Construction of the cybrid transplastomic Brassica napus plants containing Lesquerella fendleri chloroplasts

Transferring of Lesquerella fendleri genetically transformed plastids to Brassica napus plants has been performed with the somatic hybridization method. The plastome of the previously engineered transplastomic L. fendleri plants contained the aadA16gfp selective marker gene conferring spectinomycin/streptomycin resistance and green fluorescence under UV light. The protoplasts of B. napus chlorophyll-deficient plants were fused with gamma-irradiated protoplasts of L. fendleri transplastomic plants. A total of 59 green hybrid colonies have been isolated followed by spectinomycin/streptomycin selection. Shoot regeneration has been observed for two cell lines. Morphologically normal plants have been regenerated for one of them. PCR and isozyme analyses showed that the plants were transplastomic cybrids containing B. napus nuclei and L. fendleri transformed chloroplasts.     

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.     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.     

Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues.

Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.     

Comparison of a Brassica oleracea genetic map with the genome of Arabidopsis thaliana

Brassica oleracea is closely related to the model plant, Arabidopsis thaliana. Despite this relationship, it has been difficult to both identify the most closely related segments between the genomes and determine the degree of genome replication within B. oleracea relative to A. thaliana. These difficulties have arisen in part because both species have replicated genomes, and the criteria used to identify orthologous regions between the genomes are often ambiguous. In this report, we compare the positions of sequenced Brassica loci with a known position on a B. oleracea genetic map to the positions of their putative orthologs within the A. thaliana genome. We use explicit criteria to distinguish orthologous from paralogous loci. In addition, we develop a conservative algorithm to identify collinear loci between the genomes and a permutation test to evaluate the significance of these regions. The algorithm identified 34 significant A. thaliana regions that are collinear with >28% of the B. oleracea genetic map. These regions have a mean of 3.3 markers spanning 2.1 Mbp of the A. thaliana genome and 2.5 cM of the B. oleracea genetic map. Our findings are consistent with the hypothesis that the B. oleracea genome has been highly rearranged since divergence from A. thaliana, likely as a result of polyploidization.     

Transformation of Brassica napus by using the aadA gene as selectable marker and inheritance studies of the marker genes

An Agrobacterium tumefaciens -mediated transformation system for Brassica napus has been improved. We investigated several marker genes for transformation of Brassica napus , and the aadA gene, which confers resistance to streptomycin and spectinomycin, was found to be the most suitable. Forty-three out of 193 putative transformants in the T₁ generation were investigated by Southern blot analysis. Transformants containing a range of 1 to 10 integrated T-DNA copies per genome were found. Total DNA from 35 plants showed hybridisation to both the aadA and the nptll marker gene probes, from 5 plants only to one marker gene probe and from 3 plants DNA did not hybridise to any of the gene probes. Furthermore, more complex integration patterns such as direct repeated copies of the T-DNA, both as tandem and inverted copies, were observed. Inheritance of the marker genes in the T₂ generation was studied in 37 of the plants. This revealed that 22% of the plants that contained both marker genes, segregated as one single locus (3:1) for both genes, while 46% of the plants gave a segregation pattern corresponding to one T-DNA locus for at least one of the marker genes. Moreover, these inheritance patterns appeared to be more or less independent of the number of genes seen in the Southern blot analysis of the T, generation. In this study we show that the introduced marker genes are inherited by the T; generation in a less predictable way than was earlier reported for B. napus .     

WRR4, a broad-spectrum TIR-NB-LRR gene from Arabidopsis thaliana that confers white rust resistance in transgenic oilseed brassica crops

White blister rust caused by Albugo candida (Pers.) Kuntze is a common and often devastating disease of oilseed and vegetable brassica crops worldwide. Physiological races of the parasite have been described, including races 2, 7 and 9 from Brassica juncea , B. rapa and B. oleracea , respectively, and race 4 from Capsella bursa-pastoris (the type host). A gene named WRR4 has been characterized recently from polygenic resistance in the wild brassica relative Arabidopsis thaliana (accession Columbia) that confers broad-spectrum white rust resistance ( WRR ) to all four of the above Al. candida races. This gene encodes a TIR-NB-LRR (Toll-like/interleukin-1 receptor-nucleotide binding-leucine-rich repeat) protein which, as with other known functional members in this subclass of intracellular receptor-like proteins, requires the expression of the lipase-like defence regulator, enhanced disease susceptibility 1 ( EDS1 ). Thus, we used RNA interference-mediated suppression of EDS1 in a white rust-resistant breeding line of B. napus (transformed with a construct designed from the A. thaliana EDS1 gene) to determine whether defence signalling via EDS1 is functionally intact in this oilseed brassica. The eds1-suppressed lines were fully susceptible following inoculation with either race 2 or 7 isolates of Al. candida. We then transformed white rust-susceptible cultivars of B. juncea (susceptible to race 2) and B. napus (susceptible to race 7) with the WRR4 gene from A. thaliana . The WRR4-transformed lines were resistant to the corresponding Al. candida race for each host species. The combined data indicate that WRR4 could potentially provide a novel source of white rust resistance in oilseed and vegetable brassica crops.     

