Phylogenetic Analysis of S-Locus Genes Reveals the Complicated Evolution Relationship of S Haplotypes in Brassica

Self-incompatibility (SI) is reported to play a key role in the evolution of species as it promotes their outcrossing through the recognition and rejection of self-pollen grains. In Brassica, two S-locus genes expressed in the stigma, S-locus glycoprotein (SLG) gene and S-locus receptor kinase (SRK) gene, and one expressed in the pollen, S-locus protein 11 (SP11) gene, were linked as an S haplotype. In order to analyze the evolutionary relationships of S haplotypes in Brassica, a total of 39 SRK, 37 SLG, and 58 SP11 sequences of Brassica oleracea, Brassica rapa and Brassica napus were aligned. Two phylogenetic trees with similar pattern were constructed based on the nucleotide sequences of SRK/SLG and SP11, respectively. Class I and class II alleles were clustered into two distinct groups, and alleles from different species, including all the interspecific pairs of S haplotypes, were closely related to each other. The S-locus genes identified in B. napus were intermingled in phylogenetic trees. All these observations showed that class I and class II S haplotypes diverged ahead of the species differentiation in Brassica. The evolution and the genetic diversity of S haplotypes in Brassica were discussed. Moreover, the relationships between S haplotypes and SI phenotypes in Brassica, especially in B. napus, were also discussed.     

Analysis of self-incompatibility interactions in 30 resynthesized Brassica napus lines. I. Fluorescence microscopic studies

Thirty Brassica napus lines have been developed through interspecific hybridization of B. oleracea and B. campestris lines with defined S-allele constitutions. These lines, which represent 29 different S-allele combinations, were tested in a diallel of test-pollinations to determine the activity of the introgressed S-alleles and intergenomic dominance relationships. Some consistent trends were observed: B. oleracea S-alleles high in the dominance series (e.g. S₈, S₁₄, S₂₉) were always active in the resynthesized B. napus lines, whereas recessive S-alleles (S₂, S₁₅) lost their activity in some test combinations. The B. campestris S-alleles were active in most cases, although 2 alleles were partially inactivated by the recessive B. oleracea allele S₁₅.     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996     

Analysis of self-incompatibility interactions in 30 resynthesized Brassica napus lines. II. Expression of S-locus glycoproteins (SLGs)

Thirty resynthesized Brassica napus lines with defined S-allele constitution and the ancestral B. oleracea and B. campestris lines were used for the analysis of S- locus glycoproteins (SLGs). The aim of this study was to investigate (1) whether the S-specific glycoproteins of the diploid ancestor lines were also expressed in the amphidiploid hybrids and (2) whether the occurrence of SLG bands was correlated with the activity of the respective S-alleles, which had been tested by means of diallele pollination tests in a previous study. Stigma proteins were separated by isoelectric focusing (IEF)-gel electrophoresis, and glycoprotein bands were identified by Western blotting and Con-A/peroxidase reaction. The SLG bands of the B. campestris parent could be detected in all 30 resynthesized B. napus lines. In contrast, B. oleracea SLG bands could only be detected in 12 resynthesized B. napus lines. Only B. napus lines which carried the dominant B. oleracea S-alleles S₈ and S₂₉ showed respective SLG bands in all cases. Nine B. napus lines showed only glycoprotein bands of the B. campestris parent, although the biological functioning of the B. oleracea S-alleles was demonstrated by test-pollinations. New SLG bands different from those of the B. oleracea and B. campestris parents occurred in 16 B. napus lines. The expression level of the SLGs in B. napus was not correlated with the self-incompatibility phenotype, not only in the case of recessive S-alleles (S₂, S₁₅), but also for dominant alleles (e.g. S₁₄, S₃₂, S₄₅). Received: 22 January 1999 / Accepted: 30 January 1999     

Development of SCAR markers linked to self-incompatibility in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

