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ARS Home » Northeast Area » Kearneysville, West Virginia » Appalachian Fruit Research Laboratory » Innovative Fruit Production, Improvement, and Protection » Research » Research Project #435799

Research Project: Improving Fruit Crop Traits Through Advanced Genomic, Breeding, and Management Technologies

Location: Innovative Fruit Production, Improvement, and Protection

2020 Annual Report

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.

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
Characterized the chilling and warm requirement during dormancy to bud break for two peach mutants, one with a very late flowering trait and one that does not undergo dormancy (Objective 1A). Several hundred 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). Progress has been made on advancing breeding lines for the extremely late blooming peach and nectarine selections, as well as the high sugar traits and combinations of both with desired architecture and disease resistance traits (Objective 1B and 1C). Trees are in the field to be evaluated and those bearing flowers/fruit have been used for the next generation. Transgenic plum trees altered in architectural traits resulting in pillar trees, weeping trees, and horizontal-Lazy-trees were evaluated for responses to different training systems to see the most efficient way to maximize production with minimal labor. Fruit was also obtained and evaluated for a subset of these trees (Objective 2B and 2C). A second dwarfing trait in peach (A72 dwarf) was mapped through a pooled genome scheme and candidate genes identified, and their segregation was followed in a F2 generation. A gene responsible for the narrow leaf peach phenotype was mapped through a pooled genome scheme and candidate genes were identified. Markers for these genes were developed and used to determine which of those candidate genes was most closely aligned with the narrow leaf phenotype (Objective 2). Apple and plum trees that have the Deeper rooting 1 gene (DRO1) overexpressed were evaluated for a second year. These results confirmed the first year evaluation that in peach, the roots were deeper rooting and the leaves tended to curl, while in apple, the roots were smaller and more shallow (Objective 3A). Constructs were made that targeted the expression of three transcription factors involved in early fruit development. One of these was used to transform plum (Objective 4A). Fruit was obtained for the first time from a block of transgenic plum trees. The intent of the gene manipulations was to form softer stones. Evaluations are ongoing (Objective 4B). Fruit were also evaluated for stone defects in two F1 populations that came from ‘Stoneless’ and a second related parent, ‘Sans Noyau’ plum, both of which had partial stone formation. Approximately 60 trees bore fruit of which a quarter had noticeable stone defects. Several trees that had the least amount of stone over several years were pollinated with several different pollens from ‘Stoneless’ as well as self-pollinated to incorporate as much of the stoneless phenotype as possible. An early flowering plum was also pollinated with the most phenotypic stoneless pollen to incorporate the early flowering gene to rapidly cycle generations (Objective 4C). Supersweet nectarines were advanced in breeding and fruit analyzed throughout development to determine when the elevated sugar and which sugar is elevated (Objective 4D, see Objective 1 also). 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. Consultations with two separate groups for the deregulation of ‘HoneySweet’ in Canada and in the European Union were continued with information exchanged for preparation of the dossiers (Objective 5A). Additional work was performed to understand the action of a common harmless fungus (TC09), whose volatile production accelerates the growth of many different plants. The whole genome was sequenced and expression analysis of plants exposed and not exposed revealed that the accelerated growth was likely due to an enhanced ability to uptake and metabolize exogenously provided sugar. Several agreements were initiated to test this in various systems of growth, including work with NASA. A patent was applied for utilizing this fungus for acceleration of growth in all plants. 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). Using bioinformatic tools developed here, the resistant associated loci will be investigated.

1. Genetic and epigenetic regulation of the tree dormancy associated (DAM) genes. Genetic regulation of tree dormancy. Dormancy is a process, whereby, woody perennial plants survive the cold winter seasons and regrow in the spring. The ability to modify tree dormancy will help protect growers against seasonal weather changes including warm winters, extreme cold events, or most importantly, spring frost. Research at the Appalachian Fruit Research Station determined the individual roles of a group of six regulatory genes, the DAM genes, that control dormancy. It was determined that two of the six genes were specifically necessary for the entrance into and exit from dormancy. This knowledge provides breeding targets for extending or reducing dormancy periods will help in developing cultivars that may remain productive under adverse weather conditions such as spring frost.

2. Fungal volatiles for accelerated plant growth. The profitability of indoor or urban farming is limited by the high costs of energy required for temperature and lighting control. One approach to offset these costs is to increase crop productivity fast using technologies that work in controlled environmental production systems. Researchers at the Appalachian Fruit Research Station, Kearneysville, West Virginia, found a common harmless fungus that promotes fast plant growth in indoor environments through the production of beneficial gaseous compounds. The fungus showed no evidence of being pathogenic to plants but produced extreme increases in plant biomass upon exposure while decreasing those associated with photosynthesis and stress.

Review Publications
Singh, K., Dardick, C.D., Kundu, J. 2019. RNAi-mediated resistance against viruses in perennial fruit plants. Plants.
Collum, T.D., Stone, A.L., Sherman, D.J., Rogers, E.E., Dardick, C.D., Culver, J.N. 2019. Translating ribosome affinity purification profiling of plum pox virus (PPV) infected leaf tissues in Prunus domestica L reveals post-dormancy spatial coordination of defense responses in phloem tissues. Molecular Plant-Microbe Interactions. 33(1) 66-77.
Yu, J., Conrad, A., Decroocq, V., Zhebentyayeva, T., Williams, D., Bennett Jr, D.R., Roch, G., Audergon, J., Dardick, C.D., Liu, Z., Abbott, A., Staton, M. 2020. Distinctive gene expression patterns define endodormancy to ecodormancy transition in apricot and peach. Frontiers in Plant Science. 11:180.
Galimba, K.D., Tosetti, R., Loerich, K., Michael, L., Pabhakar, S., Dove, C., Dardick, C.D., Callahan, A.M. 2020. Identification of early fruit development reference genes in plum. PLoS One.
Waite, J.M., Collum, T.D., Dardick, C.D. 2020. AtDRO1 is nuclear localized in root tips under native conditions and impacts auxin localization. Plant Molecular Biology. 103:197-210.
Hollender, C., Hill, J., Waite, J.M., Webb, K.K., Dardick, C.D. 2020. Opposing influences of TAC1 and LAZY1 on lateral shoot orientation in Arabidopsis. Plant Molecular Biology. 10:6051.