Winter warming post floral initiation delays flowering via bud dormancy activation and affects yield in a winter annual crop.

Winter annual life history is conferred by the requirement for vernalization to promote the floral transition and control the timing of flowering. Here we show using winter oilseed rape that flowering time is controlled by inflorescence bud dormancy in addition to vernalization. Winter warming treatments given to plants in the laboratory and field increase flower bud abscisic acid levels and delay flowering in spring. We show that the promotive effect of chilling reproductive tissues on flowering time is associated with the activity of two FLC genes specifically silenced in response to winter temperatures in developing inflorescences, coupled with activation of a BRANCHED1-dependent bud dormancy transcriptional module. We show that adequate winter chilling is required for normal inflorescence development and high yields in addition to the control of flowering time. Because warming during winter flower development is associated with yield losses at the landscape scale, our work suggests that bud dormancy activation may be important for effects of climate change on winter arable crop yields.

Temperature during seed maturation controls seed vigour through ABA breakdown in the endosperm and causes a passive effect on DOG1 mRNA levels during entry into quiescence.

Temperature variation during seed set is an important modulator of seed dormancy and impacts the performance of crop seeds through effects on establishment rate. It remains unclear how changing temperature during maturation leads to dormancy and growth vigour differences in nondormant seedlings. Here we take advantage of the large seed size in Brassica oleracea to analyse effects of temperature on individual seed tissues. We show that warm temperature during seed maturation promotes seed germination, while removal of the endosperm from imbibed seeds abolishes temperature-driven effects on germination. We demonstrate that cool temperatures during early seed maturation lead to abscisic acid (ABA) retention specifically in the endosperm at desiccation. During this time temperature affects ABA dynamics in individual seed tissues and regulates ABA catabolism. We also show that warm-matured seeds preinduce a subset of germination-related programmes in the endosperm, whereas cold-matured seeds continue to store maturation-associated transcripts including DOG1 because of effects on mRNA degradation before quiescence, rather than because of the effect of temperature on transcription. We propose that effects of temperature on seed vigour are explained by endospermic ABA breakdown and the divergent relationships between temperature and mRNA breakdown and between temperature, seed moisture and the glass transition.

SeedGerm: a cost-effective phenotyping platform for automated seed imaging and machine-learning based phenotypic analysis of crop seed germination.

Efficient seed germination and establishment are important traits for field and glasshouse crops. Large-scale germination experiments are laborious and prone to observer errors, leading to the necessity for automated methods. We experimented with five crop species, including tomato, pepper, Brassica, barley, and maize, and concluded an approach for large-scale germination scoring. Here, we present the SeedGerm system, which combines cost-effective hardware and open-source software for seed germination experiments, automated seed imaging, and machine-learning based phenotypic analysis. The software can process multiple image series simultaneously and produce reliable analysis of germination- and establishment-related traits, in both comma-separated values (CSV) and processed images (PNG) formats. In this article, we describe the hardware and software design in detail. We also demonstrate that SeedGerm could match specialists' scoring of radicle emergence. Germination curves were produced based on seed-level germination timing and rates rather than a fitted curve. In particular, by scoring germination across a diverse panel of Brassica napus varieties, SeedGerm implicates a gene important in abscisic acid (ABA) signalling in seeds. We compared SeedGerm with existing methods and concluded that it could have wide utilities in large-scale seed phenotyping and testing, for both research and routine seed technology applications.

Awake1, an ABC-Type Transporter, Reveals an Essential Role for Suberin in the Control of Seed Dormancy.

