Can a lonesome poor A genome of Brassica napus survive at diploid stage?

Background: There are two strategies available for understanding structural and/or functional modifications that took place during the stabilization of polyploid species. The first one allows assessment of events that arose immediately after the formation of a polyploid species by crossing and doubling its parental genomes in order to produce synthetic forms. The second one tries to elucidate the changes that occurred since the polyploid species was created by extracting in the polyploidy one of its parental genome. Objectives: In the present study, we tried to identify the structural rearrangements that occurred since the origin (about 8000 years ago) of oilseed rape (Brassica napus, AACC, 2n=38), which is a natural hybrid between B. rapa (AA, 2n=20) and B. oleracea (CC, 2n=18). To that purpose, we produced an original plant material in which the B. napus A subgenome was extracted. Methods: We used two methods to extract the diploid AA genome from B. napus. Firstly, AAC F1 interspecific hybrids (produced by crosses between B. napus var Darmor and B. rapa) were backcrossed three times to B. napus, and AAC plants were selected at each generation. Secondly, the initial AAC F1 hybrids were crossed to B. rapa and plants with AA genomes were selected for, selfed and also backcrossed to B. napus. After four cycles of such crossing, we selected AA plants with mainly the A genome of B. napus. Using the 60k SNP Illumina microarray and the sequence of B. napus genomes var Darmor, we assessed the genomic structure of the so far extracted B. napus A subgenome. Result: We found that the backcrosses of AAC F1 interspecific hybrids to B. napus (first strategy) could not permit to eliminate the C chromosomes by selfing since the progenies were male sterile. The second strategy allowed production of AA plants with a regular meiosis. We expected more than 68% of Darmor A genome in this plant. To validate this assessment, genomic structure was established by SNP analysis using markers specific of A genome of Darmor and of the B. rapa variety used in the initial crosses. The homozygous or heterozygous stage of each marker physically anchored was determined. Additionally, CDarmor genome regions introduced by homeologous recombination were characterized. Conclusions: From this original material, it will be possible to determine the comparative evolution of the A genome in a diploid and polyploid genetic background. The first data seem to indicate that rearrangements are too large and/or too frequent to obtain 100% A genome of B. napus at the diploid stage. However, functional analyses will allow identification of the rearrangement impacts.