Location: Crop Improvement and Genetics Research2008 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. Replacing 5325-21420-003-00D (08/2008).
3. Progress Report
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. Progress in the first goal (molecular tools) in FY08 includes computational identification of specific potato and fruit tree gene families to act as sources of novel transcriptional control elements. These elements 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. For example, an international company has employed the bul409 promoter (published in FY08) in construction of non-bruising apples, and is initiating commercialization processes. Because of this, and the general utility of the promoter elements developed here, two international patents (bul409 and bul427 promoters) are being filed in FY08. This research contributes to the goals of National Program 301 - Plant Genetic Resources, Genomics, and Genetics Improvement (Problem Statement 2C: Genetic Analyses and Mapping of Important Traits). Progress in the second goal (reduction of glycoalkaloids) in FY08 includes introduction of second generation transgenes (employing intron-spliced hairpin RNA) into potato to allow field evaluation of transgene efficacy in reducing total SGAs in the 2009 growing season. This research contributes to the goals of National Program 301 - Plant Genetic Resources, Genomics, and Genetics Improvement (Problem Statement 2C: Genetic Analyses and Mapping of Important Traits). Progress on the third goal (to reduce susceptibility of commercial potato cultivars to potato viruses and Late Blight) includes introduction of second generation virus-resistance genes (employing intron-spliced hairpin RNA constructs transcribed from improved promoter elements) into potato varieties for field evaluation in the 2008 growing season. This research contributes to the goals of National Program 301 - Plant Genetic Resources, Genomics, and Genetics Improvement (Problem Statement 2C: Genetic Analyses and Mapping of Important Traits).
1. New transgenes to control potato glycoalkaloid accumulation. The accumulation of Steroidal GlycoAlkaloids (SGAs), secondary metabolites synthesized in solanaceous plants including potato, presents serious food safety issues that are currently addressed by careful screening of selections during the breeding process. However, this screening in potato acts to complicate the already difficult selection of new potato varieties and limit introduction of germplasm from wild potato relatives that have historically acted as sources for valuable genes for improved agronomic and quality traits. ARS scientists in the Crop Improvement/Utilization Research Unit in Albany, CA approach to this problem has been to inactivate genes encoding key enzymes in the biosynthetic pathways of the two primary SGAs that accumulate in potato tubers, alpha-chaconine and alpha-solanine, (of which the former has more acute physiological effects). In FY08 the USDA patent on the SGT2 gene issued (SGT2 encodes the first dedicated enzyme in the a-chaconine biosynthetic pathway) and transgenes utilizing this sequence were mobilized into the high-SGA potato Lenape. These intron-spliced hairpin RNA transgenes also employ the previously isolated SGT1 gene (solanidine galactosyltransferase, the first dedicated step in a-solanine biosynthesis) transgenes to down-regulate total SGA accumulation in potato and facilitate the introduction of improved potato varieties into the U.S. market. This research addresses National Program 301 - Plant Genetic Resources, Genomics, and Genetics Improvement, Problem Statement 2C: Genetic Analyses and Mapping of Important Traits.
5. Significant Activities that Support Special Target Populations