The mother plant plays an important dynamic role in the control of dormancy of her progeny seed in response to environmental signals. In order to further understand the mechanisms by which this dormancy control takes place in Arabidopsis (Arabidopsis thaliana), we conducted a forward genetic screen to isolate mutants that fail to enter dormancy in response to variation in temperature during seed set. We show that, for the first of these mutants, designated awake1, the maternal allele is required for entry into strongly dormant states and that awake1 mutants show seed phenotypes shown previously to be associated with the loss of suberin in the seed. We identify awake1 as an allele of ABCG20, an ATP-binding cassette transporter-encoding gene required for the transport of fatty acids during suberin deposition, and show that further suberin-deficient mutants have seed dormancy defects. Seed coat suberin composition is affected by temperature during seed maturation, but this response appears to be independent of ABCG20. We conclude that seed coat suberin is essential for seed dormancy imposition by low temperature and that the exclusion of oxygen and water from the seed by the suberin and tannin layers is important for dormancy imposition.

Intraspecific and interspecific adaptive latitudinal cline in Brassicaceae seed oil traits.

PREMISE OF THE STUDY: Nearly all seed plants rely on stored seed reserves before photosynthesis can commence. Natural selection for seed oil traits must have occurred over 319 million years of evolution since the first seed plant ancestor. Accounting for the biogeographic distribution of seed oil traits is fundamental to understanding the mechanisms of adaptive evolution in seed plants. However, the evolution of seed oils is poorly understood. We provide evidence of the adaptive nature of seed oil traits at the intraspecific and interspecific levels in Brassicaceae-an oilseed-rich and economically important plant family. METHODS: Univariate statistics, Pearson's correlation, multiple regression, generalized linear mixed model analysis, and phylogenetic autocorrelation tests on seed oil traits of 360 accessions of Arabidopsis thaliana and 216 Brassicaceae species helped provide evidence of the adaptive nature of seed oil traits. KEY RESULTS: At higher latitudes, both seed oil content and unsaturated fatty acids have selective advantages in Arabidopsis thaliana (intraspecific-level), while only unsaturated fatty acids have selective advantages across 216 Brassicaceae species (interspecific-level). The seed oil patterns fit within the theoretical framework of the gradient model. Seed oil content increases significantly from temperate to subtropical to tropical regions in Brassicaceae herbs. Absence of phylogenetic signals for seed oil traits and high seed oil content in four tribes of Brassicaceae were observed. CONCLUSIONS: Multiple seed oil traits are adaptive in nature and follow a gradient model. Consistent evolutionary patterns of seed oil traits were observed at the intraspecific and interspecific levels in Brassicaceae. Seed oil traits change with latitude and across biomes, suggesting selection. The absence of a phylogenetic signal for seed oil traits and the occurrence of high seed oil content in four Brassicaceae tribes provides evidence of the adaptive nature of seed oil traits in Brassicaceae.

Conserved microstructure of the Brassica B Genome of Brassica nigra in relation to homologous regions of Arabidopsis thaliana, B. rapa and B. oleracea.

BACKGROUND: The Brassica B genome is known to carry several important traits, yet there has been limited analyses of its underlying genome structure, especially in comparison to the closely related A and C genomes. A bacterial artificial chromosome (BAC) library of Brassica nigra was developed and screened with 17 genes from a 222 kb region of A. thaliana that had been well characterised in both the Brassica A and C genomes. RESULTS: Fingerprinting of 483 apparently non-redundant clones defined physical contigs for the corresponding regions in B. nigra. The target region is duplicated in A. thaliana and six homologous contigs were found in B. nigra resulting from the whole genome triplication event shared by the Brassiceae tribe. BACs representative of each region were sequenced to elucidate the level of microscale rearrangements across the Brassica species divide. CONCLUSIONS: Although the B genome species separated from the A/C lineage some 6 Mya, comparisons between the three paleopolyploid Brassica genomes revealed extensive conservation of gene content and sequence identity. The level of fractionation or gene loss varied across genomes and genomic regions; however, the greatest loss of genes was observed to be common to all three genomes. One large-scale chromosomal rearrangement differentiated the B genome suggesting such events could contribute to the lack of recombination observed between B genome species and those of the closely related A/C lineage.

