IDENTIFICATION OF RAPD MARKERS LINKED TO A FERTILITY RESTORER GENE FOR THE OGURA RADISH CYTOPLASMIC MALE-STERILITY OF RAPESEED (BRASSICA-NAPUS L)

Bulked segregant analysis was employed to identify random amplified po lymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in the Ogura radish cytoplasmic male sterility of rapeseed. A total of 1 38 arbitrary 10-mer oligonucleotide primers were screened on the DNA o f 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 indi vidual plants of each bulk was then used as a template for amplificati on with these six primers. DNA polymorphisms generated by four of thes e primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertil ity restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragmen ts 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 corn mon to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments tha t were com plately linked to the restorer locus have been cloned and s equenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of re storer lines used in breed ing programs and is the first step towards map-based cloning of the fe rtility restorer allele.