Brief Summary of Minutes of Annual Meeting

NE1833 Annual Meeting Report 2020

Station Reports:

CONNECTICUT

Sandra L. Anagnostakis, Emeritus, The Connecticut Agricultural Experiment Station, New Haven, CT, web page  https://www.ct.gov/caes/sla

Projects fall under Objective 1, 2 and 3:

During the past year I sent all of the important chestnut documents and materials housed at the Connecticut Agricultural Experiment Station to the National Agricultural Library in Maryland for preservation.  This included all of the USDA Plant Importation and Distribution cards, with records of where imported trees and seeds were sent within the US and to other countries. Planting records from Bell, MD included information on the germination of seeds received, and their designations.  I have recorded some of this on my web page as “Chestnut Importations into the U.S.”  There were boxes of photographs and negatives from USDA work and many from R. K. Beattie who traveled in the early 1900’s in Japan.  We had several extensive reports from the plant explorer Peter Liu, which were illustrated with photographs that he took in his searches for chestnuts in China.

I also sent all of the USDA breeding records, the CAES breeding records, all of the laboratory books (fungal stock records) and all theses in my collection, including those of Nienstaedt, Jaynes, Grente, Anagnostakis, and Hebard.

I now have grant funds to scan all of the distribution cards and make them available to anyone who wants the information.  However, the Library is closed, and no work can be done until it reopens.  The person in charge of the Special Collection (“The Chestnut Collection”) is:

Amy Morgan

Special Collections Librarian

National Agricultural Library

10301 Baltimore Avenue, Room 304

Beltsville, MD  20705

Phone: 301-504-5876

Fax: 301-504-7593  Email: NALSpecialCollections@ars.usda.gov

If others have historical documents and items relating to chestnuts, I recommend sending them to the Library’s Special Collection for keeping.

MAINE

Thomas Klak, University of New England

Project falls under Objective 3:

Transgenic Chestnut Pollen Production under High-Lights & Field Pollination

 The Chestnut restoration team at the University of New England has had good success producing transgenic (blight-tolerant) pollen from seeds and seedlings obtained from SUNY-ESF. We collected and froze T2 pollen from eight lines and distributed that to six collaborating institutions for pollination of wild type chestnuts in July 2020. The UNE team is expanding this pollen-production project in the coming year. We are attempting to produce T3 transgenic pollen during the same year of harvest, with hopes that it could be available for July 2021 pollination season. We also took our transgenic pollen to the field and pollinated about 1500 flowers on wild type trees, and this yielded approximately 600 fertile transgenic nuts. The other collaborating sites also had success numbering in the hundreds of transgenic nuts each. So in total we have increased our transgenic nut production and diversity considerably, as we continue to work under federal permits in hopes of eventual deregulation.

 Quantifying and prioritizing the determinants of wood quality in chestnut variants. 

MICHIGAN

Monique Sakalidis Michigan State University Research Update

Projects fall under Objective 2:

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. The fungus Gnomoniopsis smithogilvyi (G. smithogilvyi) causes brown rot on chestnuts pre- and post-harvest. The disease is characterized by soft, brown lesions on the kernel and is only detected when nuts are cut open. The presence of brown rot in nuts leads to unsalable nuts. Additionally, as the disease develops overtime after harvest, this can lead to the degradation of nuts that have passed quality check at harvest. Using nuts sampled in fall 2019 and 2020 we have evaluated the diversity of nut rotting organisms, particularly Gnomoniopsis species, and evaluated the effect of cold storage and host cultivar on disease incidence and severity. We have found that cold storage results in decreased disease severity and incidence and that the cultivar Colossal is most susceptible to natural and artificial infection. Future research will focus on the timing of spore production in the field and the infection pathway into the host.

MISSISSIPPI

Angus Dawe, Department of Biological Sciences, Mississippi State University

Current personnel:

Graduate students –Soum Kundu, Melanie Tran

Research Associate – Gisele Andrade (part time)

Visiting Scientist – Kum-Kang So

Projects fall under Objective 2:

1. Identifying parasitica genes associated with pathogenicity and virulence
2. ARV-1 and its potential role in sterol homeostasis
3. Impact of MAPK signaling pathways on fungal phenotype, virulence, and hypovirulence

Project details

1. Identifying 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.

2. 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 hemeostasis 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 develop an assay for sterol production in C. parasitica by modifying published protocols and using a GC/MS system in conjunction with Todd Mlsna in the Department of Chemistry at Mississippi State. Recent success using derivatization techniques to tag the appropriate class of compounds are indicating that sterol production is affected by hypovirus infection, although these results require further study and confirmation.

