Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

<p>Abstract</p> <p>Background</p> <p>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, <it>Peronosclerospora sorghi</it>. 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.</p> <p>Results</p> <p>Among the 55 primers pairs designed from clones from pathotype 3 of <it>P. sorghi</it>, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from <it>P. sorghi</it>, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (<it>P. maydis & P. philippinensis</it>), sugar cane (<it>P. sacchari</it>), pearl millet (<it>Sclerospora graminicola</it>) and rose (<it>Peronospora sparsa</it>) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to <it>P. philippinensis </it>(one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 <it>Peronosclerospora, Peronospora and Sclerospora </it>spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of <it>Peronosclerospora sorghi </it>in cluster I, five isolates of <it>P. maydis </it>and three isolates of <it>P. sacchari </it>in cluster II and five isolates of <it>Sclerospora graminicola </it>in cluster III).</p> <p>Conclusion</p> <p>To our knowledge, this is the first attempt to extensively develop SSR markers from <it>Peronosclerospora </it>genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.</p

Running Title: A Physical Map of the Soybean Genome

ABSTRACT- 2-Wu et al. We report a genome-wide, bacterial artificial chromosome (BAC) and planttransformation-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. The map consisted of 2,905 BAC/BIBAC contigs, and was estimated to span 1,408 Mb in physical length. The physical length is about 293 Mb greater than the expected 1,115-Mb genome size of the species, indicating that most, if not all, of the contigs remain overlapped. We evaluated the reliability of the map contigs using different contig assembly strategies, independent contig building methods, DNA marker screening results of the BACs and BIBACs and different fingerprinting methods, and the results showed that the map was assembled properly. Furthermore, we have integrated 781 of the contigs spanning 663 Mbp (59.5%) of the soybean genome into the existing soybean composite genetic map using 273 SSR and 115 RFLP markers. This map represents the first genome-wide, BAC/BIBAC-based physical map of soybean and will provide a powerful platform for many areas of soybean genome research, including large-scale genome sequencing, target marker development, gene mapping, and gene and quantitative trait locus (QTL) cloning. The inclusion of BIBACs in the map will further streamline the utility of the map for positional cloning of genes and QTLs, and functional analysis of soybean genomic sequences. [The supplemental material on the clone fingerprint database and contigs of the physical map is available online a