From Arabidopsis thaliana to Brassica napus: development of amplified consensus genetic markers (ACGM) for construction of a gene map

The evolution of genomes can be studied by comparing maps of homologous genes which show changes in nucleic acid sequences and chromosome rearrangements. In this study, we developed a set of 32 amplified consensus gene markers (ACGMs) that amplified gene sequences from Arabidopsis thaliana and Brassica napus. Our methodology, based on PCR, facilitated the rapid sequencing of homologous genes from various species of the same phylogenetic family and the detection of intragenic polymorphism. We found that such polymorphism principally concerned intron sequences and we used it to attribute a Brassica oleracea or Brassica rapa origin to the B. napus sequences and to map 43 rapeseed genes. We confirm that the genetic position of homologous genes varied between B. napus and A. thaliana. ACGMs are a useful tool for genome evolution studies and for the further development of single nucleotide polymorphism suitable for use in genetic mapping and genetic diversity analyses.     

Development and genetic mapping of microsatellite markers from genome survey sequences in Brassica napus

Microsatellite or simple sequence repeat (SSR) markers are routinely used for tagging genes and assessing genetic diversity. In spite of their importance, there are limited numbers of SSR markers available for Brassica crops. A total of 627 new SSR markers (designated BnGMS) were developed based on publicly available genome survey sequences and used to survey polymorphisms among six B. napus cultivars that serve as parents for established populations. Among these SSR markers, 591 (94.3%) successfully amplified at least one fragment and 434 (73.4%) detected polymorphism among the six B. napus cultivars. No correlation was observed between SSR motifs, repeat number or repeat length with polymorphism levels. A linkage map was constructed using 163 newly developed BnGMS marker loci and anchored with 164 public SSRs in a doubled haploid population. These new markers are evenly distributed over all linkage groups (LGs). Given that the majority of these SSRs are derived from bacterial artificial chromosome (BAC) end sequences, they will be useful in the assignment of their cognate BACs to LGs and facilitate the integration of physical maps with genetic maps for genome sequencing in B. napus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inheritance of Arabidopsis DNA in offspring from Brassica napus and A. thaliana somatic hybrids

 Chromosome counts and RFLP markers mapped to Arabidopsis thaliana were used to determine the proportion of eliminated chromosomes and retained A. thaliana DNA in the back-crossed (BC) progeny derived from symmetric and asymmetric somatic hybrids between Brassica napus and A. thaliana. All plants were analysed for the presence of two RFLP markers per chromosome, preferably with one located on each chromosome arm. A reduction in both A. thaliana RFLP markers and chromosome numbers was found in the BC₁ and BC₂ generations of the symmetric hybrids as well as in the BC₁ generation of the asymmetric hybrids. In the symmetric hybrids, two back-crosses to B. napus were required to reduce the frequency of retained A. thaliana loci to 42.4% and mean chromosome number to 39.4. In comparison, the BC₁ progeny of the asymmetric hybrids had 16% of the analysed A. thaliana loci present and an average of 38.4 chromosomes maintained. When the frequency of A. thaliana chromosomes with both analysed loci maintained was compared with the frequency of chromosomes with one locus lost and one kept, a reduction in the number of complete chromosomes between BC₁ and BC₂ derived from the symmetric hybrids was observed. Among the BC₁ plants in the asymmetric group the situation was different, with higher amounts of incomplete donor chromosomes compared to whole chromosomes. The results indicate that A. thaliana chromosome fragments are more often found in the progeny of irradiated hybrids, while back-crossed symmetric hybrids have more complete chromosomes. Received: 2 April 1998 / Accepted: 14 July 1998     

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.     

Arabidopsis thaliana: a source of candidate disease-resistance genes for Brassica napus.

