Genetic Analysis of a Horizontal Resistance Locus BLMR2 in Brassica napus.

Leptosphaeria maculans causes blackleg disease in Brassica napus. The blackleg disease is mainly controlled by resistance genes in B. napus. Previous studies have shown that the blackleg resistant BLMR2 locus that conferred horizontal resistance under field conditions, is located on chromosome A10 of B. napus. The purpose of this study is to fine map this locus and hence identify a candidate gene underlying horizontal resistance. The spectrum of resistance to L. maculans isolates of the resistance locus BLMR2 was analyzed using near isogenic lines, resistant, and susceptible cultivars. The results showed that this locus was horizontally resistant to all isolates tested. Sequence characterized amplified regions (SCAR), simple sequence repeats (SSR), and single nucleotide polymorphism (SNP) markers were developed in the chromosome region of BLMR2 and a fine genetic map was constructed. Two molecular markers narrowed BLMR2 in a 53.37 kb region where six genes were annotated. Among the six annotated genes, BnaA10g11280D/BnaA10g11290D encoding a cytochrome P450 protein were predicted as the candidate of BLMR2. Based on the profiling of pathogen induced transcriptome, three expressed genes in the six annotated genes were identified while only cytochrome P450 showed upregulation. The candidate corresponds to the gene involved in the indole glucosinolate biosynthesis pathway and plant basal defense in Arabidopsis thaliana. The molecular markers identified in this study will allow the quick incorporation of the BLMR2 allele in rapeseed cultivars to enhance blackleg resistance.

Transcriptional Insight Into Brassica napus Resistance Genes LepR3 and Rlm2-Mediated Defense Response Against the Leptosphaeria maculans Infection.

The phytopathogenic fungus Leptosphaeria maculans causes the blackleg disease on Brassica napus, resulting in severe loss of rapeseed production. Breeding of resistant cultivars containing race-specific resistance genes is provably effective to combat this disease. While two allelic resistance genes LepR3 and Rlm2 recognizing L. maculans avirulence genes AvrLm1 and AvrLm2 at plant apoplastic space have been cloned in B. napus, the downstream gene expression network underlying the resistance remains elusive. In this study, transgenic lines expressing LepR3 and Rlm2 were created in the susceptible "Westar" cultivar and inoculated with L. maculans isolates containing different sets of AvrLm1 and AvrLm2 for comparative transcriptomic analysis. Through grouping the RNA-seq data based on different levels of defense response, we find LepR3 and Rlm2 orchestrate a hierarchically regulated gene expression network, consisting of induced ABA acting independently of the disease reaction, activation of signal transduction pathways with gradually increasing intensity from compatible to incompatible interaction, and specifically induced enzymatic and chemical actions contributing to hypersensitive response with recognition of AvrLm1 and AvrLm2. This study provides an unconventional investigation into LepR3 and Rlm2-mediated plant defense machinery and adds novel insight into the interaction between surface-localized receptor-like proteins (RLPs) and apoplastic fungal pathogens.

Combinations of Independent Dominant Loci Conferring Clubroot Resistance in All Four Turnip Accessions (Brassica rapa) From the European Clubroot Differential Set.

Clubroot disease is devastating to Brassica crop production when susceptible cultivars are planted in infected fields. European turnips are the most resistant sources and their resistance genes have been introduced into other crops such oilseed rape (Brassica napus L.), Chinese cabbage and other Brassica vegetables. The European clubroot differential (ECD) set contains four turnip accessions (ECD1-4). These ECD turnips exhibited high levels of resistance to clubroot when they were tested under controlled environmental conditions with Canadian field isolates. Gene mapping of the clubroot resistance genes in ECD1-4 were performed and three independent dominant resistance loci were identified. Two resistance loci were mapped on chromosome A03 and the third on chromosome A08. Each ECD turnip accession contained two of these three resistance loci. Some resistance loci were homozygous in ECD accessions while others showed heterozygosity based on the segregation of clubroot resistance in 20 BC1 families derived from ECD1 to 4. Molecular markers were developed linked to each clubroot resistance loci for the resistance gene introgression in different germplasm.

