Brief Summary of Minutes of Annual Meeting

Summary of Minutes:

Because of the COVID-19 pandemic, the 2021 NE-1833 annual meeting was held virtually via Zoom. A total of 13 presentations spanned topics pertaining to all three of the project’s objectives. Presentations represented research from Maine, Michigan, Mississippi, New Jersey, New York, Pennsylvania, South Carolina, Tennessee, and Virginia, as well as from The American Chestnut Foundation, a strong partner in this project.

At the NE1833 business meeting, it was decided that the 2022 annual meeting will again be hosted by Tom Saielli and Jared Westbrook and will be held in person if possible in Charlottesville Virginia.

The current project expires in 2023. It was decided that a Request to Write a renewal proposal would be submitted to the USDA through NERA in 2022. The current format for the project has worked well, and the objectives encompass all of the research presented annually. It was decided that the format/objectives for the renewal would be similar to the current project format/objectives, and that three members would take the lead in writing, one member for each objective area.

Accomplishments

Objective 1: Develop and evaluate disease-resistant chestnuts for food and fiber through traditional and molecular approaches that incorporate knowledge of the chestnut genome.

NEW YORK

Andy Newhouse, Bill Powell - SUNY-ESF, Syracuse, NY

American Chestnut Research & Restoration Project

Work continues toward potential future release of transgenic American chestnuts created to express oxalate oxidase (OxO), an enzyme found naturally in a wide variety of plants and other organisms. Blight damage on young Darling 58 transgenic trees is approximately similar to that seen on Chinese chestnuts, which can get blight but typically tolerate it well enough to keep growing.  The lack of a direct pesticidal mechanism suggests it should be relatively evolutionarily stable.  ESF has completed a variety of environmental & nutritional tests that collectively show a lack of enhanced risks compared to traditional breeding.  Larger-scale, longer-term ecological tests have continued.  Regulatory review in the US consists of three agencies: USDA-APHIS (submitted Jan. 2020, currently in review), EPA (submitted Sept. 2021), & FDA (to be submitted fall 2021). Regulatory review in Canada consists of two agencies, the Environmental & Livestock Feed Assessments (CFIA) and Novel Food assessments (Health Canada), both submissions in preparation for 2022.  The USDA-APHIS review proceeded in 2020-21, including a public comment period for their Plant Pest Risk Assessment.  Overall, comments were strongly positive, including qualified support from major environment groups including The Nature Conservancy, Sierra Club, and Environmental Defense Fund, in contrast to more typical GE agricultural crops that often draw large numbers of negative comments. The USDA-APHIS recently posted a notice of intent to write an environmental impact statement, with a proposed due date of Aug. 2023. This statement will include reference both to the environmental impact of releasing and also of not releasing transgenic, blight-resistant trees.  Next research steps are focusing on combining OxO with other breeding and biocontrol strategies, enhancing genetic diversity through outcrossing with wild-type American chestnuts and also by introducing genes from related chestnut species, and incorporating Phytophthora resistance (possibly through backcrossing).

PENNSYLVANIA

Jill Hamilton, Department of Ecosystem Science and Management, PSU

Charles Ray, Department of Ecosystem Science and Management, PSU

Tetyana Zhebentyayeva,  Department of Ecosystem Science and Management, PSU

Chinese Chestnut Resistance to Phytophthora cinnamomi (Tetyana Zhebentyayeva and chestnut root rot research group).

This year research was focused on understanding structural organization genomic regions underlying QTL intervals for resistance to Phytophthora cinnamomi (Pc) in susceptible American and resistant Chinese chestnut, and developing procedures and protocols for gene expression analysis.  Taking advantage of a high-quality genome of Castanea dentata assembled and annotated by the Hudson Alpha Institute, the QTL intervals were delineated with flanking markers from the previous study and mined for gene composition.

