Location: Innovative Fruit Production, Improvement, and Protection
2022 Annual Report
Objectives
Objective 1. Identify and functionally analyze genes regulating plant architecture, abiotic stress tolerance, fruit quality, and disease resistance. [NP301, C1, PS1A, PS1B; C3, PS3A, PS3B]
Sub-objective 1a. Identify and functionally analyze genes regulating plant architecture in temperate, deciduous fruit crops and rootstocks.
Sub-objective 1b. Identify and functionally analyze genes regulating dormancy, cold hardiness, drought, disease resistance in apple and stone fruit crops.
Sub-objective 1c. Identify and functionally analyze genes regulating fruit development traits in stone fruits.
Objective 2. Develop and optimize advanced methods for tissue culture propagation and genetic transformation of temperate, deciduous fruit crops. [NP301, C1, PS1A, PS1B; C3, PS3A, PS3B]
Sub-objective 2a. Optimize tissue culture production of apple, pear, and stone fruit scion and rootstock genotypes.
Sub-objective 2b. Develop transgenic and gene edited lines and field plantings of fruit crops for functional analysis of genes that regulate important traits identified in Objective 1.
Sub-objective 2c. Develop CRISPR technologies for modifying important functional traits in fruit crops.
Objective 3. Use standard and rapid cycle breeding systems to generate advanced lines of germplasm for the apple and stone fruit breeding community and industry. [NP301, C1, PS1A, PS1B; C3, PS3A, PS3B]
Sub-objective 3a. Use early-flowering apple and stone fruit rapid breeding systems to introgress and or pyramid economically-important traits, such as disease resistance, from wild species and known sources of established cultivars, into commercial germplasm.
Sub-objective 3b. Utilize rapid breeding system to eliminate transgenes from Agrobacterium-based-CRISPR transformation of fruit crops.
Sub-objective 3c. Establish field plantings of select lines of stone fruit and apple germplasm developed through classical and transgenic technologies that exhibit economically-desirable traits.
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
Hundreds of transgenic lines that affect the expression of genes that control dormancy (DAM genes) were created to further the understanding of the dormancy process (Objective 1A). Approximately 2,000 trees have been transferred for greenhouse evaluation. Field planting permits are pending. Dormancy phenotypes were measured and information on the specific functions of individual DAM genes was determined. Additional phenotyping is underway. Progress has been made on advancing breeding lines for the extremely late blooming peach and nectarine selections, and high sugar traits and combinations of both with desired architecture and disease resistance traits (Objective 1B and 1C). A handful of selections were made for further study. Progeny of late blooming selection exhibited late blooming suggesting the trait is at least partially dominant. Trees from all populations were evaluated for bacterial spot resistance with many showing sufficient levels of resistance to enable future releases. Additional peach and nectarine crosses were made with new parental genotypes in attempt to obtain higher quality offspring.
Transgenic plum trees altered in architectural traits resulting in pillar, weeping, and horizontal-Lazy-trees were evaluated for fruit production and fruit quality. LAZY silenced lines yield fewer fruit and potentially lower fruit quality. Additional years of data will be required to confirm (Objective 2B and 2C).
Additional constructs were made that target expression of three transcription factors involved in early fruit development and are being used to transform plum (Objective 4A). Fruit was obtained for the first time from a block of transgenic plum trees targeting lignin production in the fruit endocarp. The intent of the gene manipulations was to form softer stones. Some of the lines clearly displayed stone defects such as loss of endocarp tissue. Evaluations are being repeated in summer 2022 (Objective 4B). Stone defects were phenotyped in two F1 populations that came from ‘Stoneless’ and a second related parent, ‘Sans Noyau’ plum, both of which had partial stone formation. A CRADA was initiated with Pairwise Inc. to identify the underlying causative gene and develop a cherry transformation system. Genetic mapping studies were conducted, and a single locus was identified that was significantly associated with the stoneless trait. Genome of stoneless selection was sequenced and assembled to enable candidate gene identification. Attempts to develop a functional cherry transformation system are in progress.
Additional work was performed to understand the action of a common harmless fungus (TC09), whose volatile production accelerates the growth of many different plants (Obj. 2b). Two full commercial licenses were issued to private companies. One company is seeking an exclusive license for specific crops. Assistance was provided in the form of protocols, data, and consultations to help troubleshoot issues as they arose. A new cooperative agreement with Aerofarms, Inc. was initiated to evaluate use of TC09 in lettuce. Work with NASA continued, focusing on the role of fungal produced CO2.
Previous work on plum genomes brought about a collaborative effort to sequence three citrus greening (HLB) resistant wild Australian lime citrus genomes (in collaboration with University of California, Riverside). Two of the genomes were finalized and annotation is underway. The third genome is being re-assembled with new data. Potential resistance genes from the genomes were identified and analyzed (USDA ARS Citrus Greening Grand Challenge Synergies program).
Scion apple breeding program to develop pre-breeding lines with stacked traits was initiated. 12 field crosses were conducted and six greenhouse crosses with transgenic rapid cycling apple lines. These crosses were aimed to develop new rapid cycling lines (M. fusca x M. domestica interspecific hybrids) and hybrids between advanced lines and elite cultivars. Advancements towards furthering the knowledge surrounding heritage and processing apple varieties were made (see VanderWeide et al. 2022) that will serve as a guide for future hybridizations to develop improved processing/cider apple varieties.