Characterization of Brassica napus L. genotypes utilizing sequence-related amplified polymorphism and genotyping by sequencing in association with cluster analysis.

Identifying parental combinations that exhibit high heterosis is a constant target for commercial Brassica napus L. hybrid development programs. Finding high heterotic parental combinations can require hundreds of test crosses and years of yield evaluation. Heterotic pool development could be used to divide breeding material into specific breeding pools and focus the number of parental combinations created. Here, we report the genotypic characterization of 79 B. napus genotypes by calculating genetic distance based on sequence-related amplified polymorphism (SRAP) and genotyping by sequencing (GBS) in association with a neighbour-joining clustering algorithm. Despite the different genotypic analyses, neighbour-joining cluster analysis based on genetic distance of SRAP and GBS produced similar clusters. Homology between SRAP and GBS clusters was approximately 77 % when manually comparing clusters and 68 % when comparing clusters using Compare2Trees. This research demonstrates that SRAP can have similar efficacy when compared to next-generation sequencing technology for heterotic pool classification. This information may provide an important breeding scaffold for the development of hybrid cultivars based upon genetic distance and cluster analysis.

Molecular cloning, functional characterization and expression of potato (Solanum tuberosum) 1-deoxy-d-xylulose 5-phosphate synthase 1 (StDXS1) in response to Phytophthora infestans.

1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the plastidial 2C-methyl-D-erythritol-4-phosphate (DOXP-MEP) pathway involved in isoprenoid biosynthesis. In this study, we cloned the complete cDNA of potato DXS gene that was designated StDXS1. StDXS1 cDNA encodes for 719 amino acid residues, with MW of 77.8 kDa, and is present in one copy in the potato genome. Phylogenetic analysis and protein sequence alignments assigned StDXS1 to a group with DXS homologues from closely related species and exhibited homodomain identity with known DXS proteins from other plant species. Late blight symptoms occurred in parallel with a reduction in StDXS1 transcript levels, which may be associated with the levels of isoprenoids that contribute to plant protection against pathogens. Subcellular localization indicated that StDXS1 targets the chloroplasts where isoprenoids are synthesized. Arabidopsis expressing StDXS1 showed a higher accumulation of carotenoids and chlorophyll as compared to wild type controls. Lower levels of ABA and GA were detected in the transgenic DXS lines as compared to control plants, which reflected on higher germination rates of the transgenic DXS lines. No changes were detected in JA or SA contents. Selected downstream genes in the DOXP-MEP pathway, especially GGPPS genes, were up-regulated in the transgenic lines.

Homoeologous GSL-ELONG gene replacement for manipulation of aliphatic glucosinolates in Brassica rapa L. by marker assisted selection.

Aliphatic glucosinolates are the predominant sulfur-rich plant secondary metabolites in economically important Brassica crops. Glucosinolates and their hydrolysis products are involved in plant-microbe, plant-insect, plant-animal, and plant-human interactions. It is, therefore, important to manipulate glucosinolate profiles and contents in Brassica species. In this study, aliphatic glucosinolates were genetically manipulated through homoeologous recombination in backcross lines followed by marker assisted selection in B. rapa. A resynthesized B. napus line, from a cross between B. rapa and B. oleracea, was backcrossed with Chinese cabbage doubled haploid line, RI16. Marker assisted selection for non-functional gene was performed in each backcross generations. Advanced backcross progenies (BC3F2) were developed to identify homoeologous gene replacement and/or introgression. Reduction in 5C aliphatic glucosinolates (gluconapoleiferin, glucoalyssin, and glucobrassicanapin) was observed in BC3F2 progenies of the recurrent parent that carried the GSL-ELONG (-) gene. The GSL-ELONG (-) positive backcross progenies were also screened by the A-genome and BraGSL-ELONG gene specific marker, which linked with 5C aliphatic glucosinolates. The A-genome specific marker was absent in the plants of advanced backcross progenies which showed reduction in 5C aliphatic glucosinolates. The results suggest that the functional allele had been replaced by the non-functional GSL-ELONG (-) allele from B. oleracea. Some advanced backcross progenies (BC3F2) positive for the GSL-ELONG (-) allele and the A-genome specific SCAR marker BraMAM1-1 did not show reduction in 5C aliphatic glucosinolates, suggesting that GSL-ELONG (-) allele is recessive. Replacement of the functional locus in the A-genome by non-functional counterpart in the C-genome reduced the content of 5C aliphatic glucosinolates in B. rapa seeds with 20 µmol/g.

