Genetic dissection of resistance to anthracnose and powdery mildew in Medicago truncatula.

Medicago truncatula was used to characterize resistance to anthracnose and powdery mildew caused by Colletotrichum trifolii and Erysiphe pisi, respectively. Two isolates of E. pisi (Ep-p from pea and Ep-a from alfalfa) and two races of C. trifolii (races 1 and 2) were used in this study. The A17 genotype was resistant and displayed a hypersensitive response after inoculation with either pathogen, while lines F83005.5 and DZA315.16 were susceptible to anthracnose and powdery mildew, respectively. To identify the genetic determinants underlying resistance in A17, two F7 recombinant inbred line (RIL) populations, LR4 (A17 x DZA315.16) and LR5 (A17 x F83005.5), were phenotyped with E. pisi isolates and C. trifolii races, respectively. Genetic analyses showed that i) resistance to anthracnose is governed mainly by a single major locus to both races, named Ct1 and located on the upper part of chromosome 4; and ii) resistance to powdery mildew involves three distinct loci, Epp1 on chromosome 4 and Epa1 and Epa2 on chromosome 5. The use of a consensus genetic map for the two RIL populations revealed that Ct1 and Epp1, although located in the same genome region, were clearly distinct. In silico analysis in this region identified the presence of several clusters of nucleotide binding site leucine-rich repeat genes. Many of these genes have atypical resistance gene analog structures and display differential expression patterns in distinct stress-related cDNA libraries.

Molecular and cytological responses of Medicago truncatula to Erysiphe pisi.

SUMMARY Powdery mildew is an economically important disease in a number of crop legumes; however, little is known about resistance to the disease in these species. To gain a better understanding of the genetics of resistance and plant responses to powdery mildew in legumes, we developed a pathosystem with Medicago truncatula and Erysiphe pisi. Screening accessions of M. truncatula identified genotypes that are highly susceptible, moderately resistant and highly resistant to the fungus. In the highly resistant genotype, fungal growth was arrested after appressorium development with no colony formation, while in the moderately resistant genotype a small number of colonies formed. Both resistant and moderately resistant genotypes produced hydrogen peroxide and fluorescent compounds at pathogen penetration sites, consistent with a hypersensitive response (HR), although the response was delayed in the moderately resistant genotype. Very little hydrogen peroxide or fluorescence was detected in the susceptible accession. Microarray analysis of E. pisi-induced early transcriptional changes detected 55 genes associated with the basal defence response that were similarly regulated in all three genotypes. These included pathogenesis-related genes and other genes involved in defence, signal transduction, senescence, cell wall metabolism and abiotic stress. Genes associated with the HR response included flavonoid pathway genes, and others involved in transport, transcription regulation and signal transduction. A total of 34 potentially novel unknown genes, including two legume-specific genes, were identified in both the basal response and the HR categories. Potential binding sites for two defence-related transcription regulators, Myb and Whirly, were identified in promoter regions of induced genes, and four novel motifs were found in promoter regions of genes repressed in the resistant interaction.

Soybean bacterial artificial chromosome contigs anchored with RFLPs: insights into genome duplication and gene clustering.

Surveying the soybean genome with 683 bacterial artificial chromosome (BAC) contiguous groups (contigs) anchored by restriction fragment length polymorphisms (RFLPs) enabled us to explore microsyntenic relationships among duplicated regions and also to examine the physical organization of hypomethylated (and presumably gene-rich) genomic regions. Numerous cases where nonhomologous RFLPs hybridized to common BAC clones indicated that RFLPs were physically clustered in soybean, apparently in less than 25% of the genome. By extension, we speculate that most of the genes are clustered in less than 275 M of the soybean genome. Approximately 40%-45% of this gene-rich portion is associated with the RFLP-anchored contigs described in this study. Similarities in genome organization among BAC contigs from duplicate genomic regions were also examined. Homoeologous BAC contigs often exhibited extensive microsynteny. Furthermore, paralogs recovered from duplicate contigs shared 86%-100% sequence identity.

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes.

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.

Comparative genomic analysis of sequences sampled from a small region on soybean (Glycine max) molecular linkage group G.

Eight DNA markers spanning an interval of approximately 10 centimorgans (cM) on soybean (Glycine max) molecular linkage group G (MLG-G) were used to identify bacterial artificial chromosome (BAC) clones. Twenty-eight BAC clones in eight distinct contiguous groups (contigs) were isolated from this genome region, along with 59 BAC clones on 17 contigs homoeologous to those on MLG-G. BAC clones in four of the MLG-G contigs were also digested to produce subclones and detailed physical maps. All of the BAC-ends were sequenced, as were the subclones, to estimate proportions in different sequence categories, compare similarities among homoeologs, and explore microsynteny with Arabidopsis. Homoeologous BAC contigs were enriched in repetitive sequences compared with those on MLG-G or the soybean genome as a whole. Fingerprint and cross-hybridization comparisons between MLG-G and homoeologous contigs revealed cases of highly similar physical organization between soybean duplicates, as did DNA sequence comparisons. Twenty-seven out of 78 total sequences on soybean MLG-G showed significant similarity to Arabidopsis. The homologs mapped to six compact genome segments in Arabidopsis, with the longest containing seven homologs spanning two million base pairs. These results extend previous observations of large-scale duplication and selective gene loss in Arabidopsis, suggesting that networks of conserved synteny between Arabidopsis and other angiosperm families can stretch over long physical distances.