Poster Sessions

About TACF Poster Sessions

TACF’s poster sessions aim to expose the public to the most current research around the American chestnut. Research posters contributed by members, chapters and students from schools around the country address topics relative to American chestnut restoration and include but are not limited to:

  • Silviculture and forest management
  • Chestnut blight
  • Phytophthora cinnamomi (ink disease)
  • Other pests and pathogens
  • Forest genetics
  • Restoration and reintroduction
  • Mineland reclamation
  • Forest and wildlife ecology
  • History
  • Timber value/use
  • Breeding and biotechnology
TACF student poster sessions

2023 Poster Session

2023 Winner: Uma Plambeck

The 2023 Poster Session and Student Flash Talks were held during the October 20th Chestnut Chat live webinar. Many thanks to all our participants and judges!

Congratulations to Uma Plambeck from the University of Tennessee for her winning poster titled Screening full-sib and half-sib families of chestnut seedlings for resistance to Cryphonectria parasitica using a small stem assay and to oxalic acid using a leaf-disc assayView poster and abstract.

2023 Student Flash Talks video

2023 Posters

2022 Poster Session

1st Place Winner

Updated North American range of the Asian chestnut gall wasp with the use of the citizen science database iNaturalist — Johnston, C, V; Parry, D; Newhouse, A, E; 2022.

Johnston, C, V; Parry, D; Newhouse, A, E; 2022 Poster

College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY

The Asian chestnut gall wasp (ACGW) is considered to be one of the most significant pests of chestnut trees around the world. This species, first observed in North America in 1974, is a specialist gall-former that causes significant damage to nearly all members of the genus Castanea. Galling causes twig dieback, reduced fruiting, and potential tree mortality of younger individuals. Yet, as the reintroduction of a plethora of blight-tolerant American chestnut saplings has become imminent, a comprehensive range map has not been published since 2015. We thus looked to develop an updated range map of this potentially devastating invasive pest with the use of the citizen science database iNaturalist. The ACGW presents a case in which distinct gall morphology and host specialization allows species identification before adult emergence. With the use of iNaturalist, we have identified a cost-effective method of performing “pseudo-surveys” on uploaded chestnut tree observations throughout North America. Although we cannot definitively determine where the species is absent, we can identify areas in which the species is or has been present by searching for current or previous-season galls on chestnut tree images uploaded to the database. In addition to the 74 ACGW iNaturalist observations, we have searched images from ~15,000 chestnut observations throughout North America, detecting gall formation in 247 said observations. These include reported infestations in 2 new states and 89 new counties. New state observations include Missouri and Rhode Island and have been confirmed through in-person surveys and gall collection. Such observations were found near the border of each state, displaying the potential of this method in locating newly developed infestations. In all, we find the ACGW to encapsulate the majority of the natural American chestnut range and recommend a shift in focus to management based future research.

2nd Place Winner

Validation of an alternative small stem assay for blight resistance in backcross hybrid chestnuts (Castanea spp.) and recommendations for its expanded use — Wingo, M, D; Conn, C, E; and Cipollini, M, L; 2022.

Wingo, M, D; Conn, C, E; and Cipollini, M, L; 2022 Poster

Department of Biology, Berry College, 2277 Martha Berry Highway, Mount Berry, Georgia