Comparative analysis between American and Chinese (Vanuxem) chestnut genomes, revealed a multiple-gene family of cysteine-rich receptor-like kinases (CRKs, DUF26/PF001657) associates with a membrane immune complex and apoplastic reactive oxygen species (ROS) signaling in plants. Based on previous and current results, this gene family and associated molecular networks have been prioritized for an in-depth transcriptome profiling in chestnut roots in response to Pc invasion. Treatment of roots with pathogen conducted this growing season in a test mode was used to finalize design of the RNA sequencing experiment (by establishing time intervals after inoculation, number of biological replicates and non-inoculated controls). Results will be used to enhance preliminary data in collaborative proposal “Understanding host resistance in the Chinese chestnut-Phytophthora cinnamomi pathosystem” at re-submission to the NSF Plant-Biotic interaction program.

Wood phenotyping and extension (Ray):  Work on identifying, labeling, and classifying the Penn State Xylarium continues. Specimens of the Xylarium were utilized to compare wood quality in early 20^th century American chestnut wood to specimen samples produced by hybrids in modern plantation trials around the region.

A research project entitled “Wood Quality in Hybrid Chestnuts” supported by the American Chestnut Foundation was completed and reported on in a TACF “Chestnut Chat” webinar. Results will be reported in the Winter 2022 issue of the TACF Chestnut Journal. Implied in the conclusions of the work, which will be published shortly, is that mature wood quality of the second-generation hybrids should be similar to original American chestnut, especially when grown in natural forest conditions.

SOUTH CAROLINA

Steven N. Jeffers – Dept. of Plant & Environmental Sciences, Clemson University

Screening Hybrid Chestnut Seedlings for Resistance to P. cinnamomi

• In Fall 2021, we recovered two isolate of cinnamomi from sites in Maryland: Maryland sites: BARC and WSSC 1
• These isolates were put into permanent storage in our lab and then used in the 2021 TACF hybrid chestnut seedling screening effort at the USDA Forest Service Resistance Screening Center at the Bent Creek Experimental Forest in Asheville, NC

Understanding Host Resistance in the Chinese Chestnut–P. cinnamomi Pathosystem

• We continue to collaborate with colleagues at Penn State, USDA-FS, Univ. of Kentucky, and Univ. of Tennessee on this project, but, so far, we have not been able to secure grant funds
• Therefore, a preliminary experiment was conducted in Aug 2020 to examine the early steps of the infection process by cinnamomi on chestnut roots using RNA sequencing
  • results from this first experiment were inconclusive due to experimental inconsistencies
• An experiment to more accurately identify when zoospores infect American chestnut roots is being planned—before conducting the RNA-seq experiment again

THE AMERICAN CHESTNUT FOUNDATION

Tom Saielli and¹ Sara Fitzsimmons²

¹The American Chestnut Foundation, 900 Natural Resources Drive, Charlottesville, VA 22902

²The American Chestnut Foundation, 206 Forest Resources Lab, University Park, PA 16802

Improved blight resistance among regional hybrid chestnuts

Advanced generation hybrid chestnut trees throughout TACF orchards in Virginia and Maryland, and Virginia Department of Forestry hybrid trees at the Lesesne State Forest, have been assessed for long-term blight tolerance and American-type traits for at least four years post inoculation, and sometimes ten years or more. Long-term measurement of the chestnuts’ physiological response to blight infection is required to truly assess the level of resistance for each tree and among various families. This is a uniquely different strategy than the former strategy established by TACF in 1983, in which selections were made at one year post inoculation and open-pollinated seeds were planted directly in seed orchards. We now realize that blight resistance is more complicated and may take years to evaluate. Over time, the trees that appear to be the “best of the best” are crossed via controlled pollinations and the seedlings pre-screened for resistance with a greenhouse assay. The best seedlings are planted in an orchard at Fortunes Cove in Central Virginia.