Two high linolenic mutants of Arabidopsis thaliana contain megabase-scale genome duplications encompassing the FAD3 locus.

Understanding the quantitative control of fatty acid desaturation during the biosynthesis of seed storage oil has become a priority area for research, as a consequence of its importance for both human health and the substitution of mineral oil for industrial applications. We have analysed the genome structure of two mutants in Arabidopsis thaliana that show substantially elevated content of the omega-3 polyunsaturated fatty acid linolenic acid in their seed oil. In one, rfc4, sequences totalling approximately 2 Mb from chromosome 2 have been duplicated and inserted into chromosome 3. In the other mutant, ife, chromosome 2 sequences totalling approximately 1.4 Mb have been duplicated and inserted into a linked position. In both cases, the duplications encompass the FAD3 locus, which encodes the linoleate desaturase responsible for the biosynthesis of linolenic acid for accumulation in seed storage oil. The results show that mutagens such as fast neutrons (used for the induction of rfc4) and T-DNA (used for the induction of ife, which is not linked to the T-DNA present in the line) can result in the duplication of very large genome segments. They also show that increasing the dosage of the FAD3-containing genomic region results in an increase in the linolenic acid content of seed oil. Consequently, screening methods for duplication of FAD3 orthologues in oil crops may be an appropriate approach for the identification of germplasm for breeding varieties with increased proportions of linolenic acid in the oil that they produce.

Local maternal control of seed size by KLUH/CYP78A5-dependent growth signaling.

Seed development in plants involves the coordinated growth of the embryo, endosperm, and maternal tissue. Several genes have been identified that influence seed size by acting maternally, such as AUXIN RESPONSE FACTOR2, APETALA2, and DA1. However, given the lack of gain-of-function effects of these genes on seed size, it is unclear whether their activity levels are limiting in WT plants and whether they could thus be used to regulate seed size in development or evolution. Also, whether the altered seed sizes reflect local gene activity or global physiological changes is unknown. Here, we demonstrate that the cytochrome P450 KLUH (KLU) regulates seed size. KLU acts locally in developing flowers to promote seed growth, and its activity level is limiting for seed growth in WT. KLU is expressed in the inner integument of developing ovules, where it non-cell autonomously stimulates cell proliferation, thus determining the growth potential of the seed coat and seed. A KLU-induced increase in seed size leads to larger seedlings and higher relative oil content of the seeds. Genetic analyses indicate that KLU acts independently of other tested maternal factors that influence integument cell proliferation. Thus, the level of KLU-dependent growth factor signaling determines size in ovules and seeds, suggesting this pathway as a target for crop improvement.

Sequence-level comparative analysis of the Brassica napus genome around two stearoyl-ACP desaturase loci.

We conducted a sequence-level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl-ACP desaturase-encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (approximately 56%) is lower than has been reported previously between A. thaliana and Brassica (approximately 66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 +/- 3.9% and 85.8 +/- 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 +/- 3.1% between the B. napus A genome and B. rapa, and 93.1 +/- 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.

D2 receptor-mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry.

Cannabinoid CB(1) receptors are highly expressed in the striatum where they are known to be co-localized with dopamine D(2) receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D(2) receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration-dependent manner (IC(50): 3.25 x 10(-8) M). The CB(1) receptor agonist WIN55212-2 (WIN; 1 microM) had no effect on single pulse dopamine release (93.9 +/- 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 +/- 4.2% vs. quinpirole + WIN, 48.2 +/- 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB(1) receptor antagonist [N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM-251, 1 microM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 +/- 7.9% control (peak 4 to 1 ratio measurement) and 66.9 +/- 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse-evoked dopamine release (4P1Hz: quinpirole, 28.4 +/- 4.8% vs. WIN + quinpirole, 52.3 +/- 1.2%; 10P5Hz: quinpirole, 29.5 +/- 1.3% vs. WIN + quinpirole, 59.4 +/-7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM-251 (1 microM). This is the first report demonstrating a functional, antagonistic interaction between CB(1) receptors and D(2) autoreceptors in regulating rat striatal dopamine release.

