A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds

Seed coat phenolic compounds represent important antinutritive fibre components that cause a considerable reduction in value of seed meals from oilseed rape (Brassica napus). The nutritionally most important fibre compound is acid detergent lignin (ADL), to which a significant contribution is made by phenylpropanoid-derived lignin precursors. In this study, we used bulked-segregant analysis in a population of recombinant inbred lines (RILs) from a cross of the Chinese oilseed rape lines GH06 (yellow seed, low ADL) and P174 (black seed, high ADL) to identify markers with tight linkage to a major quantitative trait locus (QTL) for seed ADL content. Fine mapping of the QTL was performed in a backcross population comprising 872 BC₁F₂ plants from a cross of an F₇ RIL from the above-mentioned population, which was heterozygous for this major QTL and P174. A 3:1 phenotypic segregation for seed ADL content indicated that a single, dominant, major locus causes a substantial reduction in ADL. This locus was successively narrowed to 0.75 cM using in silico markers derived from a homologous Brassica rapa sequence contig spanning the QTL. Subsequently, we located a B. rapa orthologue of the key lignin biosynthesis gene CINNAMOYL CO-A REDUCTASE 1 (CCR1) only 600 kbp (0.75 cM) upstream of the nearest linked marker. Sequencing of PCR amplicons, covering the full-length coding sequences of Bna.CCR1 homologues, revealed a locus in P174 whose sequence corresponds to the Brassica oleracea wild-type allele from chromosome C8. In GH06, however, this allele is replaced by a homologue derived from chromosome A9 that contains a loss-of-function frameshift mutation in exon 1. Genetic and physical map data infer that this loss-of-function allele has replaced a functional Bna.CCR1 locus on chromosome C8 in GH06 by homoeologous non-reciprocal translocation.     

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.     

Hybridisation between oilseed rape (Brassica napus) and tetraploid Brassica rapa under field conditions

Cross-compatible relatives of crop species contribute to the uncertainty regarding the potential risk of transgene escape from genetically modified varieties. The most successful crossing partner of oilseed rape (Brassica napus L.) is diploid Brassica rapa L. Variation of ploidy level among B. rapa cultivars has, until recently, been neglected in the context of gene flow and hybridisation with oilseed rape. We estimated the extent of hybridisation between autotetraploid B. rapa varieties (female) and B. napus (pollen donor) under experimental field conditions. Morphology, variation of relative DNA amount, and microsatellite markers were used to distinguish between intraspecific offspring of tetraploid B. rapa and interspecific hybrids with B. napus. Of 517 seed progenies of tetraploid B. rapa, 45 juvenile plants showed species specific morphological traits of oilseed rape. The detection of putative hybrids based on variation in relative DNA amounts was problematic due to the occurrence of aneuploidy. In total, 84 offspring showed relative DNA amounts deviating from tetraploid B. rapa, four of which were hexaploids. Of the 205 offspring analysed at three microsatellite loci, 67 had oilseed rape alleles. Based on molecular evidence a minimum hybridisation rate of 13.0% was estimated. A few mother plants accounted for the majority of hybrids. The mean pollen viability of hybrids between B. napus and tetraploid B. rapa (80.6%) was high in comparison with mean pollen viability of triploid hybrids between B. napus and diploid B. rapa. Therefore, the occurrence of tetraploid B. rapa should be taken into consideration when estimating the likelihood of gene flow from oilseed rape to close relatives at the landscape level. Tetraploid B. rapa is a common component of several seed mixtures and establishes feral populations in northwest Germany. Assuming a similar abundance of diploid and tetraploid B. rapa, gene flow from B. napus to tetraploid may be more likely than gene flow to diploid B. rapa.     

Screening of oilseed rape and other brassicaceous genotypes for susceptibility to Ceutorhynchus pallidactylus (Mrsh.)

Production of oilseed rape, Brassica napus L., is affected by various insect pests. The cabbage stem weevil, Ceutorhynchus pallidactylus (Mrsh.) (Col.: Curculionidae), is one of the most damaging pests in Northern and Central Europe that requires regular control measures. Host plant resistance is a key factor in integrated pest management systems. To evaluate a large number of genotypes for their susceptibility to infestation by C. pallidactylus, new screening techniques were developed for testing both, the amount of feeding and the number of eggs deposited by adult C. pallidactylus on accessions of Brassicaceae under controlled conditions. In no‐choice screening tests, the leaf area consumed by adult cabbage stem weevil was quantified on a wide spectrum of 107 brassicaceous genotypes (B. napus, Brassica rapa L. and Brassica oleracea L. cultivars, breeding lines, resynthesized rapeseed lines and wild Brassicaceae). In comparison to feeding on the standard cultivar ‘Express’, the average leaf area consumed by C. pallidactylus on nine oilseed rape cultivars, four resynthesized rapeseed lines and five other accessions [B. oleracea, Camelina alyssum (Mill.) and Lunaria annua L.] was significantly reduced by 44–90%. In dual‐choice screening tests for the evaluation of oviposition preferences on 42 genotypes, female C. pallidactylus laid significantly fewer eggs into plants of two oilseed rape cultivars, five resynthesized rapeseeds and three accessions of B. oleracea and Brassica fruticulosa Cyrillo, respectively, than into plants of the standard cv ‘Express’. Results of both laboratory screening tests were confirmed by results of additional field testing.     