Evaluate the correlation between canopy dominance and blight resistance among backcross chestnuts at Lesesne State Forest

Mature hybrid chestnut stands at the Lesesne State Forest (VDOF) in Central Virginia were established in the late 1980’s and early 1990’s, are now canopy trees averaging 70 feet tall. Within the first few decades after the orchard was established, the most susceptible trees succumbed to blight infections and died, leaving only the more resistant trees to form a forest stand. However, after four decades, there is an additional level of variation among the survivors: trees with high levels of blight resistance dominate the canopy; whereas, trees with moderate blight resistance have survived, but are now suppressed in the understory of the more resistant trees. Many of the suppressed trees are showing signs of stress and some dieback, while the dominant trees are healthy and produce large annual masts of seeds.

We hope to quantify the correlations between blight resistance and canopy dominance, and the projected implications for growth and survival, recruitment of progeny, and carbon sequestration among reintroduced hybrid chestnuts.

Objective 2: Evaluate biological approaches for controlling chestnut blight from the ecological to the molecular level by utilizing knowledge of the fungal and hypovirus genomes to investigate the mechanisms that regulate virulence and hypovirulence in C. parasitica.

MICHIGAN

Monique Sakalidis, Michigan State University, East Lansing, MI

Brown rot is a disease of chestnuts that has been reported globally in nut production areas particularly in Europe and Australia and can result in up to 91% of chestnuts infected. It has been detected annually since 2017 in Michigan, at somewhat lower levels than in Europe and Australia. In high disease incidence years, cold storage decreases disease incidence and severity; in low disease incidence years, cold storage may decrease disease incidence, but not in all cases. In 2020, brown rot was detected in up to 9% of nuts, caused by the fungus Gnomoniopsis smithogilvyi (G. smithogilvyi) among other species. The most susceptible chestnut cultivar was European X Japanese (Colossal). In addition to its role as a postharvest nut pathogen, G. smithogilvyi  was also found to be associated with canker disease in chestnut populations in Michigan and Wisconsin.

MISSISSIPPI

Angus Dawe, Department of Biological Sciences, Mississippi State University

Current personnel:

Graduate students –Soum Kundu, Melanie Tran

Research Associate – Gisele Andrade

Current Projects:

Note, due to COVID-19 limitations, progress has been negatively impacted on all projects.

1. ARV-1 and its potential role in sterol homeostasis
2. Identifying C. parasitica genes associated with pathogenicity and virulence

Identifying C. parasitica genes associated with pathogenicity and virulence. (Melanie Tran, MS student.)

This project is leveraging a set of progeny from a cross between strains EP155 (considered more virulent) and SG2-3. Virulence phenotyping of the progeny was previously performed by ACF in Meadowview (F. Hebard). Sequencing was completed of all 92 progeny in late 2019 at Mississippi State via the Genomics Core at the University of Mississippi Medical Center in Jackson, MS. In total, the run generated >800 million reads (PE 150), with QC30>81.7%. Coverage is estimated at >15-30X for almost all the samples with only minor quality issues with source DNA for a small number. Work is ongoing with these data. Melanie is building a pipeline for analysis using the MSU Biological Sciences genomics server in collaboration with Jean-Francois Gout, a computational biologist member of the faculty. Jared Westbrook (ACF) is also involved and will be assisting in relating the sequence data to the phenotype data. Limited progress was made on this project this year due to student challenges.

ARV-1 and its potential role in sterol homeostasis. (Soum Kundu, PhD student).

ARV-1 is a predicted gene in C. parasitica that shares similarity with genes that code for proteins with important roles in sterol homeostasis in other organisms. The knockout of ARV-1, serendipitously made when investigating an unrelated phenomenon, is avirulent and has a heavily impaired vegetative growth phenotype. Soum has been working to verify which of two possible genes originally knocked out is the source of the debilitated phenotype (the updated genomic arrangement was apparent with a new genome annotation). He has also developed and verified an assay for ergosterol production in C. parasitica by modifying published protocols and using a GC/MS system in collaboration with the lab of Todd Mlsna in the Department of Chemistry at Mississippi State. Recent success using derivatization techniques to tag the appropriate class of compounds show that ergosterol accumulation is much reduced in the ARV-1 mutant. When tested, the hypovirus infected strain EP713 shows a reduction of ergosterol accumulation very similar to that of the mutant, suggestion that a component of the membrane alterations induced by the hypovirus may be due to altered ergosterol presence.

