Project : USDA ARS

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Research Project: Superior Fruit Tree Cultivars for Orchard Resilience, Sustainability, and Consumer Appeal

Location: Innovative Fruit Production, Improvement, and Protection

2025 Annual Report

Objectives
Objective 1: Discover the genetic and molecular mechanisms that underlie tree architectural traits in fruit crops. Sub-objective 1A: Evaluate transgenic TAC1-silenced and LAZY1-silenced plum germplasm with different architectures for their potential use in novel growing systems. (Hypothesis) - Trees having more upright (TAC1-silenced) or horizontal (LAZY1-silenced) branch angles will be amenable to novel growing systems and enable ,high-density orchard systems. Sub-objective 1B: Integration of a narrow leaf (NL) trait into diverse canopy types to study the role of leaf size/shape in tree productivity. (Hypothesis) -The NL trait will improve light penetration and productivity, particularly within peach tree architectures having dense canopies. Objective 2: Identify genes for pitless and robust flavor traits and incorporate into stone fruits breeding. (Hypothesis)- The naturally occurring stoneless trait in plum is conferred by a single dominant mutation. Objective 3: Identify and characterize genes associated with resilience to climate change, spring frost injury, and associated abiotic stresses. Sub-objective 3A: Study the role of individual DAM genes within a set of specially designed plum transgenic lines. (Hypothesis) DAM genes have sub-functionalized in Prunus to precisely couple chilling and heat requirements to flowering time in flower or vegetative buds. Sub-objective 3B: Create a set of Apple transgenic lines with altered DAM gene expression to compare to DAM gene sub-functionalization in Prunus. (Hypothesis) DAM genes function differently in Malus compared to Prunus regarding chilling and heat requirements for flowering time. Objective 4: Breed improved pome and stone fruit cultivars that combine disease resistance, enhanced production, and high fruit quality traits. The Unit has active conventional breeding programs in stone and pome fruits that have multiple long-term goals. Specific efforts that will be accomplished within the proposed 5-year project plan are detailed below. Sub-objective 4A: Generate high-quality super sweet nectarine varieties and phenotype segregating populations for genetic mapping. Sub-objective 4B: Develop late-flowering peach/nectarine cultivars, characterized parental germplasm for breeding late flowering traits, and associated mapping populations and genomic information to facilitate future breeding efforts. Sub-objective 4C: Develop new pre-breeding apple lines and varieties with stacked traits related to fruit quality, productivity, and abiotic stress-resilience into disease-resistant backgrounds. Sub-objective 4D: Develop pear varieties with improved fruit quality, storage, and supply-chain resilience traits in disease-resistant backgrounds.

