The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1.

LepR3, found in the Brassica napus cv 'Surpass 400', provides race-specific resistance to the fungal pathogen Leptosphaeria maculans, which was overcome after great devastation in Australia in 2004. We investigated the LepR3 locus to identify the genetic basis of this resistance interaction. We employed a map-based cloning strategy, exploiting collinearity with the Arabidopsis thaliana and Brassica rapa genomes to enrich the map and locate a candidate gene. We also investigated the interaction of LepR3 with the L. maculans avirulence gene AvrLm1 using transgenics. LepR3 was found to encode a receptor-like protein (RLP). We also demonstrated that avirulence towards LepR3 is conferred by AvrLm1, which is responsible for both the Rlm1 and LepR3-dependent resistance responses in B. napus. LepR3 is the first functional B. napus disease resistance gene to be cloned. AvrLm1's interaction with two independent resistance loci, Rlm1 and LepR3, highlights the need to consider redundant phenotypes in 'gene-for-gene' interactions and offers an explanation as to why LepR3 was overcome so rapidly in parts of Australia.

Mapping genes for resistance to Leptosphaeria maculans in Brassica juncea.

Blackleg disease of crucifers, caused by the fungus Leptosphaeria maculans, is a major concern to oilseed rape producers worldwide. Brassica species containing the B genome have high levels of resistance to blackleg. Brassica juncea F2 and first-backcross (B1) populations segregating for resistance to a PG2 isolate of L. maculans were created. Segregation for resistance to L. maculans in these populations suggested that resistance was controlled by two independent genes, one dominant and one recessive in nature. A map of the B. juncea genome was constructed using segregation in the F2 population of a combination of restriction fragment length polymorphism (RFLP) and microsatel lite markers. The B. juncea map consisted of 325 loci and was aligned with previous maps of the Brassica A and B genomes. The gene controlling dominant resistance to L. maculans was positioned on linkage group J13 based on segregation for resistance in the F2 population. This position was confirmed in the B1 population in which the resistance gene was definitively mapped in the interval flanked by pN199RV and sB31143F. The provisional location of the recessive gene controlling resistance to L. maculans on linkage group J18 was identified using a subset of informative F2 individuals.

Identification of two novel genes for blackleg resistance in Brassica napus.

Blackleg, caused by Leptosphaeria maculans, is a major disease of Brassica napus. Two populations of B. napus DH lines, DHP95 and DHP96, with resistance introgressed from B. rapa subsp. sylvestris, were genetically mapped for resistance to blackleg disease with restriction fragment length polymorphism markers. Examination of the DHP95 population indicated that a locus on linkage group N2, named LepR1, was associated with blackleg resistance. In the DHP96 population, a second locus on linkage group N10, designated LepR2, was associated with resistance. We developed BC1 and F2 populations, to study the inheritance of resistance controlled by the genes. Genetic analysis indicated that LepR1 was a dominant nuclear allele, while LepR2 was an incompletely dominant nuclear resistance allele. LepR1 and LepR2 cotyledon resistance was further evaluated by testing 30 isolates from Canada, Australia, Europe, and Mexico. The isolates were from B. napus, B. juncea, and B. oleracea and represented different pathogenicity groups of L. maculans. Results indicated that LepR1 generally conferred a higher level of cotyledon resistance than LepR2. Both genes exhibited race-specific interactions with pathogen isolates; virulence on LepR1 was observed with one isolate, pl87-41, and two isolates, Lifolle 5, and Lifolle 6, were virulent on LepR2. LepR1 prevented hyphal penetration, while LepR2 reduced hyphal growth and inhibited sporulation. Callose deposition was associated with resistance for both loci.

Latent S alleles are widespread in cultivated self-compatible Brassica napus.

The genetic control of self-incompatibility in Brassica napus was investigated using crosses between resynthesized lines of B. napus and cultivars of oilseed rape. These crosses introduced eight C-genome S alleles from Brassica oleracea (S16, S22, S23, S25, S29, S35, S60, and S63) and one A-genome S allele from Brassica rapa (SRM29) into winter oilseed rape. The inheritance of S alleles was monitored using genetic markers and S phenotypes were determined in the F1, F2, first backcross (B1), and testcross (T1) generations. Two different F1 hybrids were used to develop populations of doubled haploid lines that were subjected to genetic mapping and scored for S phenotype. These investigations identified a latent S allele in at least two oilseed rape cultivars and indicated that the S phenotype of these latent alleles was masked by a suppressor system common to oilseed rape. These latent S alleles may be widespread in oilseed rape varieties and are possibly associated with the highly conserved C-genome S locus of these crop types. Segregation for S phenotype in subpopulations uniform for S genotype suggests the existence of suppressor loci that influenced the expression of the S phenotype. These suppressor loci were not linked to the S loci and possessed suppressing alleles in oilseed rape and non-suppressing alleles in the diploid parents of resynthesized B. napus lines.