Common structural and amino acid motifs among cloned plant disease-resistance genes (R genes), have made it possible to identify putative disease-resistance sequences based on DNA sequence identity. Mapping of such R-gene homologues will identify candidate disease-resistance loci to expedite map-based cloning strategies in complex crop genomes. Arabidopsis thaliana expressed sequence tags (ESTs) with homology to cloned plant R genes (R-ESTs), were mapped in both A. thaliana and Brassica napus to identify candidate R-gene loci and investigate intergenomic collinearity. Brassica R-gene homologous sequences were also mapped in B. napus. In total, 103 R-EST loci and 36 Brassica R-gene homologous loci were positioned on the N-fo-61-9 B. napus genetic map, and 48 R-EST loci positioned on the Columbia x Landsberg A. thaliana map. The mapped loci identified collinear regions between Arabidopsis and Brassica which had been observed in previous comparative mapping studies; the detection of syntenic genomic regions indicated that there was no apparent rapid divergence of the identified genomic regions housing the R-EST loci.     

Estimating genetic diversity of Arabidopsis thaliana ecotypes with amplified fragment length polymorphisms (AFLP)

The extensive natural variation of Arabidopsis thaliana ecotypes is being increasingly exploited as a source of variants of genes which control (agronomically) important traits. We have subjected 19 different Arabidopsis thaliana ecotypes to an analysis using the anplified fragment length polymorphism (AFLP) technique in order to estimate their genetic diversity. The genetic diversity was estimated applying the method of Nei and Li (1979) and a modified version of it and using 471 informative polymorphisms. The data obtained revealed that within this small set of ecotypes a group of three ecotypes and a further single ecotype exhibit considerable genetic diversity in comparison to the others. These ecotypes clustered at positions significantly separated from the bulk of the ecotypes in the generated similarity plots. The analysis demonstrated the usefulness of the AFLP method for determinating intraspecies genetic diversity as exemplified with Arabidopsis thaliana ecotypes. Results are discussed and compared with data obtained with other methods. Received: 18 June 1999 / Accepted: 28 July 1999     

Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5.

This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.     

Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

The genetics of stamenoid petal production in oilseed rape (Brassica napus) and equivalent variation in Arabidopsis thaliana

 The agronomic potential of a Brassica napus variant with petalless flowers was compromised by an associated detrimental change in leaf morphology. Genetic analysis demonstrated the cosegregation of genes controlling both morphologies. Two STAP loci controlling the production of flowers with stamenoid petals were mapped to homoeologous locations in the genome of B. napus. The STAP loci were probably duplicate genes because they exhibited an epistatic interaction such that only plants homozygous for recessive stap alleles at both loci expressed the variant phenotype. The CURLY LEAF (CLF) gene of Arabidopsis thaliana pleiotropically influences both flower and leaf morphologies. The cloned CLF gene of Arabidopsis was homologous to a polymorphic B. napus locus coincident with one of the B. napus STAP loci. The possibility that CLF is a candidate gene for STAP suggests that the variant stap alleles of B. napus exert pleiotropic effects over both flower and leaf morphologies. Received: 26 August 1996 / Accepted: 20 September 1996     

Production of Brassica olereceae (+Arabidopsis thaliana) and Brassica napus cell lines resistant to spectinomycin/streptomycin as a result of plastome genetic transformation

Plastid genetic transformation has been performed using both the PEG-treatment of protoplasts of somatic hybrids of B. oleracea carrying A. thaliana chloroplasts and the particle bombardment of regenerable calluses of B. napus sv. Westar. The chloroplast transformation vector pCB040 carried resistance (aadA) gene flanked by rapeseed plastid DNA sequences to target its insertion between the trnV-rps7 fragments. Selection of transplastomic cell lines has been performed according to their ability to grow on the medium supplied with spectinomycin and streptomycin in high concentrations. Antibiotic resistant cell lines have been obtained using the both transformation methods. The presence of the aadA gene in the A. thaliana and B. napus plastomes was confirmed by PCR analysis for two cell lines of B. oleracea (+ A. thaliana) and three lines of B. napus.     

Arabidopsis thaliana chromosome III restores fertility in a cytoplasmic male-sterile Brassica napus line with A. thaliana mitochondrial DNA