3. Impact of MAPK signaling pathways on fungal phenotype, virulence, and hypovirulence. (Kum-Kang So, visiting scientist)

Dr. So came to us from South Korea, arriving in early 2020 and unfortunately was unable to accomplish much before the COVID-related disruptions began. Her time in the US is limited to a year so she is now working hard to make up for lost time. She has a number of mutants in various components of the MAPK signaling pathway of C. parasitica from her PhD work with Dr. Dae-Hyuk Kim and she is further investigating these and transcription factors that mediate their expression. She is also looking into any correlations between the impact of these mutations and the hypovirulent phenotype caused by infection with CHV1 hypovirus.

NEW JERSEY

Bradley Hillman, Rutgers University

Projects fall under Objective 2:

We continued to examine effects of related and unrelated viruses on each other during mixed infections in C. parasitica. Coinfection with different viruses may have varying effects on the fungal host, and understanding those effects helps evaluate the potential effectiveness of viruses as biocontrol agents. Infection of the mycoreovirus MyRV2 from West Virginia C. parasitica strain C-18 was reported last year to be potentiated by coinfection with the unrelated hypovirus CHV4; MyRV2 required coinfection to be stable through repeated serial passage in culture. The N-terminal protein encoded by the CHV4 has now been confirmed to be a 24 kDa protease (p24) and has been identified as the protein responsible for potentiating co-infection of the fungus with MyRV2 by acting as a suppressor of RNA silencing. p24 is therefore similar to the well-studied p29 protein of the prototypical hypovirus CHV1 in serving as a self-cleaving protease and as a suppressor of RNA silencing. Unlike CHV1 p29, CHV4 p24 does not serve as a symptom determinant. It is not yet known whether p24 is part of an internal ribosome entry site (IRES) element, as CHV1 p29 is. This project is a collaboration with Dr. Nobuhiro Suzuki, Okayama University.

1. We proceeded with a project mapping of trees and environmental sampling of chestnut blight cankers in New Jersey, and discovery of new viruses associated with hypovirulence and biological control. We have begun to collect C. parasitica isolates from northern New Jersey, near the New York border, to look for hypovirulent isolates and compare them to isolates from the Atlantic Highlands/Middletown NJ region that we’ve collected over many years. Due to COVID-19, only three isolates were collected in 2020 from a single infected tree before collected stopped. 
2. To examine factors promoting virus invasion and adaptation to C. parasitica, we expanded a project comparing codon use among different viruses to codon use in the common host organism. The degeneracy of the genetic code dictates that the 20 amino acids are encoded by 61 codons, and many factors contribute to which codons are preferred in a given organism. Codon bias can be high (at extreme, only 20 codons used to encode the 20 amino acids) or low (at extreme, all 61 codons used equally. The expectation is that the host organism has evolved to optimize its codon use, and that viruses that are well adapted to that host organism evolve toward a similar codon use profile. Especially with RNA viruses, codon preference for translation in a given host is only one driver of nucleotide preference at a given position – RNA structure/function relationships are critical. Our hypothesis is that viruses that have been resident in C. parasitica for a longer time will display greater codon adaptation and thus grater similarity to the fungal host. Through use of a variety of metrics, our analysis of codon usage of six viruses of C. parasitica whose genomes have been completely sequenced supports the hypothesis of a long-term association with host C. parasitica of three members of the family Hypoviridae: CHV1, CHV2, and CHV4; and sorter-term association of one Hypoviridae member, CHV3, and of the two members of the Reoviridae family, MyRV1 and MyRV2. These results are generally consistent with current knowledge of the biogeography of these viruses: CHV1 and CHV2 are closely related to one another and both have been found in Asia, presumably having been there before the fungus invaded North America. In contrast, MyRV1, MyRV2, and CHV3 all have been found only in North America and presumably infected the fungus only after its arrival in North America in the late 19^th Century. The unexpected result was that CHV4, which is well established in North America, has never been found in Asia. Our results suggest that CHV4 may have a longer association with the fungus than previously thought, and Asian isolates of C. parasitica should be examined specifically for its presence.

NEW YORK

Andy Newhouse - SUNY-ESF American Chestnut Research & Restoration Project

Projects fall under Objective 1 and 3:

Summary: Transgenic American chestnuts have been created to express oxalate oxidase, an enzyme found naturally in a wide variety of plants and other organisms.  Preliminary tests have shown it is effective at reducing damage (canker size) on stems compared to wild-type American chestnuts.  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 are in progress.  Regulatory review in the US consists of three agencies: USDA, EPA, & FDA.  The USDA review has begun, and the public comment period for their Plant Pest Risk Assessment is currently open.  TACF has instructions and details on their website for how to make comments on the Federal Register.  So far, most of the comments are positive, in contrast to more typical GE agricultural crops that often draw large numbers of negative comments.  Next research steps are focusing on enhancing genetic diversity through outcrossing with wild-type American chestnuts, and incorporating Phytophthora resistance (possibly through backcrossing).

