Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones (∼4× genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified ∼2400 BACs and ordered ∼700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located ∼200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in ∼65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome.[The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF218263.]Integrated genetic and physical genome maps are extremely valuable for map-based gene isolation, comparative genome analysis, and as sources of sequenceready clones for genome sequencing projects. Various methods have been developed for assembling physical maps of complex genomes. One of the best characterized approaches uses restriction enzymes to generate large numbers of DNA fragments from genomic subclones (Brenner and Livak 1989;Gregory et al. 1997;Marra et al. 1997). These DNA fingerprints are compared to identify related clones, and to assemble overlapping clones in contigs. The utility of fingerprinting for ordering a complex genome is limited, however, due to variation in DNA migration from gel to gel, the presence of repetitive DNAs, unusual distribution of restriction sites and skewed clone representation. Moreover, fingerprinting, unless combined with other methods, does not link genomic clones directly to gen...

This cross-national study examined preparation for and psychological functioning following Hurricane Georges in the U.S. Virgin Islands, Puerto Rico, Dominican Republic, and the United States. Four to five weeks after the storm made landfall, 697 college students (222 men, 476 women) completed a questionnaire assessing demographic characteristics, preparation, social support, resource loss, and symptoms associated with acute stress disorder. Location, resource loss (especially personal characteristic resources) and social support accounted for a significant portion of psychological distress variance. The findings support the conservation of resources stress theory (Hobfoll, 1989, 1998). Implications of the findings and future research directions are discussed.

The ω‐6 fatty acid desaturase (FAD2) gene family in soybean [Glycine max (L.) Merr.] consists of at least five members in four regions of the genome and are responsible for the conversion of oleic acid to linoleic acid. Here we report the identification of two new ω‐6 fatty acid desaturase (FAD2) gene copies from soybean expressed sequence tags (ESTs). Four bacterial artificial chromosomes (BACs) containing five FAD2 genes were sequenced to investigate structural and functional conservation between duplicate loci. Sequence comparisons show that the soybean genome is a mosaic, with some duplicate regions retaining high sequence conservation in both genic and intergenic regions, while others have only the FAD2 genes in common. Genetic mapping using SSRs from within the BAC sequences showed that two BACs with high sequence homeology mapped to linkage groups I and O; these groups share syntenic markers. Another BAC mapped to linkage group L. The fourth BAC could not be mapped. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis of the five FAD2 genes showed that the FAD2‐2B and FAD2‐2C copies were the best candidates for temperature‐dependent expression changes in developing pod tissue. Semiquantitative RT‐PCR confirmed these results, with FAD2‐2C showing upward of an eightfold increase in expression in developing pods grown in cooler conditions relative to those grown in warm conditions. The implications of these results suggest a candidate gene for controlling the levels of linoleic acid in developing pods grown in cooler climates.

PlantGDB (http://www.plantgdb.org/) is a database of molecular sequence data for all plant species with significant sequencing efforts. The database organizes EST sequences into contigs that represent tentative unique genes. Contigs are annotated and, whenever possible, linked to their respective genomic DNA. Genome sequence fragments are assembled similarly. The goal of the PlantGDB web site is to establish the basis for identifying sets of genes common to all plants or specific to particular species by integrating a number of bioinformatics tools that facilitate gene prediction and cross- species comparisons. For species with large-scale genome sequencing efforts, PlantGDB provides genome browsing capabilities that integrate all available EST and cDNA evidence for current gene models (for Arabidopsis thaliana, see the AtGDB site at http://www.plantgdb.org/AtGDB/).

Background: Soybean, Glycine max (L.) Merr., is a well documented paleopolyploid. What remains relatively under characterized is the level of sequence identity in retained homeologous regions of the genome. Recently, the Department of Energy Joint Genome Institute and United States Department of Agriculture jointly announced the sequencing of the soybean genome. One of the initial concerns is to what extent sequence identity in homeologous regions would have on whole genome shotgun sequence assembly.

