STS markers linked to Phoma resistance genes of the Brassica B-genome revealed sequence homology between Brassica nigra and Brassica napus

The RFLP and AFLP techniques are laborious and expensive and therefore of limited use for marker-assisted selection, demanding a high throughput of samples in a short time. But marker-assisted selection is most useful for traits which are hard to score on single plants and influenced by environmental factors. Four RFLP and three AFLP markers have been found to be linked to genes of the B-genome of Brassica mediating resistance against Phoma lingam in oilseed rape. One RFLP and one AFLP marker were converted into three PCR-based STS markers: one of dominant, as well as one of codominant inheritance separated in a standard agarose gel and a third one of codominant inheritance to be separated in a polyacrylamide gel on an automated sequencer. As expected, the STS markers mapped at the same position as the original RFLP and AFLP markers. The STS markers are efficient in marker-assisted backcross programs of the resistant B-genome/Brassica napus recombinant lines with most of the tested oilseed rape varieties and breeding lines. More than 90% of the tested oilseed rape varieties and breeding lines exhibited no resistance marker alleles. The mapping results obtained with the markers, as well as comparative sequencing of the marker alleles, indicate synteny and homology between the B-genome resistance gene donors and B. napus in the region of the resistance genes. The location of the resistance genes in the B-genome/B. napus recombinant lines is most likely on the A genome. Thus the transfer of the B-genome resistance genes into Brassica campestris is also possible. Received: 9 December 1999 / Accepted: 21 June 2000     

Selection of stable Brassica napus-Brassica juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 2. A ‘to and fro’ strategy to localise and characterise interspecific introgressions on the B. napus genome

 A new strategy to localise and characterise interspecific introgressions in the genus Brassica is presented. It consists of the localisation of RAPD specific markers from the donor species (B. juncea) by RFLP on a genetic map of the recipient (B. napus) and on the observation of the disappearance of rapeseed markers in recombinant lines. With this method, we localised an interspecific introgression of B. juncea, which confers blackleg resistance at the cotyledon stage in B. napus, on the linkage group DY17 of the previously determined B. napus genetic map. The estimated size of the substituted B. napus fragment was 39 cM, and the resistance gene was introgressed into the rapeseed genome by homologous recombination. The significance of the different strategies used and the implication of these results in breeding programs are discussed. Received: 23 August 1997 / Accepted: 13 October 1997     

Construction of Brassica B genome synteny groups based on chromosomes extracted from three different sources by phenotypic, isozyme and molecular markers

The three B genomes of Brassica contained in B. nigra, B. carinata and B. juncea were dissected by addition in B. napus. Using phenotypic, isozyme and molecular markers we characterized 8 alien B-genome chromosomes from B. nigra and B. carinata and 7 from B. juncea by constructing synteney groups. The alien chromosomes of the three different sources showed extensive intragenomic recombinations that were detected by the presence of the same loci in more than one synteny group but flanked by different markers. In addition, intergenomic recombinations were observed. These were evident in euploid AACC plants of the rapeseed phenotype derived from the addition lines carrying a few markers from the B genome due to translocations and recombinations between non-homoeologous chromosomes. The high plasticity of the Brassica genomes may have been an powerful factor in directing their evolution by hybridization and amphiploidy.     

Resistance to Leptosphaeria maculans is conserved in a specific region of the Brassica B genome

 Offspring from asymmetric hybrids between Brassica napus and the three B-genome species Brassica nigra, Brassica juncea and Brassica carinata were analysed for the presence of B-genome markers and resistance to the fungus Leptosphaeria maculans, the causal agent of blackleg disease. Twenty five plants from each species combination were analysed in the first backcross (BC₁) generation, 30 plants in BC₂ and 60 plants in BC₃. The plants were analysed by 46 RFLP markers detecting 85 loci dispersed throughout the B. nigra genome. The plants with additional B. carinata DNA had a decrease in the presence of RFLP markers ranging from 59% in BC₁ to 36% in BC₂ and down to 11% in BC₃. Similar results were obtained in the lines with additional DNA from B. juncea where the 60% presence of RFLP markers in BC₁ was reduced to 33% in BC₂ and to 10% in BC₃. However presence of the markers were significantly lower in the B. nigra-derived material where BC₁ had 46%, BC₂ 25% and BC₃ 8%. Since at least two loci could be detected on each end of the eight linkage groups of the B genome, the degree of symmetry was estimated. After one back-cross between 0.5 and 1.25% intact chromosomes were retained, whereas in BC₂ this frequency was 0.21% for all three B-genome donor species. The maintenance of half-chromosomes ranged from 2.63% to 5.38% in BC₁ and between 0.73% and 1.15% in BC₂. No chromosome arms were found in any of the BC₃ plants. In total, four co-segregating markers for cotyledon and adult-leaf resistance to L. maculans were found which detected six loci located on linkage groups 2, 5 and 8. When the results from the three donor species were compared, one triplicate region in the B genome had preserved the resistance loci in all three species. Received: 19 January 1999 / Accepted: 30 January 1999     