NEW JERSEY

Bradley Hillman, Rutgers University, New Brunswick, NJ

1. Discovery of new viruses associated with hypovirulence and biological control in New Jersey forests proceeded with laboratory characterization of isolates that were collected from a large recovering American chestnut tree in northern New Jersey, near the New York border in 2020. An undergraduate student, Rebecca Bright, began the characterization of the isolates, and the work has continued under another student, Zarja Miovic. The following experiments were performed: 1) Isolation of the chestnut blight fungus, C. parasitica, from the recovering tree. Three new fungal isolates were collected. 2) Examination of fungal colony morphology/phenotype in culture. Two of the three new isolates were fast growing, similar to virulent, virus-free cultures, but one of the cultures grew more slowly and had less aerial mycelium in culture, similar to some hypovirulent, virus-infected cultures in our collection. 3) Isolation of isogenic cultures from single conidia (asexual spores) of the slow-growing culture and determination of colony morphology/phenotype of the single conidial isolates (SCIs). Of the total of 40 SCIs that were isolated and examined for colony morphology, 31 colonies showed rapid growth, orange coloration, and abundant aerial mycelium typical of virulent, virus-free colonies and 9 colonies showed slow growth, brown coloration, and suppressed aerial mycelium typical of hypovirulent, virus-containing colonies. 4) Initial assessment of virulence was performed on one of the rapidly-growing colonies (designated strain R1V₂) and one of the slow-growing colonies (designated strain R1V₁) by inoculation of apple fruits, including known virus-free virulent (strain EP155) and virus-containing hypovirulent (strain EP713) cultures. In two replicate trials, lesions resulting from slow-growing strain R1V₁ were similar in size to control hypovirulent strain EP713, whereas lesions resulting from fast-growing strain R1V₂ were similar in size to control virulent strain EP155. 5) Pairings of R1V₁ and R1V₂ allowing the two colonies to grow together in contact resulted in conversion of the R1V₂ phenotype to the R1V₁ phenotype, consistent with transmission of a virus. This step completes a modified Koch’s postulates, demonstrating that the infectious agent associated with hypovirulence in strain R1V₁ is transmissible and is curable, although the specific virus in R1V₁ has not yet been characterized. This represents the first hypovirulence-associated virus from northern NJ forests and could lay the groundwork for investigating its prevalence and impact on natural chestnut populations in the region.

2. We worked to finish the project comparing codon usage among different viruses in C. parasitica to codon use in the host fungus, as summarized in detail last year. We are continuing to explore the hypothesis that the hypovirus CHV4, which is the most prevalent virus of the chestnut blight fungus in North America, may have a longer association with the fungus than previously thought, and may have entered C. parasitica in Asia, before its invasion to North America in the late 19^th century. The virus has never been found in Asia. An undergraduate student in the lab, Abhishek Kashalikar, has expanded the comparisons among the viruses examined last year in the study, and has overlaid details about the geographic origins of those viruses and their different fungal hosts. In other words, he addressed the question of whether there is any notable geographic grouping associated with related viruses in the virus family Hypoviridae, which contains most of the viruses used for biocontrol of chestnut blight disease. Aside from known worldwide movement of CHV1 in C. parasitica, no pattern of phylogeographic association was identified among 51 other members of the family Hypoviridae in this study.