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 important production traits. Many of the objectives will leverage the unique plant transformation capabilities of the unit coupled with available genome sequences for several tree fruit species. For Obj 1, tree architecture will be studied by evaluating horticultural characteristics of plum tree mis-expressing the TAC1 and LAZY1 genes. Collectively, these data will provide important practical information about how IGT genes contributes to tree shape. For Obj 2,technology to breed or engineer stoneless fruits will be developed by identifying the gene responsible for a naturally occurring stoneless trait in plum. In Obj 3, regulation of flowering time will be investigated using genetic, molecular, biotechnology, and next generation sequencing-based strategies. The breeding efforts in Obj 4 will include: 1) a novel super sweet trait in peach/nectarine that confers extremely high brix will be bred to develop commercial quality super sweet varieties; 2) peaches with delayed bloom will be developed by introgressing a naturally occurring late blooming trait, 3) Apple breeding will leverage the existing rapid cycle breeding system to intogress traits from wild germplasm and them stack key traits into parental germplasm including fire blight and scab resistance along with crisp fruit texture and superior flavor, and 4) in pear, we will leverage our unique germplasm to integrate fire blight resistance with important fruit quality characteristics. 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
Plum trees with altered architectures silenced for Tiller Angle Control 1 (TAC1) or LAZY1 were trained to different high-density growing systems in test blocks and evaluated for growth, flowering, and fruiting characteristics. This work was completed and showed that fruit tree growth habits can be manipulated through breeding or genetic engineering to make them more amenable to training, thus limiting labor costs and increasing fruit productivity. A population of peach trees consisting of 167 individuals segregating for a narrow leaf trait displayed clear segregation and was used for genetic mapping. Whole genome DNA sequencing was performed on all individuals and is being used for mapping studies. In addition to Narrow Leaf, a peach trait marked by a dark red coloration on the outside of the fruit, forming a bright red ring was also mapped via sequencing. This color ring is extremely attractive and could provide consumers with a unique type of peach. It also represents an intriguing biological question about how fruit flesh develops and how red colors are spatially formed. Over 400 individuals segregating for the color ring trait were sequenced and the resulting data was used to identify a single chromosomal locus. Candidate genes are currently being assessed from the sequencing data. A novel trait in plum, called stoneless, was mapped in the previous year and the genomic region was further analyzed to identify potentially causative sequence variants. One challenge was that this entire genetic region turned out to be unique to stoneless, thus having nothing to compare it against. Therefore, we sequenced additional plum varieties in attempt to find the genetic region in germplasm that lacks the stoneless trait. This led to the identification of several Eastern European cultivars that carry the putative region but have normal stones. Analyses of these data are currently underway in attempt to find mutations that could explain the stoneless phenotype. In addition, a large gene expression study of stoneless flower buds and fruits revealed critical aspects of the physiological nature of the stoneless trait. This includes the role of ethylene and programmed cell death in developing plum ovules and provides a possible mechanism to explain the loss of the stone tissues. Transgenic plum plants silenced for one of four Dormancy Associated MADS-BOX genes (DAM3, DAM4, DAM5, and DAM6) were analyzed in growth chambers, greenhouse and orchard for budbreak, tree vigor, growth habits and flowering. These analyses conducted on over 500 trees confirmed that silencing DAM6 or DAM5 significantly impaired the ability of vegetative buds to enter into winter dormancy. The results provide a potential approach to manipulate cold-induced dormancy to expand production ranges and/or avoid negative impacts of unseasonal weather conditions. The transcriptome profiles of flower and leaf buds of EVEGROWING (a peach mutant that lacks DAM genes) vs a normal control peach “John boy” were studied to elucidate the roles of DAMs in dormancy induction in the fall, maintenance of dormancy during the winter, and exit from dormancy in spring. The results of this extremely large and complex study involving over 500 samples are currently being analyzed and prepared for publication. The findings are transforming the way we think about dormancy and will have profound impacts on breeding, horticulture, and management of perennial crops to minimize winter damage and manipulate bloom times. Fruit quality assessments were performed on peach and nectarine populations created in 2019, 2020, and 2021 to identify potentially high-quality selections. A total of 8 new selections were made that carry a novel high sugar trait. These consistently have had a superior flavor profile along with commercial quality fruit shape, color, and other critical fruit attributes. Leaf samples were collected and sent for virus testing by the Cooperative Research and Development Agreement (CRADA) partner to facilitate future field trials for these advanced selections. In addition, a very large fruited and highly productive peach selection made in 2023 was propagated and distributed to the CRADA partner for evaluation and establishment of test blocks. The genomes of two native Malus species, M. angustifolia and M. fusca, that harbor a range of high value stress resilience and disease resistance traits were assembled and annotated. Moreover, the genomes of two additional native Malus species, M. coronaria and M. ioensis, were also completed. All four genomes are being utilized to build a comprehensive apple pangenome assembly for all native Malus species. Progress was also made on generating a population of 200+ hybrid breeding lines between the native Malus and the rapid cycle breeding line T1190 and its derivatives. This past spring, cross pollinations were conducted between M. fusca and an AFRS 44 breeding line that has yielded over 30 fruits. This fruit collectively is expected to contain 30-90 hybrid seeds that will be stratified and planted. Additionally, fruit was also obtained from crosses with M. angustifolia, M. coronaria, M. ionesis, and M. sylvestris but at a much lower success rate than M. fusca. The resulting seeds will also be germinated for planting in FY26. A successful protocol for tissue culture of pear selection US 79439-004 was developed. Experiments were initiated to use this protocol to transform US 79439-004 with the transgene used in our rapid cycle apple program, BpMADS4. Transformations have been successful (recorded eight events); however, regenerating plantlets from these transformed cultures is still in progress. Interest in the US 79439-004 as a new cultivar for the pear industry has grown significantly. An ARS scientist in Kearneysville, West Virginia, was invited to present on the prospective release and the breeding program at the Washington State Horticulture Expo and Oregon State University (OSU) in December of 2024. Furthermore, advanced testing of US 79439-004 has begun with the existing CRADA partner Adams County Nursery (Aspers, Pennsylvania) and Varieties International (Dundee, Oregon). Clonal material of US 79439-004 was provided to both CRADA partners and to OSU and Washington State University under a Plant Evaluation Material Transfer Research Agreement (PEMTRA) earlier this year for advanced trialing.

Accomplishments
1. Successful Sweet Cherry Transformation and Gene-Editing. Sweet cherry production suffers from a number of serious diseases and weather-related vulnerabilities that cannot be readily addressed through conventional breeding. The availability of gene-editing and/or genetic transformation technologies would allow researchers to utilize the most advanced biotech tools to solve these problems in the most popular cherry varieties. USDA ARS scientists in Kearneysville, West Virginia, along with collaborators developed a reliable method for sweet cherry transformation and gene-editing of commercial varieties. This tremendous technological advance will provide the nearly $1 billion cherry industry with new solutions for some of the most pressing problems facing growers and ensure an abundant supply of cherries for future generations. This technology has also provided the CRADA partner with the ability to use gene-editing to create pitless fruit. The lack of pits in cherries would meet significant consumer demand for convenience, lower processing costs, and dramatically boost consumption of this highly nutritious fruit, particularly in children and elderly populations.

U.S. DEPARTMENT OF AGRICULTURE

Innovative Fruit Production, Improvement, and Protection: Kearneysville, WV