We evaluated an alternative small stem assay (AltSSA) for blight resistance in backcross hybrid chestnuts (Castanea dentata/mollissima). Whereas standard small stem assays (SSAs) are done by inoculating small incisions in stems, in our AltSSA, 4-5 mm stems are cut off, and the exposed (living) stem tips are inoculated with discs of Cryphonectria parasitica inoculum and temporarily covered with plastic sleeves. Intended primarily for forward selection, this method was designed to be easy to implement, to consistently induce cankering, and to better enable seedling recovery via the development of lateral shoots from the lower stem. After 90+ days, cankers are evaluated and removed, and seedlings are prepared for out-planting.  Previous results showed that AltSSAs performed at least as well as a common SSA method in distinguishing resistant and susceptible types. In this follow-up analysis of 35 lines of backcross seedlings studied in 2020 and 2021, we show that mean orange zone canker length (OZCL) and a multifactor PCA-based blight resistance index gave results consistent with predictions derived from the blight resistance phenotype (two methods) and percent American chestnut ancestry of the parents of each line.  As expected, based upon the apparent polygenic inheritance of blight resistance in backcross chestnuts, mean OZCL of backcross families ranged from intermediate (F1-level) to low (American chestnut-level). Consistent with prior results, canker production was near 100%, survivorship after out-planting was very high, and post-inoculation deaths were apparently unrelated to the stem tip inoculations (e.g., unrelated basal cankering).  Altogether, these results suggest that the AltSSA is a viable method for early detection of relative blight resistance in seedlings and may enable a reduction in the numbers of trees out-planted and placed under care for long-term evaluation and breeding.

Keywords: blight, canker, Castanea, chestnut, fungi, resistance breeding, small stem assay, United States

3rd Place Winner

Oxalic acid leaf disk assays may be another method of screening phenotypic variability in “American” looking Castanea hybrid trees — Harden, K, E; Craddock, J, H; 2022.

Harden, K, E; Craddock, J, H; 2022 Poster

Department of Biology, Geology, and Environmental Science, The University of Tennessee at Chattanooga, 615 McCallie Avenue, Chattanooga, TN

The TACF chestnut breeding efforts aims are to preserve Castanea dentata phenotypic characteristics, but also encode for protection from Cryphonectria parasitica. Phenotypic variability is seen in the hybridized Castanea trees in traits such as stipule persistence, stipule shape, stem and leaf hairs, twig pigmentation, and of course resistance to C. parasitica. Blight resistance in the Asian species may be due in part to their tolerance to oxalic acid.  It may be possible to characterize endogenous oxalic acid tolerance phenotypes in Castanea species and hybrids using a leaf-disk oxalic acid soak. I hypothesize that there will be measurable differences in area of leaf disk necrosis following an oxalic acid soak of leaves of C. alabamensis, C. dentata, C. mollissima, C. ozarkensis, C. pumila, and open-pollinated seedlings of selected backcross hybrids.  The assay involves 1.5 cm diameter leaf discs, soaked overnight in 50mM oxalic acid, then scanned to calculate remaining green/healthy leaf area relative to total disc area using ImageJ software. In addition to the species listed above, I plan to observe the phenotypic traits of the thirteen container- grown seedling families included in the 2022 C. parasitica small stem assay. The SSA families include open-pollinated seedlings of C. mollissima, C. dentata, and eleven selected backcross trees from the Tennessee Chapter breeding program. The BnF2 trees are expected to look morphologically like C. dentata, but this experiment will reveal their relative tolerances to OA.  Because many of these same seedlings are included in the SSA, we may be able to correlate OA tolerance with a blight resistance rating for each tree.

Poster Session Participants

Applying American chestnut biotechnology approaches for the conservation of Ozark chinquapin — Fernandes, P; Pilkey, H; Powell, W; 2022.

Fernandes, P; Pilkey, H; Powell, W; 2022 Poster

Department of Environmental Biology, College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY

The necrotrophic pathogen Cryphonectria parasitica (causal agent of chestnut blight) left the American chestnut (AC-Castanea dentata) functionally extinct in the 20th century. This host-pathogen interaction has been studied for decades, and as a result, several laboratory and field techniques are available to help bring back this tree. A blight-tolerant AC (Darling58-D58) was developed by adding a gene from wheat encoding for a detoxifying enzyme, oxalate oxidase (OxO), to counter the main virulence factor of the pathogen. With the close deregulation of D58 by the U.S. regulatory entities, the next logical step is to apply the knowledge from AC to other species impacted by the chestnut blight, such as the Ozark chinquapin (OC-Castanea ozarkensis), using classical breeding and direct transformations. In 2021, controlled crosses were performed in OC mother trees with D58 transgenic pollen. A total of 205 OC/D58 hybrid nuts were obtained from which 121 germinated and were tested for OxO presence through histochemical analysis on leaf punches. Fifty-five and 58 tested OxO-positive and OxO-negative, respectively, representing ˜50% gene inheritance, which is expected (8 remain untested). Fifty-two trees (26 OxO-positive, 26 OxO-negative) were planted in a regulated field to access morphological development, and the remaining are growing in 1-gallon pots. OxO expression levels and blight tolerance after pathogen inoculation will be accessed when plants reach the appropriate size. Also, rapid pollen production methods developed for AC will be used for future backcrosses to restore the OC phenotype. The direct genetic transformation of OC somatic embryos with OxO is ongoing parallel to breeding. The applicability of the AC’s Agrobacterium-mediated transformation protocol was confirmed by obtaining OC somatic embryos expressing a green fluorescent protein (GFP) reporter gene, and transformations with OxO are ongoing. Laboratory and field techniques developed for the AC have shown promising results and are currently being fine-tuned for the future restoration of the OC.

Keywords: Castanea dentata; Castanea ozarkensis; chestnut blight; breeding; Agrobacterium-mediated transformation

Population dynamics of the lesser chestnut weevil in the Northeastern United States — Filgueiras, C, C; Reynolds, G, R; Guthrie, C, R; 2022.

Filgueiras, C, C; Reynolds, G, R; Guthrie, C, R; 2022 Poster Currently Unavailable

Department of Biology, UNC-Asheville, 1 University Heights, Asheville, NC

The American chestnut was a tree of vital importance to the eastern United States before the introduction of the pathogenic chestnut blight which decimated the population. Through efforts to reestablish the American chestnut tree, an unwanted pest has also regained its prevalence. Curculio sayi, the lesser chestnut weevil, is a specialized seed predator that damages the chestnuts through the oviposition of eggs as well as through the introduction of toxic fungi to the tree. The goal of this project is to examine population dynamics of C. sayi through genetic analysis to establish sources of origin, rates of population dispersal, and human impact on population spread. Combined with previous data on chestnut weevil phenology, this analysis will provide insight into population variation as well as potential species divergence of C. sayi in the Northeastern United States.

Lesser chestnut weevil: lifecycle and phenology — Filgueiras, C, C1; Willett, D, S2; 2022.

Filgueiras, C, C1; Willett, D, S2;2022 Poster

1Department of Biology, UNC-Asheville, 1 University Heights, Asheville, NC
2North Carolina Institute for Climate Studies, NC State University, 151 Patton Avenue, Asheville, NC

The lesser chestnut weevil (Curculio sayi) is a re-emerging pest of American chestnut trees.  Originally documented when American chestnut trees were dominant on the east coast, the lesser chestnut weevil had mostly disappeared until resurfacing in the last decade with the re-expansion of chestnuts on the east coast.  This weevil can expand rapidly, appearing and reaching greater than 80% infestation in less than two years.  Here, we explore the lifecycle and phenology of this chestnut weevil documenting the larval, pupal, and adult stages.  We also highlight phenologies distinctive of a univoltine northern population peaking in November.  Effective means of monitoring these populations are also presented with an eye towards improving management and recognition of this re-emergent, charismatic weevil.

Assessment of soils of New York's Adirondack mountains as a potential site of American chestnut afforestation in a warming climate — Heit, M, D; Horton, T, R; Powell, W, A; 2022.

