Development of a pooled probe method for locating small gene families in a physical map of soybean using stress related paralogues and a BAC minimum tile path.

BACKGROUND: Genome analysis of soybean (Glycine max L.) has been complicated by its paleo-autopolyploid nature and conserved homeologous regions. Landmarks of expressed sequence tags (ESTs) located within a minimum tile path (MTP) of contiguous (contig) bacterial artificial chromosome (BAC) clones or radiation hybrid set can identify stress and defense related gene rich regions in the genome. A physical map of about 2,800 contigs and MTPs of 8,064 BAC clones encompass the soybean genome. That genome is being sequenced by whole genome shotgun methods so that reliable estimates of gene family size and gene locations will provide a useful tool for finishing. The aims here were to develop methods to anchor plant defense- and stress-related gene paralogues on the MTP derived from the soybean physical map, to identify gene rich regions and to correlate those with QTL for disease resistance. RESULTS: The probes included 143 ESTs from a root library selected by subtractive hybridization from a multiply disease resistant soybean cultivar 'Forrest' 14 days after inoculation with Fusarium solani f. sp. glycines (F. virguliforme). Another 166 probes were chosen from a root EST library (Gm-r1021) prepared from a non-inoculated soybean cultivar 'Williams 82' based on their homology to the known defense and stress related genes. Twelve and thirteen pooled EST probes were hybridized to high-density colony arrays of MTP BAC clones from the cv. 'Forrest' genome. The EST pools located 613 paralogues for 201 of the 309 probes used (range 1-13 per functional probe). One hundred BAC clones contained more than one kind of paralogue. Many more BACs (246) contained a single paralogue of one of the 201 probes detectable gene families. ESTs were anchored on soybean linkage groups A1, B1, C2, E, D1a+Q, G, I, M, H, and O. CONCLUSION: Estimates of gene family sizes were more similar to those made by Southern hybridization than by bioinformatics inferences from EST collections. When compared to Arabidopsis thaliana there were more 2 and 4 member paralogue families reflecting the diploidized-tetraploid nature of the soybean genome. However there were fewer families with 5 or more genes and the same number of single genes. Therefore the method can identify evolutionary patterns such as massively extensive selective gene loss or rapid divergence to regenerate the unique genes in some families.

The Soybean Genome Database (SoyGD): a browser for display of duplicated, polyploid, regions and sequence tagged sites on the integrated physical and genetic maps of Glycine max.

Genomes that have been highly conserved following increases in ploidy (by duplication or hybridization) like Glycine max (soybean) present challenges during genome analysis. At http://soybeangenome.siu.edu the Soybean Genome Database (SoyGD) genome browser has, since 2002, integrated and served the publicly available soybean physical map, bacterial artificial chromosome (BAC) fingerprint database and genetic map associated genomic data. The browser shows both build 3 and build 4 contiguous sets of clones (contigs) of the soybean physical map. Build 4 consisted of 2854 contigs that encompassed 1.05 Gb and 404 high-quality DNA markers that anchored 742 contigs. Many DNA markers anchored sets of 2-8 different contigs. Each contig in the set represented a homologous region of related sequences. GBrowse was adapted to show sets of homologous contigs at all potential anchor points, spread laterally and prevented from overlapping. About 8064 minimum tiling path (MTP2) clones provided 13,473 BAC end sequences (BES) to decorate the physical map. Analyses of BES placed 2111 gene models, 40 marker anchors and 1053 new microsatellite markers on the map. Estimated sequence tag probes from 201 low-copy gene families located 613 paralogs. The genome browser portal showed each data type as a separate track. Tetraploid, octoploid, diploid and homologous regions are shown clearly in relation to an integrated genetic and physical map.

Root response to Fusarium solani f. sp . glycines: temporal accumulation of transcripts in partially resistant and susceptible soybean.

Sudden death syndrome (SDS) of soybean is a complex of root rot disease caused by the semi-biotrophic fungus Fusarium solani f. sp. glycines (Fsg) and a leaf scorch disease caused by toxins produced by the pathogen in the roots. Development of partial rate-reducing resistance in roots to SDS was studied. The recombinant inbred line 23 (RIL23) that carried resistance conferred by six quantitative trait loci (QTL) derived from cultivars 'Essex' x 'Forrest' was compared to the susceptible cultivar Essex. Roots of RIL23 and its susceptible parent Essex were inoculated with Fsg. Transcript abundance (TA) of 191 ESTs was studied at five time points after inoculation. For most of the genes, there was an initial decrease in TA in the inoculated roots of both genotypes. By days 7 and 10 the inoculated roots of Essex failed to increase expression of the transcripts of defense-related genes. In RIL23 inoculated roots, the TA of 81 genes was increased by at least two-fold at day 3 (P=0.004), 88 genes at day 7 (P=0.0023) and 129 genes at day 10 (P=0.0026). A set of 35 genes maintained at least a two-fold higher abundance at all three time points. The increase in TA in RIL23 was in contrast to that observed in Essex where most of the ESTs showed either no change or a decreased TA. The ESTs with an increased TA had homology to the genes involved in resistance (analogs), signal transduction, plant defense, cell wall synthesis and transport of metabolites. Pathways that responded included the protein phosphorylation cascade, the phospholipase cascade and the phenolic natural products pathways, including isoflavone and cell wall synthesis.