SOUTH CAROLINA

Steven N. Jeffers, Dept. of Plant & Environmental Sciences, Clemson University

Evaluating Virulence of P. cinnamomi Isolates

• We worked with Dr. Jared Westbrook at TACF to review and evaluate the data collected in 2019 and 2020 variation in virulence among 7 subsets of isolates of cinnamomi
• Data analysis is in progress, but preliminary results suggest:
  • there may be significant differences among inoculum treatments
  • differences appear to be related to age of cultures or time in storage
  • the current strategy of screening hybrid chestnut genotypes using fresh isolates from out-planting sites seems reasonable and sound

Detection of Phytophthora spp. in Chestnut Samples

• In the past year, Sep 2020-Aug 2021, samples were received from 6 states: GA, MD, MO, TN, VA, WV
• In all, there were 9 submissions and 39 soil samples received
• Phytophthora detected in 13 samples = 33%
  • cinnamomi was detected in 8 samples from 5 states: GA, MD, MO, TN, VA
  • Phytophthora in 5 samples from WV

THE AMERICAN CHESTNUT FOUNDATION

Tom Saielli and¹ Sara Fitzsimmons²

¹The American Chestnut Foundation, 900 Natural Resources Drive, Charlottesville, VA 22902

²The American Chestnut Foundation, 206 Forest Resources Lab, University Park, PA 16802

Fungicide research

Currently, controlled pollinations take place both in situ, on wild chestnut trees and, on wild-type American chestnut established in germplasm conservation orchards (GCO). Unlike the advanced hybrid chestnuts, the GCO trees are highly susceptible to chestnut blight, often leading to significant dieback and mortality. Therefore, we are experimenting with various fungicides and biofungicides to determine if chemical treatments can control blight infections and enable GCO chestnut trees to survive and grow. Experiments are being conducted in collaboration with the Jeffers Lab at Clemson University, greenhouse experiments in Central Virginia, and field trials in GCO’s in Northern Virginia and other locations.

VIRGINIA

Laurel Rodgers, Shenandoah University

Comparison of the fungal microbiome in chestnut trees resistant to C parasitica to those that are not resistant to C parasitica

We sampled a total of 45 trees from each orchard, pulling two plugs from each for a total of 180 bark plugs. A combination of American, Chinese, and hybrid trees were collected from each orchard, with the addition of F1 trees from The Ranch (Table 1).

Table 1. Number of plugs collected from sample sites

We isolated a total of 361 fungi samples from all plugs collected. From the Ranch, we isolated 39 fungi samples from Chinese, 34 from American, 46 from hybrids, and 39 from F1 chestnut trees. At Mount Zion, we isolated 39 fungi from Chinese, 20 from American, and 144 from hybrid chestnut trees (Table 2). So far, we have sequenced and identified about twelve samples from the Ranch and thirty-seven from Mount Zion, but we still need to sequence approximately 100 from the Ranch and 150 from Mount Zion.

Table 2. Number of fungi isolated

Species identification was interrupted by the issues with our protocols in 2019 as we experienced turn over in our undergraduate researchers and again in 2020 due to the Sars-Cov2 pandemic. Identification of samples is once again in progress, but some samples have been lost due to time and contamination. Of the species we have identified thus far at Mount Zion, a majority of the fungi are parasitic fungi. However, based on brief literature searches, three species of fungi we isolated, Fimetariella rabenhorstii, Hypoxlon submonticulosum, and Albifimbria verrucaria, have been documented as being endophytes in other plant species (Table 3). However, the hybrid trees in which these samples were collected proved to have very little resistance to C. parasitica. Though the sample size is low, it seems unlikely that these three species of fungi serve as endophytes in the chestnut tree.

We were surprised to find the fungus Gnomoniopsis smithogilvyi growing within one of our hybrid trees. This is a fungus known to cause cankers on chestnuts in Europe and has begun to be documented within the US. A more extensive study will need to be completed in order to determine how wide spread G. smithogilvyi is within the United States and whether it will act as a pathogen in the American chestnut tree.

Table 3. Fungi species identified thus far at Mt. Zion (potential endophytes highlighted in blue)

Table 4: Fungi species identified thus far at The Ranch.

