Location: Sunflower Research
2013 Annual Report
A comprehensive understanding of the molecular events occurring during infection of canola by the phyto-pathogenic fungus, S. sclerotiorum will likely generate novel information regarding the white mold disease and may lead to more effective disease control measures. The primary objective of this study is to identify differences in expression patterns of host and pathogen genes during infection of canola lines either susceptible or tolerant to S. sclerotiorum using next-generation Illumina/Solexa sequencing. Insilica analysis of the resulting data for determination of gene identities will be followed by validation of expression pattern differences using quantitative real-time PCR techniques. This will enable identification of potential resistance and pathogenicity genes in the host and the fungus respectively.
The S. sclerotiorum isolate used for genome sequencing, NE152 (also known as 1980) was obtained from collaborators at the University of Nebraska for use in our studies. Experiments were conducted with the double haploid canola lines differing in their susceptibility to white mold. During the initial period of limited seed stocks, preliminary experiments were conducted to test the efficacy of the proposed approach in identifying genes. During this preliminary trial in which a small number of plants belonging to the resistant (NEP 63) and susceptible (NEP32) lines were inoculated with a highly aggressive S. sclerotiorum isolate (NE152) in a growth chamber, an established petiole inoculation technique involving potato dextrose agar (PDA) plugs with actively growing S. sclerotiorum was utilized and samples were collected at 24 and 48 hours post inoculation. Non-inoculated, control canola plants were treated with PDA plugs containing no fungus. At both the time points examined, there were no observable phenotypic differences between the susceptible and resistant canola lines and there was no difference in appearance of control petioles. However, four days post inoculation the plants from the susceptible line died whereas the resistant plants continued growing unharmed. Inoculated petioles were harvested at 24 and 48 hours post inoculation and immediately flash frozen in liquid nitrogen to prevent degradation of genetic material and were stored at -80°C. RNA extraction, mRNA purification, and cDNA library preparation was conducted for these samples using commercially available kits. The prepared libraries were submitted to the DNA Sequencing and Analysis Facility, Bio Medical 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, a 76 cycle run was completed. This initial sequencing run was performed using the pooled samples in a single lane to validate the library preparation process. This initial run generated more than two million sequence reads. After validating the library preparation protocol, large scale experiments involving more time points were conducted using two different inoculation methods, the petiole inoculation method conducted previously and a leaf based inoculation method.
This project addresses stated Sclerotinia Initiative goals including, "Pathogen and Host Genomics" as well as "Pathogen Biology and Development". One of the anticipated outcomes of this project is the identification of genes mediating pathogenicity in S. sclerotiorum and resistance in canola. The identification of these genes is likely to have relevance to other important crops that are susceptible to S. sclerotiorum infection (e.g., dry beans, soybeans, pea, and sunflower.) The data produced in this project could also lead to the generation of new functional molecular markers associated with white mold resistance which would be valuable in future molecular marker assisted breeding programs to produce agriculturally-significant crops with increased resistance to S. sclerotiorum.