Potential gene flow of two herbicide-tolerant transgenes from oilseed rape to wild B. juncea var. gracilis

Four successive reciprocal backcrosses between F₁ (obtained from wild Brassica juncea as maternal plants and transgenic glyphosate- or glufosinate-tolerant oilseed rape, B. napus, as paternal plants) or subsequent herbicide-tolerant backcross progenies and wild B. juncea were achieved by hand pollination to assess potential transgene flow. The third and forth reciprocal backcrosses produced a number of seeds per silique similar to that of self-pollinated wild B. juncea, except in plants with glufosinate-tolerant backcross progeny used as maternal plants and wild B. juncea as paternal plants, which produced fewer seeds per silique than did self-pollinated wild B. juncea. Germination percentages of reciprocal backcross progenies were high and equivalent to those of wild B. juncea. The herbicide-tolerant first reciprocal backcross progenies produced fewer siliques per plant than did wild B. juncea, but the herbicide-tolerant second or third reciprocal backcross progenies did not differ from the wild B. juncea in siliques per plant. The herbicide-tolerant second and third reciprocal backcross progenies produced an amount of seeds per silique similar to that of wild B. juncea except for with the glufosinate-tolerant first and second backcross progeny used as maternal plants and wild B. juncea as paternal plants. In the presence of herbicide selection pressure, inheritance of the glyphosate-tolerant transgene was stable across the second and third backcross generation, whereas the glufosinate-tolerant transgene was maintained, despite a lack of stabilized introgression. The occurrence of fertile, transgenic weed-like plants after only three crosses (F₁, first backcross, second backcross) suggests a potential rapid spread of transgenes from oilseed rape into its wild relative wild B. juncea. Transgene flow from glyphosate-tolerant oilseed rape might be easier than that from glufosinate-tolerant oilseed rape to wild B. juncea. The original insertion site of the transgene could affect introgression.     

The impact on biosafety of the phosphinothricin-tolerance transgene in inter-specific B. rapa×B. napus hybrids and their successive backcrosses

 There is strong evidence indicating that gene flow from transgenic B. napus into weedy wild relatives is inevitable following commercial release. Research should now focus on the transmission, stability, and impact of transgene expression after the initial hybridization event. The present study investigated the transfer of a phosphinothricin-tolerance transgene by inter-specific hybridization between B. rapa and two transgenic B. napus lines. The expression of the transgene was monitored in the F₁ hybrids and in subsequent backcross generations. The transgene was transmitted relatively easily into the F₁ hybrids and retained activity. Large differences in the transmission frequency of the transgene were noted between offspring of the two transgenic lines during backcrossing. The most plausible explanation of these results is that the line showing least transmission during backcrossing contains a transgene integrated into a C-genome chromosome. Approximately 10% of offspring retained the tolerant trait in the BC₃ and BC₄ generations. The implications of these findings for the stable introgression of transgenes carried on one of the chromosomes of the C-genome from B. napus and into B. rapa are briefly discussed. Received: 5 November 1996 / Accepted: 21 February 1997     

Loss of C-genome-specific markers during transgene introgression from Brassica napus to wild Brassica juncea

Transgene flow from engineered Brassica napus to wild weed relatives could potentially have an environmental effect. To evaluate the introgression of transgenic B. napus into wild Brassica juncea, the hybrid F₁ and backcross progenies derived from B. juncea (genome constitution AABB) and transgenic B. napus (AACC) crosses were investigated. C-genome-specific simple sequence repeat (SSR) markers corresponding to linkage groups N11–N19 in B. napus were screened and used to estimate the marker frequency in hybrid F₁ and backcross progenies. C-genome-specific markers could be stably detected in hybrid F₁ and backcross BC₁ plants, but were only rarely found in the BC₂–BC₅ generations. For example, a specific SSR marker for linkage group N12 segregated in BC₂ generation but were completely lost in BC₃–BC₅, while a specific SSR marker of linkage group N15 segregated in BC₁, BC₂ and BC₃ generations and was absent in more advanced backcrossed generations (BC₄ and BC₅). The results indicate that a certain gene regions in Brassica napus plants are transmitted at a relatively lower frequency to wild relatives, and more rapidly disappeared in subsequent backcross generations. We propose that a foreign gene or transgene that is integrated in the C-chromosome of Brassica napus could reduce the risk of introgression in nature.     

