Location: Innovative Fruit Production, Improvement, and Protection
2019 Annual Report
Objectives
Objective 1: Develop, late flowering peach germplasm that is less susceptible to bloom-time cold injury using advanced breeding technologies and discovery of the genetic and epigenetic factors underpinning flowering time, chilling requirement, and dormancy.
1.A. Studies on the genetic and epigenetic mechanisms of dormancy and flowering time.
1.B. Genetically characterize the Extreme Late Blooming (ELB) trait.
1.C. Introgress later flowering traits into peach/nectarine breeding program.
Objective 2: Develop enhanced germplasm with pillar and upright growth habits that will enable high density production and/or efficient mechanization and fill key genetic knowledge gaps regarding fruit tree scion architecture.
2.A. Study environmental regulation of TAC/LAZY genes.
2.B. Evaluate transgenic plum germplsam with altered architecture to assess potential agronomic value.
2.C. Perform crosses to combine pillar/upright trait with supersweet and commercial quality peach/nectarine traits.
Objective 3: Develop enhanced tree fruit rootstock germplasm with dwarfing, precocity, and superior performance and fill key genetic knowledge gaps regarding fruit tree root architecture.
3.A. Create transgenic plums with altered root architectures and evaluate phenotypes.
3.B. Transform pear rootstocks with candidate apple Dw1 or Dw2 genes.
Objective 4: Develop commercial high sugar peach varieties, stoneless plums, and fill knowledge gaps about agronomically important fruit developmental traits (such as flesh texture, fruit size, and sweetness).
4.A. Functionally characterize genes that control early fruit development and important fruit quality traits.
4.B. Study the impacts of down regulating lignin pathway genes on stone development.
4.C. Genetically characterized the stoneless plum trait for breeding.
4.D. Perform crosses to combine supersweet with pillar/upright and commercial quality peach/nectarine traits.
Objective 5: Develop next generation biotechnology tools for genetic improvement of fruit crops as well as associated regulatory data to ease their path to market.
5.A. Develop CRISPR gene editing systems for plum and pear.
5.B. Develop regulatory data and help prepare dossier for EU deregulation of 'HoneySweet'.
Approach
This project leverages plant breeding, genomics, genetics, molecular biology, and biotechnology strategies to address fundamental problems facing tree fruit production. The variety development and basic research activities are synergistic as the germplasm developed through the breeding efforts serve as a critical resource for identifying the genetic basis for complex traits. Many of the objectives proposed will use the unique transformation technologies developed by the investigators coupled with available genome sequences for several tree fruit species. These transformation systems have been used to develop FasTrack technology to shorten the breeding cycle for fruit tree species. Plums and pears transformed with the poplar FLOWERING LOCUS T produce flowers within the first year of growth and can be hybridized to achieve generation cycles of one to two years. Biological processes under study will include flowering time/dormancy, tree architecture, and fruit development. Regulation of flowering time in peach will be investigated using genetic, molecular and deep sequencing-based strategies. An extreme late blooming trait that avoids spring frost will be combined with commercial quality traits through conventional breeding. Tree architecture, specifically the regulation of TAC1, LAZY1, and LAZY2 expression by light, gravity, and the circadian clock, will be carried out via gene expression studies along with promoter swap experiments to determine the functional consequences of mis-expressing each gene. Collectively, these data will provide important practical information about how light regulation of IGT genes contributes to tree shape. We will continue to characterize previously created plum and apple PpeDRO1 over-expression transgenic lines and RNAi silencing lines to evaluate the impacts of over- or loss- of DRO1 function on root system architecture. In pear, we will leverage our biotechnology system to functionally characterize putative apple dwarfing/precocity genes and assess their potential to confer these traits in pear rootstocks. To study how fruit tissue determination is achieved, we have begun transcriptome-based comparisons of different fruit types, tissues, and developmental times to identify gene networks that specify properties of fleshy versus non-fleshy tissues during and after fruit set. Technology to engineer and breed for stoneless fruits will be tested using a combination of biotechnology and conventional breeding. A novel super sweet trait in peach/nectarine that confers extremely high brix (20o–30o) will be bred to develop commercial quality super sweet varieties. Methods for gene editing will be developed for plum and pear via isolation and use of novel promoters. Lastly, the research unit will continue to pursue national and international release of the transgenic plum ‘HoneySweet’ that is resistant to Plum Pox Virus (PPV). Collectively, these efforts will fill in key knowledge gaps about fundamental fruit tree developmental processes, provide new technologies for developing fruit tree germplasm with economically important traits, and lead to the development of new fruit varieties with superior traits.
Progress Report
We completed a large gene expression and metabolic profiling study of apricot flower buds and found that the production of a class of chemical compounds called phenylpropanoids was highly correlated with bloom time (Objective 1.A). We also carried out crosses using an extreme late blooming peach to better understand the genetics of this trait (Objective 1.B). Over 200 seed were collected from these crosses which will be germinated to create a segregating population.
We previously identified a set of genes including TAC1 and LAZY1 that control branch orientation. We found that TAC1 and LAZY gene expression is controlled by light and photosynthesis, suggesting a connection between branch growth angle and light availability (Objective 2.A). A field trial of plums genetically engineered to have different architectures was horticulturally evaluated. The results showed that trees with reduced LAZY1 activity not only have horizontal branches, but also exhibit defects in photosynthesis (Objective 2.B). While these trees bloomed, they failed to produce a significant crop. Thus, breeding efforts to alter branch angles via reduced LAZY1 activity may suffer from reduced crop load. In contrast, trees having reduced TAC1 expression showed upright growth and produced abundant, highly quality fruit. In peach, we germinated 1,014 seedlings (2018 crosses) and planted 1,031 seedlings (2017 crosses) that were derived from pillar types (lacking a function TAC1 gene) X supersweet nectarines. Evaluations were begun on a population of seedlings derived from similar crosses (2012-2015).
