HIGH THROUGHPUT SEQUENCING FOR ASIAN SOYBEAN RUST AND PHYTOPHTHORA
Corn Insects and Crop Genetics Research
2011 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.
Previously, we initiated high-throughput sequencing of ribonucleic acid on a variety of soybean lines known to be resistant/susceptible to important soybean diseases. We selected germplasm for sequencing to identify genes involved in Phytophthora resistance conferred by Rps8. These lines included PI 399073 (resistant/Rps8), a backcross from PI 399073 X Williams (resistant) and Williams (susceptible). Following data normalization, we identified 1,104 genes significantly differentially expressed between PI399073 and Williams. Comparison of the data from PI399073X Williams and Williams narrowed the number of differentially expressed genes to 409. We are currently evaluating these genes to determine if any correspond to the Rps8 locus. In addition, we initiated sequencing to identify genes involved in resistance to Asian Soybean Rust. We collected leaf tissue from PI 230970 that is resistant to Rpp2 and backcrossed material from a backcross of PI 54878 (resistant/Rpp2) and Williams. Thus far, only the data from PI230970 and William has been evaluated. A total of 4,788 differentially expressed genes were identified. Bioinformatic methods are being used to identify genes with homology to known resistance genes within this data. Progress on this project was monitored through telephone calls, data exchange, and written reports.