Scion pear breeding was conducted to introduce fruit quality traits into elite disease resistance backgrounds. A total of 46 crosses were made with 27 appearing to be successful. The aims are to develop populations with improved eating quality characteristics with disease and pest resistance, segregating populations for tree architecture traits (columnar x spur/dwarf) and storage conditioning requirements, red-flesh trait, and processing/perry specific traits. In addition, an advanced selection (79439-004) that exhibits highly desirable fruit quality and disease resistance was made. Clonal identity of four older replicate trees was confirmed and virus testing was performed, which showed that all four trees were virus free. To facilitate further variety evaluation, 10+ new grafted trees of 79439-004 were established as shoot tip tissue cultures.
Apple production faces many challenges from abiotic and biotic stresses. These stresses are expected to be more intensive and/or extensive due of climate change. To address these challenges, breeding apples with improved abiotic resilience and biotic resistance is required. Wild apple species could offer the genetics to solve these challenges, as many have been found to be disease resistant and/or were identified as growing in more extreme climates that are unsuitable for commercial apple production. To accelerate the process of breeding between these wild apple species and cultivated apple, we sequenced and assembled two diploid, reference-quality genomes of Malus angustifolia and Malus fusca. These genome assemblies represent the highest-quality apple genomes to date and will facilitate the identification of novel genetics related to abiotic resilience and disease resistance, and the development of DNA diagnostic tools for breeding.
Canadian import approval of ‘Honeysweet’ plum. Plum pox virus (PPV) represents a severe threat to US stone fruit production including peaches, plums, nectarines, apricots, and cherries. Scientists within the unit previously developed a biotech plum that is immune to PPV called ‘Honeysweet’. Honeysweet plum has been approved for commercial production by the relevant US regulatory agencies. Unfortunately, Honeysweet production in the US is limited by the inability of growers to export fruit to trading partners that have restrictions on biotech products. A petition was submitted to Health Canada and granted to allow import of transgenic ‘Honeysweet’ plums from the U.S. that carry a plum pox virus RNA interference (RNAi) resistance cassette. The approval represents a major step forward for the use of RNAi technology in crops and paves the way for future regulatory approvals for numerous other crops in key US international trading partners. The foreign import approval for ‘Honeysweet’ also represents a leap forward for international efforts to combat PPV and maintain an abundant and affordable supply of high-quality stone fruits.
Accomplishments
Review Publications
Singh, K., Callahan, A.M., Smith, B., Malinowski, T., Scorza, R., Jarosova, J., Beoni, E., Polak, J., Kundu, J., Dardick, C.D. 2021. Long term efficacy and safety of RNAi-mediated virus resistance in 'Honeysweet' plum. Frontiers in Plant Science. 12:726881. https://doi.org/10.3389/fpls.2021.726881.
Gottschalk, C.C., Vanderweide, J., Van Nocker, S. 2022. Meta-analysis of apple (Malus x domestica Borkh.) fruit and juice quality traits for potential use in hard cider production. Plants, People, Planet. 3.10262. https://doi.org/10.1002/ppp3.10262.
Hadden, W., Nixon, L.J., Leskey, T.C., Bergh, J. 2021. Seasonal distribution of Halyomorpha halys (Hemiptera: Pentatomidae) captures in woods-to-orchard pheromone trap transects in Virginia. Journal of Economic Entomology. 115(1):109-115. https://doi.org/10.1093/jee/toab226.
Liu, J., Artlip, T.S., Sherif, S.M., Wisniewski, M. 2021. Genetics and genomics of cold hardiness and dormancy in apple. Book Chapter. p. 247-270. https://doi.org/10.1007/978-3-030-74682-7_12.
Li, M., Galimba, K.D., Xiao, Y., Dardick, C.D., Mount, S., Callahan, A.M., Liu, Z. 2021. Comparative transcriptomic analysis of apple and peach fruits: insights into fruit type specification. The Plant Journal. 109:1614-1629. https://doi.org/10.1111/tpj.15633.
Bate, N., Dardick, C.D., De Maagd, R., Williams, R. 2021. Opportunities and challenges applying gene editing to specialty crops. In Vitro Cellular and Developmental Biology - Plants. https://doi.org/10.1007/s11627-021-10208-x.
Yu, J., Bennett Jr, D.R., Dardick, C.D., Zhebentyayeva, T., Abbott, A., Liu, Z., Staton, M. 2021. Genome-wide changes of regulatory non-coding RNAs reveal pollen development initiated at ecodormancy in peach. Frontiers in Plant Science. https://doi.org/10.3389/fmolb.2021.612881.
Ricci, A., Sabbadini, S., Prieto, H., Padilla, I.M., Dardick, C.D., Li, Z., Ralph, S., Limera, C., Mezzetti, B., Perez-Jimenez, M., Burgos, L., Petri, C. 2020. Genetic transformation in peach (Prunus persica L.): challenges and ways forward. Plants. 9(8). https://doi.org/10.3390/plants9080971.
