2012 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)
RNA was extracted from barley callus tissue collected over ten weeks of tissue culture. Three cultivars were used and ethylene produced by the callus was measured every week before tissue collection. cDNA made from the RNA was subjected to real-time quantitative PCR using primers designed for each member of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene receptor gene families. Expression of each gene is being measured to determine which members in each family are involved in tissue culture regeneration.
Transgenic Fusarium head blight (FHB) field trials continued. Homozygous collaborator lines were developed for 2012 field tests after backcrossing with an adapted cultivar. Backcrossing of our lines that showed reduced deoxynivalenol (DON) in multiple field trials has begun with the goal of transferring the transgenes into elite lines that show FHB resistance and reduced DON to see if the transgenes will provide an additive effect. Additional lines expressing genes identified by collaborators as providing some FHB/DON protection are being generated and will be advanced for field trials.
Preliminary tests of lines that show resistance to 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.
Dahleen, L.S., Bregitzer, P.P., Jackson, E.W., Mornhinweg, D.W. 2012. Association mapping of russian wheat aphid resistance in barley as a method to identify diversity in the National Small Grains Collection. Crop Science. 52:1651-1662.
Dahleen, L.S., Morgan, W.C., Mittal, S., Bregitzer, P.P., Brown, R.H., Hill, N.S. 2012. Quantitative trait loci (QTL) for Fusarium ELISA compared to QTL for Fusarium head blight resistance and deoxynivalenol content in barley. Plant Breeding. 131:237-243.
Brown, R.H., Dahleen, L.S., Bregitzer, P.P. 2012. An efficient method for flanking sequence isolation in barley. Crop Science. 52:1229-1234.