‘SI1300’ is a self-incompatible Brassica napus line generated by introgressing an S haplotype from B. rapa ‘Xishuibai’ into a rapeseed cultivar ‘Huayou No. 1’. Five S-locus specific primer pairs were employed to develop cleaved amplified polymorphic sequences (CAPS) markers linked the S haplotype of ‘SI1300’. Two segregating populations (F₂ and BC₁) from the cross between ‘SI1300’ and self-compatible European spring cultivar ‘Defender’, were generated to verify the molecular markers. CAPS analysis revealed no desirable polymorphism between self-incompatible and self-compatible plants. Twenty primer pairs were designed based on the homology-based candidate gene method, and six dominant sequence characterized amplified region (SCAR) markers linked with the S-locus were developed. Of the six markers, three were derived from the SRK and SP11 alleles of class II B. rapa S haplotypes and linked with S haplotype of ‘SI1300’. The other three markers were designed from the SLG-A10 and co-segregated with S haplotype of ‘Defender’. We successfully combined two pairs of them and characterized two multiplex PCR markers which could discriminate the homozygous and heterozygous genotypes. These markers were further validated in 24 F₃ and 22 BC₁F₂ lines of ‘SI1300 × Defender’ and another two segregating populations from the cross ‘SI1300 × Yu No. 9’. Nucleotide sequences of fragments linked with S-locus of ‘SI1300’ showed 99% identity to B. rapa class II S-60 haplotype, and fragments from ‘Defender’ were 97% and 94% identical to SLG and SRK of B. rapa class I S-47 haplotype, respectively. ‘SI1300’ was considered to carry two class II S haplotypes and the S haplotype on the A-genome derived from B. rapa ‘Xishuibai’ determines the SI phenotype, while ‘Defender’ carry a class I S haplotype derived from B. rapa and a class II S haplotype from B. oleracea. SCAR markers developed in this study will be helpful for improving SI lines and accelerating marker-assisted selection process in rapeseed SI hybrid breeding program.     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Analysis of S-locus and expression of S-alleles of self-compatible rapid-cycling Brassica oleracea ‘TO1000DH3’

Brassica oleracea is a strictly self-incompatible (SI) plant, but rapid-cycling B. oleracea ‘TO1000DH3’ is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Three S-locus genes, S-locus glycoprotein (SLG), S-locus receptor kinase (SRK) and S-locus protein 11 or S-locus cysteine-rich (SP11/SCR), have been reported to date, all of which are classified into class I and II. In this study, we investigated the molecular mechanism behind alterations of SI to SC in rapid-cycling B. olerace ‘TO1000DH3’. Class I SRK were identified by genomic DNA PCR and PCR-RFLP analysis using SRK specific markers and found to be homozygous. Cloning and sequencing of class I SRK revealed a normal kinase domain without any S-domain/transmembrane domain. Moreover, S-locus sequencing analysis revealed only an SLG sequence, but no SP11/SCR. Expression analysis showed no SRK expression in the stigma, although other genes involved in the SI recognition reaction (SLG, MLPK, ARC1, THL) were found to have normal expression in the stigma. Taken together, the above results suggest that structural aberrations such as deletion of the SI recognition genes may be responsible for the breakdown of SI in rapid-cycling B. oleracea ‘TO1000DH3’.     

Physical distances between S-locus genes in various S haplotypes of Brassica rapa and B. oleracea

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes. Received: 20 September 1999 / Accepted: 8 October 1999     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

A review of sources of resistance to turnip yellows virus (TuYV) in Brassica species

Turnip yellows virus (TuYV; previously known as beet western yellows virus) causes major diseases of Brassica species worldwide resulting in severe yield-losses in arable and vegetable crops. It has also been shown to reduce the quality of vegetables, particularly cabbage where it causes tip burn. Incidences of 100% have been recorded in commercial crops of winter oilseed rape (Brassica napus) and vegetable crops (particularly Brassica oleracea) in Europe. This review summarises the known sources of resistance to TuYV in B. napus (AACC genome), Brassica rapa (AA genome) and B. oleracea (CC genome). It also proposes names for the quantitative trait loci (QTLs) responsible for the resistances, Tu rnip Y ellows virus R esistance (TuYR), that have been mapped to at least the chromosome level in the different Brassica species. There is currently only one known source of resistance deployed commercially (TuYR1). This resistance is said to have originated in B. rapa and was introgressed into the A genome of oilseed rape via hybridisation with B. oleracea to produce allotetraploid (AACC) plants that were then backcrossed into oilseed rape. It has been utilised in the majority of known TuYV-resistant oilseed rape varieties. This has placed significant selection pressure for resistance-breaking mutations arising in TuYV. Further QTLs for resistance to TuYV (TuYR2-TuYR9) have been mapped in the genomes of B. napus, B. rapa and B. oleracea and are described here. QTLs from the latter two species have been introgressed into allotetraploid plants, providing for the first time, combined resistance from both the A and the C genomes for deployment in oilseed rape. Introgression of these new resistances into commercial oilseed rape and vegetable brassicas can be accelerated using the molecular markers that have been developed. The deployment of these resistances should lessen selection pressure for resistance-breaking isolates of TuYV and thereby prolong the effectiveness of each other and extant resistance.     