Objective 3: Investigate chestnut reestablishment in orchard and forest settings with special consideration of the current and historical knowledge of the species and its interaction with other pests and pathogens.

MAINE

Thomas Klak, University of New England, Portland, ME

Transgenic Chestnut Pollen Production under High-Lights & Field Pollination

The Chestnut restoration team at the University of New England continues to have success producing transgenic (blight-tolerant) pollen from seeds and seedlings obtained from SUNY-ESF. Pollen production in chestnut has been inconsistent year-to-year, but the UNE team expanded the pollen-production project in 2021. Green and white aphids, spider mites, mealy bugs, fungus gnats, and powdery mildew fungus are major barriers to pollen production. The total number of fertile transgenic chestnuts produced from UNE-produced pollen in 2021 exceeded the 5500+ total of 2020.

PENNSYLVANIA

Kim C. Steiner, Department of Ecosystem Science and Management, PSU

Sara F. Fitzsimmons, TACF and PSU

Breeding and field trials (Steiner): Dr. Steiner has a long-time partnership with The American Chestnut Foundation (TACF) on breeding for blight-resistance in American chestnut and on research towards restoring the species to Appalachian forests. TACF's Northcentral Regional Breeding Coordinator is based in the Steiner lab, and the Pennsylvania TACF Chapter’s statewide and regional breeding programs have been coordinated by Steiner’s PhD student S.F. Fitzsimmons. Dr. Steiner has provided oversight to the national TACF plan for breeding and restoration, as Chair of the TACF Science Cabinet from 2007 to 2012, and currently as Chair of the TACF Board of Directors.

Dr. Steiner and Sara Fitzsimmons have established a large field trial, on the Penn State campus, of many families of TACF 3^rd back-cross generation progeny that are being evaluated for blight resistance and form.

TENNESSEE

Hill Craddock, Taylor Perkins, The University of Tennessee at Chattanooga

Evolutionary genetics of chinquapin and American chestnut

• Morphological diversity of American chestnut and chinquapin
• Evidence of hybridization between the species
• Adaptive significance of gene introgression between the species is under investicgation

Breeding for Disease Resistance

• Finished selections at Ruth Cochran Orchard and Dave Cantrell Orchard

1. dentata collected from underrepresented areas in Alabama and Tennessee

• Clonal collections maintained in field plots in Indiana and in container nursery in Tennessee
• Germplasm Conservation ex situ

Phylogeography of Castanea in the southern US

• Collection trip (with Sisco and Paillet) to S. Missouri and NW Arkansas
• Annotations of 900 herbarium sheets for Perkins et al

Works in Progress

• Herbarium vouchers prepared for SERNEC imaging and digital data capture
• Nursery production of BnF2s for TN seed orchards
• Nursery production of C. dentata germplasm for GCOs

Stacy L. Clark (USDA Forest Service, Southern Research Station, Knoxville, TN), Leila Pinchot (USDA Forest Service, Northern Research Station, Delaware, OH), and Scott E. Schlarbaum (The University of Tennessee, Department of Forestry, Wildlife, and Fisheries, Knoxville, TN):

The University of Tennessee’s Tree Improvement Program (UT-TIP) chestnut activities include evaluations of historic chestnut plantings at the Norris Reservation (Tennessee Valley Authority) in TN and collaborating with the USDA Forest Service Southern and Northern Research Stations. The collaborative work includes implementation and long-term comprehensive field evaluations of chestnut research test plantings (ca. 2009-2017) in NC, PA, TN, and VA. Experimental material represents 7500 trees from various breeding generations (BC₁F₃, BC₂F₃, BC₃F₃, BC₃F₂) and parental species (American and Chinese chestnut) from The American Chestnut Foundation’s and the Connecticut Agricultural Experiment Station’s breeding programs. Evaluations of survival, growth, blight resistance, deer herbivory, and competitive ability within different silvicultural prescriptions have been conducted. Results indicate chestnuts bred for blight resistance exhibit superior competitive ability and intermediate blight resistance, but performance varies depending on seedling quality, vegetation competition, site quality, and deer browse pressure at the time of planting.