Slug herbivory on hybrids of the crop Brassica napus and its wild relative B. rapa

Some plant species can cross with each other but stay nevertheless distinct with little gene flow between them. Selective herbivory could explain this pattern when hybrids are more susceptible or intermediate between their parents. We performed choice and no-choice experiments with the slug Arion lusitanicus to test this hypothesis for the crop Brassica napus, wild B. rapa and their backcross hybrids. In both experiments slugs greatly preferred B. napus over B. rapa while average herbivory on backcross hybrids was intermediate. Concentrations of aliphatic glucosinolates in the hybrids were intermediate between the parental species. Slug herbivory potentially reduces establishment of backcross hybrids. This hypothesis needs to be tested in the field.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop × weed hybrid generations

The level of transgene expression in crop × weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T₁ single-locus insert GFP/Bacillus thuringiensis (Bt) transgenic canola (Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC₁F₁, BC₂F₁) were produced by backcrossing various GFP/Bt transgenic canola (B. napus, cv Westar) and birdseed rape (Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC₂F₂ Bulk) were generated by crossing BC₂F₁ individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC₂F₂ Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F₁ hybrid generations contained 95–97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15–29% presence in the BC₂F₂ Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC₂F₂ Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F₁, BC₁F₁ and BC₂F₁). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.Communicated by J. Dvorak     

Detection,introgression and localization of genes conferring specific resistance to <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> from <Emphasis Type="Italic">Brassica rapa</Emphasis> into <Emphasis Type="Italic">B. napus</Emphasis>

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of “new” resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.     

Hybridisation and introgression between Brassica napus and B. rapa in the Netherlands

We used flow cytometry, chromosome counting and AFLP markers to investigate gene flow from the crop plant oilseed rape, Brassica napus (AACC) to wild B. rapa (AA) in the Netherlands. From 89 B. napus source populations investigated, all near cropping fields or at transhipment sites, only 19 contained a B. rapa population within a 2.5‐km radius. During our survey we found only three populations with F1 hybrids (AAC), as recognized by their nine extra chromosomes and by flow cytometry. These hybrids were all collected in mixed populations where the two species grew in close proximity. Populations with F1 hybrids were not close to crops, but instead were located on road verges with highly disturbed soils, in which both species were probably recruited from the soil seed bank. Many plants in the F2, BC1 or higher backcrosses are expected to carry one to eight C chromosomes. However, these plants were not observed among the hybrids. We further investigated introgression with molecular markers (AFLP) and compared sympatric B. rapa populations (near populations of B. napus) with control populations of B. rapa (no B. napus within at least 7 km). We found no difference between sympatric and control populations in the number of C markers in B. rapa, nor did we find that these sympatric populations closely resembled B. napus. Our data show that hybrids occur but also suggest no recent introgression of alleles from the crop plant B. napus into wild B. rapa in the Dutch populations studied.     

Improved resistance to bacterial soft rot by protoplast fusion between Brassica rapa and B. oleracea

Erwinia soft rot is a destructive disease of Brassica rapa vegetables. Reliable sources of resistance and control methods are limited, so development of highly resistant breeding lines is desirable. Protoplasts from B. rapa and B. oleracea genotypes selected for resistance to soft rot were fused in order to combine different sources of resistance. Twelve somatic hybrids (synthetic B. napus) were obtained and confirmed by morphology, nuclear DNA content, and RAPD analysis. They were normal looking plants that easily set seeds following self-pollination and backcrossing to B. rapa. Assays of detached leaves or seedlings inoculated in a mist-chamber showed that most somatic hybrids had lower disease severity ratings than the B. rapa fusion partner and a commercial variety of B. napus. Some progeny from selfing or backcrossing of somatic hybrids to B. rapa showed much more resistance than either fusion partner. The offspring populations of the somatic hybrids (F₁–S₁ and F₁–BC₁) clearly moved to the resistant direction compared to the parents; the percentage of resistant plants increased from 21% (average of parents) to 36% (F₁–S₁) and 48% (F₁–BC₁). These results suggest that it may be possible to obtain highly resistant B. rapa lines by further backcrossing and selection. Received: June 1999 / Accepted: 29 July 1999     

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.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.     