Origins of the amphiploid species <Emphasis Type="Italic">Brassica napus</Emphasis>L. investigated by chloroplast and nuclear molecular markers

Background  

The amphiploid species Brassica napus (oilseed rape, Canola) is a globally important oil crop yielding food, biofuels and industrial compounds such as lubricants and surfactants. Identification of the likely ancestors of each of the two genomes (designated A and C) found in B. napus would facilitate incorporation of novel alleles from the wider Brassica genepool in oilseed rape crop genetic improvement programmes. Knowledge of the closest extant relatives of the genotypes involved in the initial formation of B. napus would also allow further investigation of the genetic factors required for the formation of a stable amphiploid and permit the more efficient creation of fully fertile re-synthesised B. napus. We have used a combination of chloroplast and nuclear genetic markers to investigate the closest extant relatives of the original maternal progenitors of B. napus. This was based on a comprehensive sampling of the relevant genepools, including 83 accessions of A genome B. rapa L. (both wild and cultivated types), 94 accessions of B. napus and 181 accessions of C genome wild and cultivated B. oleracea L. and related species.     

Gene Silencing of BnTT10 Family Genes Causes Retarded Pigmentation and Lignin Reduction in the Seed Coat of Brassica napus

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

Exploitation of host plant preferences in pest management strategies for oilseed rape (Brassica napus)

New control strategies for insect pests of arable agriculture are needed to reduce current dependence on synthetic insecticides, the use of which is unsustainable. We investigated the potential of a simple control strategy to protect spring‐sown oilseed rape, Brassica napus L. (Brassicaceae), from two major inflorescence pests: the pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), and the seed weevil, Ceutorhynchus assimilis (Paykull) (Coleoptera: Curculionidae), through exploitation of their host plant preferences. The strategy comprised, for the main crop, Starlight [an oilseed rape cultivar with relatively low proportions of alkenyl glucosinolates in the leaves (thereby releasing lower levels of attractive isothiocyanates than conventional cultivars)] and turnip rape, Brassica rapa (L.) (Brassicaceae), as a trap crop. We tested the system in laboratory, polytunnel semifield arena, and field experiments. The odours of Starlight were less attractive in olfactometer tests to both pests than those from a conventional cultivar, Canyon, and the plants were less heavily colonized in both polytunnel and field experiments. Turnip rape showed good potential as a trap crop for oilseed rape pests, particularly the pollen beetle as its odour was more attractive to both pests than that of oilseed rape. Polytunnel and field experiments showed the importance of relative growth stage in the system. As turnip rape flowers earlier than oilseed rape, beetles would be maintained on turnip rape past the damage‐susceptible growth stage of oilseed rape. The development of a pest control regime based on this strategy is discussed.     

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     

Transferability and genome specificity of a new set of microsatellite primers among Brassica species of the U triangle

We present a new set of 12 highly polymorphic simple sequence repeat primer sequences for use with Brassica species. These new primers, and four from A.K.S. SzewcMcFadden and colleagues, were tested in four Brassica species (B. rapa, B. napus, B. oleracea and B. nigra). Most primers successfully amplified products within all species and were polymorphic. Due to the risk of gene flow from GM oilseed rape to its wild relatives, hybrid formation in the Brassicaceae is of great interest. We identify six primer pairs as specific to the A, B or C genomes that could be used to identify such hybrids.     

A rich TILLING resource for studying gene function in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Background  

The Brassicaceae family includes the model plant Arabidopsis thaliana as well as a number of agronomically important species such as oilseed crops (in particular Brassica napus, B. juncea and B. rapa) and vegetables (eg. B. rapa and B. oleracea).     

Performance of cabbage stem flea beetle larvae (Psylliodes chrysocephala) in brassicaceous plants and the effect of glucosinolate profiles