Development of a core set of single-locus SSR markers for allotetraploid rapeseed (Brassica napus L.)

Brassica napus (AACC) is a recent allotetraploid species evolved through hybridization between two diploids, B. rapa (AA) and B. oleracea (CC). Due to extensive genome duplication and homoeology within and between the A and C genomes of B. napus, most SSR markers display multiple fragments or loci, which limit their application in genetics and breeding studies of this economically important crop. In this study, we collected 3,890 SSR markers from previous studies and also developed 5,968 SSR markers from genomic sequences of B. rapa, B. oleracea and B. napus. Of these, 2,701 markers that produced single amplicons were putative single-locus markers in the B. napus genome. Finally, a set of 230 high-quality single-locus SSR markers were established and assigned to the 19 linkage groups of B. napus using a segregating population with 154 DH individuals. A subset of 78 selected single-locus SSR markers was proved to be highly stable and could successfully discriminate each of the 45 inbred lines and hybrids. In addition, most of the 230 SSR markers showed the single-locus nature in at least one of the Brassica species of the U’s triangle besides B. napus. These results indicated that this set of single-locus SSR markers has a wide range of coverage with excellent stability and would be useful for gene tagging, sequence scaffold assignment, comparative mapping, diversity analysis, variety identification and association mapping in Brassica species.     

Sequence and expression of endogenous S-locus glycoprotein genes in self-compatible Brassica napus

Brassica napus is an amphidiploid plant which is self-compatible even though it is derived from hybridisation of the self-incompatible species B. oleracea and B. campestris. Experiments were undertaken to establish if S-locus glycoprotein (SLG) genes exist in B. napus and whether these are expressed as in self-incompatible Brassica species. Two different stigma-specific cDNA sequences homologous to SLG genes were obtained from the B. napus cultivar Westar. One of these sequences, SLG _WS1, displayed highest homology to class I SLG alleles, whereas the other, SLG _WS2, showed greatest homology to class II SLG genes. Both were expressed at high levels in Westar stigmas following a developmental pattern typical of SLG genes in the self-incompatible diploids. We infer that they represent the endogenous SLG genes at the two homoeologous S-loci. The occurrence of normally expressed SLG genes and its relevance to the self-compatible phenotype of B. napus is discussed.     

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

The self-compatibility mechanism in <Emphasis Type="Italic">Brassica napus</Emphasis> L. is applicable to F<Subscript>1</Subscript> hybrid breeding

Brassica napus, an allopolyploid species having the A genome of B. rapa and the C genome of B. oleracea, is self-compatible, although both B. rapa and B. oleracea are self-incompatible. We have previously reported that SP11/SCR alleles are not expressed in anthers, while SRK alleles are functional in the stigma in B. napus cv. ‘Westar’, which has BnS-1 similar to B. rapa S-47 and BnS-6 similar to B. oleracea S-15. This genotype is the most frequent S genotype in B. napus, and we hypothesized that the loss of the function of SP11 is the primary cause of the self-compatibility of ‘Westar’. To verify this hypothesis, we transformed ‘Westar’ plants with the SP11 allele of B. rapa S-47. All the transgenic plants and their progeny were completely self-incompatible, demonstrating self-compatibility to be due to the S haplotype having the non-functional SP11 allele in the A genome, which suppresses a functional recessive SP11 allele in the C genome. An artificially synthesized B. napus line having two recessive SP11 alleles was developed by interspecific hybridization between B. rapa and B. oleracea. This line was self-incompatible, but F₁ hybrids between this line and ‘Westar’ were self-compatible. These results suggest that the self-compatibility mechanism of ‘Westar’ is applicable to F₁ seed production in B. napus.     

