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.

Capturing cold-stress-related sequence diversity from a wild relative of common bean (Phaseolus angustissimus).

One restriction to the cultivation of common bean, Phaseolus vulgaris L., is its limited tolerance to low temperatures. In the present study, subtraction suppression hybridization was employed to enrich for stress responsive genes in both a chilling-susceptible common bean and a relatively more chilling-tolerant wild bean species, Phaseolus angustissimus. For each species, approximately 11 000 expressed sequence tags were generated. Comparative sequence analysis of the EST collection with the available annotated genome sequences of the model Fabaceae species Medicago truncatula and Glycine max identified protein homologues for approximately 65% and 80% of the Phaseolus sequences, respectively. This difference reflects the closer phylogenetic relationship between the genera Phaseolus and Glycine compared with Medicago. Annotation of the Phaseolus sequences was facilitated through this comparative analysis and indicated that several heat shock proteins, cytochrome P450s, and DNA binding factors were uniquely found among the sequences from the wild species P. angustissimus. The Phaseolus sequences have been made available on a GBrowse implementation using M. truncatula as the reference genome, providing rapid access to the sequence data and associated comparative genome data.

Introgression of B-genome chromosomes in a doubled haploid population of Brassica napus x B. carinata.

The Brassica B-genome species possess many valuable agronomic and disease resistance traits. To transfer traits from the B genome of B. carinata into B. napus, an interspecific cross between B. napus and B. carinata was performed and a doubled haploid (DH) population was generated from the BC2S3 generation. Successful production of interspecific DH lines as identified using B-genome microsatellite markers is reported. Five percent of DH lines carry either intact B-genome chromosomes or chromosomes that have deletions. All of the DH lines have linkage group J13/B7 in common. This was further confirmed using B. nigra genomic DNA in a fluorescent in situ hybridization assay where the B-genome chromosomes were visualized and distinguished from the A- and C-genome chromosomes. The 60 DH lines were also evaluated for morphological traits in the field for two seasons and were tested for resistance to blackleg, caused by Leptosphaeria maculans, under greenhouse conditions. Variation in the DH population followed a normal distribution for several agronomic traits and response to blackleg. The lines with B-genome chromosomes were significantly different (p < 0.01) from the lines without B-genome chromosomes for both morphological and seed quality traits such as days to flowering, days to maturity, and erucic acid content.

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.

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.

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.

PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression.

Self-incompatibility (SI) in Brassica species is controlled by a single polymorphic locus (S) with multiple specificities. Two stigmatically expressed genes that have been cloned from this region encode the S locus glycoprotein (SLG) and S receptor kinase (SRK). Both appear to be essential for the operation of SI. It is believed that rejection of incompatible pollen grains is effected by recognition events between an as yet unidentified S locus-encoded pollen coating-borne protein and the SLG/SRK. We previously identified a small pollen coat protein PCP7 (renamed here PCP-A1, for pollen coat protein, class A, 1) that binds with high affinity to SLGs irrespective of S genotype. Here, we report the cloning of PCP-A1 from Brassica oleracea and demonstrate that it is unlinked to the S locus. In situ localization of PCP-A1 transcripts revealed that they accumulate specifically in pollen at the late binucleate/trinucleate stage of development rather than in the tapetum, which previously was taken to be the principal source of the pollen coat. PCP-A1 is characterized by the presence of a structurally important motif consisting of eight cysteine residues shared by the plant defensins. Based on the presence of this motif and other data, homology modeling has been used to produce a putative structure for PCP-A1. Protein-protein interaction analyses demonstrate that SLG exists in monomeric and dimeric forms, both of which bind PCP-A1. Evidence is also presented for the existence of putative membrane-associated PCP-A1 binding proteins in stigmatic tissue.

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.

Alignment of the conserved C genomes of Brassica oleracea and Brassica napus.

A population of 169 microspore-derived doubled-haploid lines was produced from a highly polymorphic Brassica oleracea cross. A dense genetic linkage map of B. oleracea was then developed based on the segregation of 303 RFLP-defined loci. It is hoped that these lines will be used by other geneticists to facilitate the construction of a unified genetic map of B. oleracea. When the B. oleracea map was compared to one ofB. napus (Parkin et al. 1995), based on the same RFLP probes (Sharpe et al. 1995), good collinearity between the C-genome linkage groups of the two species was observed.

Frequent nonreciprocal translocations in the amphidiploid genome of oilseed rape (Brassica napus).

A RFLP map of Brassica napus, consisting of 277 loci arranged in 19 linkage groups, was produced from genetic segregation in a combined population of 174 doubled-haploid microspore-derived lines. The integration of this map with a B. napus map derived from a resynthesized B. napus x oilseed rape cross allowed the 10 linkage groups of the B. napus A genome and the 9 linkage groups of the C genome to be identified. Collinear patterns of marker loci on different linkage groups suggested potential partial homoeologues. RFLP patterns consistent with aberrant chromosomes were observed in 9 of the 174 doubled-haploid lines. At least 4 of these lines carried nonreciprocal, homoeologous translocations. These translocations were probably the result of homoeologous recombination in the amphidiploid genome of oilseed rape, suggesting that domesticated B. napus is unable to control chromosome pairing completely. Evidence for genome homogenization in oilseed rape is presented and its implications on genetic mapping in amphidiploid species is discussed. The level of polymorphism in the A genome was higher than that in the C genome and this might be a general property of oilseed rape crosses.

Identification of the A and C genomes of amphidiploid Brassica napus (oilseed rape).

A genetic linkage map consisting of 399 RFLP-defined loci was generated from a cross between resynthesized Brassica napus (an interspecific B. rapa x B. oleracea hybrid) and "natural" oilseed rape. The majority of loci exhibited disomic inheritance of parental alleles demonstrating that B. rapa chromosomes were each pairing exclusively with recognisable A-genome homologues in B. napus and that B. oleracea chromosomes were pairing similarly with C-genome homologues. This behaviour identified the 10 A genome and 9 C genome linkage groups of B. napus and demonstrated that the nuclear genomes of B. napus, B. rapa, and B. oleracea have remained essentially unaltered since the formation of the amphidiploid species, B. napus. A range of unusual marker patterns, which could be explained by aneuploidy and nonreciprocal translocations, were observed in the mapping population. These chromosome abnormalities were probably caused by associations between homoeologous chromosomes at meiosis in the resynthesized parent and the F1 plant leading to nondisjunction and homoeologous recombination.