Molecular mapping of Arabidopsis thaliana lipid-related orthologous genes in Brassica napus

Quantitative Trait Loci (QTL) for oil content has been previously analyzed in a SG-DH population from a cross between a Chinese cultivar and a European cultivar of Brassica napus. Eight QTL with additive and epistatic effects, and with environmental interactions were evaluated. Here we present an integrated linkage map of this population predominantly based on informative markers derived from Brassica sequences, including 249 orthologous A. thaliana genes, where nearly half (112) are acyl lipid metabolism related genes. Comparative genomic analysis between B. napus and A. thaliana revealed 33 colinearity regions. Each of the conserved A. thaliana segments is present two to six?times in the B. napus genome. Approximately half of the mapped lipid-related orthologous gene loci (76/137) were assigned in these conserved colinearity regions. QTL analysis for seed oil content was performed using the new map and phenotypic data from 11 different field trials. Nine significant QTL were identified on linkage groups A1, A5, A7, A9, C2, C3, C6 and C8, together explaining 57.79% of the total phenotypic variation. A total of 14 lipid related candidate gene loci were located in the confidence intervals of six of these QTL, of which ten were assigned in the conserved colinearity regions and felled in the most frequently overlapped QTL intervals. The information obtained from this study demonstrates the potential role of the suggested candidate genes in rapeseed kernel oil accumulation.     

Variation and selection at the CAULIFLOWER floral homeotic gene accompanying the evolution of domesticated Brassica oleracea

The evolution of plant morphologies during domestication events provides clues to the origin of crop species and the evolutionary genetics of structural diversification. The CAULIFLOWER gene, a floral regulatory locus, has been implicated in the cauliflower phenotype in both Arabidopsis thaliana and Brassica oleracea. Molecular population genetic analysis indicates that alleles carrying a nonsense mutation in exon 5 of the B. oleracea CAULIFLOWER (BoCAL) gene are segregating in both wild and domesticated B. oleracea subspecies. Alleles carrying this nonsense mutation are nearly fixed in B. oleracea ssp. botrytis (domestic cauliflower) and B. oleracea ssp. italica (broccoli), both of which show evolutionary modifications of inflorescence structures. Tests for selection indicate that the pattern of variation at this locus is consistent with positive selection at BoCAL in these two subspecies. This nonsense polymorphism, however, is also present in both B. oleracea ssp. acephala (kale) and B. oleracea ssp. oleracea (wild cabbage). These results indicate that specific alleles of BoCAL were selected by early farmers during the domestication of modified inflorescence structures in B. oleracea.     

芸薹属多倍体植物基因组进化的RAPD分析

多倍化是促进高等植物发生进化的重要力量。为了更清楚地了解多倍体在形成之后其基因组是如何进化的,利用38个随机引物对芸薹属Brassica L.禹氏三角（U’Triangle）中的多倍体物种及其祖先二倍体物种进行了研究。根据扩增出的273条带计算了遗传距离,并用UPGMA法进行了聚类分析。结果发现,二倍体物种B.campestris（AA）与B.oleracea（CC）的亲缘关系比与B.nigra（BB）的要近;异源多倍体B.napus（AACC）比起其二倍体祖先之一B.campestris（AA）与另一个     

Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers

To gain new insights into the mechanism underlying cytoplasmic male sterility (CMS), we compared the nuclear gene expression profiles of flowers of a Brassica napus CMS line with that of the fertile B. napus maintainer line using Arabidopsis thaliana flower-specific cDNA microarrays. The CMS line used has a B. napus nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of both B. napus and A. thaliana DNA. Gene expression profiling revealed that a large number of genes differed in expression between the two lines. For example, nuclear genes coding for proteins that are involved in protein import into organelles, genes expressed in stamens and pollen, as well as genes implicated in either cell-wall remodeling or architecture, were repressed in the CMS line compared with B. napus. These results show that the mt genome of the CMS line strongly influences nuclear gene expression, and thus reveal the importance of retrograde signalling between the mitochondria and the nucleus. Furthermore, flowers of the CMS line are characterized by a replacement of stamens with carpelloid organs, and thus partially resemble the APETALA3 (AP3) and PISTILLATA (PI) mutants. In accordance with this phenotype, AP3 expression was downregulated in the stamens, shortly before these organs developed carpelloid characteristics, even though it was initiated correctly. Repression of PI succeeded that of AP3 and might be a consequence of a loss of AP3 activity. These results suggest that AP3 expression in stamens depends on proper mt function and a correct nuclear-mt interaction, and that mt alterations cause the male sterility phenotype of the CMS line.     