Mapping of days to flower and seed yield in spring oilseed <Emphasis Type="Italic">Brassica napus</Emphasis> carrying genome content introgressed from <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Earliness of flowering and maturity and high seed yield are important objectives of breeding spring Brassica napus canola. Previously, we have introgressed earliness of flowering from Brassica oleracea into spring B. napus canola through interspecific crossing between these two species. In this paper, we report quantitative trait locus (QTL) mapping of days to flower and seed yield by use of publicly available markers and markers designed based on flowering time genes and a doubled haploid population, derived from crossing of the spring canola parent and an early flowering line developed from a B. napus × B. oleracea cross, tested in nine field trials for over 5 years. Five genomic regions associated with days to flower were identified on C1, C2, C3, and C6 of which the single QTL of C1 was detected in all trials; in all cases, the allele introgressed from B. oleracea reduced the number of days to flower. BLASTn search in the Brassica genomes located the physical position of the QTL markers and identified putative flowering time genes in these regions. In the case of seed yield, ten QTL from eight linkage groups were detected; however, none could be consistently detected in all trials. The QTL region of C1 associated with days to flower did not show significant association with seed yield in more than 80% of the field trials; however, in a single trial, the allele introgressed from B. oleracea exerted a negative effect on seed yield. Thus, the genomic regions and molecular markers identified in this research could potentially be used in breeding for the development of early flowering B. napus canola cultivars without affecting seed yield in a majority of the environments.     

During photosynthetic induction,biochemical and stomatal limitations differ between Brassica crops

Interventions to increase crop radiation use efficiency rely on understanding of how biochemical and stomatal limitations affect photosynthesis. When leaves transition from shade to high light, slow increases in maximum Rubisco carboxylation rate and stomatal conductance limit net CO₂ assimilation for several minutes. However, as stomata open intercellular [CO₂] increases, so electron transport rate could also become limiting. Photosynthetic limitations were evaluated in three important Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. Measurements of induction after a period of shade showed that net CO₂ assimilation by B. rapa and B. napus saturated by 10 min. A new method of analyzing limitations to induction by varying intercellular [CO₂] showed this was due to co-limitation by Rubisco and electron transport. By contrast, in B. oleracea persistent Rubisco limitation meant that CO₂ assimilation was still recovering 15 min after induction. Correspondingly, B. oleracea had the lowest Rubisco total activity. The methodology developed, and its application here, shows a means to identify the basis of variation in photosynthetic efficiency in fluctuating light, which could be exploited in breeding and bioengineering to improve crop productivity.     

Growth and mineral nutrition of six rapid-cycling Brassica species in response to seawater salinity

The growth of six rapid-cycling lines of Brassica species, B. napus, B. campestris, B. nigra, B. juncea, B. oleracea and B. carinata was inhibited by seawater salinity. Based on the change in dry matter reduction relative to the control at varying concentrations of salts (4, 8 and 12 dS m^-1), the relative salt tolerance of these species was evaluated. B. napus and B. carinata were the most tolerant and most sensitive species, respectively, while the other four species were moderately tolerant. The influence of seawater on the concentrations of 12 elements including macronutrients and micronutrients in the shoots of these Brassica plants was characterized to determine the relationship between nutritional disturbance and relative salt tolerance. It was found that seawater salinity had a significant effect on the concentrations of Ca, Mg, K, Cl, Na and total N in the shoots of these plants but only the change in Ca concentration was significantly related to the relative salt tolerance of these six rapid-cycling Brassica species according to a rank analysis of the data. This finding indicates that Ca may play a regulatory role in the responses of Brassica species to saline conditions.     

