly, fine mapping performed using 8795 F(3) individuals mapped R(11) at an interval of 0.00210 cM, co-segregating with two SNP markers, S13_225290789 and C13_181790141. Sequence analysis identified Rf5 as a pentatricopeptide repeat-encoding gene. The high-density map and diagnostic SNP markers developed in this study will accelerate the use of Rf5 and R(11) in sunflower breeding.