Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Plant Sciences and Plant Pathology
Non Technical Summary
Agronomic yield and product quality of cereal crops such as wheat are the two most important factors affecting farmer income. Agronomic yield is controlled by many factors with the rate and duration of carbon fixation in leaves a primary factor. Product quality is impacted by genes that influence seed development and determine seed composition and suitability for various product applications.Total agronomic yield in cereals is a function of the amount of carbon fixed in leaves and ultimately stored and harvested in seeds. Among the important yield limiting processes we propose to study here are tillering, plant height, and leaf and seed starch biosynthesis. Our first and second objectives are both focused on genes that impact wheat tillering. Both have the goal to identify new allelic variants that can be used to optimize wheat plant biomass for different applications. The specific genes are the major wheat dwarfing gene, termed Reduced height (Rht) and a gene that is linked to Rht, termed Teosinte Branched (TB1). Essentially all current spring and winter wheat varieties are semi-dwarf and contain one of two common Rht mutations and Rht is perhaps the most important single yield related gene known. The development and characterization of new Rht alleles will lead to a greater understanding of yield limiting processes and perhaps to greater agronomic yields.While variation in TB1 impacts tillering, most common wheat varieties do not vary substantially in TB1 function.Our recent studies examining the role of starch biosynthesis rate limiting genes have indicated that both source and sink starch biosynthetic rates limit plant growth and yield. We are translating these experiments to wheat under field conditions by examining whether natural variation in wheat leaf starch is associated with plant biomass or seed yield.Starch and carbon metabolism are not just important to overall plant growth. In fact, much of the world's population relies on starch from cereals as a source of calories. Starch comprises approximately 70% of the dry weight of cereal seeds and therefore an increased understanding of starch biosynthesis could prove useful in improving cereal product and nutritional quality.Therefore, our third objective is focused on the role of starch in seed product quality. Starch in cereals typically consists of 25% amylose and 75% amylopectin. While both amylose and amylopectin consist of glucose chains, the chains of glucose in amylose are relatively straight while those in amylopectin are highly branched. The glucose residues in amylose are also complexed with lipid with the result being that amylose is less easily digested. Decreased ease of digestion equates to a lower glycemic response or a "low net carb" effect in high amylose wheat- based foods. Aside from health effects, high amylose wheat flour would prove useful in improving the product quality of pasta and the nutritional properties of bread. Our experiments are designed to create specific levels of amylose in wheat seeds and then to measure the impact of the altered starch upon product and nutritional properties of pasta.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Objective 1. The Impact of Rht Allelic Variation upon Wheat Growth and Development. The overall goal here being to determine the degree to which wheat growth and development can be modified by Rht allelic variation. Ultimately, we hope to demonstrate that several new alleles are superior to those currently being utilized and that one or more of the new alleles are deployed in breeding programs.MilestonesCreation and testing of new Rht alleles in each of several different source populations. This has entailed creation and initial characterization of EMS lines created in the standard height variety Fortuna and in the semi-dwarf variety Alpowa for hexaploid wheat. For durum wheat, it has involved creation, identification and testing of Rht alleles in EMS populations created in Divide and Kronos.Characterization of a subset of the new Rht alleles in vitro and in segregating populations in different wheat varieties. Demonstration that one or more of the new Rht alleles is superior to currently utilized Rht dwarfing genes at the agronomic or product quality levels.Objective 2. Increasing Wheat Tillering and Yield by Optimizing TB1 Expression. The overall goal of this objective is to determine the optimal level of TB1 expression required for optimal tillering, plant biomass, and seed yield for both forage and seed yield applications in durum and hexaploid wheat.MilestonesCreation of and testing of new TB1 alleles in each of two source populations. This will entail the testing of alleles created in Kronos and Divide for durum wheat and in Alpowa and Cadenza for hexaploid wheat.We will characterize the impact of mutations in one or more TB1 alleles by creating backcross populations in semi-dwarf and standard height and semi-dwarf durum, spring, and winter wheat.We anticipate demonstrating that mutations in one or more TB1 genes is associated with increased biomass and protein content equal or higher than control varieties.Objective 3. Understanding the Role of Leaf Starch in Whole Plant Metabolism and Product Quality.The overall goal of this objective is to assess the degree to which plant growth and productivity is limited by normal levels of seed and leaf starch biosynthesis. We hope to identify key stages at which selection for increased leaf and/or seed starch biosynthesis would yield improved agronomic yield.MilestonesCompletion of yield studies designed to determine the relative role of leaf starch biosynthesis in overall plant growth.Identification of chromosome regions associated with increased leaf starch.Ability to select for increased leaf starch and improved whole plant productivity via selection for variation in genes that impact leaf starch content.Agronomic, product quality, and nutritional testing of durum seed with increased amylose, protein, and dietary fiber.Assessment of the usefulness and importance of the trait to wheat breeding programs.