Somatic Brassica napus (+) Arabidopsis thaliana hybrids with a cytoplasmic male sterility (CMS)-inducing cytoplasm were screened for fertility-restored plants. One line was selected and recurrently backcrossed with the maintainer line, B. napus, resulting in fertile/sterile segregating populations. Restriction fragment length polymorphism mapping showed the co-segregation of A. thaliana chromosome (chr) III markers with the fertility trait. As it was not possible to stabilise the fertility trait via selfings, a dihaploidisation strategy was assessed. Ninety haploid plants were regenerated and analysed with numerous simple sequence length polymorphism (SSLP) markers. Markers covering both arms of A. thaliana chr III were present in two plants, whereas no A. thaliana DNA could be detected in the other plants. Following colchicine-induced chromosome doubling only these two plants with A. thaliana DNA produced fertile offspring. In one of the two lines, however, the A. thaliana-specific DNA markers and fertility were lost in subsequent generations. The other line remained fertile after repeated selfings. Using genomic in situ hybridisation (GISH) we were able to demonstrate that this latter line possessed a disomic addition of the A. thaliana chromosome. The restored line was comparable to the maintainer line with respect to flower morphology, but the petals and stamens were slightly reduced in size. The homeotic conversion of stamens to pistil-like structures, which is typical for the CMS line, was reversed, and stamens with a normal appearance with viable pollen appeared. Flowering time was as in the CMS line—in both lines it was delayed in comparison to the maintainer line. The introgressed chromosome also contributes to several pleiotropic effects, such as reduced leaf crinkling and shorter stems. The ability to restore fertility through the introgression of nuclear genes from the main cytoplasmic donor species indicates that the CMS trait in this system mainly is due to B. napus/A. thaliana alloplasmic incompatibility and not mitochondrial DNA rearrangements. Further exploitation of the material is discussed.Communicated by C. Möllers     

Dwarfism in Brassica napus L. induced by the over-expression of a gibberellin 2-oxidase gene from Arabidopsis thaliana

Gibberellins (GAs) are endogenous hormones that play an important role in regulating plant stature by increasing cell division and elongation in stem internodes. The GA2-oxidase gene from Arabidopsis thaliana (AtGA2ox8) was introduced into Brassica napus L. by Agrobacterium-mediated floral-dip transformation with the aim of decreasing the amount of bioactive GA and hence reducing plant stature. As anticipated, the transgenic plants exhibited dwarf phenotype. Compared with the wild type, the transgenic plants had increased primary branches (by 14.1?C15.3%) and siliques (by 10.8?C15.2%), which resulted in a significant increase in the seed yield (by 9.6?C12.4%). Moreover, the contents of anthocyanin in leaves of 60-day-old transgenic plants was about 9.4-fold higher in winter and about 6.8-fold higher in summer than the wild type. These excellent agronomic traits of the transgenic plants could not only improve the lodging resistance and seed yields, but also protect them against stress. Therefore, the over-expression of AtGA2ox8 might be used to produce dwarf varieties and increase seed yield in Brassica napus L.     

Linkage mapping of genes controlling resistance to white rust (Albugo candida) in Brassica rapa (syn. campestris) and comparative mapping to Brassica napus and Arabidopsis thaliana.

Genes for resistance to white rust (Albugo candida) in oilseed Brassica rapa were mapped using a recombinant inbred (RI) population and a genetic linkage map consisting of 144 restriction fragment length polymorphism (RFLP) markers and 3 phenotypic markers. Young seedlings were evaluated by inoculating cotyledons with A. candida race 2 (AC2) and race 7 (AC7) and scoring the interaction phenotype (IP) on a 0-9 scale. The IP of each line was nearly identical for the two races and the population showed bimodal distributions, suggesting that a single major gene (or tightly linked genes) controlled resistance to the two races. The IP scores were converted to categorical resistant and susceptible scores, and these data were used to map a single Mendelian gene controlling resistance to both races on linkage group 4 where resistance to race 2 had been mapped previously. A quantitative trait loci (QTL) mapping approach using the IP scores detected the same major resistance locus for both races, plus a second minor QTL effect for AC2 on linkage group 2. These results indicate that either a dominant allele at a single locus (Acal) or two tightly linked loci control seedling resistance to both races of white rust in the biennial turnip rape cultivar Per. The map positions of white rust resistance genes in B. rapa and Brassica napus were compared and the results indicate where additional loci that have not been mapped may be located. Alignment of these maps to the physical map of the Arabidopsis genome identified regions to target for comparative fine mapping using this model organism.     