Genetic mapping of the novel Turnip mosaic virus resistance gene TuRB03 in Brassica napus.

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC(1) population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.

Homoeologous loci control the accumulation of seed glucosinolates in oilseed rape (Brassica napus).

The genetic control of seed glucosinolate content in oilseed rape was investigated using two intervarietal backcross populations. Four QTLs segregating in the population derived from a Brassica napus L. 'Victor' x Brassica napus L. 'Tapidor' cross, together accounting for 76% of the phenotypic variation, were mapped. Three of these loci also appeared to control the accumulation of seed glucosinolates in a Brassica napus L. 'Bienvenu' x 'Tapidor' cross, and accounted for 86% of the phenotypic variation. The three QTLs common to both populations mapped to homoeologous regions of the B. napus genome, suggesting that seed glucosinolate accumulation is controlled by duplicate genes. It was possible to extend the comparative analysis of QTLs controlling seed glucosinolate accumulation by aligning the published genetic maps generated by several research groups. This comparative mapping demonstrated that high-glucosinolate varieties often carry low-glucosinolate alleles at one or more of the loci controlling seed glucosinolate accumulation.

Mapping the mosaic of ancestral genotypes in a cultivar of oilseed rape (Brassica napus) selected via pedigree breeding.

Recent oilseed rape breeding has produced low glucosinolate cultivars that yield proteinaceous meal suitable for animal feed. The low glucosinolate character was introduced into modern cultivars from Brassica napus 'Bronowski', a cultivar that is agronomically inferior in most other respects. Residual segments of 'Bronowski' genotype in modern cultivars probably cause reduced yield, poorer winter hardiness, and lower oil content. The quantity and distribution of the 'Bronowski' genotype in the modern oilseed rape cultivar Brassica napus 'Tapidor' was investigated using a segregating population derived from a cross between 'Tapidor' and its high glucosinolate progenitor. This population was analyzed with 65 informative Brassica RFLP probes and a genetic linkage map, based on the segregation at 77 polymorphic loci, was constructed. The mapping identified 15 residual segments of donor genotype in 'Tapidor', which together occupy approximately 29% of the B. napus genome. Mapping the loci that control variation for the accumulation of total seed glucosinolates in the segregating population has identified three loci that together explain >90% of the variation for this character. All of these loci are in donor segments of the 'Tapidor' genome. This result shows the extent to which conventional breeding programmes have difficulty in eliminating residual segments of donor genotype from elite material.

Patterns of genome duplication within the Brassica napus genome.

The progenitor diploid genomes (A and C) of the amphidiploid Brassica napus are extensively duplicated with 73% of genomic clones detecting two or more duplicate sequences within each of the diploid genomes. This comprehensive duplication of loci is to be expected in a species that has evolved through a polyploid ancestor. The majority of the duplicate loci within each of the diploid genomes were found in distinct linkage groups as collinear blocks of linked loci, some of which had undergone a variety of rearrangements subsequent to duplication, including inversions and translocations. A number of identical rearrangements were observed in the two diploid genomes, suggesting they had occurred before the divergence of the two species. A number of linkage groups displayed an organization consistent with centric fusion and (or) fission, suggesting this mechanism may have played a role in the evolution of Brassica genomes. For almost every genetically mapped locus detected in the A genome a homologous locus was found in the C genome; the collinear arrangement of these homologous markers allowed the primary regions of homoeology between the two genomes to be identified. At least 16 gross chromosomal rearrangements differentiated the two diploid genomes during their divergence from a common ancestor.

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.

Inheritance of Race-Specific Resistance to Xanthomonas campestris pv. campestris in Brassica Genomes.

ABSTRACT The inheritance of resistance to three Xanthomonas campestris pv. campestris races was studied in crosses between resistant and susceptible lines of Brassica oleracea (C genome), B. carinata (BC genome), and B. napus (AC genome). Resistance to race 3 in the B. oleracea doubled haploid line BOH 85c and in PI 436606 was controlled by a single dominant locus (Xca3). Resistance to races 1 and 3 in the B. oleracea line Badger Inbred-16 was quantitative and recessive. Strong resistance to races 1 and 4 was controlled by a single dominant locus (Xca1) in the B. carinata line PI 199947. This resistance probably originates from the B genome. Resistance to race 4 in three B. napus lines, cv. Cobra, the rapid cycling line CrGC5, and the doubled haploid line N-o-1, was controlled by a single dominant locus (Xca4). A set of doubled haploid lines, selected from a population used previously to develop a restriction fragment length polymorphism map, was used to map this locus. Xca4 was positioned on linkage group N5 of the B. napus A genome, indicating that this resistance originated from B. rapa. Xca4 is the first major locus to be mapped that controls race-specific resistance to X. campestris pv. campestris in Brassica spp.