Reducing progoitrin and enriching glucoraphanin in Brassica napus seeds through silencing of the GSL-ALK gene family.

The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 µmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.

Genetic mapping of the Leptosphaeria maculans avirulence gene corresponding to the LepR1 resistance gene of Brassica napus.

AvrLepR1 of the fungal pathogen Leptosphaeria maculans is the avirulence gene that corresponds to Brassica LepR1, a plant gene controlling dominant, race-specific resistance to this pathogen. An in vitro cross between the virulent L. maculans isolate, 87-41, and the avirulent isolate, 99-56, was performed in order to map the AvrLepR1 gene. The disease reactions of the 94 of the resulting F(1) progenies were tested on the canola line ddm-12-6s-1, which carries LepR1. There were 44 avirulent progenies and 50 virulent progenies suggesting a 1:1 segregation ratio and that the avirulence of 99-56 on ddm-12-6s-1 is controlled by a single gene. Tetrad analysis also indicated a 1:1 segregation ratio. The AvrLepR1 gene was positioned on a genetic map of L. maculans relative to 259 sequence-related amplified polymorphism (SRAP) markers, two cloned avirulence genes (AvrLm1 and AvrLm4-7) and the mating type locus (MAT1). The genetic map consisted of 36 linkage groups, ranging in size from 13.1 to 163.7 cM, and spanned a total of 2,076.4 cM. The AvrLepR1 locus was mapped to linkage group 4, in the 13.1 cM interval flanked by the SRAP markers SBG49-110 and FT161-223. The AvrLm4-7 locus was also positioned on linkage group 4, close to but distinct from the AvrLepR1 locus, in the 5.4 cM interval flanked by FT161-223 and P1314-300. This work will make possible the further characterization and map-based cloning of AvrLepR1. A combination of genetic mapping and pathogenicity tests demonstrated that AvrLepR1 is different from each of the L. maculans avirulence genes that have been characterized previously.

Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

BACKGROUND: Sequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. To combine next generation sequencing technology with SRAP, Illumina's Solexa sequencing was used to sequence tagged SRAP PCR products. RESULTS: Three sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR reactions and the same number of tagging PCR reactions respectively to produce a pooled sample for Illumina's Solexa sequencing. After sequencing, 1.28 GB of sequence with over 13 million paired-end sequences was obtained and used to match Solexa sequences with their corresponding SRAP markers and to integrate Solexa sequences on an ultradense genetic map. The ultradense genetic bin map with 465 bins was constructed using a recombinant inbred (RI) line mapping population in B. rapa. For this ultradense genetic bin map, 9,177 SRAP markers, 1,737 integrated unique Solexa paired-end sequences and 46 SSR markers representing 10,960 independent genetic loci were assembled and 141 unique Solexa paired-end sequences were matched with their corresponding SRAP markers. The genetic map in B. rapa was aligned with the previous ultradense genetic map in B. napus through common SRAP markers in these two species. Additionally, SSR markers were used to perform alignment of the current genetic map with other five genetic maps in B. rapa and B. napus. CONCLUSION: We used SRAP to construct an ultradense genetic map with 10,960 independent genetic loci in B. rapa that is the most saturated genetic map ever constructed in this species. Using next generation sequencing, we integrated 1,878 Solexa sequences on the genetic map. These integrated sequences will be used to assemble the scaffolds in the B. rapa genome. Additionally, this genetic map may be used for gene cloning and marker development in B. rapa and B. napus.

Identification of two blackleg resistance genes and fine mapping of one of these two genes in a Brassica napus canola cultivar 'Surpass 400'.

