Location: Plant Genetic Resources Unit (PGRU)
2022 Annual Report
Objectives
Objective 1: Develop and release improved apple rootstocks by leveraging advances in marker assisted breeding, including construction of genetic maps, establishing trait associations, gene discovery for important rootstock traits (dwarfing, early bearing, yield efficient, fire blight resistant), and screening for novel alleles for important rootstock traits.
Sub objective 1A: Perform all breeding and evaluation stages involved in the 15-30 year process (timeline depending on intensity of phenotyping and need to fast-track) of developing new rootstocks with the assistance of recently developed breeding tools, such as high throughput phenotyping and marker-assisted breeding.
Sub-objective 1B: Identify and characterize novel germplasm, genes, alleles and trait loci through quantitative trait analyses leveraging new genetic-physical maps.
Objective 2: Identify and dissect important rootstock traits that modify gene activity in the scion, toward enhancing drought tolerance, tree architecture, propagation by nurseries, root growth and physiology, nutrient use efficiency, and disease resistance; incorporate this knowledge into breeding and selection protocols.
Sub-objective 2A: Identify components of rootstock induced traits that modify gene expression and metabolic/physiological profiles of grafted scions to increase tolerance to abiotic stresses, improve fruit quality and storability, increase tree productivity, disease resistance and nutrient use efficiency.
Sub-objective 2B: Validate relationships between trait components and overall apple tree performance in different rootstock-scion combinations and incorporate new knowledge into breeding and selection protocols.
Approach
The objectives of this project will be met by applying a combination of conventional breeding techniques and marker assisted breeding to select for improved rootstocks. The project will also leverage the use of aeroponics to study components of root traits that aid in nutrient uptake and water use efficiency by monitoring gene expression and other metabolic componds in apple roots.
Progress Report
Breeding apple rootstocks has field and laboratory components that occur cyclically every year and some that occur every few years because of the nature of how apple trees grow. Spring of 2022 posed some challenges with the process of making new breeding populations as commercial pollinators (rented bees and bumblebees) used to cross-pollinate the breeding parents were not readily available during peak flowering time. We were fortunate to find a local source of beehives at the tail end of pollination season that allowed us to perform some new planned crosses aimed at improving apple rootstock tolerance to viruses and woolly apple aphids. Approximately 2,500 apple rootstock hybrid seed generated from previous years was successfully germinated and the young seedlings were readily challenged with diseases that cause crown and root rot, killing approximately 40% of them. The survivors will be challenged with bacteria that causes fire blight disease resulting in survivors that should be resistant to both diseases. In order to test the productivity and field performance of new breeding lines we grafted approximately 2,000 trees which were planted in a nursery field. These trees will be harvested at the end of the season (November) and planted in a new Phase 1 field trial in 2023. In June 2022 we also planted a new replicated field trial that features three different apple scion cultivars and seven different apple rootstocks. Similar plantings of these rootstock-scion combinations were made at the USDA ARS Kearneysville location and at 7 grower cooperator trial locations in California. This field trial will be used as a demonstration block and to study various questions related to abiotic stress and the effect of climate on flowering and apple tree productivity.
A very heavy natural infection of fire blight in some of our breeding plots provided some very useful data on the rootstock resistance component for this disease and confirmed that resistant rootstocks help the survival of the whole apple tree infected by fire blight.
A major experiment was initiated in one of our greenhouses which aims to confirm resistance to the woolly apple aphid in our breeding germplasm. This experiment is quite difficult as the material has to be maintained pesticide free in order for the insects to reproduce and colonize susceptible plants. We have discovered that some apple rootstocks that were considered susceptible were found to be resistant, providing new sources of resistance beyond what had been used in the breeding program. We aim to add more germplasm to the experiment, including breeding populations that will allow us to work out the genetics of resistance to this insect.
A combination of apple viruses is suspected to affect growth and productivity of apple trees. We are utilizing the custom built aeroponic system to study the effect of this specific virus combination on root growth and gene expression. Replicates of G.935, G.890, G.969 and G.213 were grafted with virus laden and virus cleaned buds. Root tissue was harvested before inoculation and will be harvested after the grafts are fully developed into small trees.
The laboratory was able to process RNA samples that were supposed to be sequenced in 2020 and were stored in the freezer because of the pandemic. We are proceeding to analyze the sequenced samples to discover differentially expressed genes in contrasting breeding lines that display high and low levels of nutrient uptake. This and other sequencing experiments are laying the foundation for new selection parameters of new apple rootstocks and increasing our overall understanding of how apple tree roots work.
Accomplishments
1. New method of virus elimination for apple rootstocks. The Geneva apple rootstock breeding program has adopted a procedure to establish “clean” propagation material at key micropropagation laboratories prior to release of elite breeding lines into the commercial stream as rootstock cultivars. In preparation to release a set of new apple rootstocks for the U.S. industry, we collaborated with Foundation Plant Services in Davis, California, and APHIS PGQP in Beltsville, Maryland, to index plant material with what is known as High Throughput Sequencing (HTS) or Deep Sequencing, which is extremely powerful at picking up even small quantities of viral RNA. We found that some of the stocks slated for release to the public were infected by viruses. At the same time, scientists at USDA ARS National Laboratory for Genetic Resources Preservation (NLGRP), were working on discovering new ways to eradicate viruses from apple germplasm destined for cryopreservation. As a result, a collaboration ensued between the Geneva breeding program and NLGRP to investigate whether thermotherapy or cryotherapy alone or in combination could effectively eradicate apple viruses from in-vitro cultures of the apple rootstocks destined for release. Overall, thermotherapy combined with cryotherapy treatments resulted in the highest percentage of virus- and viroid-free plants.
Review Publications
Bettoni, J.C., Fazio, G., Carvalho Costa, L., Hurtado-Gonzales, O.P., Ah Rwahnih, M., Nedrow, A.K., Volk, G.M. 2022. Thermotherapy followed by shoot tip cryotherapy eradicates latent viruses and Apple hammerhead viroid from in vitro apple rootstocks. Plants. 11(5). Article e582. https://doi.org/10.3390/plants11050582.
Liu, J., Abdelfattah, A., Wasserman, B., Wisniewski, M., Droby, S., Fazio, G., Mazzola, M., Wu, X. 2022. Contrasting effects of genotype and root size on the fungal and bacterial communities associated with apple rootstocks. Horticulture Research. 9. Article uhab013. https://doi.org/10.1093/hr/uhab013.
