Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
(N/A)
Non Technical Summary
Wheat stem sawfly is a highly adaptable endemic insect species in North America, as evidenced by its initial move into spring wheat from native grasses in the early 1900s, the more recent adaptation to winter wheat in the northern and central Great Plains and now into barley. Historical solid stem resistance from one source has been deployed in spring and winter wheat fields for more than a half a century in the northern Great Plains and now is being rapidly incorporated into wheat breeding programs in the Central Plains. We are fighting an arms race with this resilient insect, and we need every weapon available to fight this pest. The outcome of this proposal will be the identification and validation of novel loci resistant to WSS in hexaploid wheat, tetraploid durum and barley. These results will improve WSS resistance breeding in all three crops, plus the durum resistance genes have the potential of being crossed into hexaploid wheat, which currently experiences the worst WSS damage. Once we identify WSS resistant loci and validate them using NILs, we will elucidate the mechanism(s) of resistance, which could lead to addition unknown sources of resistance that will further diversify our genetic toolbox for breeding WSS resistant varieties. By deploying multiple resistance mechanisms, we will make it harder for the WSS overcome plant host resistance in varieties. Limiting the spread of the WSS to other regions and crops will help reduce the threat from WSS evolution. The long-term impact of this research will be to identify, combine and deploy WSS resistance into new elite varieties, and train graduate students in how to identify and deploy host plant resistance to insect pests in new varieties.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Goals / Objectives
To improve breeding efforts for developing WSS resistant high yielding small grains varieties, we need to increase the number and diversity of WSS resistance alleles to combat this pest. Our project will identify new sources of WSS resistance in hexaploid wheat, durum and barley, develop NILs to verify their efficacy in different genetic backgrounds and deploy the WSS resistance alleles to develop improved WSS resistant high yielding wheat varieties. This work will also provide information about the genetic architecture controlling WSS resistance in durum and barley, which could prove invaluable if WSS becomes more prolific in these crops as well.Overall hypothesis or goal: Identify new genetic resources for improving WSS resistant, high yielding hexaploid wheat, tetraploid durum and barley varieties.Specific objectives:Dissect the genetic architecture of host plant wheat stem sawfly (WSS) resistance in hexaploid wheat, durum and barley to identify new WSS resistance genes.Test near-isogenic lines (NILs) to validate the efficacy of WSS resistance genes, and examine different combinations of genes to identify the best combination for improving WSS resistance.Integrate new genes into breeding programs.Train two PhD students in plant breeding and entomology.
Project Methods
Recombinant inbred line (RIL) and near-isogenic line (NIL) development.Hexaploid spring wheat: NILs will be developed from heterozygous F5 RILs using the 'heterogeneous inbred family' (HIF) strategy to validate WSS resistance QTL. Using MAB, the QTL will be incorporated into previously developed WSS resistance NILs to test WSS resistance interactions among the WSS resistance genes. The NILs will be evaluated in WSS field screening nurseries to measure WSS resistance. Rigorous laboratory bioassays will be used to understand the mechanisms of resistance controlled by the QTL.Durum wheat: Four WSS resistant durum parents will be crossed with the WSS susceptible PI 41353 parent to produce genetic mapping population consisting of ~100 RILs. The populations will be developed using single-seed descent (SSD), and RILs will be derived from F5 plants. Once analysis of the durum association mapping population is completed, additional RIL populations will be produced. After completion of the QTL analysis, NILs will be developed from heterozygous F5 RILs to validate newly discovered WSS resistance QTL. We will also test the impact of these durum QTL for resisting WSS in elite spring wheat backgrounds using hexaploid wheat/tetraploid durum populations.Barley: Preliminary results have identified an elite barley variety, Hockett, that is susceptible to WSS compared to other widely grown barley varieties. A Hockett/Craft RIL population will be phenotyped in the field for WSS resistance during the 2022 and 2023 growing seasons. Once QTL analysis is completed, we will derive NILs from heterozygous F5 RILs using the previously described HIF method to validate newly discovered high effect WSS resistance genes.Genotyping and genetic map construction.Genotype-by-sequencing (GBS) approaches are available for all three crops and will be used for this study. We will utilize the genotyping services provided by the USDA-ARS Small Grains Genotyping Laboratory that uses the TASSEL based GBS pipeline to generate SNPs. We are proposing full map construction for a minimum of eleven populations (6 hexaploid wheat, 4 durum wheat and one barley).Genetic linkage mapping will be performed using R packages R/qtl and R/ASMap that are available in the most resent version of the statistical R software. Using command functions in R/qtl, GBS markers with >20% missing