De novo genetic variation associated with retrotransposon activation, genomic rearrangements and trait variation in a recombinant inbred line population of Brassica napus derived from interspecific hybridization with Brassica rapa

Interspecific hybridization is a significant evolutionary force as well as a powerful method for crop breeding. Partial substitution of the AA subgenome in Brassica napus (AⁿAⁿCⁿCⁿ) with the Brassica rapa (A^rA^r) genome by two rounds of interspecific hybridization resulted in a new introgressed type of B. napus (A^rA^rCⁿCⁿ). In this study, we construct a population of recombinant inbred lines of the new introgressed type of B. napus. Microsatellite, intron‐based and retrotransposon markers were used to characterize this experimental population with genetic mapping, genetic map comparison and specific marker cloning analysis. Yield‐related traits were also recorded for identification of quantitative trait loci (QTLs). A remarkable range of novel genomic alterations was observed in the population, including simple sequence repeat (SSR) mutations, chromosomal rearrangements and retrotransposon activations. Most of these changes occurred immediately after interspecific hybridization, in the early stages of genome stabilization and derivation of experimental lines. These novel genomic alterations affected yield‐related traits in the introgressed B. napus to an even greater extent than the alleles alone that were introgressed from the A^r subgenome of B. rapa, suggesting that genomic changes induced by interspecific hybridization are highly significant in both genome evolution and crop improvement.     

Construction of an oilseed rape (Brassica napus L.) genetic map with SSR markers

We constructed a Brassica napus genetic map with 240 simple sequence repeats (SSR) primer pairs from private and public origins. SSR, or microsatellites, are highly polymorphic and efficient markers for the analysis of plant genomes. Our selection of primer pairs corresponded to 305 genetic loci that we were able to map. In addition, we also used 52 sequence-characterized amplified region primer pairs corresponding to 58 loci that were developed in our lab. Genotyping was performed on six F2 populations, corresponding to a total of 574 F2 individual plants, obtained according to an unbalanced diallel cross design involving six parental lines. The resulting consensus map presented 19 linkage groups ranging from 46.2 to 276.5 cM, which we were able to name after the B. napus map available at , thus enabling the identification of the A genome linkage groups originating from the B. rapa ancestor and the C genome linkage groups originating from the B. oleracea ancestor in the amphidiploid genome of B. napus. Some homoeologous regions were identified between the A and the C genomes. This map could be used to identify more markers, which would eventually be linked to genes controlling important agronomic characters in rapeseed. Furthermore, considering the good genome coverage we obtained, together with an observed homogenous distribution of the loci across the genome, this map is a powerful tool to be used in marker-assisted breeding. Electronic Supplementary Material Supplementary material is available for this article at     

Analysis of the Brassica oleracea genome by the generation of B. campestris-oleracea chromosome addition lines: characterization by isozymes and rDNA genes

Summary This study aimed at generating chromosome addition lines and disclosing genome specific markers in Brassica. These stocks will be used to study genome evolution in Brassica oleracea L., B. campestris L. and the derived amphidiploid species B. napus L. B. campestris-oleracea monosomic and disomic chromosome addition plants were generated by crossing and backcrossing the natural amphidiploid B. napus to the diploid parental species B. campestris. The pollen viability of the derived sesquidiploid and hyperploid ranged from 63% to 88%, while the monosomic and disomic addition plants had an average pollen fertility of 94% and 91%, respectively. The addition lines were genetically characterized by genome specific markers. The isozymes for 6PGD, LAP, PGI and PGM, and rDNA Eco RI restriction fragments were found to possess the desired genome specificity. Duplicated loci for several of these markers were observed in B. campestris and B. oleracea, supporting the hypothesis that these diploid species are actually secondary polyploids. A total of eight monosomic and eight disomic addition plants were identified and characterized on the basis of these markers. Another 51 plants remained uncharacterized due to the lack of additional markers. rDNA genes were found to be distributed in more than one chromosome, differing in its restriction sites. Intergenomic recombination for some of the markers was detected at frequencies between 6% and 20%, revealing the feasibility of intergenomic gene transfer.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii

Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line. Received:15 December 1998 / Accepted:30 January 1999     

A PCR based B-genome-specific marker in <Emphasis Type="Italic">Brassica</Emphasis> species

Previous hybridisation studies showed that the repetitive DNA sequence pBNBH35 from Brassica nigra (genome BB, 2n=16) bound specifically to the B-genome and not to the A- or C-genomes of Brassica species. We amplified a sub-fragment of pBNBH35 from B. nigra by PCR, cloned and sequenced this sub-fragment, and confirmed that it was a 329-bp sub-fragment of pBNBH35. PCR and hybridisation techniques were used to confirm that the pBNBH35 sub-fragment was Brassica B-genome-specific. Fluorescence in situ hybridisation (FISH) in B. nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica B-genome chromosomes and absent from the A- and C-genome chromosomes. The pBNBH35 repeat was localised to the centromeric region of each B-genome chromosome. FISH clearly distinguished the B-genome chromosomes from the A-genome chromosomes in the amphidiploid species B. juncea. This is the first known report of a B-genome repetitive marker that is present on all B-genome chromosomes. It will be a useful tool for the detection of B chromosomes in interspecific hybrids and may prove useful for phylogenetic studies in Brassica species.     

Reconstituting the genome of a young allopolyploid crop,Brassica napus,with its related species

Brassica napus (AⁿAⁿCⁿCⁿ) is an important worldwide oilseed crop, but it is a young allotetraploid with a short evolutionary history and limited genetic diversity. To significantly broaden its genetic diversity and create a novel heterotic population for sustainable rapeseed breeding, this study reconstituted the genome of B. napus by replacing it with the subgenomes from 122 accessions of Brassica rapa (A^rA^r) and 74 accessions of Brassica carinata (B^cB^cC^cC^c) and developing a novel gene pool of B. napus through five rounds of extensive recurrent selection. When compared with traditional B. napus using SSR markers and high‐throughput SNP/Indel markers through genotyping by sequencing, the newly developed gene pool and its homozygous progenies exhibited a large genetic distance, rich allelic diversity, new alleles and exotic allelic introgression across all 19 AC chromosomes. In addition to the abundant genomic variation detected in the AC genome, we also detected considerable introgression from the eight chromosomes of the B genome. Extensive trait variation and some genetic improvements were present from the early recurrent selection to later generations. This novel gene pool produced equally rich phenotypic variation and should be valuable for rapeseed genetic improvement. By reconstituting the genome of B. napus by introducing subgenomic variation within and between the related species using intense selection and recombination, the whole genome could be substantially reorganized. These results serve as an example of the manipulation of the genome of a young allopolyploid and provide insights into its rapid genome evolution affected by interspecific and intraspecific crosses.     

Brassica naponigra,a somatic hybrid resistant to Phoma lingam

Summary Brassica napus and B. nigra were combined via protoplast fusion into the novel hybrid Brassica naponigra. The heterokaryons were identified by fluorescent markers and selected by flow sorting. Thirty hybrid plants were confirmed by isozyme analysis to contain both B. nigra and B. napus chromosomes; of these, 20 plants had the sum of the parental chromosome numbers. A non-random segregation of the chloroplasts was found in the hybrids. Of 14 hybrid plants investigated, all had the B. napus type of chloroplast. The resistance to Phoma lingam found in the B. nigra cultivar used in the fusion experiments was expressed in 26 of the hybrid plants. The hybrids obtained in this study contain all of the three Brassica genomes (A, B and C) and have thus created unique possibilities for genetic exchanges between the genomes. Since most of the plants were fertile as well as resistant to P. lingam, they have been incorporated into conventional rapeseed breeding programs.     

Molecular cytogenetic identification of B genome chromosomes linked to blackleg disease resistance in Brassica napus × B. carinata interspecific hybrids

Key message

Provide evidence that the Brassica B genome chromosome B3 carries blackleg resistance gene, and also the B genome chromosomes were inherited several generations along with B. napus chromosomes.

