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

Location: Innovative Fruit Production, Improvement, and Protection

2023 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
Five year summary: Substantial progress was made in identifying and functionally evaluating genes underlying tree architecture, dormancy, and fruit development. The genes identified in peach that control tree architecture are among the first such genes ever described and have uncovered key aspects of plant gravitropism and the links between gravitropism and light sensing in both shoots and roots. The work has provided a foundation for a basic understanding of tree plant growth and development and is having broad impacts for numerous other crops such as poplar, cotton, and apple. In addition, the work on genes regulating dormancy has greatly expanded our understanding of how dormancy is regulated in deciduous trees. This includes the role of epigenetic factors such as long non-coding RNAs that are critical for regulating dormancy and potentially determining chill requirement. Lastly, we developed technology to specifically alter gene expression within the fruit endocarp and were able to alter lignin pathway genes to reduce endocarp in developing fruit. This work provides a critical first step towards our goal of developing pitless fruit in plums and cherries. The project also made substantial progress in germplasm development for peach and apple. Peach breeding focused on combining a high sugar trait with upright tree architecture and disease resistance. Numerous crosses and backcrosses were made and have led to stacking of these critical traits. This has resulted in a large number of new peach and nectarine selections that are currently being evaluated for potential variety releases. In addition, substantial advances were made in pome fruit breeding. In pear, we completed evaluation and released a new variety called ‘Bell’ which is currently being sold by our CRADA partner and is receiving significant demand. Using the apple rapid cycle breeding system, we were able to stack genes for apple scab and fire blight resistance along with fruit quality genes for flavor and texture. This germplasm represents a huge milestone in apple breeding as the rapid cycle system has allowed us to shortcut decades of breeding in a very short period of time. The parental material with stacked loci enables the rapid development of superior apple varieties and is currently being used to diversify apple breeding germplasm an incorporate critical traits from a wide variety of germplasm sources. Current year summary: Over 1,000 transgenic plum plants silenced and over-expressed for each of four Dormancy Associated MADS-BOX (DAM) genes were grown and synchronized in the greenhouse and coldbox. Plants from each of the lines were also planted in the orchard under APHIS permit. The remaining plants in greenhouse will be used for analysis of defects in dormancy onset, chilling requirement and bud break, respectively. We are currently working on enhancement of production of lateral shoots in growth chambers in order to meet a need for a large number of apical shoot tips or buds to be collected for the analysis of transcriptome profiles during dormancy induction and chilling treatment. We also carried out a few experiments in analysis of specific genes that are targeted by DAM genes during dormancy induction and chilling treatment by comparison of transcriptome profiles between EVERGROWING and Johnboy peach cultivars (described in 033 annual report). Transgenic apple plants that have altered flowering and parthenocarpy (fruit with no seeds) were created per the project plan with A. Callahan. This was done to complement and as part of research into floral development in stone fruits (Sub-Objective 1c). Gene-edited apple trees that should have altered floral organs were created and maintained in a greenhouse (in lieu of a field planting) in support of Sub-Objectives 2b and 2c. The lines have not flowered yet (typically three to four years are required to advance from the juvenile stage, but genome sequencing has confirmed that the target genes have been edited. A manuscript is in preparation with the project collaborator (A. Klocko, University of Colorado at Colorado Springs). 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. This suggests that the Lazy gene may not be a good breeding target for manipulation of tree architecture. 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. Two major steps were taken in utilizing the apple rapid cycle breeding system. 1) Genotyping of the first set of ~200 Malus angustifolia germplasm developed in 2022. This involved the extraction of gDNA and sequencing of the genotypes on an Illumina sequencing platform. 2) In 2022, we conducted crosses using rapid cycle breeding line apples with desirable germplasm for pyramiding of new traits and development of new varieties. Six new populations have been developed from those crosses in 2023: 1) a population of 62 Fuji × AFRS 42-157 progeny that aims to combine fire blight and scab resistance markers with desirable Honeycrisp-associated fruit quality markers and Fuji-associated fruit firmness marker, 2) a population of 36 Ozark Gold × AFRS 19-1G08 progeny to combine scab resistance and Honeycrisp-associated fruit quality markers into a southern, yellow colored, early ripening background from Ozark Gold, 3) a population of 62 Red Cinnamon × AFRS 19-1G08 progeny to combine scab resistance and Honeycrisp-associated fruit quality markers into a red colored, spicy flavored background from Red Cinnamon, 4) a small population of 12 Kidd’s Orange Red × AFRS 19-1G08 progeny to combine scab resistance and Honeycrisp-associated fruit quality markers into a fire blight resistant, high classic apple flavored background from Kidd’s Orange Red, 5) a population of 33 Priscilla × AFRS 19-1G08 progeny to integrate novel fire blight resistance from Priscilla into background with scab resistance and Honeycrisp-associated fruit quality traits from the AFRS breeding line, and 6) an experimental population of an apple/pear hybrid [Pyrus × Delicious] × AFRS 19-1G08. This germplasm will serve as the basis for the initiation of an apple genetic improvement program aimed at producing superior parental material for breeders as well as the selection of cultivars specifically adapted to mid-atlantic production conditions.

Accomplishments

Review Publications
Zhang, S., Gottschalk, C.C., Van Nocker, S. 2023. Conservation and divergence of expression of GA2-oxidase homeologs in apple (Malus x domestica Borkh) . Frontiers in Plant Science. 14:1117069. https://doi.org/10.3389/fpls.2023.1117069.
Goeckeritz, C., Gottschalk, C.C., Hollender, C., Van Nocker, S. 2023. Malus species with diverse bloom times exhibit variable rates of floral development. Journal of the American Society for Horticultural Science. 148:2. https://doi.org/10.21273/JASHS05236-22.
Gottschalk, C.C., Evans, B.E., Collum, T.D. 2023. Improved genome assembly resource of the plant pathogen Fusarium avenaceum. American Phytopathological Society. https://doi.org/10.1094/PHYTOFR-10-22-0117-A.
Bell, R., Schuup, J., Dardick, C.D., Demuth, M.A., Gottschalk, C.C. 2023. ‘Bell’ pear. HortScience. 58(8):832-835. https://doi.org/10.21273/HORTSCI17113-23.