Genome-wide physical maps are crucial to many aspects of advanced genome research. We report a genome-wide, bacterial artificial chromosome (BAC) and plant-transformation-competent binary large-insert plasmid clone (hereafter BIBAC)-based physical map of the soybean genome. The map was constructed from 78,001 clones from five soybean BAC and BIBAC libraries representing 9.6 haploid genomes and three cultivars, and consisted of 2905 BAC/BIBAC contigs, estimated to span 1408 Mb in physical length. We evaluated the reliability of the map contigs using different contig assembly strategies, independent contig building methods, DNA marker hybridization, and different fingerprinting methods, and the results showed that the contigs were assembled properly. Furthermore, we tested the feasibility of integrating the physical map with the existing soybean composite genetic map using 388 DNA markers. The results further confirmed the nature of the ancient tetraploid origin of soybean and indicated that it is feasible to integrate the physical map with the linkage map even though greater efforts are needed. This map represents the first genome-wide, BAC/BIBAC-based physical map of the soybean genome and would provide a platform for advanced genome research of soybean and other legume species. The inclusion of BIBACs in the map would streamline the utility of the map for positional cloning of genes and QTLs, and functional analysis of soybean genomic sequences.

Two plant-transformation-competent large-insert binary clone bacterial artificial chromosome (hereafter BIBAC) libraries were previously constructed for soybean cv. Forrest, using BamHI or HindIII. However, they are not well suited for clone-based genomic sequencing due to their larger ratio of vector to insert size (27.6 kbp:125 kbp). Therefore, we developed a larger-insert bacterial artificial chromosome (BAC) library for the genotype in a smaller vector (pECBAC1), using EcoRI. The BAC library contains 38,400 clones; about 99.1% of the clones have inserts; the average insert size is 157 kbp; and the ratio of vector to insert size is much smaller (7.5 kbp:157 kbp). Colony hybridization with probes derived from several chloroplast and mitochondrial genes showed that 0.89% and 0.45% of the clones were derived from the chloroplast and mitochondrial genomes, respectively. Considering these data, the library represents 5.4 haploid genomes of soybean. The library was hybridized with six RFLP marker probes, 5S rDNA and 18S-5.8S-25S rDNA, respectively. Each RFLP marker hybridized to about six clones, and the 5S and 18S-5.8S-25S rDNA probes collectively hybridized to 402 BACs--about 1.05% of the clones in the library. The BAC library complements the existing soybean Forrest BIBAC libraries by using different restriction enzymes and vector systems. Together, the BAC and BIBAC libraries encompass 13.2 haploid genomes, providing the most comprehensive clone resource for a single soybean genotype for public genome research. We show that the BAC library has enhanced the development of the soybean whole-genome physical map and use of three complementary BAC libraries improves genome physical mapping by fingerprint analysis of most of the clones of the library. The rDNA-containing clones were also fingerprinted to evaluate the feasibility of constructing contig maps of the rDNA regions. It was found that physical maps for the rDNA regions could not be readily constructed by fingerprint analysis, using one or two restriction enzymes. Additional data to fingerprints and/or different fingerprinting methods are needed to build contig maps for such highly tandem repetitive regions and thus, the physical map of the entire soybean genome.

Background: A recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, Peronosclerospora sorghi. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.

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Thirty-one accessions of Citrullus spp. belonging to Citrullus lanatus var. lanatus, C. lanatus var. citroides and Citrullus colocynthis were subjected to phylogenetic analysis using combined datasets of amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs). Tree topologies inferred by neighbour-joining analysis have resolved the phylogenic relationships among the species with special reference to established taxonomic classification. In this study, we have clearly resolved species boundaries of various taxa of citroides, lanatus and colocynthis into three well-supported clusters. Clustering pattern of principal component analysis with the shared polymorphisms using the subsets of data between any two taxon combinations helped to elucidate the introgression and interrelationships among the species. We report two major groups of C. lanatus taxa, one of which has undergone wide introgressions with the taxa of C. lanatus var. citroides and C. colocynthis. In this paper, we identified 583 AFLP bands that are polymorphic within the var. lanatus of C. lanatus, which is the largest set ever reported. The species-specific diagnostic SSRs and polymorphic AFLPs that are informative within and between the taxa reported in this paper would be immensely useful for future studies of these economically important genera.

Thirty-eight melon accessions, which are of primary breeding importance in the Ukraine, were analysed for diversity. These collections represent a major non-US and non-western Europe source of melon germplasm that have not yet been subjected to molecular characterization. Molecular diversity was estimated based on a robust set of 465 polymorphisms gathered by amplified fragment length polymorphisms and simple sequence repeats (SSR). In this paper, we report 12 newly developed polymorphic SSR primer pairs, and their use for molecular characterization in the Ukrainian melon collections. Based on these polymorphisms, we estimated similarity indices that ranged from 0.70 to 1.00 among various accessions. The phylogenetic tree based on the similarity indices and a three-dimensional plot of the first three vectors of the principal component analysis corresponded fairly well with the existing three classical morphotypes namelyaestivalis,europeusandhiemalis, under theconvarEuropeus, which is also known asadana. The polymorphisms generated in the current study, which are specific to the grouping of fruit types and days to maturity will be very useful for further genetic studies and marker-assisted selections.

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Chilepepper (Capsicum spp.) is the third most important vegetable crop in the United States. The market value of chile peppers for spices and condiments exceeds $650 million per year. With a growing Hispanic population across the United States, the demand for high yielding, good quality cayenne pepper continues to increase. In order to fulfill this niche market, a study has been initiated to develop pepper varieties that combine high yield potential with superior agronomic traits, including insect and disease resistance, and fruit characteristics, using molecular marker assisted breeding/selection. In preliminary trials, several F1 generations were created through inter- and intra-specific crosses among 220 germplasm lines belonging to six Capsicumsp. in the greenhouse. Selected F1 progeny, parent lines, and selected accessions were planted in single-row field plots the following summer. The crossing success was higher within species than between. The genotypic variation was significant for all parameters examined. The average percent germination (81.1) of F1 progeny was 32% and 45% higher than that of the parent lines and selected accessions, respectively. The F1 progeny were shorter in height; more vigorous in growth, flowered early, and with fewer, but heavier, fruits per plant out-yielded the parent lines and accessions by 50% and 120%, respectively. The study showed a marked heterosis in F1 progeny compared to the parent lines and accessions. Microsatellite genotyping to estimate genetic diversity and validation of markers that are linked to various traits is in progress and will be discussed in the presentation.