High-oleic acid Australian Brassica napus and B. juncea varieties produced by co-suppression of endogenous Delta12-desaturases

Genetic engineering methods have been used successfully to modify the fatty acid profile of elite Australian germplasm of Brassica napus and B. juncea. Co-suppression plasmids carrying oleate desaturase genes from each species have been constructed and transferred into Australian elite breeding lines of B. napus and B. juncea using Agrobacterium tumifaciens plant-transformation techniques. Modifications to existing Brassica transformation protocols and the use of an intron-interrupted hygromycin-resistance gene as the selectable marker have resulted in improved transformation efficiencies. Silencing of the endogenous oleate desaturase genes has resulted in substantial increases in oleic acid levels, up to 89% in B. napus and 73% in B. juncea.     

Production of asymmetric hybrids between Arabidopsis thaliana and Brassica napus utilizing an efficient protoplast culture system

Application of the protoplast culture method developed for Brassica protoplasts to protoplasts of Arabidopsis thaliana has increased the opportunities for interspecific hybridizations involving Arabidopsis. A more-efficient and much-simpler method was established compared to the earlier-reported protocol developed for A. thaliana protoplasts in which alginate beads were utilized. Mesophyll protoplasts of A. thaliana (ecotypes 'Landsberg erecta' and 'Wassilewskija') were cultured in the modified 8p liquid medium, which had been developed for Brassica protoplasts. For comparison, protoplasts were cultured in sodium alginate beads supplied with B5 medium according to the protocol for A. thaliana. The protoplasts divided with high frequencies in the 8p medium, and calli proliferated more rapidly than in the sodium alginate beads. High frequencies of shoot differentiation and regeneration were observed in calli of both ecotypes, from about 30% in the ecotype 'Wassilewskija' to about 60% for 'Landsberg erecta'. The more-rapidly the calli developed, the higher the regeneration frequencies were. Asymmetric hybrids between A. thaliana and Brassica napus were obtained by treating the protoplasts of A. thaliana with iodoacetamide (IOA) and B. napus protoplasts with UV-irradiation before fusion with polyethylene glycol (PEG). By using the culture procedure developed for Brassica protoplasts, calli developed and plants were regenerated. Although most of the plants regenerated after cell fusion were A. thaliana-like and were judged to be escapes from IOA treatment, more than ten plants showed hybrid features of both morphological and molecular characters. Among the hybrids that have flowered so far, both male-fertile and male-sterile plants have been obtained. Back-crossings to A. thaliana are now in progress as is morphological and molecular characterization of the plants.     

Dehiscence-related expression of an Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus

Pod dehiscence in Arabidopsis thaliana is accompanied by an increase in the expression of a polygalacturonase (PG). The gene encoding this mRNA has been characterized and shown to have extensive homology to a similar PG gene from Brassica napus . The A. thaliana PG promoter was fused to β -glucuronidase (GUS) and the expression of this reporter gene analysed in transgenic B. napus plants. The GUS activity was detected throughout the dehiscence zone of pods from 35 d after anthesis and expression was restricted to those cells that undergo cell separation. Expression was also detectable at the junction between the seed and the funicular tissue and in mature anthers of flowers. Transgenic plants containing the PG promoter fused to barnase were sterile as a consequence of the anthers failing to undergo dehiscence. Fertilization of PG-barnase plants resulted in the development of pods that exhibited a reduced capacity to shatter. The role of PG during cell separation processes in plants is discussed.     

Spontaneous gene flow from rapeseed (Brassica napus) to wild Brassica oleracea

Research on the environmental risks of gene flow from genetically modified (GM) crops to wild relatives has traditionally emphasized recipients yielding most hybrids. For GM rapeseed (Brassica napus), interest has centred on the 'frequently hybridizing' Brassica rapa over relatives such as Brassica oleracea, where spontaneous hybrids are unreported in the wild. In two sites, where rapeseed and wild B. oleracea grow together, we used flow cytometry and crop-specific microsatellite markers to identify one triploid F1 hybrid, together with nine diploid and two near triploid introgressants. Given the newly discovered capacity for spontaneous introgression into B. oleracea, we then surveyed associated flora and fauna to evaluate the capacity of both recipients to harm cohabitant species with acknowledged conservational importance. Only B. oleracea occupies rich communities containing species afforded legislative protection; these include one rare micromoth species that feeds on B. oleracea and warrants further assessment. We conclude that increased attention should now focus on B. oleracea and similar species that yield few crop-hybrids, but possess scope to affect rare or endangered associates.     

