Location: Crop Production and Pest Control Research
2020 Annual Report
Objectives
Objective 1: Identify new sources of resistance to Hessian fly and aphids in cereal crops for use in breeding programs to reduce damage from these pests and associated pathogens.
Sub-objective 1a. Identify germplasm accessions, from wheat and related species, that confer resistance to Hessian fly.
Sub-objective 1b. Characterize effectiveness against Hessian fly of insecticidal and antifeedant proteins from wheat and other plant sources for potential use as transgenic resistance to pyramid with and protect native resistance loci.
Sub-objective 1c. Identify and evaluate germplasm accessions that confer resistance to wheat against greenbug.
Objective 2: Characterize and evaluate plant-pest interactions at the molecular level in cereals to improve methods of control for insect pests of wheat.
Sub-objective 2a. Compare Hessian fly-wheat and greenbug-wheat interactions among different cereals/grasses to identify genes consistently associated with resistance, susceptibility, virulence and avirulence.
Sub-objective 2b. Investigate timing and composition of overlapping resistance and susceptibility responses when both virulent and avirulent Hessian fly larvae inhabit the same wheat plant, as can happen in field infestations.
Sub-objective 2c. Increased understanding of the molecular basis of the quadratrophic interactions between wheat, greenbug, Buchnera and viruses.
Objective 3: Evaluate germplasm and regional insect populations to assist cereal breeders in selecting effective sources of resistance for their breeding programs.
Sub-objective 3a. Evaluation of wheat breeding lines in regional uniform nursery tests.
Approach
Objective 1. Resistance phenotypes in new and under-utilized resistant wheat lines will be characterized using genotype-by-sequencing.
Objective 2. The Hessian fly-wheat and greenbug-wheat interactions among different cereals/grasses will be used to identify genes consistently associated with resistance, susceptibility, virulence and avirulence. This will be accomplished by analyzing Illumina HiSeq time-course data of resistant, tolerant and susceptible wheat and the Hessian fly on those hosts. Genes of interest will be verified by quantitative real-time polymerase chain reaction (qRT-PCR). We will characterize the induction of susceptibility also known as obviation. Transcript profiling and qRT-PCR will quantify the abundance of transcripts leading to biomarker genes for compatible and incompatible interactions at a variety of timepoints. In wheat, gene expression differences when infested with aphids carrying barley yellow dwarf virus (BYDV) will be characterized by whole genome mRNA profiles using high throughput sequencing and qRT-PCR. Selected genes of interest that are significantly upregulated or down regulated in both the aphid and wheat will be examined further.
Objective 3. To assist cereal breeders in selecting effective sources of resistance, we will evaluate germplasm and regional insect populations. New sources of germplasm containing resistance to Hessian fly will be identified using traditional screening procedures in a greenhouse setting. A variety of insect populations will be used to determine resistance and susceptibility of available wheat lines. The efficacy of resistance Rgene intervention will be assessed by comparing the change in frequency of phenotypic resistance to historical data.
Progress Report
Objective 1: Identify new sources of resistance to Hessian fly and aphids in cereal crops for use in breeding programs to reduce damage from these pests and associated pathogens.
Sub-objective 1a. Identify germplasm accessions, from wheat and related species, that confer resistance to Hessian fly.
Single nucleotide polymorphisms (SNPs) markers are variations of only one base pair in DNA sequences between plant genotypes. These SNP markers can be discovered by GBS (genotype-by-sequencing), a technique where sequencing of the plant DNA will reveal the different areas between genotypes. GBS was conducted on seven genotypes of Triticum durum (tetraploid) wheat to identify crossing parental pairs for the construction of mapping population. SNPs were identified from five homozygous resistant and two homozygous susceptible Triticum durum (tetraploid) wheat accessions to Biotype L Hessian fly larvae. A total of 13,692 SNPs were identified in all the resistant and susceptible accessions by GBS. Pairwise comparisons between each of the resistant and susceptible wheat accession was carried out using Fst Statistics to determine the genetic variation between the pairs. A resistant and susceptible parental pair showing greater than 5000 non-redundant/non-overlapping SNPs have been identified that are currently being grown for making crosses and generating F1 plants that will be further screened for Hessian fly resistance.
Sub-objective 1b. Characterize effectiveness against Hessian fly of insecticidal and antifeedant proteins from wheat and other plant sources for potential use as transgenic resistance to pyramid with and protect native resistance loci. HIT (Hessian fly in planta translocation) feeding assay was further improved by using liquid nutrient as the translocating medium in place of water in the feeding assay. This modification in the protocol allows for extending the experimental time points for larval data collection. Feeding assay was carried out with two Hessian fly-responsive wheat proteins along with a positive and negative control. Using blotting techniques, the toxin ingestion by the larvae has been confirmed. Larval measurements revealed negative effects on larval growth and development, thus indicating that these wheat defense proteins act as toxins. The improved technique has identified two Hessian fly-responsive proteins to serve as candidate toxins in novel approaches of insect control through breeding and transgenics.
Sub-objective 1c. Identify and evaluate germplasm accessions that confer resistance to wheat against greenbug. Wheat cultivars that have shown resistance to Hessian fly in our previous experiments, as well as new germplasm available through a collaborator from Purdue, have been collected, and in some cases, increased. Schizaphis graminum (greenbug) from 4 biotypes have been screened for resistance to greenbug on wheat containing Hessian fly resistance genes, and Newton (no resistance). The wheat was seeded at a rate of 22 seeds in randomized half-rows. Susceptible wheat ‘Custer’ was planted in ‘check’ rows at the ends and middle of each flat to check for even infestation. TAM 110 was used as a reference for full resistance. Plants were scored as resistant or susceptible. Two wheat lines appear to show resistance/tolerance to greenbug infestations in those lines already screened. More lines are currently being screened with three replicates per testing flat.
