2012 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: Host plant resistance is one of the most effective and sustainable options for managing fire blight, a devastating disease of apple and pear. To determine if specific genes for fire blight resistance could be identified in 'Splendour' apple for use in apple breeding programs, a subset of a 'Honeycrisp' × 'Splendour' population were evaluated for fire blight resistance in greenhouse trials. Resistance to fire blight could be identified and two mapping populations were established for this purpose. This research is being conducted to identify markers for the resistance genes that will be made available to U.S. tree fruit breeding programs to facilitate the development of fire blight resistant apple and pear cultivars by marker assisted breeding. Because genetic diversity is often lost during crop domestication, accessions of M. sieversii, the progenitor of the domesticated apple, are likely to be a valuable resource for new sources of disease resistance. From a USDA-ARS collection of over 1,000 wild apple accessions, 200 were selected as potential sources of disease resistance for apple scion breeding based upon previous genetic characterization, observations on natural fire blight and apple scab infection, and fruit quality traits. These accessions were propagated and planted at a USDA-ARS research orchard and will be used for genomic studies of fire blight resistance. This research is being conducted to provide the necessary tools to develop apple cultivars that will reduce the use of chemicals, ensure the productivity of planted orchards, and provide resistance to any unforeseen introduction of foreign strains of the fire blight organism. The development of drought tolerant apple varieties will be essential, given projected changes in climate and water availability for irrigation. Selections of drought tolerant and drought susceptible M. sieversii are being clonally propagated on their own roots to assess their response to different types of water limitations. This material will be used to identify genes that contribute to drought tolerance and may serve as molecular markers for breeders or used for genetic enhancement of drought tolerance in apple via genetic transformation. Early bud break due to warm spring temperatures followed by severe frost events have resulted in devastating losses in fruit crops. Global climate change scenarios predict a continuation of erratic weather patterns. Field evaluations of transgenic apples expressing a gene that increases cold hardiness and delays bud break in apple are continuing. Additionally, reciprocal grafting experiments have been initiated to determine if the transgenic apple can be used as rootstock and effect many different cultivars. Breeding new apple varieties takes 25-30 years due to its long juvenile phase resulting in 3-5 years between crosses. Development of an early-flowering apple would greatly increase apple breeding efficiency. A total of 10 transgenic lines in 'M.26' and 'Royal Gala' have been produced using a gene that induces early flowering. Initial characterization of transgenic early flowering lines has begun. 4.Accomplishments 1. Adapting apples to climate change. Apple varieties are needed that are adapted to erratic spring weather patterns (warm temperatures followed by deep freezes) that result in devastating losses in fruit production and cause severe injury to plants. ARS researchers at Kearneysville, West Virginia are conducting field studies of a transgenic apple rootstock overexpressing a gene that increases cold hardiness and delays bud break. The studies, entering into their third year, have shown that the trees consistently go dormant earlier in the fall, maintain dormancy longer in the spring, and have increased cold hardiness. This rootstock demonstrates how apple biotechnology can be used to adapt fruit trees to global climate change. Review Publications Wen, Z., Gao, M., Wang, Q., Xu, H., Walter, M., Xu, W., Bassett, C.L., Wang, X. 2012. Characterization and expression analysis of a Retinoblastoma-related gene from Chinese wild Vitis pseudoreticulata. Plant Molecular Biology Reporter. DOI: 10.1007/s11105-011-0410-6. Bassett, C.L. 2012. Cajal bodies and plant RNA metabolism. Critical Reviews in Plant Sciences. DOI: 10.1080/07352689.2011.645431. Wisniewski, M.E. 2012. Understanding plant cold hardiness: an opinion. Physiologia Plantarum. DOI: 10.111/J.1399-3054. Norelli, J.L., Gardner, S., De Silva, N., Fazio, G., Peil, A., Malnoy, M., Homer, M., Bowatta, D., Carlisle, C., Wan, Y., Bassett, C.L., Baldo, A.M., Celton, J., Aldwinckle, H., Bus, V. 2012. Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus 'Robusta 5' accessions. Biomed Central (BMC) Plant Biology. 13:25.