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In breeding of winter rape (Brassica napus L. var. napus) using the OGU-INRA system, based on cytoplasmic male sterility (CMS), it is necessary to distinguish pollen-sterile lines, carrying CMS factors and the recessive fertility restorer alleles rfrf, from fertile lines, carrying at least one dominant restorer gene allele (Rfrf or RfRf). To grow plants till the flowering stage takes much time. The method was therefore modified using isozyme markers of glucose-6-phosphate isomerase (PGI) to distinguish male sterile (MS) from male-fertile lines in early stages. Since the restorer gene is tightly linked to the markers and the PGI isozymes can be distinguished by electrophoresis, the markers can be used to identify MS rape plants in early stages. Also, homozygous and heterozygous fertility-restored plants can be separated this way. In our work we tried to optimise the distinction of pollen-fertile and pollen-sterile rape plants with PGI isozyme markers, using vertical polyacrylamide gel electrophoresis (native-PAGE). The method will be used for the breeding of rapeseed with the OGU-INRA system.

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene ( Rfo) used in the Ogura 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. [ABSTRACT FROM AUTHOR]