Blackleg resistant cultivars have been developed through conventional breeding methods and are successfully used globally to control this disease in canola production. To clone blackleg resistance genes and to understand the mechanism underlying the resistance, a blackleg resistant canola cultivar 'Surpass 400' was used to develop a gene mapping population. A previously reported high density genetic map was used to find a resistance gene region that corresponded to linkage group N10 in B. napus. Differential interactions between the resistant lines and a pathogen isolate were discovered with two resistance genes BLMR1 and BLMR2 identified through linkage analysis of five genome-specific molecular markers. BLMR1 provides resistance through the hypersensitive response that protects inoculated cotyledons from becoming infected, Unlike BLMR1, BLMR2 slows down the development of individual infection loci. BLMR1 and BLMR2 segregated independently in two large F(3)BC(2) populations. Fine mapping of BLMR1 was performed with 12 genome-specific molecular markers. The closest marker with a genetic distance of 0.13 cM to BLMR1 was identified, which lays a solid foundation for cloning BLMR1.

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.

Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa.

A glabrous, yellow-seeded doubled haploid (DH) line and a hairy, black-seeded DH line in Chinese cabbage (B. rapa) were used as parents to develop a DH line population that segregated for both hairiness and seed coat color traits. The data showed that both traits completely co-segregated each other, suggesting that one Mendelian locus controlled both hairiness and seed coat color in this population. A fine genetic map was constructed and a SNP marker that was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) in Arabidopsis showed complete linkage to both the hairiness and seed coat color gene, suggesting that the Brassica TTG1 ortholog shared the same gene function as its Arabidopsis counterpart. Further sequence analysis of the alleles from hairless, yellow-seeded and hairy, black-seeded DH lines in B. rapa showed that a 94-base deletion was found in the hairless, yellow-seeded DH lines. A nonfunctional truncated protein in the hairless, yellow-seeded DH lines in B. rapa was suggested by the coding sequence of the TTG1 ortholog. Both of the TTG1 homologs from the black and yellow seeded B. rapa lines were used to transform an Arabidopsis ttg1 mutant and the results showed that the TTG1 homolog from the black seeded B. rapa recovered the Arabidopsis ttg1 mutant, while the yellow seeded homolog did not, suggesting that the deletion in the Brassica TTG1 homolog had led to the yellow seeded natural mutant. This was the first identified gene in Brassica species that simultaneously controlled both hairiness and seed coat color traits.

High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding.

A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.

Development of SRAP, SNP and multiplexed SCAR molecular markers for the major seed coat color gene in Brassica rapa L.

Seed coat color inheritance in B. rapa was studied in F(1), F(2), F(3), and BC(1) progenies from a cross of a Canadian brown-seeded variety 'SPAN' and a Bangladeshi yellow sarson variety 'BARI-6'. A pollen effect was found when the yellow sarson line was used as the maternal parent. Seed coat color segregated into brown, yellow-brown and bright yellow classes. Segregation was under digenic control where the brown or yellow-brown color was dominant over bright yellow seed coat color. A sequence related amplified polymorphism (SRAP) marker linked closely to a major seed coat color gene (Br1/br1) was developed. This dominant SRAP molecular marker was successfully converted into single nucleotide polymorphism (SNP) markers and sequence characterized amplification region (SCAR) markers after the extended flanking sequence of the SRAP was obtained with chromosome walking. In total, 24 SNPs were identified with more than 2-kb sequence. A 12-bp deletion allowed the development of a SCAR marker linked closely to the Br1 gene. Using the five-fluorescence dye set supplied by ABI, four labeled M13 primers were integrated with different SCAR primers to increase the throughput of SCAR marker detection. Using multiplexed SCAR markers targeting insertions and deletions in a genome shows great potential for marker assisted selection in plant breeding.

High-density Brassica oleracea linkage map: identification of useful new linkages.