Project Methods
Objective 1.The Impact of Rht Allelic Variation upon Wheat Growth and Development.Intermediate plant heights (between that observed in Rht-B1b or Rht-D1b and WT or between B1b/D1b double mutants and Rht-B1b or Rht-D1b semi-dwarf alleles) are desirable for various reasons. For instance, plants that are somewhat taller than semi-dwarf plants may be more suited for growth under dryland conditions with normal fertilizer inputs, while shorter plants may be more suited for growth under irrigated, higher fertilizer practices. In this objective, we will continue integrating unique Rht allelic combinations into elite wheat varieties. Plant height phenotypes within the zones of interestwill be obtained by intercrossing Rht variant alleles alone and in combination with Rht-B1b. We will cross select alleles into spring and winter varieties for preliminary testing and transfer to breeding programs.Quality AnalysisOur results demonstrate that in addition to reducing plant height and increasing yield, Rht-B1b and Rht-D1b modify flour milling and baking properties and micronutrient content. In this objective we will replicate this study to examine milling and baking properties for the BC2F2 populations that we generate in Objective 1. This will provide important information demonstrating how each new variant allele impacts milling and baking, as well as the dosage effect in our double and triple mutant combinations. Currently, further studies are needed to investigate how the semi-dwarfing alleles reduce protein content without reducing dough strength, as well as why they are associated with decreased micronutrient content. Further information is also needed on the impact they have on alpha amylase activity and dormancy.Objective 2. Increasing Wheat Tillering and Yield by Optimizing TB1 Expression.We will test the impact of each new TB1 allele upon wheat plant growth and productivity under multiple environments. We will also examine biological mechanisms behind observed growth changes in variant lines.Identification of additional stop codon alleles in hexaploid wheat: Two stop codon mutations were identified in hexaploid wheat using the wheat TILLING database in the standard height variety Cadenza. We will identify additional mutations by screening two additional EMS populations. These populations were selected to account for the tight linkage and lack of recombination between Rht and TB1. Identification of TB1 nonsense alleles in a Rht-D1b semi-dwarf and in a standard height variety, along with intercrossing of alleles in both populations will allow us to test if TB1 mutation conditioned tiller number increases are additive with Rht-D1b) tiller number increases. The first EMS population is in the semi-dwarf variety, Alpowa. Alpowa carries the Rht-D1b allele, so to obtain a TB1 knockout linked to Rht-D1b we will screen our 2,500 line population that carries a mutation discovery rate of 1 in 12 kb. The TB1 coding sequence is ~1,000 bp and screening of 2,500 kb would result in ~208 mutations of which ~5% or ~10 would be stop mutations (Our goal is to simply identify several stop mutations in each gene). From our screening of Cadenza, we already have stop mutations in TB1-A and TB1-D and therefore the main focus on screening the Alpowa mutation will be TB1-B and TB1-D stops (with the TB1-D stops from Cadenza linked to wild type Rht-D1a and TB1-D stops from Alpowa linked to a semi-dwarfing Rht-D1b). This will enable us to create populations that are either semi-dwarf or tall and that vary in function for all three TB1 genes. For Alpowa, DNA was extracted from M1:2 leaves at the four-leaf stage for each of the 2500 M1:2 plants. Each of the three TB1 genes will be amplified using genome specific primers followed by direct sequencing. We have used this approach for multiple projects to identify novel alleles (Feiz et al. 2009, Hogg et al. 2013).Progress:We have streamlined our mutation screening process, therefore identification of new mutant lines of interest will be completed soon. After identifying the mutations, we will narrow our focus to two or three of the most promising alleles representing each genome for both standard height and semi-dwarf populations.Objective 3.Understanding the Role of Leaf Starch in Whole Plant Metabolism and Product Quality.Test newly identified SSIIa alleles and combinations to determine their relative impact upon plant yield related characters.Purpose:We will carry out detailed characterization of plant productivity for durum lines carrying variant SSIIa alleles. Yield trials will be grown under multiple environments and will include all genotypes. This will determine the allelic combinations best suited to increase amylose and dietary fiber without decreasing yield.Plant Material:The mutant lines to be tested include 3 SSIIa-A missense alleles and 6 SSIIa-B missense alleles. The SSIIa-A and -B variant alleles being tested were combined with the SSIIa-B or -A confirmed null alleles described in Hogg et al., (2013, 2015). We believe this will result in durum in which amylose content is greatly increased, yet the desirable allele retains some SSIIa function to improve seed size and product quality over a full SSIIa null. The mutations of interest will be crossed to Divide and Mountrail and advanced to the BC2F2 generation.This objective is designed to provide important information on how the new starch synthase variant alleles and allele combinations impact pasta quality and nutrition. The durum isolines developed under Objective 1 carrying single missense mutations in SSIIa-A or SSIIa-B will be analyzed, as well as lines carrying the ssIIa-A null in combination with missense ssIIa-B alleles.?Pasta Analysis:For each genotype, pasta quality will be measured for two subsamples of spaghetti for replicated growing conditions. Noodle color of uncooked and cooked spaghetti will be measured with a Minolta model CR310 color difference meter (Konica Minolta, Ramsey, NJ, U.S.A.) according to AACCI Approved Method 14-22.01. Optimal cooking time, cook loss, and noodle firmness will be measured by following AACCI Approved Method 66-50.01. Spaghetti strands will be cooked in boiling deionized water (500 mL) until they reach their appropriate minimum cook times. Noodles will be drained into a beaker and rinsed with 50 mL of deionized water. The cooked pasta will be weighed to determine water absorption, [(g cooked - g uncooked)/g cooked]. The collected cook and rinse water will be evaporated in a forced-air oven at 110°C to determine % cooking loss (g dried solids × 4). Cooked spaghetti firmness will be determined by measuring the force (g) required to shear five cooked pasta strands with a TA.XT2 texture analyzer (Texture Technologies, Scarsdale, NY, U.S.A.) equipped with a TA-47 pasta probe. For each subsample, texture measurements will be collected in duplicate and averaged. Cooked pasta firmness will be determined at the following time points past optimal cooking time: +2, +4, +6, +8, and +10 min.