The power of model-to-crop translation illustrated by reducing seed loss from pod shatter in oilseed rape.

KEY MESSAGE: Elucidation of key regulators in Arabidopsis fruit patterning has facilitated knowledge-translation into crop species to address yield loss caused by premature seed dispersal (pod shatter). In the 1980s, plant scientists descended on a small weed Arabidopsis thaliana (thale cress) and developed it into a powerful model system to study plant biology. The massive advances in genetics and genomics since then have allowed us to obtain incredibly detailed knowledge on specific biological processes of Arabidopsis growth and development, its genome sequence and the function of many of the individual genes. This wealth of information provides immense potential for translation into crops to improve their performance and address issues of global importance such as food security. Here, we describe how fundamental insight into the genetic mechanism by which seed dispersal occurs in members of the Brassicaceae family can be exploited to reduce seed loss in oilseed rape (Brassica napus). We demonstrate that by exploiting data on gene function in model species, it is possible to adjust the pod-opening process in oilseed rape, thereby significantly increasing yield. Specifically, we identified mutations in multiple paralogues of the INDEHISCENT and GA4 genes in B. napus and have overcome genetic redundancy by combining mutant alleles. Finally, we present novel software for the analysis of pod shatter data that is applicable to any crop for which seed dispersal is a serious problem. These findings highlight the tremendous potential of fundamental research in guiding strategies for crop improvement.

Vernalization and Floral Transition in Autumn Drive Winter Annual Life History in Oilseed Rape.

Plants with winter annual life history germinate in summer or autumn and require a period of prolonged winter cold to initiate flowering, known as vernalization. In the Brassicaceae, the requirement for vernalization is conferred by high expression of orthologs of the FLOWERING LOCUS C (FLC) gene, the expression of which is known to be silenced by prolonged exposure to winter-like temperatures [1]. Based on a wealth of vernalization experiments, typically carried out in the range of 5°C-10°C, we would expect field environments during winter to induce flowering in crops with winter annual life history. Here, we show that, in the case of winter oilseed rape, expression of multiple FLC orthologs declines not during winter but predominantly during October when the average air temperature is 10°C-15°C. We further demonstrate that plants proceed through the floral transition in early November and overwinter as inflorescence meristems, which complete floral development in spring. To validate the importance of pre-winter temperatures in flowering time control, we artificially simulated climate warming in field trial plots in October. We found that increasing the temperature by 5°C in October results in raised FLC expression and delays the floral transition by 3 weeks but only has a mild effect on flowering date the following spring. Our work shows that winter annuals overwinter as a floral bud in a manner that resembles perennials and highlights the importance of studying signaling events in the field for understanding how plants transition to flowering under real environmental conditions.

Transcriptional changes in prostate of men on active surveillance after a 12-mo glucoraphanin-rich broccoli intervention-results from the Effect of Sulforaphane on prostate CAncer PrEvention (ESCAPE) randomized controlled trial.