Abstract

Blackleg disease caused by fungus Leptosphaeria maculans causes significant yield losses in Brassica napus. Brassica carinata possesses excellent resistance to this disease. To introgress blackleg resistance, crosses between B. napus cv. Westar and B. carinata were done. The interspecific-hybrids were backcrossed twice to Westar and self-pollinated three times to produce BC₂S₃ families. Doubled haploid lines (DH1) were produced from one blackleg resistant family. SSR markers were used to study the association between B genome chromosome(s) and blackleg resistance. The entire B3 chromosome of B. carinata was associated with blackleg resistance in DH1. A second DH population (DH2) was produced from F₁s of resistant DH1 lines crossed to blackleg susceptible B. napus cv. Polo where resistance was found to be associated with SSR markers from the middle to bottom of the B3 and top of the B8 chromosomes. The results demonstrated that the B3 chromosome carried gene(s) for blackleg resistance. Genomic in situ hybridization (GISH) and GISH-like analysis of the DH2 lines revealed that susceptible lines, in addition to B. napus chromosomes, possessed one pair of B genome chromosomes (2n = 40), while resistant lines had either one (2n = 40) or two pairs (2n = 42) of B chromosomes. The molecular and GISH data suggested that the B chromosome in the susceptible lines was B7, while it was difficult to confirm the identity of the B chromosomes in the resistant lines. Also, B chromosomes were found to be inherited over several generations along with B. napus chromosomes.     

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.     

Inheritance of rapeseed (Brassica napus)-specific RAPD markers and a transgene in the cross B. juncea x (B. juncea x B. napus)

We have examined the inheritance of 20 rapeseed (Brassica napus)-specific RAPD (randomly amplified polymorphic DNA) markers from transgenic, herbicide-tolerant rapeseed in 54 plants of the BC₁ generation from the cross B. junceax(B. junceaxB. napus). Hybridization between B. juncea and B. napus, with B. juncea as the female parent, was successful both in controlled crosses and spontaneously in the field. The controlled backcrossing of selected hybrids to B. juncea, again with B. juncea as the female parent, also resulted in many seeds. The BC₁ plants contained from 0 to 20 of the rapeseed RAPD markers, and the frequency of inheritance of individual RAPD markers ranged from 19% to 93%. The transgene was found in 52% of the plants analyzed. Five synteny groups of RAPD markers were identified. In the hybrids pollen fertility was 0–28%. The hybrids with the highest pollen fertility were selected as male parents for backcrossing, and pollen fertility in the BC₁ plants was improved (24–90%) compared to that of the hybrids.     

Development of a SCAR (sequence characterised amplified region) marker for molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus L.

 A SCAR (sequence characterised amplified region) has been developed for optimal tagging of the dwarf Bzh gene in Brassica napus. A RAPD marker named OPMO7-730 was previously found closely linked to the dwarf locus at 0.8±0.7 cM. The DNA band corresponding to this marker was cloned and sequenced, and specific PCR primers were designed. The PCR test allowed us to amplify the locus corresponding to OPM07-730. With a restriction endonuclease digest and optimal electrophoresis conditions, three allelic forms of this marker have been recovered on the 40 B. napus accessions tested. The usefullness of this marker in breeding dwarf rapeseed lines is discussed. Received: 20 April 1998 / Accepted: 29 April 1998     

Evaluation of nematode-resistant sugar beet (Beta vulgaris L.) lines by molecular analysis

 Thirty sugar beet (Beta vulgaris) lines conferring complete resistance to the beet cyst nematode (BCN, Heterodera schachtii) originating from interspecific crosses with wild beets of the section Procumbentes (B. procumbens, B. webbiana and B. patellaris) were investigated by morphology and wild beet-specific molecular markers. The beet lines carrying chromosome mutations consisted of monosomic additions (2n=18+1), fragment additions (2n=18+fragment) and translocations (2n=18) from the wild beets. Genome-specific single-copy, satellite and repetitive probes were applied to study the origin, chromosomal assignment and presence of nematode resistance genes. Within the wild beet species at least three different resistance genes located on different chromosomes were distinguished: Hs1 on the homoelogous chromosomes I of each species, Hs2 on the homoelogous chromosomes VII of B. procumbens and B. webbiana and Hs3 on chromosome VIII of B. webbiana. A clear distinction between the three chromosomes was possible by morphological and molecular means. The translocation lines were separated into two different groups: one containing the resistance gene Hs1 from chromosome I and the other carrying a different nematode resistance gene. The molecular data combined with sequence analyses of Hs1 of the three wild beet species revealed a clear distinction between B. procumbens and B. webbiana. The evolutionary and taxonomical relationship of these species supporting the idea of three different species originating from a common ancestor is discussed. Received: 6 April 1998 / Accepted: 22 April 1998     