Heit, M, D; Horton, T, R; Powell, W, A; 2022 Poster Currently Unavailable

College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY

Advances in genetic engineering biotechnologies have augmented American chestnut (Castanea dentata) reforestation efforts by allowing for the generation of blight-tolerant transgenic chestnut lines, including the Darling-58 tree expressing the oxalate oxidase (OxO) gene. Though planting of the OxO-positive tree is still federally regulated, and much restoration still relies on many hybrid and back-crossed lines, advances in the regulatory process have allowed the American Chestnut Research and Restoration Project to advance to site selection for its blight-tolerant tree. One avenue of interest for the transgenic C. dentata is an afforestation scheme in the Adirondack mountains, where the modern chestnut has not historically grown due to cold intolerance. As the climate warms, low-elevation areas of the Adirondacks may become warm enough to serve as C. dentata afforestation sites. Since C. dentata has been absent from the northeastern forest overstory throughout the development of the techniques used to evaluate ectomycorrhizal fungal (EMF) diversity on a molecular level, the tree’s typical EMF community assemblage is not thoroughly understood. This study aims specifically to evaluate chestnut performance and EMF diversity in soils along a known east-west nitrogen (N) deposition gradient present in the Adirondacks, as ambient N may relieve the N-limitation conditions under which EMF typically dominate. The microhabitat of 12 sites distributed across the region (N, C/N ratio, Ca2+, ammonium and nitrate) will also be evaluated. Non-transgenic wild-type chestnuts were planted in soils mixed with inoculum from these sites (N=190). Seedlings were grown in a plant growth chamber for 7 months; EMF abundance and diversity across sites is being assessed via DNA extraction and species identification using restriction fragment length polymorphism (RFLP) typing of the fungal internal transcribed spacer (ITS) sequence. Further, dry root:shoot biomass ratio and foliar nutrient composition will be assessed as possible covariates for chestnut performance. Previous studies have designated C. dentata as an appropriate species for post-logging and mine reclamation sites, which are common in the Adirondacks, and thus these sites may be an appropriate choice for land managers of the future. If C. dentata can find fungal partners to support its growth in the Adirondacks, the transgenic tree may be one step closer to finding a new home.

Tree growth, vine growth, and resource investment for the establishment of an American chestnut - hybrid wine grape agroforestry system — Kehas-Dewaghe, E, M; Diemont, S.A, W; Powell, W, A; 2022.

Kehas-Dewaghe, E, M; Diemont, S.A, W; Powell, W, A; 2022 Poster

College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY

Sustainably designed agricultural systems are receiving increasing research attention not only for their role in reducing ecological damages resulting from modern conventional agricultural practices, but as tools for restoring species and landscapes subject to negative anthropogenic impacts. Agroforestry systems intentionally integrating tree crops with arable crops, pasture, or livestock systems in multi-species polycultures provide beneficial ecosystem services and potential avenues for species restoration in temperate forest and agricultural landscapes. This study investigates the effects of establishing agroforestry plantings consisting of arable plots surrounded by co-plantings of blight-tolerant American chestnuts (Castanea dentata) developed by the American Chestnut Project at SUNY ESF, and two hybrid wine grape (Vitis X) varieties on growth performance in chestnut and grape. Such assemblages are a rare but traditional form of viticulture in Europe. We use energy analysis, a method of analyzing renewable and nonrenewable energy and material resources invested in a system, to examine the resource use burden of establishing and maintaining these plantings. Conventional monoculture plantings of chestnut and grape concurrently established adjacent to the agroforestry plantings provide a basis for comparison of growth performance and resource use between agroforestry and conventional systems. Biweekly height and stem diameter measurements collected from agroforestry and conventional plantings during 2021 and 2022 growing seasons and grape vine dormant pruning weights taken in March 2021 will be used to assess growth performance in chestnut and grape. Preliminary results presented here indicate that co-planting American chestnut and wine grapes in the agroforestry system has a positive effect on chestnut seedling growth, while no detrimental effects were observed in wine grape growth.

Rooting American chestnut cuttings — Liang, H; 2022.