Quantitative trait analysis of seed yield and other complex traits in hybrid spring rapeseed (Brassica napus L.): 1. Identification of genomic regions from winter germplasm

The introgression of winter germplasm into spring canola (Brassica napus L.) represents a novel approach to improve seed yield of hybrid spring canola. In this study, quantitative trait loci (QTL) for seed yield and other traits were genetically mapped to determine the effects of genomic regions introgressed from winter germplasm into spring canola. Plant materials used comprised of two populations of doubled haploid (DH) lines having winter germplasm introgression from two related French winter cultivars and their testcrosses with a spring line used in commercial hybrids. These populations were evaluated for 2 years at two locations (Wisconsin, USA and Saskatchewan, Canada). Genetic linkage maps based on RFLP loci were constructed for each DH population. Six QTL were detected in the testcross populations for which the winter alleles increased seed yield. One of these QTL explained 11 and 19% of the phenotypic variation in the two Canadian environments. The winter allele for another QTL that increased seed yield was linked in coupling to a QTL allele for high glucosinolate content, suggesting that the transition of rapeseed into canola could have resulted in the loss of favorable seed yield alleles. Most QTL for which the introgressed allele decreased seed yield of hybrids mapped to genomic regions having homoeologous non-reciprocal transpositions. This suggests that allelic configurations created by these rearrangements might make an important contribution to genetic variation for complex traits in oilseed B. napus and could account for a portion of the heterotic effects in hybrids. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

A comparative study of DNA synthesis in cotyledon protoplast cultures of Brassica napus,B. campestris and B. oleracea using an immunocytochemical technique

The present study was designed (1) to observe the characterization of 5-bromo-2′-dexoyuridine (BrdU) incorporation into cultured Brassica cotyledon protoplasts and (2) to investigate the genetic differences in the levels of nuclear DNA synthesis (expressed by the percentage of nuclei labelled with BrdU) in cotyledon protoplast cultures from 12 cultivars of three Brassica species (Brassica napus, B. campestris and B. oleracea) at an early stage using immunocytochemistry. Nuclei labelled with BrdU were different from those showing only staining with 4′-6′-diamidino-2-phenylindole (DAPI) under fluorescence and light microscopy. Two to 5% of nuclei were labelled with BrdU after 1 h of culture, indicating that nuclear DNA synthesis occurred at a very early stage of culture. The percentage of nuclei labelled with BrdU increased with time over the length of the culture period. The mean percentage of nuclei labelled with BrdU in the 12 cultivars was about 25% at 24 h after culture initiation. The curve of the increase in percentage of nuclei labelled with BrdU exhibited an S-shape from 1 to 24 h. However, cultivar differences in percentages of nuclei labelled with BrdU were very significant over the time course of 1-24 h from initial culture, with cultivars Eureka (B. napus), Global (B. napus), Narc 82 (B. napus), Bunyip (B. campestris) and Sugar Loaf (B. oleracea) having a consistently higher percentage of nuclei labelled with BrdU than the other cultivars. Species differences were also significant, with cultivars of B. napus showing much higher percentages than the tested cultivars of B. campestris and B. oleracea. The results indicate that the differences in nuclear DNA synthesis in Brassica cotyledon protoplast cultures were most likely at both intra- and interspecies levels.     

Mapping loci controlling flowering time in Brassica oleracea

The timing of the transition from vegetative to reproductive phase is a major determinant of the morphology and value of Brassica oleracea crops. Quantitative trait loci (QTLs) controlling flowering time in B. oleracea were mapped using restriction fragment length polymorphism (RFLP) loci and flowering data of F₃ families derived from a cabbage by broccoli cross. Plants were grown in the field, and a total of 15 surveys were made throughout the experiment at 5–15 day intervals, in which plants were inspected for the presence of flower buds or open flowers. The flowering traits used for data analysis were the proportion of annual plants (PF) within each F₃ family at the end of the experiment, and a flowering-time index (FT) that combined both qualitative (annual/biennial) and quantitative (days to flowering) information. Two QTLs on different linkage groups were found associated with both PF and FT and one additional QTL was found associated only with FT. When combined in a multi-locus model, all three QTLs explained 54.1% of the phenotypic variation in FT. Epistasis was found between two genomic regions associated with FT. Comparisons of map positions of QTLs in B. oleracea with those in B. napus and B. rapa provided no evidence for conservation of genomic regions associated with flowering time between these species.