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United States Department of Agriculture

Agricultural Research Service

Research Project: USING FUNCTIONAL AND APPLIED GENOMICS TO IMPROVE STRESS AND DISEASE RESISTANCE IN FRUIT TREES
2008 Annual Report


1a.Objectives (from AD-416)
Utilize functional genomic/proteomic approaches to identify genes or proteins in fruit crops that confer resistance or susceptibility to freezing or water stress and fire blight. Develop and evaluate new transgenic technologies needed to control gene flow and expression in transgenic apple trees. Evaluate genetically engineered rootstocks as (1) a means of enhancing resistance to diseases and (2) a means of indirectly influencing gene expression in the scion for the improvement of performance or quality.


1b.Approach (from AD-416)
Transcript profiling will be used to identify genes associated with environmental stress, fire blight resistance, and susceptibility, while proteomic approaches will be used to better understand the relationship between gene expression and cognate protein levels. RNAi-induced gene silencing will be used to elucidate the role of specific candidate genes in resistance and adaptation. The ability to use genetically-engineered rootstocks of apple for scion trait modification will be explored by investigating graft-transmissible gene silencing. The use of floral-specific promoters to confine and regulate the expression of dehydrin genes (responsible for cold and dehydration tolerance) to flowers of fruit crops in order to improve frost tolerance will also be investigated.


3.Progress Report
Genes, down- and up- regulated, in response to fire blight (Erwinia amylovora) challenge were identified in the highly susceptible apple rootstock, 'Malling.26', and the highly resistance apple rootstock, 'Geneva 41', using different molecular technologies. In collaboration with scientists at HortResearch, New Zealand, genetic markers for resistance-related genes were identified and mapped in the apple genome. Several markers were found to map to known genetic locations for fire blight resistance. These markers have potential to be used by apple breeders to select for fire blight resistance. This research falls within NP 302- Plant Molecular and Biological Processes and addresses Component 2B- Plant Interactions with Their Environment and Component 3A – Applying Genomics to Crop Improvement.

ARS scientists in collaboration with the University of Illinois used a molecular technique known as expressed sequence tag analysis (EST) to identify genes in different tissues of apple that respond to drought. This research falls within NP 302- Plant Molecular and Biological Processes and addresses Component 2B- Plant Interactions with Their Environment.

The role of a cold-binding-transcription-factor (CBF) in fruit trees is unclear. We have isolated an apple CBF and using transformation technologies have created several apple lines that either overexpress or repress (silence) the CBF gene. These lines are now being evaluated for the effect of CBF overexpression or silencing on cold hardiness and gene expression. This research falls within NP 302- Plant Molecular and Biological Processes and addresses Component 2B- Plant Interactions with Their Environment.

'Royal Gala' apple containing a gene to produce GUS protein was grafted onto apple rootstocks engineered to silence expression of the GUS gene and to non-silencing rootstock. To determine if the gene silencing effect could spread from the rootstock to the scion, the amount of GUS protein in the scion was then measured. Our analysis indicated that graft-transmissible gene silence will not be a useful mechanism for modification of fruiting apple varieties by genetically engineered rootstocks. This research falls within NP 302- Plant Molecular and Biological Processes and addresses Component 3A – Improving and Assessing Genetic Engineering Technology.

Six flower-specific constructs were designed to express a single or multiple dehydrin genes which have been shown to protect plants from freezing and dehydration. The contructs have put into Arabidopsis thaliana to evaluate their ability to protect flower parts from frost damage. This research falls within NP 302- Plant Molecular and Biological Processes and addresses Component 3A – Improving and Assessing Genetic Engineering Technology.


4.Accomplishments
1. Identification of DNA Markers for Genes that Confer Resistance to Fire Blight Disease in Apple. Fire blight is a destructive disease of apple and pear trees that is estimated to cost the US fruit industry over $100 million a year in crop losses and disease control. ARS scientists working in collaboration with scientists from Cornell University, The Pennsylvania State University and HortResearch, New Zealand, identified gene-specific DNA markers for fire blight resistance in apple. DNA markers derived from the candidate resistance genes were mapped in a population of apple seedling segregating for fire blight resistance. To date, of 28 candidate fire blight resistance gene markers that have been mapped, 6 have co-located with known genetic loci for fire blight resistance. This research will facilitate new methods of marker-assisted selection to efficiently breed superior apple cultivars with fire blight resistance. This accomplishment addresses National Program 302 'Plant Biological and Molecular Processes', Problem Statement 2B 'Understanding Plant Interactions with Their Environment' and Component 3A – Applying Genomics to Crop Improvement.

2. A 40,000 Gene Apple Microarray for Functional Genomic Studies in the Rosaceae. In order to better understand and manipulate plant development and important economic traits, it is essential to identify the coordinated induction or repression of numerous genes that are involved in various biochemical pathways. In collaboration with the University of Illinois, an apple microchip was developed consisting of 40,000 gene probes that can be used to better understand which genes and which biochemical pathways are involved in such things as growth, fruiting, fruit quality, disease and stress resistance, etc. This tool will allow scientists to study the genetic regulation of traits important to the apple industry and will also be able to be used for pear, peach, plum, apricot, and other Rosaceous plant species. This accomplishment addresses National Program 302 'Plant Biological and Molecular Processes', Problem Statement 2B 'Understanding Plant Interactions with Their Environment' and Component 3A – Applying Genomics to Crop Improvement.

3. Expression of Dehydrin Genes in Flowers. Last year, nearly 98% of the South Carolina peach crop was lost due to cold snaps occurring in late March and early April. The flower is the most sensitive to cold damage and represents a potential target for genetic manipulation. ARS scientists constructed plant vectors carrying peach dehydrin genes under the control of a carpel-specific promoter from petunia. Since dehydrins have been shown to have cryoprotective activity, boosting expression only in the carpels may protect flowers from frost damage. It is anticipated that expression of one or more dehydrins in the carpels of fruit trees will provide adequate protection from episodes of late spring frost. This accomplishment addresses National Program 302 'Plant Biological and Molecular Processes', Problem Statement 3A: Improving and Assessing Genetic Engineering Technology.


5.Significant Activities that Support Special Target Populations
NONE


6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings1

Review Publications
Norelli, J.L., Farrell, Jr., R.E., Bassett, C.L., Baldo, A.M., Lalli, D., Aldwinckle, H.S., Wisniewski, M.E. 2008. Rapid transcriptional response of apple to fire blight disease revealed by cDNA suppression subtractive hybridization analysis. Tree Genetics & Genomics. 5: p. 27-40.

Wisniewski, M.E., Bassett, C.L., Norelli, J.L., Macarisin, D., Artlip, T.S., Gasic, K., Korban, S. 2007. Expressed sequence tag analysis of the response of apple (Malus x domestica) to low temperature and water deficit. Physiologia Plantarum. 133:298-317.

Wang, Y., Wisniewski, M.E., Meilan, R., Uratsu, S.L., Cui, M., Dandekar, A., Fuchigami, L. 2007. Ectopic expression of mn-sod in lycopersicon esculentum leads to enhanced tolerance to salt and oxidative stress. Journal of Applied Horticulture. 9(1):3-8.

Renaut, J., Wisniewski, M.E., Bassett, C.L., Artlip, T.S., Henry-Michel, C., Witters, E., Laukens, K., Hausman, J. 2008. Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (Prunus persica L. Batsch). Tree Genetics and Genomes. DOI 10:1007/s11295-008-0134-4.

Last Modified: 12/19/2014
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