No abstract

The heavy selection pressure due to intensive breeding of Brassica napus has created a narrow gene pool, limiting the ability to produce improved varieties through crosses between B. napus cultivars. One mechanism that has contributed to the adaptation of important agronomic traits in the allotetraploid B. napus has been chromosomal rearrangements resulting from homoeologous recombination between the constituent A and C diploid genomes. Determining the rate and distribution of such events in natural B. napus will assist efforts to understand and potentially manipulate this phenomenon. The Brassica high-density 60K SNP array, which provides genome-wide coverage for assessment of recombination events, was used to assay 254 individuals derived from 11 diverse cultivated spring type B. napus. These analyses identified reciprocal allele gain and loss between the A and C genomes and allowed visualization of de novo homoeologous recombination events across the B. napus genome. The events ranged from loss/gain of 0.09 Mb to entire chromosomes, with almost 5% aneuploidy observed across all gametes. There was a bias toward sub-telomeric exchanges leading to genome homogenization at chromosome termini. The A genome replaced the C genome in 66% of events, and also featured more dominantly in gain of whole chromosomes. These analyses indicate de novo homoeologous recombination is a continuous source of variation in established Brassica napus and the rate of observed events appears to vary with genetic background. The Brassica 60K SNP array will be a useful tool in further study and manipulation of this phenomenon.

Seventy-nine recombinant backcross substitution lines from a cross between Brassica oleracea var. italica and Brassica oleracea var. alboglabra were grown in ®eld trials over ®ve years along with the alboglabra recurrent parent. Plants were scored for the days from sowing to the opening of the ®rst ower, and lines that¯owered signi®cantly earlier or later than the recurrent parent were identi®ed. Based on the lengths of the substitutions, evidence for 11 QTL on chromosomes O1, O2, O3, O5 and O9 was found, ®ve of which mapped to similar regions to ®ve of the six found in a previous analysis of doubled haploid lines from the same cross. Several of the QTL were linked closely in repulsion.Keywords: Brassica,¯owering time, QTL analysis, substitution lines. IntroductionBrassica oleracea is an agriculturally important species including many vegetables such as cabbage, broccoli and cauli¯ower. It is closely related to the model dicotyledonous plant Arabidopsis thaliana and so it is expected that information concerning control of basic biological processes in A. thaliana will be transferable to Brassica crops.Information should also be transferable between di erent species within the Brassica genus, as the three diploid genomes, A, B and C, of B. rapa, B. nigra and B. oleracea, respectively, are thought to have derived from the same ancestor. These genomes reveal striking conservation of content, although chromosome duplication and translocation have occurred during divergence (Lagercrantz & Lydiate, 1996). The amphidiploid B. napus (oilseed rape) genome is made up of the A and C genomes probably from close relatives of B. rapa and B. oleracea. (U, 1935; Parkin et al., 1995).Flowering time is not only of scienti®c interest because it facilitates the understanding of plant development, it is also important in agriculture, because its modi®cation may enable the geographical range of the Brassica crop to be extended. For example, cultivation of B. napus is usually restricted to temperate latitudes, but the development of early¯owering cultivars would allow the crop to be grown in low-rainfall regions such as the Western Australian wheat belts (Thurling & Depittayanan, 1992), and more northern regions of Canada (Murphy & Scarth, 1994).Genes a ecting¯owering time have been identi®ed in A. thaliana by mutagenesis (Koornneef et al., 1991). For example, mutations of the CONSTANS gene cause delayed¯owering under long days, but not short, and the gene has been cloned by chromosome walking (Putterill et al., 1993(Putterill et al., , 1995.Two regions in¯uencing¯owering time in B. nigra (on LG2 and LG8) have been found to be homologous to the CONSTANS gene region (Lagercrantz et al., 1996). These regions have also been shown to carry quantitative trait loci (QTL) in the A genome of B. napus, and show large-scale colinearity between regions on chromosomes O2, O3 and O9 of B. oleracea.Previous studies mapping QTL for¯owering time in B. oleracea have been reported. For example, Kennard et al. (1994) found signi®cant QTL using singl...

The two genomes (A and C) of the allopolyploid Brassica napus have been clearly distinguished using genomic in situ hybridization (GISH) despite the fact that the two extant diploids, B. rapa (A, n ¼ 10) and B. oleracea (C, n ¼ 9), representing the progenitor genomes, are closely related. Using DNA from B. oleracea as the probe, with B. rapa DNA and the intergenic spacer of the B. oleracea 45S rDNA as the block, hybridization occurred on 9 of the 19 chromosome pairs along the majority of their length. The pattern of hybridization confirms that the two genomes have remained distinct in B. napus line DH12075, with no significant genome homogenization and no large-scale translocations between the genomes. Fluorescence in situ hybridization (FISH)-with 45S rDNA and a BAC that hybridizes to the pericentromeric heterochromatin of several chromosomes-followed by GISH allowed identification of six chromosomes and also three chromosome groups. Our procedure was used on the B. napus cultivar Westar, which has an interstitial reciprocal translocation. Two translocated segments were detected in pollen mother cells at the pachytene stage of meiosis. Using B. oleracea chromosome-specific BACs as FISH probes followed by GISH, the chromosomes involved were confirmed to be A7 and C6.

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.