2012 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 12, both laboratory and greenhouse experiments were conducted. Greenhouse experiments were performed by University of Nebraska scientists to assess the resistance/susceptibility of 5 different switchgrass tetraploid populations to insect damage by greenbugs and the yellow sugarcane aphid. These experiments were performed with plants at the two-leaf stage and five leaf stage. Consistent damage by greenbugs and yellow sugarcane aphids was observed in two populations and consistent resistance was observed in one population. Limited or no damage was seen in plants challenged with several other aphids. Work will now focus using greenbugs and the yellow sugarcane aphid to obtain plant tissues for a number of downstream uses including microscopy, biochemistry and RNA studies. In conjunction with the yearly visit by a collaborator, experimental design and collection schedule for plants were established. Leaf samples from several switchgrass genotypes identified as resistant or susceptible towards greenbug feeding were fixed and observed by scanning electron microscopy. Plants scored as resistant or susceptible have been isolated from three switchgrass populations and will be allowed to intermate in the greenhouse in 2012. Seeds obtained from these crosses will be tested for insect resistance. Plants will be maintained in the greenhouses until spring 2013, when they will be transplanted to the field.
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 in-person meetings for project personnel located in Lincoln, NE. Two team meetings of all personnel were held in Lincoln on April 27, 2012 and on June 22, 2012.