We constructed a 1,257-marker, high-density genetic map of Brassica oleracea spanning 703 cM in nine linkage groups, designated LG1-LG9. It was developed in an F2 segregating population of 143 individuals obtained by crossing double haploid plants of broccoli "Early-Big" and cauliflower "An-Nan Early". These markers are randomly distributed throughout the map, which includes a total of 1,062 genomic SRAP markers, 155 cDNA SRAP markers, 26 SSR markers, 3 broccoli BAC end sequences and 11 known Brassica genes: BoGSL-ALK, BoGSL-ELONG, BoGSL-PROa, BoGSL-PROb, BoCS-lyase, BoGS-OH, BoCYP79F1, BoS-GT (glucosinolate pathway), BoDM1 (resistance to downy mildew), BoCALa, BoAP1a (inflorescence architecture). BoDM1 and BoGSL-ELONG are linked on LG 2 at 0.8 cM, making it possible to use the glucosinolate gene as a marker for the disease resistance gene. By QTL analysis, we found three segments involved in curd formation in cauliflower. The map was aligned to the C genome linkage groups and chromosomes of B. oleracea and B. napus, and anchored to the physical map of A. thaliana. This map adds over 1,000 new markers to Brassica molecular tools.

An ultradense genetic recombination map for Brassica napus, consisting of 13551 SRAP markers.

Sequence related amplified polymorphism (SRAP) was used to construct an ultradense genetic recombination map for a doubled haploid (DH) population in B. napus. A total of 1,634 primer combinations including 12 fluorescently labeled primers and 442 unlabeled ones produced 13,551 mapped SRAP markers. All these SRAPs were assembled in 1,055 bins that were placed onto 19 linkage groups. Ten of the nineteen linkage groups were assigned to the A genome and the remaining nine to the C genome on the basis of the differential SRAP PCR amplification in two DH lines of B. rapa and B. oleracea. Furthermore, all 19 linkage groups were assigned to their corresponding N1-N19 groups of B. napus by comparison with 55 SSR markers used to construct previous maps in this species. In total, 1,663 crossovers were detected, resulting in a map length span of 1604.8 cM. The marker density is 8.45 SRAPs per cM, and there could be more than one marker in 100 kb physical distance. There are four linkage groups in the A genome with more than 800 SRAP markers each, and three linkage groups in the C genome with more 1,000 SRAP markers each. Our studies suggest that a single SRAP map might be applicable to the three Brassica species, B. napus, B. oleracea and B. rapa. The use of this ultra high-density genetic recombination map in marker development and map-based gene cloning is discussed.

Arabidopsis thaliana UBC13: implication of error-free DNA damage tolerance and Lys63-linked polyubiquitylation in plants.

Ubiquitylation is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitylation requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets specific proteins for degradation. Recently polyubiquitylation through a noncanonical Lys63 chain has been reported, and is required for error-free DNA damage tolerance (or postreplication repair) in yeast. To date, Ubc13 is the only known ubiquitin-conjugating enzyme (Ubc) capable of catalyzing the Lys63-linked polyubiquitylation reaction and this function requires interaction with the Ubc variant Mms2. No information is available on either Lys63-linked ubiquitylation or error-free damage tolerance in plants. We thus cloned and functionally characterized two Arabidopsis thaliana UBC13 genes, AtUBC13A and AtUBC13B. The two genes are highly conserved with respect to chromosomal structure and protein sequence, suggesting that they are derived from a recent gene duplication event. Both AtUbc13 proteins are able to physically interact with yeast or human Mms2, implying that plants also employ the Lys63-linked polyubiquitylation reaction. Furthermore, AtUBC13 genes are able to functionally complement the yeast ubc13 null mutant for spontaneous mutagenesis and sensitivity to DNA damaging agents, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The AtUBC13 genes appear to express ubiquitously and are not induced by various conditions tested.

Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana.