We previously identified a gene in peach called Deeper Rooting 1 (DRO1) that controls lateral root orientation. Transgenic plum and apple trees designed to over-express peach DRO1 were evaluated in the greenhouse. Plum trees had larger and deeper root systems and exhibited upward leaf curling accompanied with more upright branch growth. In contrast, apple trees over-expressing DRO1 had more shallow root systems and were smaller. The results for apple were surprisingly different from plum yet consistent with what we previously observed in the model plant Arabidopsis thaliana. We also found that DRO1 protein localizes to the cell nucleus and likely moves back and forth between the nucleus and plasma membrane. Collectively, these data provide important information into how DRO1 functions in plant roots and gives insights into developing deep rooting technologies for tree rootstocks.
We completed a large gene expression profiling study to compare fruit development between peach, apple, raspberry, and strawberry. This dataset includes gene expression information in numerous tissues throughout development including ovary, hypanthium, receptacle, and seed. This massive data resource is now being mined for insights into fruit development and has been made available to the broader fruit research community. In addition, a fruit collection series was taken throughout the season for supersweet nectarines to better understand the physiological basis of this trait. The fruit will be subject to sugar analyses in order to determine which sugars hyper-accumulate and when. Also, a dozen new supersweet crosses were carried in 2019 and over 1,000 seedlings were planted from supersweet crosses performed in 2018. These trees will be evaluated for fruit quality and disease resistance in future years to identify commercial quality supersweet selections.
We previously identified a common harmless fungus that can stimulate plant growth through the production of gaseous, volatile compounds. We found that the fungus (called TC09) can stimulate the growth of peach tissue culture seedlings and reduces the time it takes to regenerate plants from tissue culture and move them to soil. This technology stands to remove weeks to months from the regeneration and tissue culture process and could have significant impacts on the production of tree rootstocks, as well as other clonally propagated plants.
Accomplishments
1. Prune plum genome sequenced. Prunes have numerous beneficial health properties including the prevention of cardiovascular disease, diabetes and obesity, and promotion of digestive health. The prune plum genome is complex having originated as a hybrid between at least two different wild plum ancestors. This complexity of the prune plum genome poses a significant barrier to breeding and biotechnology applications. ARS researchers in Kearneysville, West Virginia, produced the first reference prune plum genome for ‘Improved French’, a clonal variety that accounts for >90% of worldwide prune production. The genome was annotated and made publicly available through the Genome Database for Rosaceae. The complete genome provides breeders and researchers with a critical tool to develop new varieties for the industry and ensure the long-term sustainability of prune production.
2. The evolution of fruit. Many of the fruits and nuts we commonly consume are derived from plants in the Rosaceae family. These include apples, pears, peaches, plums, apricots, cherries, blackberries, raspberries, strawberries, and almonds. Despite the fact they are closely related plant species, the sweet fleshy parts of these fruits form from different parts of the flower. Understanding how different fruit types evolved, particularly the edible fruit flesh, will fill critical knowledge gaps necessary to improve fruit quality in these species. ARS researchers in Kearneysville, West Virginia, created the first developmentally staged fruit collection to allow comparisons between different fruit types. Gene expression profiling was conducted on the entire tissue set resulting in a comprehensive picture of gene activity throughout flower/fruit development in peach, apple, strawberry and raspberry. This dataset has been used to identify candidate genes that control fleshy tissue development and has been made available to the broader fruit research community.
Review Publications
Conrad, A., Yu, J., Staton, M., Audergon, J., Decroocq, V., Knagge, K., Chen, H., Zhebentyayeva, T., Liu, Z., Dardick, C.D., Nelson, D., Abbott, A. 2019. Association of the phenylpropanoid pathway with dormancy and adaptive trait variation in apricot (Prunus armeniaca). Tree Physiology. https://doi.org/10.1093/treephys/tpz053.
Zhebentyayeva, T., Shankar, V., Scorza, R., Callahan, A.M., Ravelonandro, M., Castro, S., Dejong, T., Saski, C.A., Dardick, C.D. 2019. Genetic characterization of worldwide Prunus domestica (plum) germplasm using sequence-based genotyping. Horticulture Research. https://doi.org/10.1038/s41438-018-0090-6.
Xia, R., Chen, C., Zeng, Z., Liu, Z. 2018. Small RNAs, emerging regulators critical for the development of horticultural traits. Horticulture Research. https://doi.org/10.1038/s41438-018-0072-8.
Galimba, K.D., Bullock, D.G., Dardick, C.D., Liu, Z., Callahan, A.M. 2019. Gibberellic acid induced parthenocarpic ‘Honeycrisp’ apples (Malus domestica) exhibit reduced ovary width and lower acidity. Horticulture Research. https://doi.org/10.1038/s41438-019-0124-8.
Callahan, A.M., Dardick, C.D., Scorza, R. 2019. Multilocation comparison of fruit composition for 'HoneySweet', an RNAi based plum pox virus resistant plum. PLoS One. 14(3):e0213993. https://doi.org/10.1371/journal.pone.0213993.
Li, Z., Janisiewicz, W.J., Liu, Z., Callahan, A.M., Evans, B.E., Jurick II, W.M., Dardick, C.D. 2019. Exposure in vitro to an environmentally isolated strain TC09 of Cladosporium sphaerospermum triggers plant growth promotion, early flowering and fruit yield increase. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2018.01959.
