Map and sequence-based chromosome walking towards cloning of the male fertility restoration gene Rf5 linked to R11 in sunflower

Abstract

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 R11 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 R11 using 192 F2 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 R11. Fine mapping using 3517 F3 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 F3 individuals mapped R11 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 R11 in sunflower breeding.

Mutation and sequencing‐based cloning and functional studies of a rust resistance gene in sunflower (Helianthus annuus)

“…The R 11 rust resistance gene was previously fine‐mapped to an approximately 60‐kb target region of the XRQr1.0 genome assembly on chromosome HanXRQChr13 spanning nucleotides 181 784 941–181 844 941 and containing three predicted genes, HanXRQChr13g0422111 , HanXRQChr13g0422121 and HanXRQChr13g0422131 (Ma et al., 2021). Due to the repetitive nature of this genomic region, Xgene lockdown probe pools were used for target enrichment and target region sequencing using the PacBio Sequel System II for the rust‐resistant line HA‐R9 and the three EMS‐induced susceptible mutants.…”

“…(2017) and Ma et al. (2017, 2021). Sequences flanking the mutation sites were extracted from the samples in which the mutation had been detected (Table S19).…”

“…Three EMS-induced point mutations were evaluated using a PCRbased SNP detection approach following the methods described by Long et al (2017) and Ma et al (2017Ma et al ( , 2021. Sequences flanking the mutation sites were extracted from the samples in which the mutation had been detected (Table S19).…”

“…Only seven R genes ( R 11 , R 12 , R 13a , R 13b , R 14 , R 15 and R 16 ) remain effective in conferring resistance to all P. helianthi races identified in North America (Gong et al., 2013; Liu et al., 2019; Ma et al., 2018; Qi et al., 2012; Zhang et al., 2016). The rust R gene R 11 in sunflower line HA‐R9 shows broad‐spectrum resistance to P. helianthi virulent races, and was recently fine‐mapped to a region of less than 50 kb on chromosome 13 of the sunflower XRQr1.0 reference genome assembly, approximately 12 Mb away from a cluster of six other rust R genes ( R 4 , R 13a , R 13b , R 16 –R 18 ) on the same chromosome (Ma et al., 2021; Qi et al., 2021). Three potential candidate genes ( HanXRQChr13g0422111 , HanXRQChr13g0422121 and HanXRQChr13g0422131 ) for R 11 in the target genomic region were identified in the XRQr1.0 genomic sequences, providing a foundation for cloning this gene (Ma et al., 2021).…”

Genetic Insight into Disease Resistance Gene Clusters by Using Sequencing-Based Fine Mapping in Sunflower (Helianthus annuus L.)

“…An R gene cluster with nine rust and eight DM R genes located on the lower end of sunflower chromosome 13 represents the largest R -gene cluster in sunflower. This R gene cluster can be further divided into two sub-clusters, sub-cluster I containing three rust R genes ( P u6 , R adv , and R 11 ) and three fertility restorer genes ( Rf1 , Rf5 , and Rf7 ) and sub-cluster II including six rust R genes ( R 4 , R 13a , R 13b , and R 16 – R 18 ) and eight DM R genes ( Pl 5 , Pl 8 , Pl 21 , Pl 22 , Pl 31 , Pl 32 , Pl 34 , and Pl 36 ) [ 36 , 37 , 38 , 40 , 42 , 64 , 73 , 80 ]. Six rust R genes ( R 4 , R 13a , R 13b , and R 16 – R 18 ) in sub-cluster II could be differentiated with race-specific resistance, except for the three, R 13a , R 13b , and R 16 , that exhibit resistance to all of the P. helianthi races that have been identified in North America thus far [ 73 ].…”