No abstract

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

The U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS) tetraploid cherry (Prunus L. sp.) collection at Geneva, N.Y., contains ≈75 accessions of sour cherry (P. cerasus L.), ground cherry (P. fruticosa Pall.), and their hybrids. Accurate and unambiguous identification of these accessions is essential for germplasm preservation and use. Simple sequence repeats (SSRs) are currently the markers of choice for germplasm fingerprinting because they characteristically display high levels of polymorphism. Recently SSR primer pairs from sweet cherry (P. avium L.), sour cherry, and peach [(P. persica L. Batsch (Peach Group)] have been reported. Ten SSR primer pairs were tested on 59 tetraploid cherry accessions to determine if they could differentiate among the accessions. Scorable SSR fragments were produced with all primer-accession combinations. The cherry accessions exhibited high levels of polymorphism with 4 to 16 different putative alleles amplified per primer pair. Most of the putative alleles were rare with frequencies <0.05. Heterozygosity values ranged from 0.679 to 1.00, while gene diversity values ranged from 0.655 to 0.906. The primer pairs differentiated all but two of the 59 cherry accessions. Based upon the ability of the SSR data to differentiate the cherry accessions and the high level of gene diversity, we propose that all the tetraploid cherry accessions in the USDA/ARS collection be fingerprinted to provide a mechanism to verify the identity of the individual accessions. The fingerprinting data are available on the World Wide Web (http://www.ars-grin.gov/gen/cherry.html) so that other curators and scientists working with cherry can verify identities and novel types in their collections and contribute to a global database.

A diverse collection of 133 Malus species and hybrids from the USDA Plant Genetic Resources Unit's core subset collection was screened with five simple sequence repeat (SSR) primer pairs in order to determine genetic identities and overall levels of genetic variation. The number of amplification products (alleles) per locus (primer pair) in this collection ranged from 6 to 39, with some genotypes showing complex banding patterns of up to four products per locus, suggesting that duplication events may have occurred within the genome. Five primer sets unequivocally differentiated all but 10 pairs of genotypes in the collection, with seven of these 10 being pairs of the same species. Within three of the species holdings surveyed, M. honanensis, M. sargentii, and M. sikkimensis, no genetic variation was revealed with the SSR markers. The discrimination power for the combined loci in this collection was nearly one, which indicates that the likelihood of two genetically different accessions sharing the same alleles at all the loci included in this study would be nearly impossible. Coupled with results from a previous survey of M. × domestica accessions, this finding suggests that with five SSR primer pairs, the majority of the Malus holdings could be assigned a unique fingerprint identity. The average direct count heterozygosity over all loci was 0.620, ranging in value from 0.293 to 0.871 over individual loci. These heterozygosity counts will be compared with a survey of naturally occurring M. sieversii to determine whether current repository holdings are representative of the overall levels of diversity occurring in Malus. Information generated with this study, coupled with passport and horticultural data will inform curatorial decisions regarding deaccessioning of duplicate holdings and plans for future germplasm collections.

Random amplified polymorphic DNA (RAPD) markers have been used for many types of genetic analyses, including genome mapping, genotype fingerprinting, phylogeny reconstruction, and measuring genetic similarities. They suffer from one potential limitation, however, because the PCR that is used to produce informative amplification products often produces artifactual products as well. Optimization of PCR protocols to eliminate artifactual bands completely is often too costly or too timeconsuming to be practical. Other methods for handling RAPD artifacts, such as deleting inconsistent or faint bands or using only those bands that are reproducible, introduce false negatives into the data. Simply ignoring artifacts and using all bands introduces false positives. When RAPD data are used to compute genetic similarity coefficients, such artifacts can cause significant bias in the estimation. The three coefficients most widely used with RAPD data, the simple matching coefficient, Jaccard's coefficient and Nei and Li's coefficient, differ in the amount of bias produced by a given level of artifactual bands. The simple matching coefficient and Nei and Li's coefficient always exhibit less percent bias than Jaccard's coefficient. For closely related organisms, Nei and Li's coefficient displays less percent bias than the simple matching coefficient. If new DNA samples possessing RAPD markers not present in the previously analyzed samples are added to a study, values of the simple matching coefficient will need to be computed for all samples, not just the new ones. Jaccard's and Nei and Li's coefficients, however, will not need to be recomputed. Furthermore, only Nei and Li's coefficient has a direct biological meaning (it is an estimate of the expected proportion of amplified fragments shared by two samples hecause they were inherited from a common ancestor). On the basis of these results, Nei and Li's coefficient is recommended for routine computation of genetic similarities using RAPD data, particularly if PCR artifacts are present.Random amplified polymorphic DNA (RAPD) markers ~ are well-established genetic tools. They have been used for genomic mapping and linkage analysis, ~3-6~ phylogeny reconstruction, ~7' 8~ genotype fingerprinting and identification, ~9-~4~ and determining genetic relationships, similarities, and genetic variation. (ls--17) RAPD bands are produced by PCR, using a single random primer that amplifies segments of DNA flanked by two primer-binding regions that theoretically are exactly complementary to the primer. The primer-binding sites must be close enough that amplification proceeds over the entire DNA segment spanning them. Because of base pair mismatch, though, a single base change in the genomic DNA can prevent amplification. ~2~ Most often, polymorphisms between different DNA samples occur when a segment that is amplified in a sample whose primer-binding site is exactly complementary to the primer is not amplified in another sample whose priming site is not an exact complement to the primer/2'18...