Amphidiploid Brassica juncea contains conserved progenitor genomes.

To perform a detailed study of genome evolution in the natural Brassica amphidiploid B. juncea, we have constructed two linkage maps based on RFLP (restriction fragment length polymorphism) markers; one generated from a cross between a resynthesized B. juncea (a chromosome doubled interspecific B. rapa x B. nigra hybrid) and a natural B. juncea cultivar, the other from a cross between two B. juncea cultivars. By using a common cultivar in both crosses, the two maps could be unambiguously integrated. All loci exhibited disomic inheritance of parental alleles in the natural x resynthesized cross, showing that B. rapa chromosomes paired exclusively with their A-genome homologues in B. juncea and that B. nigra chromosomes likewise paired with their B-genome homologues. The maps derived from the two crosses were also perfectly collinear. Furthermore, these maps were collinear with maps of the diploid progenitor species (B. nigra and B. rapa) produced using the same set of RFLP probes. These data indicate that the genome of B. juncea has remained essentially unchanged since polyploid formation. Our observations appear to refute the suggestion that the formation of polyploid genomes is accompanied by rapid change in genome structure.

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.

Independent deletions of a pathogen-resistance gene in Brassica and Arabidopsis.

Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with approximately 81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.

The association of flowering time quantitative trait loci with duplicated regions and candidate loci in Brassica oleracea.

A population of 150 doubled haploid lines of rapid cycling Brassica oleracea, derived from an F1 from a var. alboglabra x var. italica cross, was scored for flowering time in two trials. Using information on 82 mapped molecular markers, spread evenly across the nine linkage groups, QTL were identified at six locations; one each on linkage groups O2 and O3 and two each on linkage groups O5 and O9. In total, these QTL explained 58 and 93% of the genetical variation in the two trials. Three of these QTL, on linkage groups O2, O3, and O9, were situated in regions showing considerable homology both with each other and with chromosome regions of B. nigra that have been shown to affect flowering time. These same regions are all homologous to a single tract of Arabidopsis chromosome 5, which contains a number of the flowering-related genes, one or more of which may be candidates for the QTL found in Brassica.

Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome.

Arabidopsis thaliana (the model dicotyledonous plant) is closely related to Brassica crop species. Genome collinearity, or conservation of marker order, between Brassica napus (oilseed rape) and A. thaliana was assessed over a 7.5-Mbp region of the long arm of A. thaliana chromosome 4, equivalent to 30 cM. Estimates of copy number indicated that sequences present in a single copy in the haploid genome of A. thaliana (n = 5) were present in 2-8 copies in the haploid genome of B. napus (n = 19), while sequences present in multiple copies in A. thaliana were present in over 10 copies in B. napus. Genetic mapping in B. napus of DNA markers derived from a segment of A. thaliana chromosome 4 revealed duplicated homologous segments in the B. napus genome. Physical mapping in A. thaliana of homologues of Brassica clones derived from these regions confirmed the identity of six duplicated segments with substantial homology to the 7.5-Mbp region of chromosome 4 in A. thaliana. These six duplicated Brassica regions (on average 22 cM in length) are collinear, except that two of the six copies contain the same large internal inversion. These results have encouraging implications for the feasibility of shuttling between the physical map of A. thaliana and genetic maps of Brassica species, for identifying candidate genes and for map based gene cloning in Brassica crops.

Conserved patterns of chromosome pairing and recombination in Brassica napus crosses.

The patterns of chromosome pairing and recombination in two contrasting Brassica napus F1 hybrids were deduced. One hybrid was from a winter oilseed rape (WOSR) x spring oilseed rape cross, the other from a resynthesized B. napus x WOSR cross. Segregation at 211 equivalent loci assayed in the population derived from each hybrid produced two collinear genetic maps. Alignment of the maps indicated that B. napus chromosomes behaved reproducibly as 19 homologous pairs and that the 19 distinct chromosomes of B. napus each recombined with unique chromosomes from the interspecific hybrid between Brassica rapa and Brassica oleracea. This result indicated that the genomes of the diploid progenitors of amphidiploid B. napus have remained essentially unaltered since the formation of the species and that the progenitor genomes were similar to those of modern-day B. rapa and B. oleracea. The frequency and distribution of crossovers were almost indistinguishable in the two populations, suggesting that the recombination machinery of B. napus could cope easily with different degrees of genetic divergence between homologous chromosomes. Efficient recombination in wide crosses will facilitate the introgression of novel alleles into oilseed rape from B. rapa and B. oleracea (via resynthesized B. napus) and reduce linkage drag.