THE AMERICAN CHESTNUT FOUNDATION

Tom Saielli and¹ Sara Fitzsimmons²

¹The American Chestnut Foundation, 900 Natural Resources Drive, Charlottesville, VA 22902

²The American Chestnut Foundation, 206 Forest Resources Lab, University Park, PA 16802

Ecological Studies on American chestnut hybrids

Understory trials at Lesesne State forest hybrid orchard

The original hybrid orchard at Lesesne State Forest was established in 1960 with American x Chinese x Japanese hybrids from the Connecticut Agriculture Research Station and now consists of a closed canopy stand of chestnut trees that produce tens of thousands of viable seeds every year. To date, no recruitment has ever been observed under the canopy in this orchard. We hypothesize that significant understory vegetation suppresses the chestnut seeds, preventing seedling establishment.

In 2022 we will conduct experimental vegetation management to see if recruitment can be promoted. Treatments include cut and burn understory, cut and herbicide understory, no removal of understory.

Forest silviculture studies: mixed hybrid chestnuts and oaks under three canopy treatments

We are evaluating the influence of overstory treatments on three open-source American chestnut genotypes include BC₁F₂, BC₂F₂ and BC₃F₃seedlings, LSA x LSA seedlings (large surviving American chestnuts) and two oak species (white oak and southern red oak). Study locations will include a low elevation site at W. Kerr Scott Dam and Reservoir, in Wilkesboro, NC and a high elevation site at Boone NC (Gilley or Blackburn Vannoy). Silviculture treatments include: old field (open site), pine forest (with release planned in 2-3 years), and shelterwood (60% leaf basal area +/- 10%, with release after 2-3 years). Percent canopy removed for “release will be TBD but will be adequate to provide suitable light availability for established seedlings but may not involve a full cutover. The following measurements will be made: height, diameter and mortality and assessment of blight resistance once natural blight infections become visible.

The expected results of this research will be to identify silviculture strategies that lead to successful re-establishment of chestnut populations while minimizing management efforts (i.e. aggressive vegetation management). We also hope to quantify how genotype and levels of resistance influence species establishment and long-term competition, as well as recruitment in the understory. The goal of this research is to contribute towards the development of better silviculture and breeding strategies, with a focus on competitive, timber-type, American chestnut trees that are blight resistant (enough) and significantly ‘American’ in all other traits.

Fred V. Hebard, Virginia Chapter, The American Chestnut Foundation

The method of Ellis et al (2018, doi: 10.1111/1755-0998.12782) for identifying parents of open-pollinated progeny was applied to B3-F2s bred at Meadowview.  Optimizing input parameters led to identification of male parents in 45% of progeny.  When the most likely choice of male parent was unidentified, but the second most likely choice, identified, was only slightly less probable, the male parent was identified in 81% of progeny.  It is thought that most of the potential male parents had been genotyped, so that the 81% identification rate was more accurate.  This is based on the relative geographic separation of parents; 95% of genotyped male parents were within 200 meters of the female but ungenotyped parents were 1000 meters away.   The failure of the program to identify more than 45% of parents is thought to be due to the severe linkage disequilibrium in backcross chestnut trees.

Preliminary  GWAS were run using 13,750 SNPs against 755 Clapper and 918 Graves B3-F2 trees.  Both B3-F2 groups were segregating for blight resistance but only Graves for PRR resistance.    The populations had been selected for blight resistance and PRR resistance by Jared Westbrook and team at TACF.  The SNP alleles had called as being descended from Chinese or American chestnut.  In these GWAS, the binary trait of selected or not was tested against the binary state of SNP markers.  There were significant associations for blight resistance on five chromosomes in both Clapper and Graves families, but only two chromosomes were in common.  Furthermore, the sites of maxima on one common chromosome were 31 Mbp apart.  So blight resistance for Clapper and Graves was associated with eight of the 12 chromosomes in chestnut.  In contrast, PRR resistance was found on only one chromosome in Graves, the same result as obtained by Zhebentyayeva with QTLs.