<Emphasis Type="Italic">Brassica napus</Emphasis> lines with rearranged <Emphasis Type="Italic">Arabidopsis</Emphasis> mitochondria display CMS and a range of developmental aberrations

Numerous Brassica napus (+) Arabidopsis thaliana somatic hybrids were screened for male sterility and aberrant flower phenotypes. Nine hybrids were selected and backcrossed recurrently to B. napus. The resulting lines displayed stable maternal inheritance of flower phenotypes. Nuclear and organellar genomes were characterized molecularly using RFLP analysis. No DNA from A. thaliana was found in the nuclear genome after six back-crosses, whilst the mitochondrial genomes contained rearranged DNA from both A. thaliana and B. napus. Each line tested had a unique RFLP pattern of the mitochondrial DNA (mtDNA) that remained unchanged between the BC(3) and BC(6) generation. The plastid genomes consisted of B. napus DNA. Five lines of the BC(5) generation were subjected to more comprehensive investigations of growth, morphology and fertility. On the basis of these investigations, the five CMS lines could be assigned to two groups, one represented by three lines displaying reduced vegetative development, complete male sterility, and homeotic conversions of stamens into feminized structures. The second group, represented by the other two lines, were not completely male-sterile but still displayed severely affected flower morphologies. These two lines did not display any reduction in vegetative development. For both groups only stamens and petals suffered from the morphological and functional aberrations, while the sepals and pistils displayed normal morphology. All plants were fully female-fertile. Different rearrangements of the mitochondrial genome disturbed nuclear-mitochondrial interactions and led to various types of aberrant growth and flower development. The existence of numerous CMS lines with different mitochondrial patterns involving a species with a sequenced genome offers new opportunities to investigate the genetic regulation of CMS and its associated developmental perturbations.     

Glucosinolate genetics and the attraction of the aphid parasitoid Diaeretiella rapae to Brassica

The control of insect pests using parasitoids and carnivores has been successfully applied in protected cropping systems, orchards and forestry. Their success in annual field crops has been more limited due largely to the difficulties of attracting and maintaining a sufficient density of parasitoids in the crop before the levls of the insect herbivores become economically damaging. Parasitoids are known to be attracted to host-plant volatiles; thus, manipulating the host-plant chemistry may provide a means of enhancing the attraction of parasitoids to their prey. In this study we describe the differential attraction of the braconid wasp Diaeretiella rapae to two near-isogenic lines of Brassica oleracea which differ in a gene which alters the chemical structure of the isothiocyanates which are emitted following tissue damage. We demonstrate that, by enhancing the production of but-3-enyl isothiocyanate in B. oleracea and Brassica napus (oilseed rape), we can increase the attraction of D. rapae to these plants under standard field conditions.     

Contrasting genome organisation: two regions of the Brassica oleracea genome compared with collinear regions of the Arabidopsis thaliana genome.

Brassica crop species are of worldwide importance and are closely related to the model plant Arabidopsis thaliana for which the complete genome sequence has recently been established. We investigated collinearity of marker order by comparing two contrasting regions of the Brassica oleracea genome with homologous regions of A. thaliana. Although there is widespread replication of marker loci in both A. thaliana and B. oleracea, we found that a combination of genetic markers mapped in B. oleracea, including RFLPs, CAPS, and SSRs allowed comparison and interpretation of medium-scale chromosomal organisation and rearrangements. The interpretation of data was facilitated by hybridising probes onto the whole A. thaliana genome, as represented by BAC contigs. Twenty marker loci were sampled from the whole length of the shortest B. oleracea linkage group, 06, and 21 from a 30.4-cM section of the longest linkage group, 03. There is evidence of locus duplication on linkage group 06. Locus order is well conserved between a putative duplicated region of 10.5 cM and a discrete region comprising 25 cM of A. thaliana chromosome I. This was supported by evidence from seven paralogous loci, three of which were duplicated in a 30.6-cM region of linkage group 06. The pattern of locus order for the remainder of linkage group 06 and the sampled section of linkage group 03 was more complex when compared with the A. thaliana genome. Although there was some conservation of locus order between markers on linkage group 03 and approximately 9 cM of A. thaliana chromosome I, this was superimposed upon a complex pattern of additional loci that were replicated in both A. thaliana and B. oleracea. The results are discussed in the context of the ability to use collinear information to assist map-based cloning.