Location: Sunflower Research
2012 Annual Report
The primary objective of the project is to characterize the differential gene expression patterns between canola (Brassica napus) lines susceptible and resistant to infection by Sclerotinia sclerotiorum using next-generation, Illumina-based DNA sequencing to identify putative resistance genes. Selected doubled haploid canola lines (NEP 32 and NEP 63) differing in their susceptibility to white mold disease used in this study were generated at (NDSU) from B. napus PI Ames 26628 in a previous project supported by the Sclerotinia Initiative. Plants were maintained in a greenhouse and bagged during flowering to prevent cross pollination, and seeds were harvested once seed pods were sufficiently mature. Due to limited seed stocks, preliminary experiments were conducted in which a small number of plants were inoculated with a highly aggressive S. sclerotiorum isolate (NE162) in a growth chamber for each of the selected time points (i.e., 8, 16, 24, and 48 hours post inoculation); susceptible line (NEP 32) compared to resistant line (NEP 63). Harvested petioles were flash frozen in liquid nitrogen to prevent degradation of genetic material and were stored at -80°C. For the samples from the 24 and 48 hour post inoculation time points, RNA extraction, mRNA purification, and cDNA library preparation were completed using commercially available kits. The prepared libraries were submitted to the DNA Sequencing and Analysis Facility, BioMedical Genomics Center at the University of Minnesota for next generation sequencing using the Illumina GA IIx instrument. After passing an initial quality control KAPA qPCR assay, libraries were pooled and a single read, 76 cycle run was completed. From the sequencing runs, a total of 153,851,612 and 109,899,384 quality filter passed 76 bp short reads were generated from petiole inoculated and leaf inoculated libraries respectively. From the inoculated libraries, reads from fungal origin (pathogen) were filtered out by aligning to S. sclerotiorum genome using software program Bowtie. The remaining reads were considered to be plant origin. Initially it was proposed to use A.thalianagenome as a reference for aligning short reads generated from sequencing, but from the preliminary analysis it was found that only very low number of reads from the libraries had matches with A. thaliana which necessitated searching for alternate sources to use as a reference. Ultimately, the Brassica EST assembly, 95k unigene set from the Brassica genome gateway (http://brassica.bbsrc.ac.uk/ ), which represents all the publicly available ESTs from A, B, and C genomes of Brassica spp., was used as reference to anchor reads. Read counts that were uniquely aligned (only one best match in the reference) were obtained; reads with more than one best match in the reference were excluded. Read count data obtained from the alignment of libraries was used to determine differential expression patterns using software package DESeq. Analysis of the massive data set generated from sequencing is in progress. So far, comparisons have been made to determine genes or ESTs differentially expressed in resistant line NEP63 compared to the susceptible line NEP32 during infection process. Preliminary analysis of the petiole inoculation libraries revealed a total of 4350 differentially expressed ESTs, of which 1946 are up-regulated. Interestingly, more than 400 ESTs seem to be turned on exclusively in resistant line, 436 ESTs are up-regulated by 10 fold change. Functional categorization of these differentially expressed genes is essential to understand the mechanism underlying the differences in resistance response in the two canola lines. Efforts in this direction are in progress. Initial searches suggest that many of the ESTs have no known function, and thus can be considered as potential candidates for identification of novel resistance genes. From the preliminary analysis of the leaf inoculated libraries, a total of 477 ESTs were found to be differentially expressed in the resistant line NEP63, 320 of them were up-regulated and 157 were down-regulated. However, from the leaf inoculated conditions no genes that are exclusively turned on in resistant line have been found in the initial analysis. Functional categorization of the up-regulated gene set was performed using Blast2Go. Of the 320 ESTs only 163 have a known role in biological processes. 24 of the 163 ESTs were found to be involved in defense response. Response to stress, hormone mediated signaling pathways, transcription factors were other categories of ESTs that may be involved in regulation of a resistant response. Analysis of petiole inoculation libraries has also been initiated. Comparison of gene expression between inoculated and non-inoculated conditions will provide an insight into the various responses of the host to pathogen infection. Additionally, comparison of fungal genes that are expressed during infection process and in culture will allow us to gain understanding of mechanisms employed by S. sclerotiorum during pathogenesis. Analysis of the above two mentioned comparisons have been initiated and continue at present. Our results will contribute to the development of new functional molecular markers associated with white mold resistance that will have utility in plant breeding programs.