Comparison of three microsatellite analysis methods for detecting genetic diversity in Phytophthora sojae (Stramenopila: Oomycete)

Analysis of an organism’s genetic diversity requires a method that gives reliable, reproducible results. Microsatellites are robust markers, however, detection of allele sizes can be difficult with some systems as well as consistency among laboratories. In this study, our two laboratories used 219 isolates of Phytophthora sojae to compare three microsatellite methods. Two capillary electrophoresis methods, the Applied Biosystems 3730 Genetic Analyzer and the CEQ 8000 Genetic Analysis system, detected an average of 2.4-fold more alleles compared to gel electrophoresis with a mean of 8.8 and 3.6 alleles per locus using capillary and gel methods, respectively. The two capillary methods were comparable, although allele sizes differed consistently by an average of 3.2 bp across isolates. Differences between capillary methods could be overcome if reference standard DNA genotypes are shared between collaborating laboratories

Phenotypic Characterization of a Major Quantitative Disease Resistance Locus for Partial Resistance to Phytophthora sojae

Major quantitative disease resistance loci (QDRLs) are rare in the Phytophthora sojae (Kaufmann and Gerdemann)–soybean [Glycine max (L). Merr.] pathosystem. A major QDRL on chromosome 18 (QDRL-18) was identified in PI 427105B and PI 427106. QDRL-18 represents a valuable resistance source for breeding programs. Thus, our objectives were to determine its isolate specificity and measure its effect on yield and resistance to both P. sojae and other soybean pathogens. We characterized near isogenic lines (NILs) developed from F7 recombinant inbred lines heterozygous at QDRL-18; NILs represent introgressions from PI 427105B, PI 427106, and susceptible ‘OX20- 8’. The introgressions from PI 427105B and PI 427106 increased resistance to P. sojae by 11 to 20% and 35 to 40%, respectively, based on laboratory and greenhouse assays, and increased yield by 13 to 29% under disease conditions. The resistant introgression from PI 427105B was also effective against seven P. sojae isolates with no isolate specificity detected. Based on quantitative polymerase chain reaction assays, NILs with the susceptible introgression had significantly higher relative levels of P. sojae colonization 48 h after inoculation. No pleiotropic effects for resistance to either soybean cyst nematode or Fusarium graminearum were detected. This information improves soybean breeders’ ability to make informed decisions regarding the deployment of QDRL-18 in their respective breeding programs

An Immunofluorescence Assay to Detect Urediniospores of \u3ci\u3ePhakopsora pachyrhizi\u3c/i\u3e

An indirect immunofluorescence spore assay (IFSA) was developed to detect urediniospores of Phakopsora pachyrhizi, utilizing rabbit polyclonal antisera produced in response to intact nongerminated (SBR1A) or germinated (SBR2) urediniospores of P. pachyrhizi. Both antisera were specific to Phakopsora spp. and did not react with other common soybean pathogens or healthy soybean leaf tissue in enzyme-linked immunosorbent assay (ELISA). SBR1A and SBR2 bound to P. pachyrhizi and P. meibomiae urediniospores were detected with goat anti-rabbit Alexa Fluor 488-tagged antiserum using a Leica DM IRB epifluorescent microscope with an I3 blue filter (excitation 450 to 490 nm, emission 515 nm). The assay was performed on standard glass microscope slides; double-sided tape was superior to a thin coating of petroleum jelly both in retaining spores and in immunofluorescence. The IFSA was used to confirm the identity of P. pachyrhizi urediniospores captured on glass slides from passive air samplers from Georgia, Kentucky, and Ohio during 2006

Infection and genotype remodel the entire soybean transcriptome

<p>Abstract</p> <p>Background</p> <p>High throughput methods, such as high density oligonucleotide microarray measurements of mRNA levels, are popular and critical to genome scale analysis and systems biology. However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge. Many researchers still use an arbitrary cut off such as two-fold in order to identify changes that may be biologically significant. We have used a very large-scale microarray experiment involving 72 biological replicates to analyze the response of soybean plants to infection by the pathogen <it>Phytophthora sojae </it>and to analyze transcriptional modulation as a result of genotypic variation.</p> <p>Results</p> <p>With the unprecedented level of statistical sensitivity provided by the high degree of replication, we show unambiguously that almost the entire plant genome (97 to 99% of all detectable genes) undergoes transcriptional modulation in response to infection and genetic variation. The majority of the transcriptional differences are less than two-fold in magnitude. We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%. Our results are consistent through two different normalization methods and two different statistical analysis procedures.</p> <p>Conclusion</p> <p>Our findings demonstrate that the entire plant genome undergoes transcriptional modulation in response to infection and genetic variation. The pervasive low-magnitude remodeling of the transcriptome may be an integral component of physiological adaptation in soybean, and in all eukaryotes.</p