Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Abstract

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.

Identification of AFLP markers linked to fertility restorer genes for tournefortii cytoplasmic male-sterility system in Brassica napus

“…In B. napus, with a predicted genomic length of 2,826 cM (Landry et al 1991), the genomic segment carrying the Rft1 gene may constitute 1.28% of the donor genome. RFLP/RAPD markers linked to the Rf gene(s) for pol (Jean et al 1997) and ogu (Delourme et al 1994) have been reported previously. Jean et al (1997) also showed that the pol restorer gene(s) Rfp1 and Rfp2, previously designated as non-allelic based on elaborate genetic studies, were allelic because one RFLP marker, cRF 16, showed identical polymorphic fragments in both the Rfp1 and Rfp2 restorer lines indicating perfect linkage with the marker.…”

“…To achieve this, several research laboratories are striving to generate markers within the genomic region of interest using such marker techniques as RAPD, RFLP and AFLP. Four RAPD markers, OPC 02 1150 , OPD 02 1000 , OPF 06 1200 and OPG 02 700 , linked to the Rfo locus for the ogura CMS system (Delourme et al 1994) have been identified. These have been cloned and sequenced to develop co-dominant sequence-characterized amplified region (SCAR) markers.…”

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

“…This has to be done so far in the bloom stage, several months after sowing. Since numerous markers (Delourme et al, 1994;Murayama et al, 1999) linked tightly to the Rf gene have been identified, we can allow the recognition of MS plants in early growth stages. This would shorten the breeding process and make it cheaper.…”

Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.)

“…Recently, successful isolation of an Rf gene from petunia by a positional cloning strategy has been reported (Bentoila et al 2002). Molecular markers surrounding the ogura Rf locus (Rfo) have been isolated and mapped after introgression into B. napus (Delourme et al 1994(Delourme et al , 1998. In these reports, the authors suggested that the radish restorer line would be preferable for constructing a fine molecular map of the Rfo region, because recombination might be suppressed around the introgressed radish genome fragment in the B. napus restorer line.…”