High‐throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus,Brassica rapa and Brassica oleracea

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole‐genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C‐genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts.     

Identifying the chromosomes of the A- and C-genome diploid Brassica species B. rapa (syn. campestris) and B. oleracea in their amphidiploid B. napus

Oilseed rape (Brassica napus L.) is an amphidiploid species that originated from a spontaneous hybridisation of Brassica rapa L. (syn. campestris) and Brassica oleracea L., and contains the complete diploid chromosome sets of both parental genomes. The metaphase chromosomes of the highly homoeologous A genome of B. rapa and the C genome of B. oleracea cannot be reliably distinguished in B. napus because of their morphological similarity. Fluorescence in situ hybridisation (FISH) with 5S and 25S ribosomal DNA probes to prometaphase chromosomes, in combination with DAPI staining, allows more dependable identification of Brassica chromosomes. By comparing rDNA hybridisation and DAPI staining patterns from B. rapa and B. oleracea prometaphase chromosomes with those from B. napus, we were able to identify the putative homologues of B. napus chromosomes in the diploid chromosome sets of B. rapa and B. oleracea, respectively. In some cases, differences were observed between the rDNA hybridisation patterns of chromosomes in the diploid species and their putative homologue in B. napus, indicating locus losses or alterations in rDNA copy number. The ability to reliably identify A and C genome chromosomes in B. napus is discussed with respect to evolutionary and breeding aspects. Received: 13 July 2001 / Accepted: 23 August 2001     

Abiotic stress and transgenics: Implications for reproductive success and crop-to-wild gene flow in Brassicas

Various abiotic and biotic stressors affect crop and weed plant performance in agroecosystems. Ozone (O₃) tolerance in plants is partly regulated by the genotype and phenotypical properties, and it varies greatly in related species of wild and crop backgrounds. Thus, a continuous increase in atmospheric O₃ concentration could change population dynamics of sexually compatible crop and weed species, and consequently affect crop-to-wild gene flow in the future. One way to build resistance against a biotic stressor, in this case insect-mediated herbivory, in crop plants is transgene-mediated insecticidal toxin production. In this study we aimed to describe how the physiological and phenological responses in a crop Brassica and its weedy relatives functioned to affect their comparative O₃ tolerance. Furthermore, we studied how harbouring a transgene affects these responses in B. napus and B. rapa × transgenic B. napus BC₂F₂ backcross hybrid plants to reveal any within-plant trade-offs among toxin production, growth and O₃ tolerance. We found a higher number of O₃ symptoms but more effective compensatory assimilate allocation directed to reproduction for wild B. rapa compared to crop B. napus under elevated O₃. This result suggested that the invasion-orientated strategy of producing a high number of seeds when vegetative growth is limited might improve the performance of weedy species under elevated O₃. The probabilities for crop-to-wild transgene flow could be increased through higher seed production in hybrids under elevated O₃, but the germination of hybrid seeds in particular was hampered by O₃. The presence of transgenes did not perturb fecundity, within-plant biomass allocation or O₃ tolerance of B. napus.     

Development of a population for substantial new type Brassica napus diversified at both A/C genomes

Intersubgenomic heterosis in rapeseed has been revealed in previous studies by using traditional Brassica napus (AⁿAⁿCⁿCⁿ) to cross partial new type B. napus with A^r/C^c introgression from the genomes of B. rapa and B. carinata, respectively. To further enlarge the genetic basis of B. napus and to facilitate a sustained heterosis breeding in rapeseed, it is crucial to create a population for substantial new type B. napus diversified at both A/C genomes. In this experiment, hundreds of artificial hexaploid plants (A^rA^rB^cB^cC^cC^c) involving hundreds of B. carinata/B. rapa combinations were first crossed with elite lines of partial new type B. napus. The pentaploid plants (AABCC) were open-pollinated in isolated conditions, and their offspring were successively self-pollinated and intensively selected for two generations. Thereafter, a population of substantial new type B. napus mainly with a genomic composition of A^rA^rC^cC^c harbouring genetic diversity from 25 original cultivars of B. rapa and 72 accessions of B. carinata was constructed. The population was cytologically verified to have the correct chromosome constitution of AACC and differed genetically from traditional B. napus, in terms of the genome components of A^r/C^c and B^c as well as the novel genetic variations induced by the interspecific hybridisation process. Synchronously, rich phenotypic variation with plenty of novel valuable traits was observed in the population. The origin of the novel variations and the value of the population are discussed.     

