John Frampton

The project aims to develop CRISPR-based genome editing using somatic embryogenesis (SE) to enable the strategic engineering of superior clonal Fraser fir Christmas trees. Fraser fir is one of North Carolina������������������s most important specialty crops. Developing novel genomic tools and genome editing technologies for Fraser fir will have a transformative impact on the North Carolina Christmas tree industry. We propose four major objectives to develop the CRISPR-based SE system: (1) Streamline SE platform: we will improve the final steps of the Fraser fir SE process to establish a complete platform from cryostorage to whole tree regeneration; (2) Identify superior SE clonal lines: we will produce 30 independent Fraser fir clonal lines and select the top three lines for genome editing; (3) Assemble CRISPR-ribonucleoprotein (RNP) complexes: we will assemble and test the mutagenic function of up to ten CRISPR-RNP complexes in vitro for Fraser fir genome editing; (4) In vivo validation of CRISPR-based genome editing: We will deliver the CRISPR-RNP complexes into Fraser fir protoplasts and embryogenic cell masses for transgene-free genome editing. Subsequent to the funding period, the CRISPR-based SE system will be used for engineering superior clonal seedlings for field trials in the North Carolina Mountains.

Fraser fir Christmas trees are one of North Carolina������������������s most important specialty crops. The Camcore and Christmas Tree Genetics programs in the Department of Forestry and Environmental Resources at North Carolina State University will evaluate and select Fraser fir clones with increased tolerance to the balsam woolly adelgid to mitigate the impact of this pest and reduce overall pesticide usage for North Carolina������������������s Christmas tree industry. The results will inform decisions on seed usage from existing Fraser fir seed orchards and breeding designs for developing additional sources of adelgid tolerance. Outcomes will be reported to stakeholders through presentations at the North Carolina Christmas Tree Growers Association (NCCTGA) and an article in Limbs and Needles, the official trade magazine of the NCCTGA.

North Carolina State University will support improvement of disease- and pest-resistant Christmas tree planting stock by developing a genetic linkage map of Fraser fir showing the locations and functions of genes. This map will permit adoption of advanced breeding strategies based on recent advances in technology. The outcome of this effort will enable tree breeders to identify and select individuals likely to show improved resistance to important diseases such as Phytophthora root rot and pests such as balsam woolly adelgid. The genetic linkage map will be created by DNA sequence analysis of genes expressed in progeny of a single tree, and the functions of the genes annotated by comparison of the gene coding sequences to public databases of information about previously-described plant gene families. These comparisons will allow identification of Fraser fir genes similar to genes related to disease- and pest-resistance in other plant species, which in turn will enable surveys of other fir species known to be resistant to Phytophthora root rot or balsam woolly adelgid infestation, to identify genes likely involved in those traits in fir trees. That information can then guide breeders to identify Fraser fir trees with the desired characteristics for production of planting stock to be made available to North Carolina Christmas tree growers.

Leyland cypress is a cloned hybrid conifer propagated via rooted cuttings and commonly used throughout the Southeast in the ornamental and Christmas tree industries. Needle blight caused by Passalora sequoiae can significantly damage and kill infected Leyland cypress trees and compels Christmas tree growers to apply pesticides every 2-4 weeks during the growing season. The use of Leyland cypress planting stock resistant to this disease could reduce the need of pesticide applications and increase industry profits. The proposed research will investigate the putative resistance of a Leyland cypress ���������������Leighton Green������������������ sport discovered during the spring of 2014 by Louisiana Christmas tree grower, Clarke Gernon of Shady Pond Tree Farm. The following activities will be pursued: 1) develop techniques to artificially infect Leyland cypress cuttings with Passalora and understand the effect of inoculum concentration on disease development, 2) travel to Shady Pond to observe disease symptoms and morphology of the two Leyland cypress types, 3) inoculate typical versus putatively resistant cuttings under control conditions with inoculum from both ENC and LA, and 4) establish three research field trials in ENC with interested growers willing to NOT spray for Passalora.

The proposed work will leverage efforts of the Collaborative Fir Germplasm Evaluation (CoFirGE) Project that in 2013 established replicated genetic plantings of Turkish and Trojan fir in Connecticut, Michigan, North Carolina, Oregon, Pennsylvania, and Washington. These plantings were established in these Christmas tree production regions in the U.S. and Denmark to identify regionally adapted sources of Turkish and Trojan fir that also produce superior Christmas trees. The CoFirGE Project represents a collaborative effort involving the Connecticut Agricultural Experiment Station, Michigan State University, North Carolina State University, Oregon State University, Pennsylvania State University, University of Copenhagen and Washington State University. Funding to support the collection of seed from Turkish and Trojan fir stands in Turkey in 2010 and the production of seedlings from these and proven sources of balsam, Fraser, grand, Korean, noble and white fir was provided by the Connecticut Christmas Tree Growers Association, Danish Christmas Tree Growers Association, Michigan Christmas Tree Association, North Carolina Christmas Tree Association, Pacific Northwest Christmas Tree Association, and Pennsylvania Christmas Tree Growers Association. Each of the replicated plantings includes progeny from 60 Turkish fir (3 provenances) and 40 Trojan fir (2 provenances) trees and seedlings from proven Christmas tree sources of balsam, Fraser, grand, Korean, noble, Nordmann (3 provenances and 2 Danish seed orchards) and white fir. The two North Carolina plantings are at the NCDA&CS Upper Mountain and Mountain Research Stations. During this project we will collect growth and quality data from trees in the two NC CoFirGE studies prior to 2017 tree growth. Parameters measured will include total height and state of bud break. Data loggers will be installed at each site to record ambient temperatures and the temperature data will be used to calculate the growing degree days associated with bud break during subsequent years. This information will provide information on genetic variation in the time of bud break, which is helpful in assessing the potential risk of damage from late spring frosts as well as determining how uniformly trees from a given source break buds.