BACKGROUND: Epidemiological evidence suggests that consumption of cruciferous vegetables is associated with reduced risk of prostate cancer progression, largely attributed to the biological activity of glucosinolate degradation products, such as sulforaphane derived from glucoraphanin. Because there are few therapeutic interventions for men on active surveillance for prostate cancer to reduce the risk of cancer progression, dietary approaches are an appealing option for patients. OBJECTIVE: We evaluated whether consumption of a glucoraphanin-rich broccoli soup for 1 y leads to changes in gene expression in prostate tissue of men with localized prostate cancer. METHODS: Forty-nine men on active surveillance completed a 3-arm parallel randomized double-blinded intervention study for 12 mo and underwent transperineal template biopsy procedures and dietary assessment at the start and end of the study. Patients received a weekly 300 mL portion of soup made from a standard broccoli (control) or from 1 of 2 experimental broccoli genotypes with enhanced concentrations of glucoraphanin, delivering 3 and 7 times that of the control, respectively. Gene expression in tissues from each patient obtained before and after the dietary intervention was quantified by RNA sequencing followed by gene set enrichment analyses. RESULTS: In the control arm, there were several hundred changes in gene expression in nonneoplastic tissue during the 12 mo. These were associated with an increase in expression of potentially oncogenic pathways including inflammation processes and epithelial-mesenchymal transition. Changes in gene expression and associated oncogenic pathways were attenuated in men on the glucoraphanin-rich broccoli soup in a dose-dependent manner. Although the study was not powered to assess clinical progression, an inverse association between consumption of cruciferous vegetables and cancer progression was observed. CONCLUSION: Consuming glucoraphanin-rich broccoli soup affected gene expression in the prostate of men on active surveillance, consistent with a reduction in the risk of cancer progression. This trial was registered at clinicaltrials.gov as NCT01950143.

A Role for Adenosine A1 Receptors in GABA and NMDA-Receptor Mediated Modulation of Dopamine Release: Studies Using Fast Cyclic Voltammetry.

In the striatum many neurotransmitters including GABA, glutamate, acetylcholine, dopamine, nitric oxide and adenosine interact to regulate synaptic transmission. Dopamine release in the striatum is regulated by a number of pre- and postsynaptic receptors including adenosine. We have recently shown using isolated rat striatal slices, and the technique of fast cyclic voltammetry, that adenosine A1 receptor-mediated inhibition of dopamine release is modulated by dopamine D1 receptors. In the present study we have investigated the influence of NMDA and GABA receptor activation on the modulation of electrically stimulated dopamine release by adenosine. Application of the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), concentration-dependently inhibited dopamine release to a maxiumum of 50%. Perfusion of the glutamate receptor agonist, NMDA, in low magnesium, caused a rapid and concentration-dependent inhibition of dopamine release. Prior perfusion with the adenosine A1 receptor antagonist, DPCPX, significantly reduced the effect of 5 mM and 10 mM NMDA on dopamine release. The GABAA receptor agonist, isoguvacine, had a significant concentration-dependent inhibitory effect on dopamine release which was reversed by prior application of the GABAA receptor antagonist, picrotoxin, but not DPCPX. Finally inhibition of dopamine release by CPA (1mM) was significantly enhanced by prior perfusion with picrotoxin. These data demonstrate an important role for GABA, NMDA and adenosine in the modulation of dopamine release.

Natural variation for seed oil composition in Arabidopsis thaliana.

The biochemical pathways involved in the biosynthesis and accumulation of storage lipids in seeds have been extensively studied. However, the regulatory mechanisms of those pathways, their environmental interactions and the ecological implications of variation are poorly understood. We have initiated a new approach: the analysis of natural variation in Arabidopsis thaliana. Three hundred and sixty accessions were surveyed for content of oil, very long chain fatty acids (VLCFAs) and polyunsaturated fatty acids (PUFAs) in their seeds. The results revealed extensive natural variation. A core set of accessions, the seeds of which reproducibly contain extreme amounts of oil, VLCFAs and PUFAs have been identified. Reproducible oil content ranged from 34.6 to 46.0% of seed dry weight. VLCFA content ranged from 13.0 to 21.2% of total fatty acids. PUFA content, ranged from 53.3 to 66.1% of total fatty acids. Interactions were also identified for PUFA and VLCFA content of seeds with vernalisation of plants. Mapping of the regions of the genome involved in controlling the traits was conducted in an F(2) population and indicated that natural variation at the loci FAE1 and FAD3 might be involved in the regulation of VLCFA and PUFA content, respectively. A set of accessions, which capture a broad range of the natural variation for these traits available in A. thaliana, has been selected to form a core set which can be used to further dissect the genetics of the regulation of seed lipid traits and to identify the genes involved.

