2010 Annual Report
1a.Objectives (from AD-416)
1)Develop new promoter elements from potato that will allow refined expression profiles (tissue and/or developmental specificity) of transgenes to improve agronomic and quality properties of dicotyledonous crop species.
2)Discover and develop new molecular tools (promoters, terminators) from fruit trees. In particular, to isolate transcriptional control elements and polyadenylation signals from plum and apple.
3)Refine down-regulation technologies to improve general applications to metabolic regulation as well as improve design characteristics of pathogen-resistance transgenes.
1b.Approach (from AD-416)
Available EST, microarray and genomic DNA databases will serve as bioinformatics data sources to identify potato gene families, and specific family members, with requisite expression profiles to serve as sources of valuable transcriptional control elements. Putative elements will be isolated from a BAC library, assembled into marker-gene fusions, and function characterized in transgenic potatoes.
Polyubiquitin genes from apple and plum will serve as sources of transcriptional control elements for direction of commercial levels of transgene expression in these fruit trees. Appropriate polyubiquitin genes for these control sequences will be identified using EST databases to identify specific, highly constitutively transcribed family members. The molecular source of elements will include both BAC libraries and PCR-amplification. Putative promoter elements will be fused to standard marker and delivered to collaborators at the USDA/ARS Appalachian Fruit Research Station for introduction and characterization in apple.
Glycoalkaloids will be reduced in potatoes by suppression of both branches of the SGA pathway using small inverted hairpin structures of double stranded RNA-generating constructs. Small interfering RNAs (siRNAs) will be produced by these constructs specific for both the Sgt1 and Sgt2 gene family members responsible for the initial steps in each of the two SGA biosynthetic branches, resulting in gene inactivation. Suppression constructs will be tested for efficacy via standard genetic transformation, but will ultimately be adapted for intragenic transformation and eventual commercial application. Transgenic potato tubers producing siRNAs will be evaluated for transgene expression and SGA accumulation.
The major problem areas being addressed are development of new molecular tools for potato and fruit tree improvement and construction of transgenic potatoes with improved postharvest and disease susceptibility properties. The project has three specific goals: .
1)to discover and develop new molecular tools (promoters, terminators) for improvement of dicotyledonous crop species including intragenic modifications,.
2)to reduce levels of glycoalkaloid toxicants in potatoes, and.
3)to reduce susceptibility of commercial potato cultivars to potato viruses and Late Blight.
Transcriptional control elements developed as part of the first goal (molecular tools) are expected to have specific application in construction of intragenic potatoes and fruit trees, as well as broader applications in transgene construction for dicotyledonous crops. Progress in this goal in FY10 includes acquisition and application of genomic sequence from citrus (variety Carrizo). This genomic sequence has been made available to the entire citrus research community via a USDA web site (USDA Public Citrus Genome Database). The database has been employed in computational identification of citrus tree transcriptional control elements for transgenes to confer resistance to Huanglongbing (HLB), also known as citrus greening disease. In addition, a citrus intragenic transformation vector has been designed and constructed. Experiments carried out by collaborators at the ARS U. S. Horticultural Research Laboratory (Fort Pierce, FL) indicate that citrus pDNA sequences (which replace Agrobacterium-tDNA borders) function in the bacterium. Finally, antibacterial transgenes previously developed in our laboratory, transcribed from potato control sequences, have been entered into field evaluations by collaborators at the ARS U. S. Horticultural Research Laboratory.
Progress in the second goal (reduction of glycoalkaloids) in FY10 includes demonstration that initial siRNA construct assemblies failed to prevent glycoalkaloid accumulation in transgenic potatoes. Production of siRNAs derived from successful intron-spliced hairpin transgenes was characterized to define parameters for effective gene down-regulation in potato. These experiments indicated new parameters for re-designed siRNA transgene assemblies to eliminate glycoalkaloids. Requisite components for metabolite diversion were isolated and introduced into potato in FY10.
Progress on the third goal (to reduce susceptibility of commercial potato cultivars to potato viruses and Late Blight) includes initial field release Potato Virus Y-resistant transgenics (employing intron-spliced hairpin RNA constructs transcribed from improved promoter elements) in New York. A novel multi-virus siRNA construct targeting Potato Virus Y, Potato Virus A, Potato Virus X and Potato Leaf Roll Virus has been completed and introduced in potato varieties in preparation for greenhouse evaluation of PVY/PVA/PVX/PLRV-resistance. In addition, an intragenic late blight-resistance construct has been successfully introduced into potato (marker-free, Agrobacterium sequence-free) in preparation for field evaluation.
Construction of “intragenic” potatoes containing a late blight-resistance gene. The commercial introduction of crop plants improved using molecular genetic techniques has been limited by problems associated with public perceptions of transgenic foods. These limitations are being addressed in part by development of novel methods for in vitro genetic modification, referred to as "intragenic" technology. This method of gene introduction results in “all native” transgenic lines, i.e. lines that contain no foreign DNA. ARS scientists at the Western Regional Research Center (WRRC, Albany, California), in cooperation with scientists at the J.R. Simplot Company (Boise, ID) have successfully developed “intragenic” potatoes that contain a gene from wild potatoes known to confer resistance to the most devastating of potato diseases, late blight (cause of the Irish potato famine). These potatoes do not contain any non-potato DNA, and were generated without using selection (i.e. they do not contain antibiotic/herbicide resistance genes). “Intragenic” lines with late blight-resistance in the greenhouse and under field conditions will be one of the options for preventing losses to late blight.
Development of a citrus genome database and its application in citrus improvement. The limited amount of publicly accessible citrus genomic DNA sequence has significantly impeded application of biotechnology to citrus improvement. In order to support the utilization of this technology to address critical issues in citrus production ARS scientists at the Western Regional Research Center (WRRC, Albany, California), and the ARS U. S. Horticultural Research Laboratory (Fort Pierce, Florida), are sequencing the genome of the citrus rootstock Carrizo, and making this sequence available on the web (http://citrus.pw.usda.gov/). While this database will be useful in a broad array of applications, researchers at the WRRC are employing it to identify citrus DNA sequences useful in expressing HLB-resistance genes. For example, this data has been employed to identify DNA sequences to allow high-level expression of introduced genes in vascular tissue, where the causal agent of HLB is found. Given that HLB currently represents the most serious threat to citrus production in the U.S. and no resistance genes have yet been identified, the successful development of resistant trees offers a potential solution crucial in sustaining this crop.
Oosumi, T., Rockhold, D.R., Maccree, M.M., Deahl, K.L., Mc Cue, K.F., Belknap, W.R. 2009. Gene Rpi-bt1 from Solanum bulbocastanum confers resistance to late blight in transgenic potatoes. American Journal of Potato Research. 86(6):456-465.
Vogel, J.P., Garvin, D.F., Gu, Y.Q., Lazo, G.R., Anderson, O.D., Bragg, J.N., Chingcuanco, D.L., Weng, Y., Belknap, W.R., Thomson, J.G., Dardick, C.D., Baxter, I.R. 2010. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature. 463:763-768.