PENNSYLVANIA

Charles D. Ray, Penn State
Gary Carver, TACF
Sara Fitzsimmons, Penn State/TACF
Michael Wiemann, USDA Forest Products Lab

Projects fall under Objective 3:

The objective of this project is to determine which wood properties in chestnut variants result in statistically significant differences between variants. Based on our findings, these properties and the statistical parameters of investigation will be prioritized for future, long-term chestnut wood studies. 
The criteria for prioritization will include importance to wood quality for commercial applications and relationship to desirable growth characteristics. Findings to date include: 1) chestnut color is distinctly differentiable from other common wood species, 2) juvenile and mature wood in chestnut are distinctly differentiable, allowing determination of chestnut specimens to be categorized as being either from a mature tree or from an immature stem, and 3) within juvenile/mature wood classifications, chestnut color is not differentiable between Castanea species. This hints that at least one major reason that chestnut specimens culled from restoration plantations look different than traditional chestnut lumber is that they are from juvenile wood, and that mature wood from these same plantations may grow to closely resemble and assume other properties of traditional C. dentata.

SOUTH CAROLINA

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

Projects fall under Objective 1 and 2:

Screening Hybrid Chestnut Seedlings for Resistance to P. cinnamomi

• Previously, screening was conducted in SC for 14 years (2004-2017) in collaboration with TACF
  • All trials were conducted at Chestnut Return Farms in Seneca, SC using local isolates
• Operation turned over to TACF in 2018
  • Moved to USDA Forest Service Resistance Screening Center at the Bent Creek Experimental Forest in Asheville, NC
  • Katie McKeever (USDA FS) and Jared Westbrook (TACF) are now supervising this project
  • Our lab at Clemson provides isolates used as inoculum and assists with inoculation and final evaluation of seedlings
• 2019: New TACF strategy for screening – Use different isolates of cinnamomi each year
  • Each year, survivors from the screening trials are planted outside in the field in predetermined locations were cinnamomi is already present to determine if seedlings will survive under natural environmental conditions
  • Use isolates of cinnamomi from the out-planting field location to ensure that we do not move unique genotypes of pathogen to new locations
  • Field location identified during fall/winter prior to annual screening, and isolates from soil are recovered, identified, and stored at Clemson
• Sources of isolates used as inoculum for screening and field location for out-planting
  • 2004-2018: Two SC isolates from Chestnut Return Farms in Seneca, SC
  • 2019: Two NC isolates from Mountain Island Educational State Forest; Stanley, NC
  • 2020: One GA isolate from the Austin Flint North Ridge site in GA
• Advantage of new strategy:
  • Each year, hybrid seedlings are screened with different isolates, so seedlings are exposed to diverse genotypes of cinnamomi
• Potential disadvantage of new strategy:
  • Virulence of isolates may vary from year to year; therefore, screening rigor may vary from year to year – so this needs to be evaluated

Evaluating Virulence of P. cinnamomi Isolates

• Previous experiment in the Jeffers lab on American chestnut showed similar virulence among five groups of isolates of cinnamomi from chestnut trees collected at different geographic locations in the Southeast
• Experiment conducted in 2019 & 2020 in collaboration with TACF & USDA-FS and conducted at the Bent Creek RSC
• Experimental design
  • Eight cinnamomi inoculum treatments—including the five treatments used previously on American chestnut plus two additional treatments composed of isolates from ornamental plants and a non-inoculated control treatment
  • 2-3 isolates in each treatment with each treatment in a separate, isolated tub
  • Number of hybrid chestnut families inoculated: 8 in 2019 and 33 in 2020
  • All plants scored weekly by recording days to mortality = survival time
• Results from 2019: Data have not been analyzed statistically there may be significant differences among inoculum treatments
  • Hybrid chestnut families have different levels of resistance, which may help identify differences in virulence among isolates
• Experiment is being repeated in 2020 using different hybrid chestnut families

Detection of Phytophthora spp. in Chestnut Samples

• Conducted In collaboration with TACF: We continue to assay soils and symptomatic chestnut seedlings for Phytophthora – including soils where chestnuts are growing or might be planted
• Protocol is a simple baiting bioassay or direct isolation
• In the past year: Sep 2019 – Aug 2020
  • Samples received from 6 states: AL, GA, NC, PA, SC, TN – including nine submissions that contained 28 soil samples; this number of samples received is down compared to previous years
  • Phytophthora detected in 4/28 samples = 14%; all isolates recovered were P. cinnamomi