Liang, H; 2022 Poster

Department of Genetics and Biochemistry, 154 Robert F. Poole Agricultural Center, Clemson University, Clemson SC

American chestnut (Castanea dentata) (AC) is notoriously recalcitrant to adventitious rooting (AR) from stem cuttings. This is a major limitation for clonal propagation of elite genotypes and has hindered the breeding process and germplasm conservation. The overarching goals of the project are to overcome the difficulty of rooting American chestnut cuttings and develop an easy-to apply and efficient rooted cutting system for the heritage tree. Our results show that rooting in semi-lignified shoots of the current season’s growth is feasible with hormone treatments, and American chestnut has the opposite endogenous hormone distribution between leaf and stem when compared to easy-to-root poplar.

Speed breeding Darling 58 seedlings to expand genetically-diverse pollen production and to advance post-clonal generations — May, V; Klak, T; 2022.

May, V; Klak, T; 2022 Poster

School of Marine and Environmental Programs, University of New England (UNE), 11 Hills Beach Road, Biddeford Maine

At UNE, we have focused for three plus years on improving techniques to produce Darling 58 pollen while pursuing three objectives. We have sought to increase (a) pollen quantity and (b) genetic diversity, while (c) reducing the time between seedling sowing and pollen production. Indoor greenhouses, where seedlings are grown under intense hi-light conditions for more than a year, are vulnerable to outbreaks of many pests and pathogens which we have continually strived to recognize and control. Other greenhouse threats to seedling health we’ve experienced include electricity outages, erratic temperatures, and overly confined conditions. Other challenges come from long-term storage of viable pollen at -80C. Nevertheless, as of the second half of 2022, we can report some significant progress regarding our three objectives: (a) We are producing Darling 58 pollen in unprecedented quantities; our daily pollen collection is enough to yield as much as thousand fertile nuts. (b) We are collecting pollen simultaneously from more than a dozen seedlings, themselves the offspring of wild-type trees representing a good portion of the native range (from Maine to Virginia and New York). (c) We’ve been able to reduce the months required for a seedling to produce pollen, so that pollen from an increasing number of seedlings is available to pollinate wild-type trees within the same chestnut year. That is, nuts pollinated in July grow into mature seedlings that produce pollen we’ve applied the following July. Since the Darling 58 began as a clone (T0), our methods have advanced pollen through an entire generation per year (now producing T3 & T4 pollen). All of this speed breeding is confined to USDA permit, with hope for federal deregulation in the near future. Then we’ll be able distribute Darling 58 pollen to many more collaborators from across the native range.

Keywords: Hi-light treatments; the chestnut year; pests and pathogens; Darling 58 generations

The American chestnut founder line transformation project — Merkle, S, A; Tull, A, R; Gladfelter, H, J; Remko, S, E; 2022.

Merkle, S, A; Tull, A, R; Gladfelter, H, J; Remko, S, E; 2022. May, V; Klak, T; 2022 Poster Currently Unavailable

University of Georgia, 104 Caldwell Hall, Athens, GA

Production of American chestnut trees expressing the wheat oxalate oxidase gene (OxO) to provide resistance to the chestnut blight fungus has been adopted by The American Chestnut Foundation (TACF). The primary path chosen by TACF for spreading the transgene to multiple genetic backgrounds for restoration is via pollinating American chestnut trees with pollen produced by Darling58 transgenic OxO trees. An alternative approach is to directly insert the OxO gene into multiple American chestnut genotypes representing the natural genetic diversity of the tree. The resulting trees would already be adapted for growth in their native regions. We began pursuing this approach by initiating new embryogenic culture lines (“Founder Lines”) from nuts collected by TACF cooperators from large surviving American chestnut trees (LSAs) growing in different parts of the range from Maine to Georgia. In 2020, over 100 new embryogenic cultures representing eight source trees from five regions (New England, Pennsylvania, Maryland, Virginia, Georgia) were captured. Copies of all the new Founder Lines were placed in cryostorage. Then, the cultures were screened for their abilities to produce abundant somatic embryos and high-quality somatic seedlings, to facilitate choosing those to target for transformation with OxO.  The selected Founder Lines tested so far for sensitivity to the selection agent geneticin showed a range of sensitivities to the antibiotic in liquid medium, so selection needed to be customized for each line. Transformation experiments with these lines using the pFHI-OXO and pWIN3.12-OXO vectors are underway, and the first putative transgenic events are growing in selection medium. Once the presence of the OxO transgene in the colonies is confirmed using PCR and an OxO enzyme assay, copies of the transgenic events will be cryostored and multiple events in each background will be grown up for somatic embryo and plantlet production.

