2011 Annual Report
1a.Objectives (from AD-416)
(1) Develop efficient phenotyping tools for insect resistance of switchgrass to greenbugs, chinch bugs and other key piercing-sucking insects and determine the categories of resistance; (2) Obtain biochemical, physiological and anatomical insights into insect-bioenergy grass interactions and determine potential insect resistance mechanisms among defined switchgrass populations; (3) Generate and evaluate diverse segregating populations of switchgrass to assess for insect herbivory using phenotyping tools; (4) Use selected susceptible and resistant switchgrass genotypes to define transcriptional changes before and during insect feeding in order to identify candidate resistance genes; (5) Perform association mapping on developed switchgrass population to correlate candidate resistance genes with insect feeding; and (6) Utilize methods in RNA profiling of insects to uncover key transcriptional regulatory mechanisms that govern host range in grass-feeding insects.
1b.Approach (from AD-416)
The research goals of this project are to identify plants with enhanced resistance to piercing-sucking insects using defined populations of switchgrass and new screening protocols to identify sources of both resistant and susceptible genotypes using five populations of tetraploid switchgrasses and two piercing-sucking insects. During Obj. 1 we will develop the protocols to identify resistant and susceptible switchgrass genotypes to the green bug and the chinch bug. Selected plants will be clonally propagated to obtain materials for Obj. 2. Anatomical, physiological, biochemical and molecular tools will be used to assess insect-plant interactions. Select plants will also be intermated to develop F1 populations for Obj. 3. In this objective (3), marker populations will be screened for insect resistance at the seedling stage. Resistant and susceptible plants will be identified, removed from insect pressure and allowed to grow to obtain clonally propagated materials. During Obj. 4 harvested plant materials will be pooled for biochemical analyses and for the generation of mRNA with subsequent next-generation DNA sequencing to obtain potential candidate genes using a combination of studies conducted in the previous Objectives along with detailed bioinformatic analyses. These data will be used to perform associating mapping on switchgrass marker populations to correlate candidate resistance genes to insect feeding to support Obj. 5. For Obj. 6 insects reared on appropriate switchgrass and/or other control plants will be collected. mRNA isolated from these insects will be subjected for RNA profiling to uncover key transcriptional regulatory mechanisms that govern host range in grass-feeding insects.
This work is being performed through a USDA/NIFA competitive grant awarded in October, 2010 with an official start date of February 1, 2011 through January 31, 2015. For FY 11, both laboratory and greenhouse experiments were conducted. All team members were cognizant of the planned experiments and had approved implementation. Communication was by email and telephone between the ADODR and appropriate project personnel at distant locations (University of Nebraska-Kearney; University of Nebraska -Panhandle Research & Extension Center, Scottsbluff; University of San Diego), and by in-person meetings for project personnel located in Lincoln, NE. One team meeting of all personnel was held in Lincoln on June 21, 2011.
The University of San Diego collaborator was in Lincoln for two weeks (June 20-July 3, 2011). Experimental design and collection schedule for plants were established. Initial leaf samples from several switchgrass genotypes was collected and fixed for future studies.
Greenhouse experiments were conducted to assess the resistance/susceptibility of 5 different switchgrass tetraploid populations to insect damage by greenbugs and the common chinch bug. These experiments were performed with plants at the one leaf and two-leaf stage. Experiments are in the process of being repeated. Consistent damage by greenbugs was observed in two populations and consistent resistance was observed in one population. Limited or no damage was seen in plants challenged with chinch bugs. Immediate work at Lincoln will use greenbugs to obtain plant tissues provided by UNL researchers for a number of downstream uses including microscopy, biochemistry and RNA studies. Antibodies have been made to a switchgrass hemoglobin, a protein known to be involved in stress responses of plants. Antibodies will be used to understand changes in the physiological status of plants challenged with insects.
These laboratory and greenhouse experiments will serve as a foundational base for achieving goals stated in Objectives 1 and 2.