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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR GENETIC TOOLS FOR POTATO AND FRUIT TREE IMPROVEMENT

Location: Crop Improvement & Utilization Research

2012 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.


3.Progress Report:
Progress was made on all three objectives, all of which fall under National Program 301, Component 3: Crop Biological and Molecular Processes; Problem Statement 3A: Fundamental knowledge of plant biological and molecular processes. The project has three objectives:.
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. Development of molecular tools for citrus improvement was introduced into the research program at the direction of the National Program Leader.

Progress in the first objective included development of a citrus genomic DNA database as well as several requisite constructs for intragenic modification of citrus and potatoes. Over the past year a 15X genome of the citrus rootstock variety Carrizo was completed. This genome contains the only known source of resistance to Huanglongbing disease in citrus that is currently threatening citrus production worldwide. This database is being employed to identify potential huanglongbing-resistance genes.

This year we constructed additional components for potato and citrus “intragenic” toolboxes to allow direct genetic modification of these crops without introduction of non-native DNA. We identified and demonstrated functionality of potato sequences to allow introduction of novel sequences into the genome without introducing any sequences from bacteria. We also completed construction of a novel citrus intragenic vector, currently being evaluated by ARS cooperators in Fort Pierce, Florida. The expression of novel potato and citrus promoters was evaluated using marker gene fusions in transgenic plants.

Progress in the second goal (reduction of glycoalkaloids) in FY12 includes construction and evaluation of novel siRNA constructs to down-regulate component enzymes of the glycoalkaloid biosynthetic pathway. Five constructs designed to down-regulate genes that carry out addition of galactose or glucose to the aglycone (solanidine galactosyltransferase SGT1 and solanidine glucosyltransferase SGT2) were evaluated. The suppression of both branches of the pathway was not observed in any of the dual constructs.

Progress on the third goal (to reduce crop disease susceptibility included completion of first year field evaluation of transgenic potatoes expressing a multiple virus-resistance transgene (PVY, PVA, PVX and PLRV) showed successful conference of resistance to two of target pathogens (PVY and PLRV). Over the past year we, in cooperation with ARS scientists in Wapato, WA, have continued development of a bacterial potato disease (Zebra Chip) for evaluating potential transgenic resistance strategies.


4.Accomplishments
1. Successful field trial of transgenic potatoes expressing a gene conferring resistance to multiple viruses. Potato pathogens such as Potato Virus Y, Potato Virus X, Potato Virus A and Potato Leaf Roll Virus seriously impact potato growers and processors in the U.S by way of production losses and rejection of seed lots by seed certification agencies. ARS scientists in Albany, California, constructed an artificial gene designed to confer resistance to all four viruses and introduced it into Premier Russet, Norkotah Russet and Atlantic potatoes. These potatoes were entered into a field trial in cooperation with ARS scientists in Aberdeen, Idaho, to determine virus susceptibility. The transgenic potatoes demonstrated field resistance to both Potato Virus Y and Potato Leaf Roll Virus indicating the success of this strategy for introducing multiple virus resistance.

2. Completion of a draft citrus genome. The sequencing of the complete genome of crop plants provides a foundational scientific tool for characterizing crop gene structure and evolution as well as a valuable resource for identification of DNA sequences for crop improvement. ARS scientists in Albany, California, completed a draft genome of the citrus rootstock Carrizo with sufficient coverage (15X) to allow assembly of approximately 80% of the genome. The Carrizo genome contains the only known source of resistance to Huanglongbing disease in citrus, a disease that has the capacity to eliminate the juice processing industry in Florida. Potential huanglongbing-resistance genes have been identified in the Carrizo genome and are being prepared for testing in cooperation with ARS scientists in Fort Pierce, Florida.


Review Publications
Mc Cue, K.F., Rockhold, D.R., Chhan, A., Belknap, W.R. 2011. Structure of two solanum tuberosum steroidal glycoalkaloid glycosyltransferase genes and expression of their promoters in transgenic potatoes. American Journal of Potato Research. 88(6):485-492 doi:10.1007/s12230-011-9215-2.

Belknap, W.R., Wang, Y., Wu, J., Huo, N., Rockhold, D.R., Gu, Y.Q., Stover, E.W. 2011. Characterizing the citrus variety Carrizo genome through 454 shotgun sequencing. Genome. 54:1005-1015. doi:10.1139/G11-070.

Last Modified: 9/21/2014
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