Characterization of Darling 58 transgenic American chestnut — Newhouse, A, E; Matthews, D, F; Ostlund, S, J; Wood, H, K; Del Grosso, A, R; Mott, J, C; LoPiccolo, K, R; Powell, W, A; 2022.

Newhouse, A, E; Matthews, D, F; Ostlund, S, J; Wood, H, K; Del Grosso, A, R; Mott, J, C; LoPiccolo, K, R; Powell, W, A; 2022 Poster

College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY

Transgenic American chestnuts have been engineered to express oxalate oxidase (OxO), which enhances their tolerance to the harmful effects of oxalic acid produced by the chestnut blight fungus, Cryphonectria parasitica. The required government regulatory review of these trees involves both environmental tests and detailed characterizations of the tree itself. Environmental interactions have been well documented in a variety of outlets, but recent molecular and organismal analyses of Darling 58 have not yet been published outside of the government regulatory documents. Many of the following characterizations had previously been hypothesized or established in general terms, but newer techniques and continued growth of early-generation trees have allowed us to perform more detailed experiments. For example, older techniques determined that Darling 58 has a single copy of the OxO transgene and that it is not in or near any existing genes, and also suggested that a small section of DNA near the insertion site was inverted during the engineering process. (Such inversions are not unusual during transformation, and they are even more common with traditional breeding.) A newer genome sequencing method (PacBio) confirmed these previous findings and allowed us to read the entire insertion site at an even higher resolution, down to every individual DNA base pair. We have also compared OxO transgene expression between several families of Darling 58 offspring: there is some variation between individuals, but no strong correlations to tree growth, family background, or severity of natural cankers. Finally, we inoculated several 4-year-old Darling 58 offspring (and closely related non-transgenic controls) with a strong strain of Cryphonectria parasitica to directly compare their responses to chestnut blight. As shown in previous assays on younger Darling 58 trees, canker size and severity were both dramatically reduced on transgenic trees compared to non-transgenic controls.

A small stem assay using hypovirulent Cryphonectria parasitica to screen Castanea dentata backcross F2 families may set the stage for long-term survival — Nguyen, T, D; Craddock, J, H; 2022.

Nguyen, T, D; Craddock, J, H; 2022 Poster

Department of Biology, Geology, and Environmental Science, The University of Tennessee at Chattanooga, 615 McCallie Avenue, Chattanooga, TN

By combining the results of blight resistance breeding and the application of hypovirulence as a biocontrol, populations of resistant hybrid trees could be deployed together with a less pathogenic strain of Cryphonectria parasitica (Murr.) Barr. I used a small stem assay to screen seedlings in thirteen half-sibling backcross F2 families with an attenuated strain of C. parasitica containing the Cryphonectria parasitica hypovirus-1 (CHV-1) Euro7 virus. The experiment was set up as a randomized complete block design in 2-gallon containers. Measurements of canker length and morphology will be gathered at four, eight, 12-, and 16- weeks post-inoculation. A secondary experiment was conducted to further understand the in vivo effects of hypovirulence. Trees from five of the thirteen half-sibling families were inoculated with isogenic virulent (virus-free) and hypovirulent (virus-containing) strains of the fungus. Virulence of the virus-free (V) and virus-containing (H) strains was measured using the Granny Smith apple assay. The possibility of in vivo canker conversion from V to H will be tested later (after eight) weeks by inoculating half of the V-infected trees with the H strain. Measurements of canker length and morphology will be gathered weekly for another eight weeks post-inoculation. Only preliminary results are presented here as the SSA portion of this project is expected to continue through November 2022. All surviving trees will be planted in an experimental orchard in Middle Tennessee to create a potentially long-lasting population of disease tolerant trees together with an effective, long-term biocontrol for chestnut blight.