data will be dropped from linkage map construction. A chi-square test will identify markers with segregation ratios significantly different from the expected Mendelian segregation ratio of 1:1, and markers with a P < 1.0 × 10−7 significance threshold will be removed. Cosegregating markers will be removed using the findDupMarker function producing a final set of markers that will be used to construct a linkage map using R/ASMap command functions. Command line arguments used to construct the linkage groups included p.value = 1 × 10−6 to specify the significance threshold used for constructing linkage groups, and the dist.fun = Kosambi to calculate genetic distances between markers. The PlotRF function visually assess the final marker order of each linkage group. Appropriate reference genomes will be used to further verify proper marker order.Standard interval, composite interval, and multiple interval mapping will be implemented with extended Haley-Knott regression in R/qtl using the RIL means. The model search algorithm command line function stepwiseqtl selects the best model for identifying QTL. The stepwiseqtl function uses forward selection to identify additional additive QTL, or QTL interactions to a predefined fixed number of QTL identified by standard interval mapping, followed by backward elimination to the null model. The model with the highest penalized logarithm of odds (LOD) score is the best model. Quantitative trait locus significance level is determined by a permutation test with 1000 replicates specific to the one-dimensional QTL scan to establish appropriate LOD cutoffs. The LOD cutoffs corresponded to a P < 0.05.Association analysis will be done using the Genome Association and Prediction Integrated Tool (GAPIT), an R package that has an integrated set of association analysis methods to identify significant associations between markers and phenotype. Initial association analysis will be conducted using the default compressed mixed linear model (CMLM) approach. Kinship matrix and principal components will be calculated to control for population structure. To control for false positive associations, a false discovery rate of 0.05 will be used. The SNPs with significant association will be converted to KASP markers, for NIL development and MAS in breeding programs.PhenotypingWe will plant the genetic mapping populations at two WSS infested field locations each year. We will use a three rep, random complete block (RCB) experimental design, and each entry will be planted in hill plots. At the end of the growing season, the hill plots will be scored for WSS stem cutting, and then collected for further evaluation in the WSS laboratory during the winter months. We will also plant the genetic mapping populations at our research farm located near Bozeman, MT for solid stem evaluation and seed increase.Stem dissection from field-infested populations. Analyses of stems from mature plants are useful for determining the amount of infestation as well as the degree of larval mortality in infested stems. However, several aspects of WSS behavior contribute to these parameters. Once NILs are developed for QTL of interest, we can deploy laboratory and greenhouse protocols that allow dissection of WSS interaction with host plant resistance genes.?Host preference test. Choice tests will be conducted in the greenhouse using cages that can hold multiple pots. Pots containing plants with elongating stems that contrast for alleles at a putative resistance QTL will be infested with newly emerged adult female WSS, which have been reared from field collected populations of overwintering immature larvae. These choice tests will be accompanied by concurrent no-choice tests in single caged pots for each line of interest. Oviposition behaviors will be scored to determine the number of times females inserted the ovipositor into stems before making a decision to deposit an egg. These tests measure both initial preference during flight and detailed post-alignment oviposition parameters.Y-tube olfactometry and comparison of volatiles. To test for antixenosis based on volatile compounds, a closed-system Y-tube olfactometer is used to test whether QTLs have an effect on volatiles released by plants. Two airstreams deliver purified air to a pair of glass chambers. Each glass chamber encloses a single plant as the odor source. A pair of resistant and susceptible NILs or a pair of parental lines are used as test stimuli for each bioassay. Females placed at the basal section of the Y-tube are observed until they occupy one of the two arms of the olfactometer. A total of 50 females are used to test for preferences in each NIL or parental pair. We will also use a volatile collection system and GC-MS to identify the amounts of known volatile attractant and repellent compounds per each paired comparison. Each experiment will use six plants and will be repeated twice.Statistical Analysis. We will analyze phenotypic data using the latest version of the R software. Linear mixed-effects models will be used to determine the significance of fixed effects including cultivars, alleles, and location on wheat stem sawfly resistance traits. Replications will be treated as a random effect. Means will be obtained from each location and across all locations for all RIL population to be used in QTL analysis. Separate mixed model analyses will be used to examine WSS resistance differences among checks and between contrasting alleles in NIL pairs from each recurrent parent.