Expressed sequence tags (ESTs) currently encompass more entries in the public databases than any other form of sequence data. Thus, EST data sets provide a vast resource for gene identification and expression profiling. We have mapped the complete set of 176,915 publicly available Arabidopsis EST sequences onto the Arabidopsis genome using GeneSeqer, a spliced alignment program incorporating sequence similarity and splice site scoring. About 96% of the available ESTs could be properly aligned with a genomic locus, with the remaining ESTs deriving from organelle genomes and non-Arabidopsis sources or displaying insufficient sequence quality for alignment. The mapping provides verified sets of EST clusters for evaluation of EST clustering programs. Analysis of the spliced alignments suggests corrections to current gene structure annotation and provides examples of alternative and non-canonical pre-mRNA splicing. All results of this study were parsed into a database and are accessible via a flexible Web interface at http://www.plantgdb.org/AtGDB/.The efforts of an international collaboration to obtain the complete genome sequence of the flowering plant Arabidopsis resulted in the release and annotation of 115.4 Mb of the genome (estimated at 125 Mb) in December of 2000 (Arabidopsis Genome Initiative, 2000). At that time, 25,498 protein-coding genes were identified in the five haploid chromosomes, but only 9% of these genes had been characterized experimentally, and only 69% could be functionally classified by similarity to proteins of known functions. In the interim, sequencing and annotation has progressed. The most current release of the Arabidopsis genome available at GenBank provides 117.3 Mb and 27,288 annotated protein-coding genes (see Data Sets in "Materials and Methods"). Annotation of the Arabidopsis genome and functional characterization of all the genes is an ongoing effort. Initial, high-throughput computational gene structure prediction has likely been successful in identifying most gene locations; however, these methods still suffer from limitations in predicting the precise gene structure for an entire gene, detection of intergenic regions, and identification of non-coding exon sequences (Pavy et al., 1999;Brendel and Zhu, 2002). Recent studies have concentrated on sequencing of full-length cDNAs to improve genome annotation Seki et al., 2002).Expressed sequence tags (ESTs) are single-pass sequencing reads of cDNA clones that have become a widely employed method for gene identification, expression profiling, and polymorphism analysis. Presently, more than 13.4 million EST entries have been deposited into the National Center for Biotechnology Information (NCBI) dbEST public database, including Arabidopsis with 176,915 ESTs and 21 other species with EST sets of more than 100,000 entries (http://www.ncbi.nlm.nih.gov/dbEST/ dbEST_summary.html). In the absence of a wholegenome sequencing project for a particular species, clustering of ESTs into contigs that represent unique genes is one of the most promi...

PlantGDB (http://www.plantgdb.org/) is a database of plant molecular sequences. Expressed sequence tag (EST) sequences are assembled into contigs that represent tentative unique genes. EST contigs are functionally annotated with information derived from known protein sequences that are highly similar to the putative translation products. Tentative Gene Ontology terms are assigned to match those of the similar sequences identified. Genome survey sequences are assembled similarly. The resulting genome survey sequence contigs are matched to ESTs and conserved protein homologs to identify putative full-length open reading frame-containing genes, which are subsequently provisionally classified according to established gene family designations. For Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), the exon-intron boundaries for gene structures are annotated by spliced alignment of ESTs and full-length cDNAs to their respective complete genome sequences. Unique genome browsers have been developed to present all available EST and cDNA evidence for current transcript models (for Arabidopsis, see the AtGDB site at http://www.plantgdb.org/AtGDB/; for rice, see the OsGDB site at http://www.plantgdb.org/OsGDB/). In addition, a number of bioinformatic tools have been integrated at PlantGDB that enable researchers to carry out sequence analyses on-site using both their own data and data residing within the database.

The paleopolyploid soybean genome was investigated by sequencing homeologous BAC clones anchored by duplicate N-hydroxycinnamoyl/benzoyltransferase (HCBT) genes. The homeologous BACs were genetically mapped to linkage groups C1 and C2. Annotation of the 173,747-and 98,760-bp BACs showed that gene conservation in both order and orientation is high between homeologous regions with only a single gene insertion/deletion and local tandem duplications differing between the regions. The nucleotide sequence conservation extends into intergenic regions as well, probably due to conserved regulatory sequences. Most of the homeologs appear to have a role in either transcription/DNA binding or cellular signaling, suggesting a potential preference for retention of duplicate genes with these functions. Reverse transcriptase-PCR analysis of homeologs showed that in the tissues sampled, most homeologs have not diverged greatly in their transcription profiles. However, four cases of changes in transcription were identified, primarily in the HCBT gene cluster. Because a mapped locus corresponds to a soybean cyst nematode (SCN) QTL, the potential role of HCBT genes in response to SCN is discussed. These results are the first sequenced-based analysis of homeologous BACs in soybean, a diploidized paleopolyploid.