Chromosome walking at the fertility restorer locus Rf1 in sunflower (Helianthus annuus L.)

In Sunflower hybrids are obtained by using the cytoplasmic male sterility/fertility restoration system, which is due to a nuclear-cytoplasmic incompatibility. Hybrid breeding is based on the cytoplasmic male sterility, PET1. Male fertility can be restored by the nuclear restorer gene Rf1. The objective of this study was to use a map-based cloning strategy to isolate the Rf1 gene. Seventy-one BAC-clones were identified. Twenty-seven BAC-ends were isolated. Contigs were formed, each representing a unique genomic region. The two BAC libraries used have a too low coverage in the region of Rf1 gene.Bei der Sonnenblume werden Hybriden auf der Grundlage der cytoplasmatischen männlichen Sterilität erzeugt. Diese basieren auf dem PET1-Plasma. Die Restauration der Pollenfertilität wird über das Restorergen Rf1 vererbt. Auf Grundlage der markergestützten Klonierung sollte das Rf1-Gen isoliert wurden. Einundsiebzig BAC-Klone wurden identifiziert. Siebenundzwanzig BAC-Enden wurden isoliert. Von den gebildeten Contigs schien jeder eine einzigartige genomische Region darzustellen. Die zwei benutzten BAC-Bibliotheken erwiesen sich als zu klein, um eine vollständige Abdeckung des Rf1-Genbereichs zu erreichen

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

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 lt 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

Association studies and marker development for the fertility restorer gene Rf1 in sunflower

Fertility restoration is an essential part of hybrid breeding based on cytoplasmic male sterility. In sunflower, the CMS PET1 originating from an interspecific hybridization between Helianthus petiolaris and H. annuus is world-wide used in hybrid breeding. The restorer gene Rf1 is responsible for restoring fertility in the hybrids by specifically reducing the co-transcript of atp1 and the CMS-specific orfH522 in the anthers. From the mode of action (differences in RNA stability) Rf1 could be a pentatricopeptide repeat gene. Blasting BAC-end sequences and sequences of markers linked to the restorer gene Rf1 two physical locations (encompassing 30 Mb and 3.9 Mb, respectively) on linkage group 13 could be identified in the sunflower genome sequence of HanXRQ. These two regions contained nine possible candidate genes for Rf1: seven pentatricopeptide repeat genes, one aldehyde dehydrogenase and one poly(A) polymerase 3 gene. Using a next generation sequencing approach, the nine candidate genes were sequenced with 120 x coverage in an association panel of 59 lines (27 maintainer and 32 restorer lines) and 210 single nucleotide polymorphisms (SNPs) as well as 67 insertions/deletions (InDels) could be identified

Molecular markers for detection of Rf1 gene developed from BAC-end sequences in sunflower

Sunflower is the second most common crop among hybrids, worldwide. Development of cytoplasmic male sterile and fertility restoration lines enables creation of hybrids. Sunflower hybrid breeding most frequently relies on the combination of the CMS PET1 cytoplasm and the fertility restoration gene Rf1. Use of molecular markers can accelerate creation of restorer lines. Previous work on this subject included mapping of the Rf1 gene on chromosome 13 and development of bacterial artificial chromosome (BAC) libraries for the restorer line RHA325 and the maintainer line HA383, which enabled positioning of BAC clones surrounding the Rf1 gene in the cross RHA325 x HA342. In this study, BAC-end sequences were used to derive primers in order to amplify selected regions from RHA325 and the maintainer line, HA342. While the majority of primer combinations were monomorphic, some were polymorphic between RHA325 and HA342. Previously reported markers for detection of Rf1 gene and newly developed ones based on BAC-end sequences were further tested on hybrids and its components (A, B and R lines) created at the Institute of Field and Vegetable Crops, Novi Sad