Targeted isolation, sequence analysis, and physical mapping of nonTIR NBS-LRR genes in soybean.

Most cloned plant disease resistance genes (R-genes) code for proteins belonging to the nucleotide binding site (NBS) leucine-rich repeat (LRR) superfamily. NBS-LRRs can be divided into two classes based on the presence of a TIR domain ( Toll and interleukin receptor-like sequence) or a coiled coil motif (nonTIR) in their N-terminus. We used conserved motifs specific to nonTIR-NBS-LRR sequences in a targeted PCR approach to generate nearly 50 genomic soybean sequences with strong homology to known resistance gene analogs (RGAs) of the nonTIR class. Phylogenetic analysis classified these sequences into four main subclasses. A representative clone from each subclass was used for genetic mapping, bacterial artificial chromosome (BAC) library screening, and construction of RGA-containing BAC contigs. Of the 14 RGAs that could be mapped genetically, 12 localized to a 25-cM region of soybean linkage group F already known to contain several classical disease resistance loci. A majority of the genomic region encompassing the RGAs was physically isolated in eight BAC contigs, together spanning more than 1 Mb of genomic sequence with at least 12 RGA copies. Phylogenetic and sequence analysis, together with genetic and physical mapping, provided insights into the genome organization and evolution of this large cluster of soybean RGAs.

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

Targeted comparative genome analysis and qualitative mapping of a major partial-resistance gene to the soybean cyst nematode.

A major partial-resistance locus to the soybean cyst nematode (Heterodera glycines Ichinohe; SCN) was identified on linkage group 'G' of soybean [Glycine max (L.) Merr.] using restriction fragment length polymorphisms (RFLPs). This locus explained 51.4% (LOD=10.35) of the total phenotypic variation in disease response in soybean Plant Introduction (PI) 209332, 52.7% (LOD=15.58) in PI 90763, 40.0% (LOD=10.50) in PI 88788, and 28.1% (LOD=6.94) in 'Peking'. Initially, the region around this major resistance locus was poorly populated with DNA markers. To increase marker density in this genomic region, first random, and later targeted, comparative mapping with RFLPs from mungbean [Vigna radiata (L.) R. Wilcz.] and common bean (Phaseolus vulgaris L.) was performed, eventually leading to one RFLP marker every 2.6 centimorgans (cM). Even with this marker density, the inability to resolve SCN disease response into discrete Mendelian categories posed a major limitation to mapping. Thus, qualitative scoring of SCN disease response was carried out in an F5∶6 recombinant inbred population derived from 'Evans'xPI 209332 using a 30% disease index cut-off for resistance. Using the computer program JoinMap, an integrated map of the region of interest was created, placing the SCN resistance locus 4.6 cM from RFLP marker B53 and 2.8 cM from Bng30. This study demonstrates how a combination of molecularmapping strategies, including comparative genome analysis, join mapping, and qualitative scoring of a quantitative trait, potentially provide the necessary tools for high-resolution mapping around a quantitative-trait locus.

Genetic dissection of oligogenic resistance to bacterial wilt in tomato.

To study resistance to bacterial wilt (caused by Pseudomonas solanacearum) in tomato, we analyzed 71 F2 individuals from a cross between a resistant and a susceptible parent with 79 DNA markers. F2 plants were inoculated by two methods: bacteria were injected into shoots of cuttings or poured into soil surrounding wounded roots. Disease responses were scored on a scale of 0 to 5. Statistical comparisons between DNA marker genotypes and disease phenotypes identified three genomic regions correlated with resistance. In plants inoculated through roots, genomic regions on chromosomes 6 and 10 were correlated with resistance. In plants inoculated through shoots, a region on chromosome 7 was significant, as were the regions on chromosomes 6 and 10. The relative impact of resistance loci on disease response differed between shoot and root inoculations. To confirm the existence of a partial resistance gene on chromosome 6, an F2 individual homozygous for the resistant parent's alleles on chromosomes 7 and 10, but heterozygous for markers on chromosome 6, was selfed. Analysis of the F3 progeny confirmed that a partial resistance locus was located on chromosome 6, very close to CT184. The presence of a partial resistance locus on chromosome 10 was similarly confirmed by analysis of progeny of another F2 plant chosen on the basis of its marker phenotype.

Mapping oligogenic resistance to powdery mildew in mungbean with RFLPs.

We have used restriction fragment length polymorphisms (RFLPs) to map genes in mungbean (Vigna radiata) that confer partial resistance to the powdery mildew fungus, Erysiphe polygoni. DNA genotypes for 145 RFLP loci spanning 1570 centimorgans of the mungbean genome were assayed in a population of 58 F2 plants. This population was derived from a cross between a moderately powdery mildew resistant ("VC3980A") and a susceptible ("TC1966") mungbean parent. F3 lines derived from the F2 plants were assayed in the field for powdery mildew response and the results were compared to the RFLP genotype data, thereby identifying loci associated with powdery mildew response. A total of three genomic regions were found to have an effect on powdery mildew response, together explaining 58% of the total variation. At 65 days after planting, two genomic regions were significantly associated with powdery mildew resistance. For both loci, the allele from "VC3890A" was associated with increased resistance. At 85 days, a third genomic region was also associated with powdery mildew response. For this locus, the allele from the susceptible parent ("TC1966") was the one associated with higher levels of powdery mildew resistance. These results indicate that putative partial resistance loci for powdery mildew in mungbean can be identified with DNA markers, even in a population of modest size analyzed at a single location in a single year.

Molecular taxonomic relationships in the genus Vigna based on RFLP analysis.

The taxonomy of the genus Vigna has been primarily based on morphological attributes. We have used 27 genomic clones from soybean, common bean, mungbean and cowpea to examine restriction fragment length polymorphism (RFLP) among 44 accessions of different species belonging to four subgenera of the genus Vigna. One accession each of common bean (Phaseolus vulgaris) and soybean (Glycine max) was included in the study. Total DNA from the various genotypes was digested with one restriction enzyme (Eco RV). Results of a numerical taxonomic analysis showed a high level of genetic variation within the genus with a remarkably higher amount of variation associated with Vigna sp. from Africa relative to those from Asia. The distinctness of the Asiatic grams in subgenus Ceratotropis, cowpea in section Catiang, bambara groundnut (V. subterranean) and members of the subgenus Plectotropis was elucidated by this study. Members of the subgenus Plectotropis were closer in genome homology to those of subgenus Vigna section Catiang than to those of subgenus Ceratotropis. The relative positions of some genotypes to one another on the dendrogram and minimum spanning tree were discussed in regard to hybridisations aimed generating well-saturated genomic maps and interspecies transfer of desirable genes.

Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data.

Genome relationships between mungbean (Vigna tradiata) and cowpea (V. Unguiculata) based on the linkage arrangement of random genomic restriction fragment length polymorphism (RFLP) markers have been investigated. A common set of probes derived from cowpea, common bean (Phaseolus vulgaris), mungbean, and soybean (Glycine max) PstI genomic libraries were used to construct the genetic linkage maps. In both species, a single F2 population from a cross between an improved cultivar and a putative wild progenitor species was used to follow the segregation of the RFLP markers. Approximately 90% of the probes hybridized to both mungbean and cowpea DNA, indicating a high degree of similarity in the nucleotide sequences among these species. A higher level of polymorphism was detected in the mungbean population (75.7%) than in the cowpea population (41.2%). Loci exhibiting duplications, null phenotypes, and distorted segregation ratios were detected in both populations. Random genomic DNA RFLP loci account for about 89% of the currently mapped markers with a few cDNA and RAPD markers added. The current mungbean map is comprised of 171 loci/loci clusters distributed in 14 linkage groups spanning a total of 1570cM. On the other hand, 97 markers covered 684 cM and defined 10 linkage groups in the current cowpea map. The mungbean and cowpea genomes were compared on the basis of the copy number and linkage arrangement of 53 markers mapped in common between the two species. Results indicate that nucleotide sequences are conserved, but variation in copy number were detected and several rearrangements in linkage orders appeared to have occurred since the divergence of the two species. Entire linkage groups were not conserved, but several large linkage blocks were maintained in both genomes.

Evidence for orthologous seed weight genes in cowpea and mung bean based on RFLP mapping.

A well saturated genomic map is a necessity for a breeding program based on marker assisted selection. To this end, we are developing genomic maps for cowpea (Vigna unguiculata 2N = 22) and mung bean (Vigna radiata 2N = 22) based on restriction fragment length polymorphism (RFLP) markers. Using these maps, we have located major quantitative trait loci (QTLs) for seed weight in both species. Two unlinked genomic regions in cowpea contained QTLs accounting for 52.7% of the variation for seed weight. In mung bean there were four unlinked genomic regions accounting for 49.7% of the variation for seed weight. In both cowpea and mung bean the genomic region with the greatest effect on seed weight spanned the same RFLP markers in the same linkage order. This suggests that the QTLs in this genomic region have remained conserved through evolution. This inference is supported by the observation that a significant interaction (i.e., epistasis) was detected between the QTL(s) in the conserved region and an unlinked RFLP marker locus in both species.

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata, L. Wilczek).

Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F2 progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F2 population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.

Toxic aspergilli from pistachio nuts.

Pistachio nut samples taken during various stages of development from orchards in Iran, showed that contamination with fungi occurred mainly during the later stages of nut development. Members of the genera Aspergillus and Penicillium occurred most frequently. Of the Aspergilli, the species A. niger, A. flavus and A. fischeri var. spinosus occurred most frequently, followed by A. terreus, A. tamarii and A. nidulans. Twenty-two isolates comprising 13 species were tested for toxicity to ducklings. Isolates of known toxic fungi included A. flavus, A. niger, A. parasiticus, A. ochraceus, A. versicolor, A. nidulans and A. terreus. The toxicity of A. fischeri var. spinosus is reported. Chemical analysis showed that all isolates of A. flavus and A. parasiticus produced aflatoxin B1, the isolates of A. versicolor and A. nidulans produced sterigmatocystin while the toxic isolate of A. ochraceus did not produce ochratoxins.