Genetic diversity in Arabidopsis thaliana L. Heynh. investigated by cleaved amplified polymorphic sequence (CAPS) and inter-simple sequence repeat (ISSR) markers

In this study, we investigated genetic diversity among 37 accessions in Arabidopsis thaliana from Eurasia, North Africa and North America using morphological traits and two polymerase chain reaction (PCR)-based marker systems: cleaved amplified polymorphic sequences (CAPS) and inter-simple sequence repeats (ISSR). Cluster analysis based on genetic similarities calculated from CAPS data grouped the accessions roughly according to their geographical origin: one large group contained accessions from Western, Northern and Southern Europe as well as North Africa, a second group consisted of Eastern European and Asian continental accessions. North American accessions were interspersed into these groups. Contrary to the CAPS analysis, the dendrogram obtained from the ISSR data did not reflect the geographical origin of the accessions, and the calculated genetic distances did not match the CAPS results. This could be attributable to an uneven genomic distribution of ISSR markers as substantiated by a database search for ISSR binding sites in A. thaliana genomic DNA sequence files, or to the ISSR's different mode of evolution. We recommend CAPS markers for diversity analysis in A. thaliana because a careful selection of markers can ascertain an even representation of the entire genome.     

Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana and Brassica napus contain complex mixtures of aliphatic monomers derived from non-extractable lipid polyesters. Most of the monomers are deposited in the seed coat, and their compositions suggest the presence of both cutin and suberin layers. The location of these polyesters within the seed coat, and their contributions to permeability of the seed coat and other functional properties are unknown. Polyester deposition was followed over Brassica seed development and distinct temporal patterns of monomer accumulation were observed. Octadecadiene-1,18-dioate, the major leaf cutin monomer, was transiently deposited. In contrast, the saturated dicarboxylates maintained a constant level during seed desiccation, whereas the fatty alcohols and saturated omega-hydroxy fatty acids continually increased. Dissection and analysis of Brassica seed coats showed that suberization is not specific to the chalaza. Analysis of the Arabidopsis ap2-7 mutant suggested that suberin monomers are preferentially associated with the outer integument. Several Arabidopsis knockout mutant lines for genes involved in polyester biosynthesis (att1, fatB and gpat5) were examined for seed monomer load and composition. The variance in polyester monomers of these mutants is correlated with dye penetration assays. Furthermore, stable transgenic plants expressing promoter::YFP fusions showed ATT1 promoter activity in the inner integument, whereas GPAT5 promoter is active in the outer integument. Together, the Arabidopsis data indicated that there is a suberized layer associated with the outer integument and a cutin-like polyester layer associated with the inner seed coat.     

Reproductive Development in Oilseed Rape (Brassica napus cv. Bienvenu)

The reproductive development of oilseed rape (Brassica napusL. cv. Bienvenu) was studied using light and scanning electronmicroscopy. By using the two techniques on comparable samples,internal events such as microspore development were relatedto the morphology of the developing floral parts, and this inturn was linked to the growth stage of the plant. Reproductive development, oilseed rape, Brassica napus, growth stage     

Comparative sequence analysis for Brassica oleracea with similar sequences in B. rapa and Arabidopsis thaliana

We sequenced five BAC clones of Brassica oleracea doubled haploid ‘Early Big' broccoli containing major genes in the aliphatic glucosinolate pathway, and comparatively analyzed them with similar sequences in A. thaliana and B. rapa. Additionally, we included in the analysis published sequences from three other B. oleracea BAC clones and a contig of this species corresponding to segments in A. thaliana chromosomes IV and V. A total of 2,946 kb of B. oleracea, 1,069 kb of B. rapa sequence and 2,607 kb of A. thaliana sequence were compared and analyzed. We found conserved collinearity for gene order and content restricted to specific chromosomal segments, but breaks in collinearity were frequent resulting in gene absence likely not due to gene loss but rearrangements. B. oleracea has the lowest gene density of the three species, followed by B. rapa. The genome expansion of the Brassica species, B. oleracea in particular, is due to larger introns and gene spacers resulting from frequent insertion of DNA transposons and retrotransposons. These findings are discussed in relation to the possible origin and evolution of the Brassica genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Assessment of genetic variation within Indian mustard (Brassica juncea) germplasm using random amplified polymorphic DNA markers

Genetic diversity among 45 Indian mustard (Brassica Juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession 'Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77-0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop.