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 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

Re-annotation of the physical map of Glycine max for polyploid-like regions by BAC end sequence driven whole genome shotgun read assembly

<p>Abstract</p> <p>Background</p> <p>Many of the world's most important food crops have either polyploid genomes or homeologous regions derived from segmental shuffling following polyploid formation. The soybean (<it>Glycine max</it>) genome has been shown to be composed of approximately four thousand short interspersed homeologous regions with 1, 2 or 4 copies per haploid genome by RFLP analysis, microsatellite anchors to BACs and by contigs formed from BAC fingerprints. Despite these similar regions,, the genome has been sequenced by whole genome shotgun sequence (WGS). Here the aim was to use BAC end sequences (BES) derived from three minimum tile paths (MTP) to examine the extent and homogeneity of polyploid-like regions within contigs and the extent of correlation between the polyploid-like regions inferred from fingerprinting and the polyploid-like sequences inferred from WGS matches.</p> <p>Results</p> <p>Results show that when sequence divergence was 1–10%, the copy number of homeologous regions could be identified from sequence variation in WGS reads overlapping BES. Homeolog sequence variants (HSVs) were single nucleotide polymorphisms (SNPs; 89%) and single nucleotide indels (SNIs 10%). Larger indels were rare but present (1%). Simulations that had predicted fingerprints of homeologous regions could be separated when divergence exceeded 2% were shown to be false. We show that a 5–10% sequence divergence is necessary to separate homeologs by fingerprinting. BES compared to WGS traces showed polyploid-like regions with less than 1% sequence divergence exist at 2.3% of the locations assayed.</p> <p>Conclusion</p> <p>The use of HSVs like SNPs and SNIs to characterize BACs wil improve contig building methods. The implications for bioinformatic and functional annotation of polyploid and paleopolyploid genomes show that a combined approach of BAC fingerprint based physical maps, WGS sequence and HSV-based partitioning of BAC clones from homeologous regions to separate contigs will allow reliable de-convolution and positioning of sequence scaffolds (see BES_scaffolds section of SoyGD). This approach will assist genome annotation for paleopolyploid and true polyploid genomes such as soybean and many important cereal and fruit crops.</p

SMDB: Soybean Marker DataBase

Soybean Marker Database (SMDB) is a repository of important genomic information for soybean. At present several genomic databases are available for plants. Some of the important oilseeds plant databases are ATPID database, Castor Bean Genome Database, CGPDB, SoyBase, Legume Information System (LIS), Brassica database, Sinbase, etc. To gain comprehensive information from varied amount of resources, we developed  this database which provides general as well as specific information at universal level. Along with this it also furnishes gene level information for various functional categories such as transcription factor, disease resistant varieties, heat shock protein, genetically modified strain of soybean. The bunch of information available to researchers today increases in tremendous manner. Hence understanding the plant genome specific databases for acquiring specific information is the demand of time for crop improvement and  research programmes. SMDB is designed for the purpose of exploring potential gene differences in different plant genotypes, including genetically modified and disease resistant crops beneficial to the farmer who cultivate this crop. SMDB is publicly accessible for academic and research purpose at: http://www.bioinfoindia.org/smdb/

Three minimum tile paths from bacterial artificial chromosome libraries of the soybean (Glycine max cv. 'Forrest'): tools for structural and functional genomics

BACKGROUND: The creation of minimally redundant tile paths (hereafter MTP) from contiguous sets of overlapping clones (hereafter contigs) in physical maps is a critical step for structural and functional genomics. Build 4 of the physical map of soybean (Glycine max L. Merr. cv. 'Forrest') showed the 1 Gbp haploid genome was composed of 0.7 Gbp diploid, 0.1 Gbp tetraploid and 0.2 Gbp octoploid regions. Therefore, the size of the unique genome was about 0.8 Gbp. The aim here was to create MTP sub-libraries from the soybean cv. Forrest physical map builds 2 to 4. RESULTS: The first MTP, named MTP2, was 14,208 clones (of mean insert size 140 kbp) picked from the 5,597 contigs of build 2. MTP2 was constructed from three BAC libraries (BamHI (B), HindIII (H) and EcoRI (E) inserts). MTP2 encompassed the contigs of build 3 that derived from build 2 by a series of contig merges. MTP2 encompassed 2 Gbp compared to the soybean haploid genome of 1 Gbp and does not distinguish regions by ploidy. The second and third MTPs, called MTP4BH and MTP4E, were each based on build 4. Each was semi-automatically selected from 2,854 contigs. MTP4BH was 4,608 B and H insert clones of mean size 173 kbp in the large (27.6 kbp) T-DNA vector pCLD04541. MTP4BH was suitable for plant transformation and functional genomics. MTP4E was 4,608 BAC clones with large inserts (mean 175 kbp) in the small (7.5 kbp) pECBAC1 vector. MTP4E was suitable for DNA sequencing. MTP4BH and MTP4E clones each encompassed about 0.8 Gbp, the 0.7 Gbp diploid regions and 0.05 Gbp each from the tetraploid and octoploid regions. MTP2 and MTP4BH were used for BAC-end sequencing, EST integration, micro-satellite integration into the physical map and high information content fingerprinting. MTP4E will be used for genome sequence by pooled genomic clone index. CONCLUSION: Each MTP and associated BES will be useful to deconvolute and ultimately finish the whole genome shotgun sequence of soybean

Soybean Genomics: Developments through the Use of Cultivar “Forrest”

Legume crops are particularly important due to their ability to support symbiotic nitrogen fixation, a key to sustainable crop production and reduced carbon emissions. Soybean (Glycine max) has a special position as a major source of increased protein and oil production in the common grass-legume rotation. The cultivar “Forrest” has saved US growers billions of dollars in crop losses due to resistances programmed into the genome. Moreover, since Forrest grows well in the north-south transition zone, breeders have used this cultivar as a bridge between the southern and northern US gene pools. Investment in Forrest genomics resulted in the development of the following research tools: (i) a genetic map, (ii) three RIL populations (96 > n > 975), (iii) ∼200 NILs, (iv) 115 220 BACs and BIBACs, (v) a physical map, (vi) 4 different minimum tiling path (MTP) sets, (vii) 25 123 BAC end sequences (BESs) that encompass 18.5 Mbp spaced out from the MTPs, and 2 000 microsatellite markers within them (viii) a map of 2408 regions each found at a single position in the genome and 2104 regions found in 2 or 4 similar copies at different genomic locations (each of >150 kbp), (ix) a map of homoeologous regions among both sets of regions, (x) a set of transcript abundance measurements that address biotic stress resistance, (xi) methods for transformation, (xii) methods for RNAi, (xiii) a TILLING resource for directed mutant isolation, and (xiv) analyses of conserved synteny with other sequenced genomes. The SoyGD portal at sprovides access to the data. To date these resources assisted in the genomic analysis of soybean nodulation and disease resistance. This review summarizes the resources and their uses

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

Using A Minimum Tile Path For Plant Transformations Encompassing the Entire Soybean Genome

Genomes like Glycine max (soybean) that have been highly conserved following increases in ploidy represent a frontier for genome analysis. Many soybean QTL analyzed to date have been composed of gene clusters each with contributing a portion of the trait rather than alleles of single genes. At the Soybean Genome Database (SoyGD) http://soybeangenome.siu.edu the genome browser that integrates and served the publicly available soybean physical map, BAC fingerprint database and genetic map associated genomic data shows a minimum tile of transformation ready BIBAC-like clones in pCLD04541 (pV41; oriV; tra; bom). Sequence resources made available through the DOE genome sequencing project have allowed the minimum tile to be revised and new functional analyses to be made. There are 3,840 MTP clones that appeared to encompass 90% of the genome (see http://soybeangenome.siu.edu/cgi-bin/gbrowse/BES_scaffolds). The BIBAC-like clones (tetR) from E. coli DH10 B were transferred en masse to Agrobacterium tumefaciens by triparental matings with EHA105 (rifR) mediated by pRK2013 (oriP) in DH10B (kanR) in 384 well plates. Although not necessary the extra helper plasmid boosted efficiency 10 fold. Individual A. tumefaciens rifampicin and tetracyclin resistant strains were used for transformation of Arabidopsis thaliana flowers in 384 well arrays. Initially kanamycin selection was used to isolate transgenic plants. Because the BACs were already tetR the recA mutants of A. tumefaciens could not be used (Tn3 insertions). Consequent to this and partial transconjugation events only some inserts are transferred completely while other transformed lines contain a substitution series of deleted inserts anchored on the Ti-left border (LB). These are maintained as kanR mixtures of seed. Phenotypes found for lines transgenic for particular BACs that were repeated include seed composition (protein, oil), development (growth, senescence) and disease resistance (suddean death syndrome (SDS) and soybean cyst nematode (SCN)

New Approaches to Selecting Resistance or Tolerance to SDS and Fusarium Root Rot

Fusarial rots are a significant problem worldwide affecting roots (and sometimes fruits) of most major crops including soybean, maize and wheat. Cultivar variation in partial resistance or tolerance is widespread and significant. Different cultivars of the soybean [Glycine max (L.) Merr.] have both resistance/tolerance to the leaf scorch known as Sudden Death Syndrome (SDS) and to the infection and root rot by the causal organism, Fusarium virguliforme (ex. F. solani f. sp glycines) hence the syndrome is composed of two diseases (1-3). Thirteen loci have been identified from analysis of 7 different crosses (2). Using new strains and new methods resistance loci in ‘Hartwig’ and ‘Forrest’, resistant cultivars clearly showed two loci underlie root resistance (lower LG G and D2) and four to eleven loci underlie leaf scorch resistance, depending on the cross made(eg, C2, F, I and upper G in ExF). Transcript abundance analysis of roots in response to F. virguliforme shows an orthologous set of transcripts accumulate during infection of resistant soybean cultivars and Arabidopsis thaliana that include the pathways leading to phenylpropanoid metabolism and its control, guanyl cylase a common second messenger and several transcription factors. Guanyl cyclase is also implicated in resistance in maize. In root disease resistance the genes implicated were known to be stress related. Therefore, A. thaliana is partially resistant and can be used to test both transgenes and mutants in candidate genes. Trangenics show fine maps to BACs have isolated some genes. For example, by fine mapping in NILs candidate genes underlying the controlling loci programming root resistance was a multi-stress resistance protein (lower G; Rfs1). For leaf scorch (Rfs4) an ascorbate peroxidase (C2) has been targeted. Also, Rfs2, a receptor like kinase (G) has been used to generate stable transgenic soybeans. Identification of the genes and loci conferring SDS resistance has provided options to breed improved cultivars with resistance to SDS

An Integrated High-density Linkage Map of Soybean with RFLP, SSR, STS, and AFLP Markers Using A Single F2 Population

Soybean [Glycine max (L.) Merrill] is the most important leguminous crop in the world due to its high contents of high-quality protein and oil for human and animal consumption as well as for industrial uses. An accurate and saturated genetic linkage map of soybean is an essential tool for studies on modern soybean genomics. In order to update the linkage map of a F2 population derived from a cross between Misuzudaizu and Moshidou Gong 503 and to make it more informative and useful to the soybean genome research community, a total of 318 AFLP, 121 SSR, 108 RFLP, and 126 STS markers were newly developed and integrated into the framework of the previously described linkage map. The updated genetic map is composed of 509 RFLP, 318 SSR, 318 AFLP, 97 AFLP-derived STS, 29 BAC-end or EST-derived STS, 1 RAPD, and five morphological markers, covering a map distance of 3080 cM (Kosambi function) in 20 linkage groups (LGs). To our knowledge, this is presently the densest linkage map developed from a single F2 population in soybean. The average intermarker distance was reduced to 2.41 from 5.78 cM in the earlier version of the linkage map. Most SSR and RFLP markers were relatively evenly distributed among different LGs in contrast to the moderately clustered AFLP markers. The number of gaps of more than 25 cM was reduced to 6 from 19 in the earlier version of the linkage map. The coverage of the linkage map was extended since 17 markers were mapped beyond the distal ends of the previous linkage map. In particular, 17 markers were tagged in a 5.7 cM interval between CE47M5a and Satt100 on LG C2, where several important QTLs were clustered. This newly updated soybean linkage map will enable to streamline positional cloning of agronomically important trait locus genes, and promote the development of physical maps, genome sequencing, and other genomic research activities