Conservation of American chestnut genotypes through nut grafting — Parker C, K, Nance W, L, Nelson C, D; 2022.

Parker C, K, Nance W, L, Nelson C, D; 2022 Poster

USDA Forest Service, Southern Research Station, Southern Institute of Foreign Genetics, 23332 Old Mississippi 67, Saucier, MS

Surviving native American chestnuts (Castanea dentata) are very rare in the state of Mississippi and the few survivors are at extreme risk of mortality primarily due to chestnut blight (caused by Cryphonectria parasitica) which is prevalent across the state.  We expect these rare trees may have many genes controlling the expression of locally adapted traits for the Mississippi climate as well as similar climatic zones across the southeast.  To preserve these genotypes before they are permanently lost, we urgently need a fast, inexpensive, and reliable propagation method.

In the past couple years, we tried several propagation methods, with a modified nut-grafting technique (based on the work of Don Kines and Carl Mayfield) proving to be the best. For example, last year we attempted around 100 nut-grafts of American chestnut scions on Chinese chestnut nuts with a success rate of 70-80% (depending on genotype). This year we attempted to scale up our nut-grafting system aiming to nut-graft more than 1,000 Mississippi American chestnut scions on Chinese chestnut nuts. Although success rates were lower this year (overall ~60%), we still produced more than 600 grafts that are currently more than 6 months old.

Our poster presentation will describe our modified nut-grafting protocol and production system in detail, including overall success rates as well as success rates by genotype.  We also offer ideas for further improvements and expansion of our system as well as ideas for incorporating large numbers of grafts in breeding and restoration efforts.

Using whole genome sequences to understand admixture and selection in the North American Castanea species — Perkins, M, T1; Sandercock, M, A2; Paul Sisco, P, H3; Paillet, F, L4; Revord, R, S5 ; Westbrook, J, W6; Holliday, J, A7; Craddock, J, H1 ;2022.

Perkins, M, T1; Sandercock, M, A2; Paul Sisco, P, H3; Paillet, F, L4; Revord, R, S5 ; Westbrook, J, W6; Holliday, J, A7; Craddock, J, H1 ;2022 Poster

1Department of Biology, Geology, and Environmental Science, The University of Tennessee at Chattanooga, 615 McCallie Avenue, Chattanooga, TN
2Genetics, Bioinformatics, and Computational Biology, Department of Biological Sciences, 926 West Campus Drive, Virginia Tech, Blacksburg, VA
3The American Chestnut Foundation — Carolinas Chapter, Asheville, NC
4Department of Geosciences, University of Arkansas, 340 North Campus Walk, Fayetteville, AR
5Center for Agroforestry, University of Missouri, 302 ABNR Building 1111, Rollins Street, Columbia, MO
6The American Chestnut Foundation, 50 North Merrimon Avenue, Suite 115, Asheville, NC
7Department of Forest Resources and Environmental Conservation, 310 West Campus Drive, Virginia Tech, Blacksburg, VA

American chestnut (Castanea dentata) and the chinquapins (C. pumila sensu lato) are an evolutionary sister species pair that represent a promising system for studies of admixture and adaptation in wild plants. Since the 1920s, botanists and geneticists have hypothesized that hybridization between American chestnut and the chinquapins is common and that this process has been involved in the origins of some Castanea taxa and populations. Rigorously testing these hypotheses, however, has been difficult until the recent availability of high-throughput DNA sequencing technologies and associated computational tools. Here, we present preliminary results of our analyses of whole genome sequencing data from 255 plants representing all North American Castanea species and subspecific taxa. Population structure analysis and D statistics tentatively indicate that introgression between C. dentata and the chinquapins has been rare. In contrast, admixture between the different chinquapin taxa—Allegheny chinquapin (C. pumila var. pumila), Ozark chinquapin (C. pumila var. ozarkensis), and Alabama chinquapin (C. alabamensis)—may have been more frequent, but is still limited to only a few sympatric sites. Future work using this data set will include estimation of local ancestry (i.e., chromosome-scale) and tests for evidence of natural selection at introgressed ancestry tracts.