The GWAS were preliminary in that a separate association of resistance and marker state was run for each marker-disease-Chinese ancestor combination rather than a more inclusive model.  GWAS analysis with more inclusive models and further data cleansing is indicated

There were no tall, narrow peaks in Manhatten plots of the results.  This is attributed to the strong linkage disequilibrium in many of the B3 parents.  This should be dissected further.  There is scant evidence for or against multiple loci for resistance within a chromosome.

2021 Publications

Aulia, A., Hyodo, K., Hisano, S., Kondo, H., Hillman, B.I., Suzuki, N. 2021. Identification of an RNA silencing suppressor encoded by a symptomless fungal hypovirus, Cryphonectria hypovirus 4. Biology 10, 100. https://doi.org/10.3390/biology10020100

Callahan AM., Zhebentyayeva T.N., Humann JL, Saski CA, Galimba KD, Georgi L.L, Scorza R., Main D, Dardick CD (2021) Defining the ‘HoneySweet’ insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica). Horticulture Research 8:8. https://doi.org/10.1038/s41438-020-00438-2.

Groppi A, Liu S, Cornille A, Decroocq S, Bui QT, Tricon D, Cruaud C, Arribat S, Belser S, Marande W, Salse J, Huneau C, Rodde N, Rhalloussi W, Cauet S, Istace B, Deni E,, Carrère S, Audergon J-M, Roch G, Lambert P, Zhebentyayeva T., Liu W-S, Bouchez O, Lopez-Roques C, Serre R-F, Debuchy R, Tran J, Wincker P, Chen X, Pétriacq P, Barre A, Nikolski M, Aury J-M, Abbott AGA, Giraud G, Decroocq V (2021) Population genomics of apricots unravels domestication history and adaptive events. Nature Communication 12, 3956. https://doi.org/10.1038/s41467-021-24283-6.

Hillman, B.I., and Milgroom, M.G. 2021. The ecology and evolution of fungal viruses. pp. 139-182 in: Studies in Viral Ecology, 2^nd Ed. C.J. Hurst, editor. John Wiley & Sons, NY.  https://doi.org/10.1002/9781119608370.ch5 (this book chapter was cited last year as in press)

Perkins M.T., Zhebentyayeva T.N., Sisco P., Craddock H (2021) Genome-wide sequence-based genotyping supports a nonhybrid origin of Castanea alabamensis.  Systematic Botany 46(3): (in press); preprint BioRxiv.org https://www.biorxiv.org/content/10.1101/680371v1.

Perkins M.T., Zhebentyayeva T.N., Sisco P., Craddock H.(2021) Castanea alabamensis: rediscovery of a lost American chestnut relative. Chestnut (The Journal of the American Chestnut Foundation) 35(3):30-33.

Pina A, Irisarri P., Errea P., Zhebentyayeva T. (2021) Mapping quantitative trait loci (QTLs) associated with graft (in)- compatibility in apricot (Prunus armeniaca L.). Front. Plant Sci. 12: 622906. doi: 10.3389/fpls.2021.622906.

Suzuki, N., Cornejo, C., Aulia, A., Shahi, S., Hillman, B.I., Rigling, D. 2021. In-tree behavior of diverse viruses harbored in the chestnut blight fungus, Cryphonectria parasitica. Journal of Virology 95 (6), e01962-20. https://doi.org/10.1128/JVI.01962-20

Yu J., Bennett D., Dardick C., Zhebentyayeva T., Abbott A., Liu Z., Staton M. (2021) Genome-Wide Changes of Regulatory Non-coding RNAs Reveal Pollen Development Initiated at Ecodormancy in Peach. Frontiers in Molecular Biosciences, 8:612881. doi: 10.3389/fmolb.2021.612881.