Characterisation of resistance to turnip mosaic virus in oilseed rape (Brassica napus) and genetic mapping of TuRB01

Turnip mosaic virus (TuMV) is the major virus infecting Brassica crops. A dominant gene, TuRB01, that confers extreme resistance to some isolates of TuMV on Brassica napus (oilseed rape), has been mapped genetically. The mapping employed a set of doubled-haploid lines extracted from a population used previously to develop a reference RFLP map of the B. napus genome. The positioning of TuRB01 on linkage group N6 of the B. napus A–genome indicated that the gene probably originated from Brassica rapa. Resistance phenotypes were confirmed by indirect plate-trapped antigen ELISA using a monoclonal antibody raised against TuMV. The specificity of TuRB01 was determined using a wide range of TuMV isolates, including representatives of the European and American/Taiwanese pathotyping systems. Some isolates of TuMV that did not normally infect B. napus plants possessing TuRB01 produced mutant viruses able to overcome the action of the resistance gene. TuRB01 is the first gene for host resistance to TuMV to be mapped in a Brassica crop. A second locus, TuRB02, that appeared to control the degree of susceptibility to the TuMV isolate CHN 1 in a quantitative manner, was identified on the C-genome linkage group N14. The mapping of other complementary genes and the selective combining of such genes, using marker-assisted breeding, will make durable resistance to TuMV a realisable breeding objective. Received: 14 December 1998 / Accepted: 10 April 1999     

Annotation and characterization of Cd-responsive metal transporter genes in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis>)

In higher plants, heavy metal transporters are responsible for metal uptake, translocation and homeostasis. These metals include essential metals such as zinc (Zn) or manganese (Mn) and non-essential metals like cadmium (Cd) or lead (Pb). Although a few heavy metal transporters have been well identified in model plants (e.g. Arabidopsis and rice), little is known about their functionality in rapeseed (Brassica napus). B. napus is an important oil crop ranking the third largest sources of vegetable oil over the world. Importantly, B. napus has long been considered as a desirable candidate for phytoremediation owning to its massive dry weight productivity and moderate to high Cd accumulation. In this study, 270 metal transporter genes (MTGs) from B. napus genome were identified and annotated using bioinformatics and high-throughput sequencing. Most of the MTGs (74.8%, 202/270) were validated by RNA-sequencing (RNA-seq) the seedling libraries. Based on the sequence identity, nine superfamilies including YSL, OPT, NRAMP, COPT, ZIP, CDF/MTP, HMA, MRP and PDR have been classified. RNA-sequencing profiled 202 non-redundant MTGs from B. napus seedlings, of which, 108 MTGs were differentially expressed and 62 genes were significantly induced under Cd stress. These differentially expressed genes (DEGs) are dispersed in the rapeseed genome. Some of the genes were well confirmed by qRT-PCR. Analysis of the genomic distribution of MTGs on B. napus chromosomes revealed that their evolutional expansion was probably through localized allele duplications.     

Genetic analyses of the host-pathogen system Turnip yellows virus (TuYV)—rapeseed (Brassica napus L.) and development of molecular markers for TuYV-resistance

The aphid transmitted Turnip yellows virus (TuYV) has become a serious pathogen in many rapeseed (Brassica napus L.) growing areas. Three-years’ field trials were carried out to get detailed information on the genetics of TuYV resistance derived from the resynthesised B. napus line ‘R54’ and to develop closely linked markers. F₁ plants and segregating doubled-haploid (DH) populations derived from crosses to susceptible cultivars were analysed using artificial inoculation with virus-bearing aphids, followed by DAS-ELISA. Assuming a threshold of E ₄₀₅ = 0.1 in ELISA carried out in December, the results led to the conclusion that pre-winter inhibition of TuYV is inherited in a monogenic dominant manner. However, the virus titre in most resistant lines increased during the growing period, indicating that the resistance is incomplete and that the level of the virus titre is influenced by environmental factors. Bulked-segregant marker analysis for this resistance locus identified two closely linked SSR markers along with six closely linked and three co-segregating AFLP markers. Two AFLP markers were converted into co-dominant STS markers, facilitating efficient marker-based selection for TuYV resistance. Effective markers are particularly valuable with respect to breeding for TuYV resistance, because artificial inoculation procedures using virus-bearing aphids are extremely difficult to integrate into practical rapeseed breeding programs.