Ross Whetten

I am proposing to request funds to support a post-doc and sequencing costs associated with genome sequencing of Fraser fir.

This project will be a collaboration between the Christmas Tree Genetics Program and the Molecular Tree Breeding Lab in the Department of Forestry and Environmental Resources at North Carolina State University. Our goal is to accelerate the genetic improvement of Fraser fir against the important regulatory pest Elongate Hemlock Scale (EHS). Fraser fir is one of North Carolina������������������s most important specialty crops generating annual revenues exceeding $100 million. The development of novel genomic tools and technologies will have a positive, transformative impact on the North Carolina Christmas tree industry. Our project builds on resources developed by the NCSU Christmas Tree Genetics Program in collaboration with the NCDA and NC Christmas tree growers over the past 4+ decades. We propose four major objectives in this proposal: (1) evaluation of genetic variability in Fraser fir and select Abies spp. response to Elongate Hemlock Scale infestation; (2) histological evaluation of EHS feeding on Fraser fir and Abies spp. foliage; (3) biochemical and molecular response of Abies spp. to Elongate Hemlock Scale infestation; (4) development of molecular resources to identify defense characteristics of EHS resilient Abies genotypes; and (5) synthesis and dissemination of results to NC Christmas tree stakeholders. Subsequent to the funding period, these efforts will benefit the North Carolina Christmas tree community and contribute to the genetic conservation of native Fraser fir populations in the Appalachian Mountains. We expect project deliverables will help address key knowledge gaps of pest resilience in Fraser fir and push conventional conifer breeding strategies and integration with genomic information into a new era.

Loblolly pine is the most abundant commercially grown tree species in North Carolina with over 100,000 acres of pine plantations established each year in the state. In addition to the conventional forest products industry, loblolly pine serves as a promising source for renewable energy in the form of woody biomass. Large genetic differences exist for growth, disease resistance, and stem form. By planting genetically superior trees with desirable traits, it may be possible to substantially increase the amount and quality of biomass produced at a given site. The goal of this project is to evaluate different planting stock (families) in combination with different thinning regimes in order to inform forest landowners how best to maximize their returns when supplying both the bioenergy and sawtimber markets. This project was initiated in 2012, with the planting of a high spacing density (1037 trees/acre) long-term field trial in the NC Piedmont. The trial includes 10 of the best Coastal and 10 of the best Piedmont families with varying degrees of adaptation, growth, and wood characteristics. Different thinning regimes will be explored using eight year measurements, and the predicted financial returns from the thinnings as well as projected sawtimber production will be evaluated.

This project will be a collaboration between the Christmas Tree Genetics Program, the Forest Health and Conservation Program, and the Molecular Tree Breeding Lab in the Department of Forestry and Environmental Resources at North Carolina State University. Our goal is to accelerate the genetic improvement of Fraser fir against the tree-killing pathogen Phytophthora root rot and insect pest balsam woolly adelgid. Fraser fir is one of North Carolina������������������s most important specialty crops generating annual revenues exceeding $100 million. The development of novel genomic tools and technologies will have a positive, transformative impact on the North Carolina Christmas tree industry. Our project builds on resources developed by the NCSU Christmas Tree Genetics Program in collaboration with the NCDA and NC Christmas tree growers over the past 4+ decades. We propose five major objectives in this proposal: (1) genomic resource development of Fraser fir responses to Phytophthora and BWA; (2) identification of Phytophthora and BWA elicitors; (3) evaluation of Fraser fir responses to isolated elicitors; (4) population level analysis of key pest responsive genes in existing NCSU Fraser fir breeding program resources; and (5) synthesis and dissemination of results to NC Christmas tree stakeholders. Subsequent to the funding period, these efforts will benefit the North Carolina Christmas tree community and contribute to the genetic conservation of native Fraser fir populations in the Appalachian Mountains. We expect project deliverables will help address key knowledge gaps of pest resilience in Fraser fir and push conventional conifer breeding strategies and integration with genomic information into a new era.

Plant breeding is beginning to incorporate new tools based on genomic resources and methods, although the rate of availability and adoption of new tools and resources varies from species to species. Loblolly pine (Pinus taeda L.), the most-planted timber crop species in the United States, now has a draft sequence assembly of the 23-billion-basepair genome, and researchers are actively seeking ways to exploit this resource for purposes of applied breeding. The objective of this proposal is to test the hypothesis that specific regions of the pine genome are enriched in sequence variants that affect phenotype, and therefore have practical value for applied pine breeding purposes. A variety of methods will be used to add value to the current draft assembly, with the goal of creating a more useful resource for pine breeders to use in increasing the productivity and adaptability of loblolly pine. Preliminary data showing the feasibility of each of these methods in loblolly pine are available. The same or similar methods could be applied to other agricultural species, so benefits of this research will extend to crop and livestock breeders.