Chalcids (Megastigmus spp.) are wasps within the superfamily Chalcidoidea, part of the order Hymenoptera. Megastigmus specularis infests Fraser and balsam fir seed along the entire Appalachian Mountain chain. Recently, this chalcid has been discovered in seed harvested from the Big Springs Fraser Fir Seed Orchard in Ashe County. Seeds from this orchard, which is owned and operated by the N.C. Premium Fraser Fir Seed Cooperative, will be used for investigations to better understand chalcid infestation patterns. Specifically, this research will: 1) assess clonal variation in Fraser fir seed infested by chalcids, 2), study developmental and infestation patters of the chalcid, and 3) determine the taxonomic classification of chalcids and other insects reared from Fraser fir seed.

Fraser fir Christmas trees are one of North Carolina������������������s largest specialty crops, producing revenue of over $100 million annually. Genetic improvement efforts are underway using traditional plant breeding methods to increase growth, quality, pest resistance, and post-harvest needle retention. However, progress is slow due to intrinsic challenges of breeding a coniferous species with a long generation cycle (8-15 years). Somatic embryogenesis is a tissue culture cloning technique that generates genetically identical individuals. It is an important biotechnology tools and a key step in the development of genetic transformation and propagation protocols to produce genetically engineered planting stock in the future. The goal of this project is to improve somatic embryogenesis methods for Fraser fir and thus, provide a pathway for large-scale clonal propagation and genetic engineering in the future. Progress will be evaluated and success measured by comparison to the current state of the technology for each stage of this process.

Next-generation sequencing technology will be employed to accelerate the development and use of genetic information to improve firs for use as Christmas trees, an important specialty crop for which American consumers spend over $1 billion annually. The initial focus will be on improving postharvest needle retention, because surveys indicate that messiness is a major reason consumers do not purchase real Christmas trees. A two-step process will be used to identify single nucleotide polymorphic markers (SNPs) with predictive power: 1) candidate genes will be identified via RNA sequencing (RNA Seq) and 2) SNPs in candidate genes for will be screened for association with phenotypes by targeted sequencing of genomic DNA. Additional traits of interest that will be addressed are resistance to Phytophthora root rot, a disease that reduces productivity and increases costs in all fir production regions and adaptability of two promising exotic species that will be evaluated across all fir production regions in a related ongoing collaborative project. Consumer surveys and focus groups are included in this proposal to help identify their preferences and hence, traits to improve in the future. An integrated outreach program will educate growers about producing and using genetically improved planting stock as well as consumer preferences. Breeding of conifers requires a continuous long-term effort, so the applicants wish that this be considered as a long-term project. In the next phase of funding, other conifer species important for Christmas tree production such as Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) and pine species (Pinus spp.) will be integrated into the project plus new traits identified as important from consumer and grower input during the first phase.

Phytophthora root rot, also known as ink disease in chestnuts, is caused by several water mold species in the genus Phytophthora. In the southern United States, Phytophthora cinnamomi is the primary species responsible and usually kills afflicted American chestnut trees. While not ubiquitous, P. cinnamomi is widespread in the Southern Piedmont and Appalachians throughout the pre-blight range of chestnut. In this region, Phytophthora root rot has hindered the deployment of B3F3 restoration chestnuts by The American Chestnut Foundation. Although this planting stock has undergone seven generations of breeding and selection for resistance to chestnut blight caused by Cryphonectria parasitica, efforts to develop Phytophthora resistance have only recently commenced but are necessary to ensure successful chestnut restoration in the southern part of its range. Objective The objective of this project is to evaluate 95 advanced generation (B3F3) families from The American Chestnut Foundation������������������s breeding population for resistance to P. cinnamomi.

Fraser fir cultured as Christmas trees is one of North Carolina?s largest specialty crops with an annual revenue exceeding $100 million. The most severe disease in Fraser fir nurseries and plantations is Phytophthora root rot which afflicts the industry with an estimated $6 million in losses annually. Because Fraser fir has no innate resistance, the NCSU Christmas Tree Genetics Program has begun evaluation of two exotic species, Turkish and Trojan fir, that show promise as quality Christmas tree species and also possess some Phytophthora resistance. Genetic control of resistance to a single Phytophthora genotype has been confirmed. The proposed research will assess resistance in these fir species to a broader array of Phytophthora genotypes and use next generation sequencing technology to develop DNA markers to select for resistance in genetic field trials that will be established in 2013-2014. The technology and knowledge generated will be applied toward developing Phytophthora-resistant fir planting stock targeted for sites with known disease problems and will ultimately abate the adverse impact of this disease on the state?s Christmas tree industry.