Gene BoGSL-PRO is associated with presence of 3-carbon side-chain glucosinolates (GSL). This gene is a member of the methylthioalkylmalate synthase (MAM) gene family. A BAC clone of Brassica oleracea, B21F5, containing this gene, was sequenced, annotated and compared to its corresponding region in Arabidopsis thaliana. Twelve protein-coding genes and 10 transposable elements were found in this clone. The corresponding region in A. thaliana chromosome I has 14 genes and no transposable elements. Analysis of MAM gene family in both species, which also include genes controlling 4-carbon side-chain GSL, separated the genes in two groups based on exon numbers and function. Phylogenetic analysis of the amino acid sequences encoded by these genes suggest that these two groups were produced by a duplication that must have occurred before the divergence of the Rosid and Asterid lineages of angiosperms. Comparison with putative orthologs from several prokaryotes further suggest that the members of the gene family with 10 exons, which encode proteins involved in 4-carbon side-chain GSL biosynthesis, were derived via truncation of the 3' end from ancestral genes more similar in length to those with 12 exons, which encode proteins involved in 3-carbon side-chain GSL biosynthesis. Lower gene density in B. oleracea compared to A. thaliana is due in part to presence of transposable elements (TE) mostly in inter-genic regions.

Comparative analysis of a transposon-rich Brassica oleracea BAC clone with its corresponding sequence in A. thaliana.

We compared the sequence of a 96.7 Kb-long BAC clone (B 19 N 3) from Brassica oleracea (broccoli) with its corresponding regions in Arabidopsis thaliana. B 19 N 3 contains eight genes and 15 transposable elements (TEs). The first two genes in this clone, Bo 1 and Bo 2, have its corresponding region at the end of chromosome V of Arabidopsis (24 Mb). The third gene, Bo 3, corresponds to an ortholog at the opposite end (2.6 Mb) of the same chromosome. The other five genes, Bo 4 to Bo 8 also have a corresponding region on the same chromosome but at 7.7 Mb . These five genes are colinear with those found in the corresponding region of Arabidopsis, which contains, however, 15 genes. Therefore, a cluster of 10 genes is missing in B. oleracea clone (B 19 N 3). All five genes in common have the same order and orientation in the genomes of both species. Their 36 exons constituting the eight homologous genes have high conservation in size and sequence identity in both species. Among these, there is a major gene involved in aliphatic glucosinolate biosynthesis, Bo GSL-ELONG (Bo 4). Similar to A. thaliana, this gene, has a tandem duplicate, Bo 5. A contig for this region was constructed by primer walking and BAC-end-sequencing, revealing general gene colinearity between both species. During the 20 million years separating A. thaliana from B. oleracea from a common ancestor both genomes have diverged by chromosomal rearrangements and differential TE activity. These events, in addition to changes in chromosome number are responsible for the evolution of the genomes of both species. In spite of these changes, both species conserve general colinearity for their corresponding genes.

Comparative analysis of a Brassica BAC clone containing several major aliphatic glucosinolate genes with its corresponding Arabidopsis sequence.

We compared the sequence of a 101-kb-long bacterial artificial chromosome (BAC) clone (B21H13) from Brassica oleracea with its homologous region in Arabidopsis thaliana. This clone contains a gene family involved in the synthesis of aliphatic glucosinolates. The A. thaliana homologs for this gene family are located on chromosome IV and correspond to three 2-oxoglutarate-dependent dioxygenase (AOP) genes. We found that B21H13 harbors 23 genes, whereas the equivalent region in Arabidopsis contains 37 genes. All 23 common genes have the same order and orientation in both Brassica and Arabidopsis. The 16 missing genes in the broccoli BAC clone were arranged in two major blocks of 5 and 7 contiguous genes, two singletons, and a twosome. The 118 exons comprising these 23 genes have high conservation between the two species. The arrangement of the AOP gene family in A. thaliana is as follows: AOP3 (GS-OHP) - AOP2 (GS-ALK) - pseudogene - AOP1. In contrast, in B. oleracea (broccoli and collard), two of the genes are duplicated and the third, AOP3, is missing. The remaining genes are arranged as follows: Bo-AOP2.1 (BoGSL-ALKa) - pseudogene - AOP2.2 (BoGSL-ALKb) - AOP1.1 - AOP1.2. When the survey was expanded to other Brassica accessions, we found variation in copy number and sequence for the Brassica AOP2 homologs. This study confirms that extensive rearrangements have taken place during the evolution of the Brassicacea at both gene and chromosomal levels.