Objective 2: Characterize and evaluate plant-pest interactions at the molecular level in cereals to improve methods of control for insect pests of wheat.
Sub-objective 2a. Compare Hessian fly-wheat and greenbug-wheat interactions among different cereals/grasses to identify genes consistently associated with resistance, susceptibility, virulence and avirulence. Screening of several diploid and tetraploid wheat accessions with two virulent Hessian fly biotypes has led to the identification of two tolerant wheat lines. Detailed molecular studies with Hessian fly-responsive biomarker genes revealed tolerant wheat having intermediate response to resistant and susceptible wheat. The identification of Hessian fly-tolerant wheat varieties offers a more durable form of resistance as they do not rely on gene-for-gene resistance that can breakdown due to development of virulent Hessian fly biotypes. Data analysis is currently underway to identify differences in gene expression profiles between tolerant, resistant and susceptible wheats. RNA-seq expression data obtained previously from two different Hessian fly-resistant and -susceptible wheat accessions as well as Hessian fly larvae feeding on host (wheat) and nonhost (Brachypodium) plants were analyzed. Additional stress-responsive genes and pathways were identified and have been validated by qRT-PCR. Further, comparisons of the expression profiles observed in different types of resistant and susceptible plants were carried out to identify novel resistance mechanisms playing a critical role in plant defense. Manuscripts documenting gene expression profiles of select genes and pathways are in progress.
Sub-objective 2b. Investigate timing and composition of overlapping resistance and susceptibility responses when both virulent and avirulent Hessian fly larvae inhabit the same wheat plant, as can happen in field infestations.
List of genes associated with resistance and susceptibility have been identified from RNA-seq data analysis done under Sub-objective 2a.
Sub-objective 2c. Increased understanding of the molecular basis of the quadratrophic interactions between wheat, greenbug, Buchnera and viruses. While greenbug feeding damage can also be an issue for wheat plants, they are also efficient vectors of viruses. Experiments in which wheat was inoculated with cereal yellow dwarf virus - RPV strain (CYDV-RPV) were conducted. Bird cherry-oat aphid feeding on both wheat with and without the virus were collected, and mRNA was collected from both the aphid and sent for sequencing. That data has been returned, and analysis is currently being undertaken with a manuscript soon to be submitted. The same experimental design is now being created to evaluate greenbug in similar conditions. In the subsequent experiments with greenbug, both the aphid and wheat mRNA will be analyzed.
Objective 3. Evaluate germplasm and regional insect populations to assist cereal breeders in selecting effective sources of resistance for their breeding programs.
Sub-objective 3a. Evaluation of wheat breeding lines in regional uniform nursery tests. Effective sources of resistance to Hessian fly is limited, therefore there is a continual need to identify new genetic sources of resistance in wheat. This work is performed across the nation, and ARS researchers at West Lafayette, Indiana, evaluated Hessian fly-infested wheat lines from three nurseries. These include Uniform Eastern Soft Red Winter Wheat (USERWW), Uniform Southern Soft Red Winter Wheat (USSRWW), and Uniform Bread Wheat Trial (UBWT) nurseries. A total of 48 wheat lines were screened against HF biotypes B, C, D, and L. Evaluated wheat lines infested with 4 Hessian fly biotypes from 2 nurseries managed by different cooperators including Gulf Atlantic Wheat (52), Fusarium Head Blight (50), Mason-Dixon Wheat (68), Uniform Eastern Soft White Winter Wheat (20), Southern Universities Wheat (91) and Virginia State Wheat Trial (148) nurseries.
Accomplishments
1. Diploid wheat: An ideal tool to characterize genes in wheat-Hessian fly interactions. Despite identification of hundreds of Hessian fly-responsive genes in common bread wheat, studies to understand the role these genes play in defense and make use of these genes to develop resistant wheat varieties have been challenging. This is largely because bread wheat has two copies of 3 huge genomes that makes testing genes very difficult, labor-intensive, time-consuming and costly research. Using molecular studies, we identified two Hessian fly-resistant diploid wheat that has two copies of only one genome, as an alternate and ideal tool to characterize candidate genes effortlessly and in a cost-effective manner. This strategy can also be adopted by researchers and breeders working with other important economically important pests of wheat and other cereal crops.
2. High cost for genotype by sequencing (GBS) is still an issue for many researchers. ARS researchers at West Lafayette, Indiana, developed a strategy of fluorescently tagging multiple sequence strands for individual specimens, and then pooling them for sequencing. Using this technique, reduced overall costs of GBS by nearly forty percent. We analyzed 52 gene loci that had previously been identified as secreted salivary gland proteins for the Hessian fly. Secreted salivary gland proteins are of particular interest as they are the point of contact between the fly and wheat, reprogramming the wheat to make it susceptible. Using this multipooling sequencing strategy, researchers are able to rapidly and inexpensively identify virulent genotypes in natural populations of Hessian fly in the US. Using 52 loci annotated as secreted salivary proteins in the Hessian fly, we developed and tested a multipooling sequencing strategy to allow rapid identification of individual genotypes in a population.
Review Publications
Nemacheck, J.A., Schemerhorn, B.J., Scofield, S.R., Subramanyam, S.N. 2019. Phenotypic and molecular characterization of Hessian fly resistance in diploid wheat, Aegilops tauschii. Biomed Central (BMC) Plant Biology. 19:439. https://doi.org/10.1186/s12870-019-2058-6.
Crane, Y.M., Crane, C.F., Schemerhorn, B.J. 2019. Relationship of secreted salivary protein variants to virulence in Hessian fly (Mayetiola destructor (Say)). Advances in Entomology. 8: 15-33. https://doi.org/10.4236/ae.2020.81002.