Emergency chestnut c-sections: using immature embryo rescue of American chestnut (Castanea dentata) to conserve germplasm and accelerate breeding — Pilkey, H, C; Powell, W, A; 2022.

Pilkey, H, C; Powell, W, A; 2022 Poster

College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY

Conserving the germplasm and facilitating the breeding of the American chestnut (Castanea dentata) is a top priority as the tree is brought back from the edge of extinction. As the restoration continues, methods have been developed to protect zygotic embryos from risks, such as chestnut blight, that could lead to premature death. Embryo cultures can be utilized by slicing open the seed, extracting immature embryos, bypassing winter dormancy, and then germinating the embryos in vitro. To determine the optimal time to extract the embryos and establish them in vitro, four post-pollination extraction times were tested: 2 weeks, 4 weeks, 6 weeks, and 8 weeks. To evaluate vigor, plants were propagated and brought through the stages of plant tissue culture. Rooting and potting survivorship were evaluated. Dry biomass measurements were taken 60 days post-acclimatization. Embryos extracted at the 8-week time had the highest germination rate (38.46 %). Germination was not observed until the 4-week extraction time point. While the time between extraction and germination was significantly reduced in the 4- and 6-week extraction time points compared to the 8-well extraction, rooting survivorship and biomass measurements were not significantly different. Trees from the 4-, 6-, and 8-week extraction time points were planted in the field in less than one year after extraction. To date, this method has also been used intentionally initiate immature embryos from mother trees of interest for accelerated breeding. Embryo rescue has also been used to produce trees from indoor pollinations in greenhouses and growth chambers where the mother trees were too small to bring the nuts to maturity.

Applying remote-sensing techniques for phenotyping chestnut trees — Kobayashi, Y. C; Hession, W. C; 2022.

Kobayashi, Y. C; Hession, W. C; 2022 Poster Currently Unavailable

Department of Biological Systems Engineering, Virginia Tech, 155 Ag-Quad Lane, Blacksburg, Virginia

The structure of a chestnut tree varies between the American chestnut tree, Chinese chestnut tree, and a hybridized chestnut tree. Phenotyping a hybridized chestnut tree can be difficult as the effects of the blight on an American chestnut tree can alter the tree structure to appear as if it’s a Chinese chestnut tree. The current practice of taking field measurements in an orchard is both time consuming and not extensive. Remote-sensing applications through the use a light detection and ranging (lidar) sensor can cover more area in a shorter amount of time and collect far more measurements. A lidar produces a point cloud which can be thought up as a 3D space with which many points make up an object in the area that was scanned. With a larger dataset, machine learning algorithms can quantify the variation in the tree structure. We collected data using both drone lidar scanning [DLS] (aerial) and mobile lidar scanning [MLS] (ground) at two research orchards belonging to The American Chestnut Foundation in Meadowview, Virginia. At one site, data was collected using only DLS for leaf off season. For both sites, data was collected using DLS and MLS during leaf on season. We found that during leaf off season, we were able to extract measurements such as tree height, crown diameter, and stem/branch length and diameter. During leaf on season, the canopy obstructed the stem and branches so only tree height and crown diameter were measured. While MLS was able to capture the stem and branches, it requires further processing to separate the stems and branches from the leaves in the point cloud. Further research would assist in determining the characteristics of a tree which can be attributed to amount of American or Chinese heritage in hybridized trees.