The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae), is one of the most important pests in European winter oilseed rape production. Adult beetles feed on young leaves whereas larvae mine within the petioles and stems. Larval infestation can cause significant crop damage. In this study, the host quality for CSFB of four oilseed rape (Brassica napus L.) cultivars and seven other brassicaceous species with different glucosinolate (GSL) profiles was assessed under controlled conditions. Larval instar weights and mortality were measured after 14 and 21 days of feeding in the petioles of test plants. To study the impact of GSL on the performance of larvae, the GSL contents in petioles from non-infested and infested plants were analysed before, and 21 days after, the start of larval infestation. Larval performance was not significantly different between the four cultivars of oilseed rape, but differed considerably among the other brassicaceous species tested. In comparison to the weight of larvae in the standard B. napus cv. Robust, the larval weight was higher in turnip rape (Brassica rapa L. var. silvestris) and significantly reduced in white mustard (Sinapis alba L.), oil radish (Raphanus sativa L. var. oleiformis), and cabbage (Brassica oleracea L. convar. capitata var. alba). The duration of larval development increased in white mustard and oilseed radish. The GSL profiles of the petioles showed little difference between non-infested and infested plants of oilseed rape whereas the content of aliphatic GSL increased in the infested turnip rape plants. In contrast, the aliphatic and benzenic GSL decreased in infested Indian rape (B. rapa subsp. dichotoma Roxb.). Larval weight was not correlated with the total GSL content of plants, neither before infestation nor 21 days after. Larval weight was positively correlated with progoitrin and 4-hydroxyglucobrassicin. White mustard, which provides inferior host quality for larval development, has the potential to introduce insect resistance into high-yielding oilseed rape cultivars in breeding programmes.     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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 (～56%) is lower than has been reported previously between A. thaliana and Brassica (～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.     

The extracellular pollen coat in members of the Brassicaceae: composition, biosynthesis, and functions in pollination

Summary. I have used cellular and molecular genetic and bioinformatic approaches to characterise the components of the pollen coat in plants of the family Brassicaceae, including Arabidopsis thaliana and several brassicas including Brassica napus, B. oleracea, and B. rapa. The pollen coat in these species is mostly made up of a unique mixture of lipids that is highly enriched in acylated compounds, such as sterol esters and phospholipids. These acyl lipids are characterised by an unusually high degree of saturation. The fatty acids typically contain 70–90% saturated acyl residues such as myristate, palmitate, and stearate. The major sterol components of the pollen coat are saturated fatty acyl esters of stigmasterol, campesterol, and campestdienol. In addition to lipids, the second major component of the pollen coat is a specific group of proteins that is dominated by a family of proteins that we term pollenins. Although pollenins are by far the major protein components of the pollen coat of members of the Brassicaceae, proteomic analysis reveals that there are several additional protein components, including lipases, protein kinases, a pectin esterase, and a caleosin. The biosynthesis of these lipids and proteins and their significance for overall pollen function are reviewed and discussed. Correspondence and reprints: Biotechnology Unit, School of Applied Sciences, University of Glamorgan, Pontypridd CF37 1DL, Wales, United Kingdom.     

Brassicaceae INDEHISCENT genes specify valve margin cell fate and repress replum formation

Members of the Brassicaceae family, including Arabidopsis thaliana and oilseed rape (Brassica napus), produce dry fruits that open upon maturity along a specialised tissue called the valve margin. Proper development of the valve margin in Arabidopsis is dependent on the INDEHISCENT (IND) gene, the role of which in genetic and hormonal regulation has been thoroughly characterised. Here we perform phylogenetic comparison of IND genes in Arabidopsis and Brassica to identify conserved regulatory sequences that are responsible for specific expression at the valve margin. In addition we have taken a comparative development approach to demonstrate that the BraA.IND.a and BolC.IND.a genes from B. rapa and B. oleracea share identical function with Arabidopsis IND since ethyl methanesulphonate (EMS) mutant alleles and silenced transgenic lines have valve margin defects. Furthermore we show that the degree of these defects can be fine‐tuned for crop improvement. Wild‐type Arabidopsis produces an outer replum composed of about six cell files at the medial region of the fruits, whereas Brassica fruits lack this tissue. A strong loss‐of‐function braA.ind.a mutant gained outer replum tissue in addition to its defect in valve margin development. An enlargement of replum size was also observed in the Arabidopsis ind mutant suggesting a general role of Brassicaceae IND genes in preventing valve margin cells from adopting replum identity.     

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.     

Maternal Inheritance of Chloroplasts between <Emphasis Type="Italic">Brassica rapa</Emphasis> and F<Subscript>1</Subscript>-hybrids Demonstrated by cpDNA Markers Specific to Oilseed Rape and <Emphasis Type="Italic">B. rapa</Emphasis>

Controlled reciprocal crosses between B. rapa and F₁-hybrids (B. napus (♀) × B. rapa), giving 20 pair-crossings, were made to reveal possible irregularities in chloroplast inheritance during production of BC₁s. Despite the close relationship of chloroplasts in B. rapa and B. napus, development of PCR-based molecular markers specific to B. rapa chloroplasts and B. napus chloroplasts was successful. Offspring from each cross were investigated and among these, we found no irregular chloroplast inheritance, since their plastid genotypes in all cases were identical to that of their mother. With a certainty of 95% our data indicate that the probability that chloroplasts are being inherited paternally is less than 0.015. In oilseed rape, pollen-mediated transgene-dispersal poses a well-known risk. Our results support development of transplastomic oilseed rape as an approach to reduce transgene dispersal.