2013 Annual Report
1a.Objectives (from AD-416):
To map genes for Fusarium head blight (FHB) resistance and low mycotoxin contamination, and for pests such as Russian wheat aphid; Develop improved green plant regeneration techniques from barley tissue cultures for genotype-independent genetic engineering of disease and pest resistance; Develop transgenic barley lines
expressing combinations of pathogen response and anti-DON genes, test gene expression, and evaluate for effects on Fusarium graminearum and other fungal pathogens.
1b.Approach (from AD-416):
Single quantitative trait locus (QTL) lines for loci involved in FHB and mycotoxin resistance from Zhedar 2 will be developed by backcrossing, fine-mapped using all available molecular markers, and characterized in replicated field trials. Candidate genes from collaborative projects will be verified by inserting the genes into susceptible cultivars via transformation, and testing homozygous progeny in the field. RWA resistance genes in ARS germplasm releases will be mapped using a variety of molecular markers. The involvement of ethylene in tissue culture regeneration will be tested by manipulating ethylene levels with inhibitors and precursors. Optimal timing for ethylene exposure will be determined and tested using commercial barley cultivars. Genes responsible for ethylene perception, synthesis and regulation will be isolated and characterized by following their expression through the tissue culture process. Antifungal and antitoxin genes will be inserted into a commercial barley cultivar using particle bombardment. Resulting lines will be tested in the lab for gene expression using northern and western analyses, and in the field for reaction FHB and other diseases. BSL1 & BSL1-P: Obj. 1 – No IBC approval required; Obj. 2 – Certified 8/1/2010 (8/1/2010-7/31/2013); Obj. 3 – Certified 6/11/2009 (6/11/2009-6/10/2012)
This is the final report for the project 5442-21000-035-00D. Research continues under the new project 5442-21000-036-00D.
An updated linkage map of a 3-way cross with the resistant source Zhedar 2 was used to verify quantitative trait loci (QTL) for Fusarium head blight (FHB) resistance and low deoxynivalenol (DON), and to identify regions associated with Fusarium graminearum ELISA variation. Markers associated with QTL for FHB and DON also were associated with large mean differences for ELISA measurements, providing another tool for predicting levels of resistance in barley. Seed from lines developed with individual QTLs were provided to other barley researchers who had funding and personnel to conduct detailed analyses.
The Russian wheat aphid (RWA) is a major limitation for producing barley in the western US as aphid infestations cause major yield losses. Pesticide application to control the aphids is costly and has limited effects because the aphid causes leaf rolling, which protects the aphid from any spray exposure. So far only three aphid resistance genes have been identified, although 96 resistant barley lines have been identified. ARS researchers in Fargo, ND, Aberdeen, ID, and Stillwater, OK, used genetic mapping techniques to identify as many as 15 new genes in these resistant lines. DNA markers linked to these genes will be used to develop resistant cultivars with a variety of genes that are less likely to be defeated by the aphid. The availability of additional genes for aphid resistance provides better choices for barley breeders to incorporate resistance that will protect barley yield and quality as the Russian wheat aphid expands its habitat. Preliminary tests of lines that show resistance to US RWA biotype 1 were conducted to determine whether they show differential reactions to international biotypes. Initial results indicate some biotype-specific reactions that will be further investigated.
Transgenic barley lines expressing antifungal genes were tested in replicated field trials. Forty eight lines were tested for multiple years. Two lines were identified from these tests as having 25-30% less DON than Conlon and are being hybridized to new barley cultivars showing FHB resistance and low DON to see if the effects are additive. Homozygous collaborator lines were developed for field tests after backcrossing with an adapted cultivar. Additional lines expressing genes identified by collaborators as providing some FHB/DON protection are being generated and will be advanced for field trials. These include genes identified through virus-induced gene silencing in wheat and by overexpression in Physcomitrella.
Mapping barley gene families involved in ethylene biosynthesis and reception. Ethylene is a plant hormone involved in response to disease and in regeneration from tissue cultures. Ethylene biosynthesis and reception is controlled by multiple genes in three families, ACC synthase, ACC oxidase, and ethylene receptors. Each gene within a family plays a specific role in response to specific internal or external factors. ARS researchers at Fargo, ND identified 8 members of the ACC oxidase gene family, 5 genes for ACC synthase, and 8 genes for the ethylene receptor gene family in barley. The complete gene sequence and chromosomal location was determined for each gene within a family. Unique primers were designed for each member of the gene families for future gene expression studies. These resources can be used to identify specific ACC synthase, ACC oxidase, and ethylene receptor genes involved in disease response and tissue culture regeneration to better understand the processes and potentially to manipulate gene expression to improve both disease resistance and regeneration.
Dahleen, L.S., Tyagi, N., Bregitzer, P.P., Brown, R.H., Morgan, W.C. 2012. Developing tools for investigating the multiple roles of ethylene: Identification and mapping genes for ethylene biosynthesis and reception in barley. Molecular Genetics and Genomics. 287:793-802.