Development and Validation of Markers for the Fertility Restorer Gene Rf1 in Sunflower

Abstract

Hybrid breeding in sunflowers based on CMS PET1 requires development of restorer lines carrying, in most cases, the restorer gene Rf1. Markers for marker-assisted selection have been developed, but there is still need for closer, more versatile, and co-dominant markers linked to Rf1. Homology searches against the reference sunflower genome using sequences of cloned markers, as well as Bacterial Artificial Chromosome (BAC)-end sequences of clones hybridizing to them, allowed the identification of two genomic regions of 30 and 3.9 Mb, respectively, as possible physical locations of the restorer gene Rf1 on linkage group 13. Nine potential candidate genes, encoding six pentatricopeptide repeat proteins, one tetratricopeptide-like helical domain, a probable aldehyde dehydrogenase 22A1, and a probable poly(A) polymerase 3 (PAPS3), were identified in these two genomic regions. Amplicon targeted next generation sequencing of these nine candidate genes for Rf1 was performed in an association panel consisting of 27 maintainer and 32 restorer lines and revealed the presence of 210 Single Nucleotide Polymorphisms (SNPs) and 67 Insertions/Deletions (INDELs). Association studies showed significant associations of 10 SNPs with fertility restoration (p-value < 10−4), narrowing Rf1 down to three candidate genes. Three new markers, one co-dominant marker 67N04_P and two dominant markers, PPR621.5R for restorer, and PPR621.5M for maintainer lines were developed and verified in the association panel of 59 sunflower lines. The versatility of the three newly developed markers, as well as of three existing markers for the restorer gene Rf1 (HRG01 and HRG02, Cleaved Amplified Polymorphic Sequence (CAPS)-marker H13), was analyzed in a large association panel consisting of 557 accessions.

Mapping of the New Fertility Restorer Gene Rf-PET2 Close to Rf1 on Linkage Group 13 in Sunflower (Helianthus annuus L.)

“…In addition, a new STS-marker STS67N4 was mapped closely to the restorer gene Rf1. This marker was derived from the BAC-end sequences of BAC 67N4, which had originally been identified by hybridization of OP-K13_456 to the BAC library of RHA 325 [36].…”

“…A new polymorphic STS-marker STS3948_145 was derived from the AFLP marker E39M48_205R, which could be mapped in both populations. From E62M52_249A, linked to Rf1, an overgo probe was designed and used for hybridization to the BAC library of HA 383 identifying the BAC clones 006N12, 447N06, and 480G04 [36].…”

“…Potential candidate genes in the area are shown according to Goryunov et al [37]. The three PPR genes that showed SNPs significantly associated with fertility restoration by Rf1 are marked in red [36].…”

“…The linkage map around the Rf1 gene consisted of 35 amplified fragment length polymorphism (AFLP) markers, 7 random amplified polymorphic DNA (RAPD) markers, and ORS1030 [34]. Using sequences of cloned markers and bacterial artificial chromosome (BAC) clones hybridizing to these markers, two regions in the sunflower genome sequence HanXRQ v1r1 [35] could be identified as potential physical locations of the Rf1 gene on LG13 [36]. In the 30-Mb region and the 3.9-Mb region nine potential candidate genes for Rf1 were annotated: seven pentatricopeptide repeat (PPR) genes, one gene for a poly(A) polymerase 3 (PAPS3), and one for an aldehyde dehydrogenase 21A.…”

“…These SNPs narrowed the potential candidates for Rf1 down to three genes, PPR841, PPR861, and PPR621. All three genes are based in the 3.9-Mb region between 169,655,088 (ORS1030) and 173,581,392 (OP-H13) [36]. Genome-wide association studies using whole sunflower genome sequences [37][38][39] and data from SNP arrays [40] had also identified the distal region of LG13 for Rf1 but could not tag a specific gene due to the insufficient resolution of the whole genome analyses.…”