1a. Objectives (from AD-416):
Objective 1: Identify and develop barley and oat germplasm with improved stress resistance (rust resistance, winter-hardiness) and enhanced end-use quality (malt, ß-glucan content). Subobjective 1A: Improve productivity and quality of barley and oat germplasm, with emphasis on malting quality (barley) and food quality (oat). Subobjective 1B: Develop winter malting cultivars with improved quality and winter survival. Subobjective 1C: Introgress resistance to barley stripe rust (BSR) into improved barley germplasm. Objective 2: Develop methods to facilitate accelerated breeding for adaptive traits and utilization of germplasm diversity in barley and oat. Subobjective 2A: Identify SNP markers linked to resistance genes for BSR and oat crown rust (OCR) for use in marker-assisted selection (MAS). Subobjective 2B: Validation of SNP markers linked to ß-glucan content, malt extract, and diastatic power. Objective 3: Develop novel biotechnological approaches for the production of genetically engineered barley and oat. Subobjective 3A: Develop Ds-based and/or recombination-mediated cassette exchange (RMCE) gene delivery systems. Subobjective 3B: Develop transgenic barley lines resistant to Fusarium infection and/or deoxynivalenol (DON) accumulation
1b. Approach (from AD-416):
The three objectives are separate but complementary. Objective 1: Germplasm improvement is based on modified pedigree breeding with single seed descent, multi-location in-house and collaborative test locations, off-site winter nurseries, and winter greenhouse facilities to minimize time for varietal development. Multiple breeding targets, corresponding to specific end uses, include: spring malting barley, spring barley, and oat for food and feed. The process is similar for barley and oat, except for the specific set of targeted characteristics. Procedures are based primarily on phenotypic measurements and laboratory analyses of grain quality. Markers are being developed for key traits to enable more efficient allelic selection for trait improvement and will be used as they are developed. Failure to make adequate progress will be corrected by incorporating alternative germplasm, increasing numbers of crosses, and by increasing test environment numbers and/or quality to increase the frequency of obtaining superior progeny. Objective 2: Based on the hypothesis that SNP markers closely linked to genes for OCR and BSR resistance genes, and for malting quality, will exist. OCR resistance will be assessed and mapped within five oat populations with markers from the oat 6K SNP iSelect Illumina array. BSR resistance will be assessed and mapped within four barley populations, and malting quality in two barley populations, with markers from the barley 9K SNP iSelect Illumina array. Phenotyping will be based on multi-year, multi-location field and/or greenhouse trials. Markers identified will be validated as useful by examination of allelic effects in alternative existing or newly-created populations. Multiple genomic databases will be examined to assist the identification of candidate genes underlying QTLs. Failure to identify useful QTLs or candidate gene may require development and testing of additional populations, novel markers, or analysis of new genomic resources as they become available. Objective 3: Based on the hypothesis that Ds-mediated transposition will produce plants with single-copy loci in regions suitable for high expression, the goal is to produce "clean" transgenic plants without with intact, single-copy transgenes free of extraneous DNA derived from bacterial vectors or selectable markers genes. This system utilizes two very short (~600 bp in total length) sequences derived from another food crop, maize, that when flanking other sequences can transpose--along with the intervening sequences--to new location. Transposition is controlled by introducing the relevant enzymatic activity via crosses to Ac transposase--expressing plants. Vectors will be constructed and introduced via Agrobacterium-mediated delivery. Research will concentrate on commercial cultivars, and genes with activity against Fusarium head blight or that suppress mycotoxin production will be introduced. Failure of proper gene expression is guarded against by using. The use of Ds transposition to deliver genes tends to promote good expression, and multiple candidate genes are available, some of which have imparted useful levels of resistance in preliminary work.
3. Progress Report:
This project was approved in March, and work towards meeting the objectives from continued Project 5366-21000-028-00D. For more information see new project. A critical vacancy has been filled with the arrival of a new scientist. Germplasm development (objective 1) activities included evaluation of barley and oat germplasm lines from the National Small Grains Collection and from collaborating breeders for increased winter hardiness, a critical need for expanding the utility of winter malting barley. To enhance winter hardiness screening, a new testing environment was secured by establishing a collaborative agreement with a cereal producer in Idaho Falls. The development of new methods and tools for accelerating the development of improved germplasm (objective 2) made progress in several areas. Three barley populations were assessed for barley stripe rust resistance in the field by cooperators in Washington, Oregon, and California. In addition, seedling resistance assays for barley stripe rust were conducted on two populations in Aberdeen, Idaho, using growth chambers and greenhouse facilities. Initial analysis is underway to ‘map’ the location of stripe rust resistance in these populations and find markers that can be used to speed breeding for barley stripe rust resistance. Several steps were taken necessary for the implementation of Ds-mediated and RMCE-mediated transgene insertion into barley. Transgenic plants were produced in two cultivars, Golden Promise and Conlon, by introducing Ds-bordered TAG vectors that will function as the site-specific recombination site in the RMCE system. A transformation vector was constructed that will enable insertion of various transgenes within a modified Ds to enable direct Ds-delivery of transgenes, and genetic sequences targeting Tri12 were inserted in this vector. A collaborative effort was established with a scientist at Michigan State University with expertise in Fusarium development and genetics to facilitate the evaluation of transgenic plants and to identify additional genes that may impart resistance to Fusarium head blight.