HIGH THROUGHPUT SEQUENCING FOR ASIAN SOYBEAN RUST AND PHYTOPHTHORA
Corn Insects and Crop Genetics Research
2010 Annual Report
1a.Objectives (from AD-416)
The objectives are to identify differentially expressed genes in Rpp2 and rpp2 gentoypes, and Rps8 and rps8 genotypes during post-infection response to Asian Soybean Rust and Phytophthora Root and Stem Rot pathogens, respectively.
1b.Approach (from AD-416)
Soybean lines resistant (Rpp2) or susceptible (rpp2) to Asian Soybean Rust will be inoculated with the rust pathogen by collaborators in Brazil. Tissue samples will be collected at time zero, plus four other timepoints. RNA will be isolated from tissue representing each time point. Libraries will be made from each time point and provided to the cooperator. The cooperator will generate Solexa Digital Gene Expression data for each library. In addition, the cooperator will generate Solexa Whole Transcriptome Shotgun data from two pooled libraries representing resistant and sensitive lines. Soybean lines resistant (Rps8) or susceptible (rps8) to Phytophthora sojae will be inoculated with the Phytophthora pathogen. Tissue samples will be collected at time zero, plus three other timepoints. RNA will be isolated from tissue representing each time point. Libraries will be made from each timepoint and provided to the cooperator. The cooperator will generate Solexa Digital Gene Expression data for each library. In addition, the cooperator will generate Solexa Whole Transcriptome Shotgun data from libraries representing resistant and sensitive lines. The cooperator will generate a database of gene expression based on transcript counts and will provide this information to the ADODR. Differentially expressed genes will be overlain onto the soybean whole genome sequence and correlated with known positions of resistance genes.
We initiated high-throughput sequencing on a variety of soybean lines known to be resistant/suceptible to important soybean diseases. Given previous experiences, it was assumed that genes responsible for resistance are turned on all of the time and are not induced by pathogens. This assumption made it much simpler to harvest tissue for analyses. We harvested hypocotyl or leaf tissue for all of the experiments. In addition, whenever possible backcross lines were used to diminish differences caused by the genetic background of the genes of interest. We selected germplasm for sequencing to identify genes involved in Phytophthora resistance conferred by Rps8 and Rps2. These lines included PI 399073(resistant/Rps8), a backcross from PI 399073 X Williams (susceptible), L76-1988 (resistant/Rps2), L82-2024 (susceptible, and Williams 82 (susceptible). In addition we initiated sequencing to identify genes involved in resistance to Asian Soybean Rust. We collected tissue from PI 230970 that is resistant to Rpp2 and backcrossed material from a backcross of PI 54878 (resistant/Rpp2) and Williams. Ribo Nucleic Acid was isolated from all biological samples and this was sent to collaborators at the National Center for Genome Resources for sequencing. Solexa transcriptome sequence was returned to Ames, IA and the data is currently being analyzed. Progress on this project was monitored through telephone calls, data exchange and written reports.