Comparison of flowering time genes in Brassica rapa, B. napus and Arabidopsis thaliana.

The major difference between annual and biennial cultivars of oilseed Brassica napus and B. rapa is conferred by genes controlling vernalization-responsive flowering time. These genes were compared between the species by aligning the map positions of flowering time quantitative trait loci (QTLs) detected in a segregating population of each species. The results suggest that two major QTLs identified in B. rapa correspond to two major QTLs identified in B. napus. Since B. rapa is one of the hypothesized diploid parents of the amphidiploid B. napus, the vernalization requirement of B. napus probably originated from B. rapa. Brassica genes also were compared to flowering time genes in Arabidopsis thaliana by mapping RFLP loci with the same probes in both B. napus and Arabidopsis. The region containing one pair of Brassica QTLs was collinear with the top of chromosome 5 in A. thaliana where flowering time genes FLC, FY and CO are located. The region containing the second pair of QTLs showed fractured collinearity with several regions of the Arabidopsis genome, including the top of chromosome 4 where FRI is located. Thus, these Brassica genes may correspond to two genes (FLC and FRI) that regulate flowering time in the latest flowering ecotypes of Arabidopsis.

Indistinguishable patterns of recombination resulting from male and female meioses in Brassica napus (oilseed rape).

An F1 individual derived from a cross between two distinct lines of spring oilseed rape (Brassica napus) was used to produce a pair of complementary backcross populations, each consisting of 90 individuals. The F1 donated male gametes to the Male population and female gametes to the Female population. Genetic maps were generated from both populations and aligned using 117 common loci to form an integrated genome map of B. napus with 243 RFLP-defined loci. A comparison of the frequency and distribution of crossovers in the two populations of F1 gametes (assayed in the Male and Female populations) detected no differences. The genetic maps derived from the Male and Female populations each consisted of 19 linkage groups spanning 1544 and 1577 cM, respectively. The maps were aligned with other B. napus maps, and all 19 equivalent linkage groups were unambiguously assigned. The genetic size and general organisation of the new maps were comparable with those of pre-existing B. napus maps in most respects, except that the levels of polymorphism in the constituent A and C genomes were unusually similar in the new cross.

Comparative genome mapping in Brassica.

A Brassica nigra genetic linkage map was developed from a highly polymorphic cross analyzed with a set of low copy number Brassica RFLP probes. The Brassica genome is extensively duplicated with eight distinct sets of chromosomal segments, each present in three copies, covering virtually the whole genome. Thus, B. nigra could be descended from a hexaploid ancestor. A comparative analysis of B. nigra, B. oleracea and B. rapa genomes, based on maps developed using a common set of RFLP probes, was also performed. The three genomes have distinct chromosomal structures differentiated by a large number of rearrangements, but collinear regions involving virtually the whole of each the three genomes were identified. The genic contents of B. nigra, B. oleracea and B. rapa were basically equivalent and differences in chromosome number (8, 9 and 10, respectively) are probably the result of chromosome fusions and/ or fissions. The strong conservation of overall genic content across the three Brassica genomes mirrors the conservation of genic content observed over a much longer evolutionary span in cereals. However, the rate of chromosomal rearrangement in crucifers is much higher than that observed in cereal genomes.

The construction of a substitution library of recombinant backcross lines in Brassica oleracea for the precision mapping of quantitative trait loci.

The currently available methods for locating quantitative trait loci (QTLs) and measuring their effects in segregating populations lack precision unless individual QTLs have very high heritabilities. The use of recombinant backcross lines containing short regions of donor chromosome introgressed into a constant recipient background permits QTLs to be located with greater precision. The present paper describes the use of molecular markers to introgress defined short regions of chromosome from a donor doubled haploid calabrese line of Brassica oleracea (var. italica) into a recipient short generation variety (Brassica oleracea var. alboglabra). We demonstrate that in just two or three generations of backcrossing, combined with selection for mapped molecular markers, the generation of a library of recombinant backcross lines is feasible. The possible use and refinement of these lines are discussed. Key words : backcrossing, Brassica oleracea, introgression, molecular markers, near-isogenic lines, QTL mapping, recombinant backcross lines, substitution lines.