Location: Innovative Fruit Production, Improvement, and Protection
2024 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
Horticultural characteristics of plum trees silenced for tiller angle control TAC1 and LAZY1 were characterized in a randomized block for fruit quality, stem mechanical strength, and bud break. The results showed that LAZY1 trees had reduced mechanical strength in 1 year old branches. This could have important implications for tree training systems based on lazy1 mutants. In addition, TAC1 and LAZY1 silenced plums were crossed and approximately 40 hybrid trees were evaluated for growth habit. Approximately half of the trees showed the pillar growth habit suggesting it is dominant to the LAZY1 mutant phenotype. This finding is contrary to previous results in Arabidopsis where lazy1 mutants were epistatic to tac1 phenotypes.
Additional narrow leaf (NL) seedlings from multiple populations were evaluated in 2024. Each individual was scored for leaf width and fruit quality (if available). The population displayed clear segregation for the NL trait and DNA was extracted for genetic mapping. Sequencing was performed on the NL segregants and is currently being used for mapping studies.
A total of 167 individuals segregating for the stoneless trait were re-sequenced using the Illumina platform. The reads were assembled to the stoneless genome (see FY23 report) and use for SNP and INDEL calling. These were used for mapping studies which resulted in the unambiguous detecting of a single locus strongly associated with the stoneless trait. The mapped region was further analyzed to identify potentially causative sequence variants. In addition, a bud/flower/early fruit development tissue series (over 400 samples) was collected and used for RNAseq experiments. The data is being processed and analyzed to identify the physiological nature of the stoneless trait.
Two ongoing projects are being conducted to characterize dormancy associated MADS box gene DAM functions in chilling requirements, bud break, flowering time, and climate adaptation: 1. Continue phenotypic analysis of the transgenic plum plants harboring RNAi-silenced four DAMs in growth chambers, greenhouse and orchard, including bud break, tree vigor, growth habits and their response to high temperature and drought stresses particularly under field conditions in various seasons. A total of 500 trees were evaluated constantly. These analyses confirmed that silencing DAM6 and DAM4's ncRNA impairs trees' growth vigor and promotes senescence, and silencing DAM3 and DAM4 leads to early bud breaks in both greenhouses and orchards. As of now, we have collected leaf samples from 310 trees for analysis by RNA-seq to confirm that RNAi down-regulates DAMs in these plants. RNA isolation and construction of cDNA libraries for RNA-seq are in progress. 2. Analyze the transcriptome profiles of EVEGROWING and “John boy” peaches to elucidate the roles of DAMs in dormancy induction and exit, and gene regulatory networks they regulate. The results of this study will lay a foundation for the late analysis of transgenic plums and the identification of specific target genes by each DAM. We have done all dormancy induction, chilling and warming treatments, and collected over 300 floral bud samples previously. To date, we have generated 70 RNA-seq datasets and are currently working to complete the remaining 170 samples. Meanwhile, we investigated the defect of dormancy induction of EVERGROWING by cold temperatures and short photoperiods. We also documented about 200 images of various phenotypes of the treated floral buds and processed them into six figures in three manuscript drafts. Together, the analyses of DAM functions in RNAi transgenic lines and in EVERGROWING mutant will help identify key genes and regulatory networks that control chilling requirement, bud breaks, flowering time, stress tolerances (heat, drought) and climate adaption in peach plants.
We successfully achieved part of 24th month milestone of hybridization of rapid cycle lines with two native species to generate interspecific hybrids. This was conducted using PI 589975 Malus fusca and PI 613880 M. angustifolia. Currently, only crosses with PI 589975 have yielded seedling trees. However, we are conducting additional crosses to increase population numbers of hybrids PI 589975 and retrying PI 613880 this summer. As a supplement, we have generated hybrids of M. sylvestris and M. sieversii which are wild, progenitor species but are not native to the US. We have also achieved our 36th month goal of assembling genomes for two native Malus species (M. fusca and M. angustifolia). Objective 4(d) is to develop pear varieties with improved fruit-quality, storage, and supply-chain resilience traits in disease-resistant backgrounds. We have successfully evaluated 85 different genotypes of germplasm for fruit quality and storage requirements. We expect to conduct a third and final year of this analysis which includes supply chain resiliency testing during the fall of FY24/FY25. We have also achieved our 36th month goal of establishing tissue cultures of US 79439-004. The resulting protocol includes not only culturing but rooting for clonal propagation of the prospective pear release US 79439-004.
Accomplishments
Review Publications
Jacobson, S., Bondarchuk, N., Nguyen, A., Canada, A., Artlip, T.S., Welser, P.J., Klocko, A. 2023. Apple CRISPR-Cas9 – a recipe for successful targeting of AGAMOUS genes in domestic apple. Plants. 12(21):3693. https://doi.org/10.3390/plants12213693.
Song, G., Liu, Z., Zhong, G. 2024. Regulatory frameworks involved in the floral induction, formation and developmental programming of woody horticultural plants: a case study on blueberries. Frontiers in Plant Science. 15:1336892. https://doi.org/10.3389/fpls.2024.1336892.
Singh, K., Huff, M., Liu, J., Park, J., Rickman, T., Keremane, M.L., Krueger, R., Kunta, M., Roose, M., Dardick, C.D., Staton, M., Ramadugu, C. 2024. Chromosome-scale, de novo, phased genome assemblies of three Australian limes, Citrus australasica, C. inodora, and C. glauca, towards finding insights into disease resistance to citrus huanglongbing. Plants. https://doi.org/10.3390/plants13111460.
Kangben, F., Kumar, S., Li, Z., Sreedasyam, A., Dardick, C.D., Jones, D., Saski, C. 2024. Phylogenetic and functional analysis of tiller angle control homeologs in allotetraploid cotton. Frontiers in Plant Science. 14:1320638. https://doi.org/10.3389/fpls.2023.1320638.
Liu, J., Bennett Jr, D.R., Demuth, M.A., Burchard, E.A., Artlip, T.S., Dardick, C.D., Liu, Z. 2024. euAP2a, a key gene that regulates flowering time in peach (Prunus persica) by modulating thermo-responsive transcription programming. Horticulture Research. 11(5). https://doi.org/10.1093/hr/uhae076.
Buell, R., Dardick, C.D., Parrott, W., Schmitz, R., Shih, P., Tsai, C., Urbanowicz, B. 2023. Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials. Frontiers in Plant Science. 14:1288826. https://doi.org/10.3389/fpls.2023.1288826.
Zhou, J., Li, M., Li, Y., Xiao, Y., Li, X., Gao, S., Sadowski, N., Timp, W., Dardick, C.D., Callahan, A.M., Mount, S., Liu, Z. 2023. Comparison of red raspberry and wild strawberry fruits reveals mechanisms of fruit type specification. Plant Physiology. 193(2):1016-1035. https://doi.org/10.1093/plphys/kiad409.