Within-population variation in hybridisation and transgene transfer between wild Brassica rapa and Brassica napus in the UK

Several studies in Europe and North America have shown that cultivated Brassica napus will readily hybridise with wild Brassica rapa but at widely different frequencies. To understand the implications of this phenomenon with regard to transgene flow, we examined the rate at which cultivated B. napus cv. Westar containing a capsid (coat protein, CP)‐coding sequence from Turnip mosaic virus (Potyvirus) hybridised under glasshouse conditions with wild B. rapa from Culham, in Oxfordshire, UK. We found that the hybridisation rate, as judged using simple sequence repeat (SSR)‐PCR and primer oligonucleotides specific for either the C or the A genomes in progeny from individual crosses varied from 5% to 100%. In hybrids (F₁ progeny), transgene transfer was always observed (inferred by SSR‐PCR) when hybrids were detected. Our observations revealed a hitherto unrecorded source of variability in transgene flow to wild UK B. rapa.     

Inheritance of oilseed rape (Brassica napus) RAPD markers in a backcross progeny with Brassica campestris

Different cultivars/transgenic lines of oilseed rape (Brassica napus) were crossed (as females) with different cultivars/populations of Brassica campestris. All cross combinations produced seed, with an average seed set per pollination of 9.8. Backcrossing of selected interspecific hybrids (as females) to B. campestris resulted in a much lower seed set, average 0.7 seed per pollination. In the single backcross progeny where a large enough population (92 plants) was obtained for analysis, 33 B. napus specific RAPD markers were investigated to determine the extent of transfer of oilseed rape genetic material into this population. Markers were transferred to the backcross generation with frequencies ranging from 26% to 91%. Almost all of the markers (30/33) were transferred in a frequency not significantly different from 50%. Analysis of the pairwise segregation of markers revealed that 23 markers could be assigned to six linkage groups, most probably reflecting six B. napus C-chromosomes. The presence of backcross plants with recombinant genotypes suggests that complex genetic processes can take place during interspecific hybridisation and backcrossing in these Brassica species. The implications of our results for the possible choice of integration sites of transgenes in oilseed rape are discussed.     

Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species

Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F₁, F₂ and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F₂ progeny, and eight were unable to produce progeny when the F₁ was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.     

RFLP and AFLP Analysis of Inter- and Intraspecific Variation of Brassica rapa and B. napus Shows that B. rapa Is an Important Genetic Resource for B. napus Improvement

Fingerprinting of 29 accessions of oilseed rape, including seven accessions of Brassica napus, and 22 accessions of B. rapa (B. campestris) from Europe, North America, and China was analyzed using RFLP and AFLP markers. In total, 1 477 polymorphic RFLP bands and 183 polymorphic AFLP bands from 166 enzyme-probe combinations and two pairs of AFLP primers, respectively, were scored for the 29 accessions. On average, RFLP analysis showed that the Arabidopsis EST probe detected more polymorphic bands in Brassica than the random genomic probe performed. More polymorphic RFLP markers were detected with the digestion of EcoR I or BamH I than HindIII. According to the number of bands amplified from each accession, the copy numbers of each gene in the genomes of B. rapa and B. napus were estimated. The average copy numbers in B. rapa of China, B. rapa of Europe, and B. napus, were 3.2, 3.1, and 2.9, respectively. Genetic distance based on the AFLP data was well correlated with that based on the RFLP data (r = 0.72, P<0.001), but 0.39 smaller on average. Genetic diversity analysis showed that Chinese B. rapa was more polymorphic than Chinese B. napus and European materials. Some European B. napus accessions were clustered into European B. rapa, which were distinctly different from Chinese B. napus. The larger variations of Chinese accessions of B. rapa suggest that they are valuable in oilseed rape breeding. Novel strategies to use intersubgenomic heterosis between genome of B. rapa (A^rA^r) and genome of B. napus (AⁿAⁿCⁿCⁿ) were elucidated.