Location: Grain, Forage, and Bioenergy Research2013 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.
3. Progress Report:
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, 2016. For FY 13, both laboratory and greenhouse experiments were conducted. One of the goals of the USDA-ARS Forage and Bioenergy project at Lincoln, Nebraska is to develop improved switchgrass plants for forage and bioenergy. However, it is unclear if these improved plants will be resistant to insect herbivory. The goal of this project performed through a USDA/NIFA competitive grant is to understand plant responses to herbivory to piercing-sucking insects to ultimately develop switchgrass plants with improved insect resistance. Greenhouse experiments documented the relative susceptibility and resistance of four tetraploid switchgrass populations to greenbugs and the yellow sugarcane aphid. These experiments showed that the upland cv Summer was most susceptible to damage by piercing-sucking insects. In contrast the lowland cv Kanlow was most resistant. Progeny plants derived from crosses between cv Summer and cv Kanlow plants displayed varying levels of susceptibility. Switchgrass plants were not affected by several other aphids tested. Clones of resistant and susceptible plants were subsequently transferred to isolation crossing blocks in the field. Each block contained 10-15 plants of a single population belonging to a single category (resistant or susceptible). Plants will be allowed to intermate and seeds (if available) will be harvested on an individual plant basis for further insect feeding and genetic trials at the end of the 2013 growing season. In other experiments, leaves from cv Summer plants challenged with greenbugs were harvested. RNA extracted from leaf tissues has been submitted for DNA sequencing using a next-generation sequencing platform (Illumina Hi-Seq 2000, University of Nebraska-Medical Center). Other work is underway or planned for completion within FY13 to address biochemical and anatomical changes associated with aphid feeding of switchgrass plants. Insect-related work focused on obtaining the microRNA (miRNA) profiles in the greenbug and the yellow sugarcane aphid. These data are currently being analyzed using bioinformatic tools. MiRNAs are important regulatory molecules in cells, and could be used as defensive measures in insects. RNA was also isolated from these two aphids for next-generation sequencing of insect transcriptomes when feeding on preferred host plants. Both of these datasets will be firsts in this area of research and will be used to understand how the aphid transcriptome changes in response to feeding on resistant versus susceptible switchgrass plants. 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-Omaha; University of Nebraska -Panhandle Research & Extension Center, Scottsbluff; University of San Diego) and by several in-person meetings for project personnel located in Lincoln, NE. One team meeting of all personnel was held in Lincoln on June 21, 2013.