Associative transcriptomics study dissects the genetic architecture of seed glucosinolate content in Brassica napus.

Breeding new varieties with low seed glucosinolate (GS) concentrations has long been a prime target in Brassica napus. In this study, a novel association mapping methodology termed 'associative transcriptomics' (AT) was applied to a panel of 101 B. napus lines to define genetic regions and also candidate genes controlling total seed GS contents. Over 100,000 informative single-nucleotide polymorphisms (SNPs) and gene expression markers (GEMs) were developed for AT analysis, which led to the identification of 10 SNP and 7 GEM association peaks. Within these peaks, 26 genes were inferred to be involved in GS biosynthesis. A weighted gene co-expression network analysis provided additional 40 candidate genes. The transcript abundance in leaves of two candidate genes, BnaA.GTR2a located on chromosome A2 and BnaC.HAG3b on C9, was correlated with seed GS content, explaining 18.8 and 16.8% of phenotypic variation, respectively. Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b. These deletion polymorphisms were then successfully converted into polymerase chain reaction-based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines.

Six new recombinant inbred populations for the study of quantitative traits in Arabidopsis thaliana.

Quantitative approaches are now widely used to study the genetic architecture of complex traits. However, most studies have been conducted in single mapping populations, which sample only a fraction of the natural allelic variation available within a gene pool and can identify only a subset of the loci controlling the traits. To enable the progress towards an understanding of the global genetic architecture of a broad range of complex traits, we have developed and characterised six new Arabidopsis thaliana recombinant inbred populations. To evaluate the utility of these populations for integrating analyses from multiple populations, we identified quantitative trait loci (QTL) controlling flowering time in vernalized plants growing in 16 h days. We used the physical positions of markers to align the linkage maps of our populations with those of six existing populations. We identified seven QTL in genomic locations coinciding with those identified in previous studies and in addition a further eight QTL were identified.

Origin and maintenance of a broad-spectrum disease resistance locus in Arabidopsis.

The broad-spectrum mildew resistance genes RPW8.1 and RPW8.2 define a unique type of plant disease resistance (R) gene, and so far homologous sequences have been found in Arabidopsis thaliana only, which suggests a recent origin. In addition to RPW8.1 and RPW8.2, the RPW8 locus contains three homologs of RPW8, HR1, HR2, and HR3, which do not contribute to powdery mildew resistance. To investigate whether RPW8 has originated recently, and if so the processes involved, we have isolated and analyzed the syntenic RPW8 loci from Arabidopsis lyrata, and from Brassica rapa and B. oleracea. The A. lyrata locus contains four genes orthologous to HR1, HR2, HR3, and RPW8.2, respectively. Two syntenic loci have been characterized in Brassica; one locus contains three genes and is present in both B. oleracea and B. rapa, and the other locus contains a single gene and is detected in B. rapa only. The Brassica homologs have highest similarity to HR3. Sequence analyses suggested that the RPW8 gene family in Brassicaceae originated from an HR3-like ancestor gene through a series of duplications and that RPW8.1 and RPW8.2 evolved from functional diversification through positive selection several MYA. Examination of the sequence polymorphism of 32 A. thaliana accessions at the RPW8 locus and their disease reaction phenotypes revealed that the polymorphic RPW8 locus defines a major source of resistance to powdery mildew diseases. A possible evolutionary mechanism by which functional polymorphism at the AtRPW8 locus has been maintained in contemporary populations of A. thaliana is discussed.

Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives.

The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting from the hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). Using clones from the JBnB library, we have analysed the microstructure of the corresponding segments of the B. napus genome. The results show that there has been little or no change to the microstructure of the analysed segments of the Brassica A and C genomes as a consequence of the hybridization event forming natural B. napus. The observations indicate that, upon polyploid formation, these segments of the genome did not undergo a burst of evolution discernible at the scale of microstructure.