Fraser fir (Abies fraseri [Pursch] Poir.) Christmas trees are one of North Carolina������������������s most important specialty crops. The North Carolina Christmas tree industry produces annual revenues exceeding $100 million from sales of Fraser fir trees and greenery alone. Genetic improvement efforts for the species led by the North Carolina State University Christmas Tree Genetics Program have been underway since the mid-1990s, using traditional breeding methods to increase growth, quality, pest resistance, and post-harvest needle retention. Recently, breeding efforts have expanded to include modern biotechnology approaches for improving propagation techniques, shortening the breeding cycle, and increasing opportunities for genetic modification. Among insects affecting Fraser fir production in North Carolina the exotic-invasive Balsam Woolly Adelgid (BWA, Adelges piceae [Ratzeburg]) is the most important. This pest, native to Europe, was introduced to the United States in the early 1900s and was responsible for the death of approximately 70% of mature Fraser firs throughout native stands in the Southern Appalachian Mountains. The adelgid also infests Christmas tree plantations where it affects tree growth, form, and survival, costing North Carolina growers $1.53 million annually to protect their plantations with chemical insecticides. Increasing the level of adelgid resistance in Fraser fir breeding lines offers the best opportunity to produce planting stock that is more resilient against this pest and reduce overall pesticide use in North Carolina Christmas tree plantations. In 2014, 312 grafts from 30 elite Frasier fir clones in the North Carolina Premium Fraser Fir Seed Coop Orchard in Ashe County were established on potted rootstocks. At the same time, 81 grafts from seven potentially adelgid-resistant Fraser fir clones, originating from surviving trees in the Great Smoky Mountains National Park and maintained in a clone bank at the NCDA&CS Upper Mountain Research Station, were also established. In 2016, 348 of the potted clones were arranged in a replicated study design at the NCDA&CS Mountain Research Station and artificially infested with the balsam woolly adelgid. The other 48 clones were designated as uninfested controls. Phenotypic assessments made in 2018 indicated significant variation among the clones with respect to their physical responses to adelgid infestation. A number of the clones had significant levels of twig gouting and loss of apical dominance, characteristics associated with adelgid susceptibility. A few of the clones showed no physical response to infestation suggesting they may be partially resistant or tolerant to the adelgid. A companion study funded by the NCDA Specialty Crops Block Grant Program is assessing the volatile chemistry of these clones in relation to their responses to adelgid infestation, with the goal of identifying chemical markers indicative of increased adelgid resistance that could be utilized in breeding. This proposal seeks funding for a preliminary study to test the use of RNA-Seq next-generation gene sequencing to identify differential gene expression between putatively adelgid resistant and susceptible Fraser fir clones. If successful, this technology will be applicable to the use of modern genomic approaches for incorporating adelgid resistance into Fraser fir breeding lines.

The Christmas tree industry in North America is mainly supplied with Fraser fir seeds from natural stands and genetically improved material from clonal seed orchards (CSO). Trees derived from CSOs have better quality and more desirable marketable characteristics than trees that originate from seeds collected in natural stands. These attributes provide a considerable increase in income for Christmas tree plantation owners. Chalcidoidea (chalcid wasps) are a megadiverse group of seed feeders, and at least 49 species from the genus Megastigmus are associated with conifers. Previous work by our group --funded by the NC Christmas Tree Association-- showed that chalcid wasps were the only insects present in infested seeds from a specific CSO, and that they had a significant impact on seed yields. Our preliminary results suggested that there may be genetic differences in chalcid infestation rates among clones in the CSO, and that these differences could affect the cost of planting stock for Christmas tree growers due to downstream impacts on the viability of seeds from the same clone during long-term storage. Additionally, we found a possible effect of pesticide treatment on chalcid infestation rates. This proposal seeks funding to determine clone-specific infestation rates before and after pesticide treatment to test these hypotheses and to identify candidate clones with reduced susceptibility. Ultimately, the proposed research will contribute basic and practical knowledge to improve seed quality and leverage chalcid development to control future infestations in other fir orchards in North Carolina.

Loblolly pine (Pinus taeda L.) is the primary woody bioenergy feedstock for North Carolina. There are over 2.6 million acres of pine plantations in NC, and almost all have been established with loblolly pine genotypes from the NCSU Cooperative Tree Improvement Program. The 5-year-old Loblolly Pine Biomass Genetics/Cropping Study at the NCDA&CS Umstead Farm at Butner, NC is a unique field laboratory where we are evaluating the genetic differences in traits that impact biomass/bioenergy traits. In this field trial, we planted 10 of the best Coastal and 10 of the best Piedmont loblolly pine varieties with varying degrees of adaptation, growth, and wood characteristics. At age 3 years, Coastal families grew faster but suffered more cold damage and stem malformations than the better adapted Piedmont families. Funding is sought to continue this critical experiment and to better understand the genetic basis of variation in biomass/biofuel traits and improve pine varieties for biomass production. Trees will be at the ideal age to collect wood samples and measure density, strength, and moisture content to project dry weight yields and biomass/bioenergy value.

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.

The goal for this partnership is to plant, develop and document the information and tools needed to demonstrate the sustainable production of biomass for bioenergy across the Southern US. Specifically, this program will develop and demonstrate sustainable, flexible, integrated biomass production solutions that create innovative deployment scenarios to reliably produce and supply biomass feedstocks that are optimized for performance in leading conversion technologies. Research and development activities will target specific barriers in each step of the supply chain that are identified as critical to regional economic and/or environmental sustainability. Education, extension and outreach activities will be integrated so that the results of this work will reach target audiences with appropriate real-world examples.