Location: Chemistry Research
Project Number: 6036-11210-001-007-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 2, 2019
End Date: Oct 30, 2021
Objective:
The main objective of the proposed research project is to identify southern leaf blight (SLB) resistance genes and work toward elucidating their function. The specific objectives are:
1. Infect 10 highly resistant and 10 highly susceptible maize inbred lines with southern leaf blight (SLB).
2. Perform RNAseq analysis and combine results with the PI's global metabolomics data to perform multi-omic analyses.
3. Identify and functionally characterize candidate genes involved in maize resistance to SLB using mutants in genes of interest from publicly available collections such as the UniformMu population.
Approach:
The primary goal of this research is to identify southern leaf blight (SLB) resistance genes and work toward elucidating their function. To accomplish this goal we will (1) perform inoculations on 12 highly resistant and 12 highly susceptible maize inbred lines (Wisser et al., 2011), (2) perform RNAseq and multi-omics analyses, and (3) identify and functionally characterize candidate genes. The procedure will be as follows:
Objective 1: Infect 10 highly resistant and 10 highly susceptible maize inbred lines (n=4; Wisser et al., 2011) with southern leaf blight (SLB).
Thirty-four-day-old plants will be injected 25 times through the developing stems and leaves along outer-leaf midribs.
Objective 2: Perform RNAseq analysis and combine results with Dr. Christensen’s global metabolomics data to perform multi-omic analyses.
Sample collection and processing: Collected and frozen tissues from objective 1 will be used in replicates.
Total RNA will be isolated, dissolved in nuclease free water, and quantified. Known amounts of RNA will be used for library construction and Illumina sequencing.
cDNA synthesis: Four µl of buffer mix and 1µl of enzyme will be added to 11µl of fragmented and primed mRNA and incubated for 25°C for 10 min; 42°C for 50 min and 70°C for 15min.
PCR: PCR will be performed to selectively enrich the fragments that have adaptor ligated on both ends and to amplify the amount of DNA in the library.
High throughput RNA-Seq: High throughput RNA-Seq will be done on a sequencing instrument.
Sequence files generated will be analyzed in Gainesville Florida at the CMAVE lab. Sequence alignments will be generated and an accurate measurement of gene expression will be taken. Its output will be directly parsed and used for differential expression analysis. Gene ontology (GO) tool will be used to determine the biological process with the most homologous gene.
Multi-omics Analysis: Generated RNAseq results will be combined with the global metabolomics data generated by the PI’s lab from the same tissue used for RNAseq analysis. Statistical, functional, and integrative analysis will be performed using Metaboanalyst software.
In summary this section will involve: 1/RNA extractions, 2/Library building, 3/Sequencing, 4/RNAseq analysis, 5/Statistical analysis, and 6/Multi-omics analysis.
Objective 3, Identify and functionally characterize candidate genes involved in maize resistance to SLB using mutants in genes of interest from publicly available collections; perform targeted mutagenesis.
RNAseq and mulit-omics analyses will be used to identify candidate genes associated with SLB resistance. Mutants in candidate genes will be isolated from publicly available collections. In cases where important candidate genes are not represented by transposon-tagged alleles in public collections, targeted mutagenesis will be performed by transgenic delivery of a CRISPR/Cas9 system.