Fungicides for Phytophthora Root Rot (PRR) on American Chestnut Seedlings

• Rational: TACF is encouraging the planting of Germplasm Conservation Orchards (GCO) to collect and preserve existing American chestnut genotypes
  • Trees planted in GCOs in states where cinnamomi is present are at risk of infection and mortality, so fungicides could be used to protect susceptible seedlings and trees
• Objective: Evaluate the efficacy of registered oomycete-specific fungicides to manage PRR on American chestnut seedlings
• Trial conducted in a greenhouse at Clemson University in 2019-2020
  • Experimental Design: 10 treatments: 2 controls + 8 fungicides with 8 replicate seedlings/treatment; Fungicides were applied following label rates & application intervals
• Results were published in Plant Disease Management Reports, an online publication
• Overall conclusions: Fungicides vary in efficacy at protecting American chestnut seedlings
  • Promising active ingredients: K salts of phosphorous acid (phosphonates), fosetyl-AL (similar to phosphonates), mefenoxam
  • Experiment is being repeated in 2020 but results do not appear to be consistent, which may be due to “modified operations” at Clemson during the pandemic
• Therefore, this experiment will be conducted again in 2021

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

• This is a multi-state/university collaboration involving Clemson, Penn State, USDA-FS, Univ. of Kentucky, and Univ. of Tennessee
• A grant proposal was submitted to NSF/NIFA Plant Biotic Interactions Program in 2019
  • Project was led by Tatyana Zhebentyayeva at Penn State
  • Our proposal was not funded – in part because we needed more preliminary data
• Therefore, an experiment was conducted in Aug 2020 at Clemson University to examine the early steps of the infection process and determine when plants become infected and what genes are being regulated
  • Roots of American and Chinese chestnut seedlings in an aqueous environment were inoculated with zoospores of cinnamomi
• Current status: Frozen root samples are still waiting to be analyzed
• We have plans to repeat this experiment with minor adjustments to the protocol

TENNESSEE

Hill Craddock, The University of Tennessee at Chattanooga Department of Biology Geology and Environmental Science

Projects fall under Objective 1:

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

UTC graduates

• Masters Theses: Meg Miller & Trent Deason
• Undergraduate Honors Theses: Hannah Crawford, Colton Jones (Peyden Valentine)

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)

Projects fall under Objective 1 and 3:

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

Projects fall under Objective 1 and 3:

Summary: The most up to date research on traditionally bred hybrid chestnuts indicates that there is a tradeoff between blight resistance and percent American chestnut germplasm, how do we balance “resistant enough” with “American enough”? ​We propose ecological studies to assess ecological fitness among various hybrid genotypes with a range of American vs. Chinese germplasm. Studies could be performed in labs and greenhouses and at least one or more field studies. Variables of interest may include, but will not be limited to: leaf and seed herbivory, wood and leaf litter decomposition, associations with shoot and root microbes, etc. TACF seeks to fully understand how ecologically fit various hybrids may or may not be and use that information to inform future breeding strategies. TACF seeks partners to assist with these studies.

Fred Hebard, The American Chestnut Foundation

Projects fall under Objective 1 and 3:

The American Chestnut Foundation has been conducting a vigorous program of backcrossing the blight resistance of Chinese chestnut into American chestnut.  At the main breeding facility in Meadowview, VA, around 60,000 nuts of B3-F2 progeny in about 30 American backgrounds from two sources of blight resistance were planted in two seed orchards.  Blight resistance of B3-F2s and their B3-F3 progeny was intermediate between that of Chinese and American chestnut, as opposed to equaling the resistance of Chinese chestnut (Steiner et al 2017).   The following study sought to find reasons resistance was not higher.

The lower-than-expected level of blight resistance found in B3-F2s might be explained, at least in part, by two complementary hypotheses.  The first is that there are numerous genes for blight resistance and many were lost during backcrossing, so that B3 parents were not heterozygous at all loci conferring resistance.  This would make it impossible to obtain B3-F2s homozygous resistant at those loci.  The second hypothesis is that chestnut has a preference to be heterozygous, and that homozygotes are disfavored by recessive lethal genes and other mechanisms.  Again, this would result in fewer loci homozygous resistant.  Such homozygous deficiency might be exacerbated because the B3-F2s were identical by descent at all Chinese loci, being bred within two sources of blight resistance both derived from a single B1.

To test these hypotheses, and others, it would be very helpful to infer the male parent of the open-pollinated B3-F2 progeny.  The 60,000 B3-F2 seed had been winnowed down by mortality and by selection using phenotyping and progeny testing. Two-thousand, six-hundred, seventy-one were genotyped by sequencing (GBS).  An additional 189 potential parents were genotyped.  For this study, 938 SNP markers (single nucleotide polymorphisms) were used for Ellis’ method of hierarchal clustering. The objective was to infer the most likely male parent of chestnut B3-F2 progeny resulting from open pollination of B3s.  About 80% of likely male parents were from the same source of blight resistance as the female parent.  These were the subjects of the study.  They included 826 B3-F2s from the Clapper source of blight resistance, 699 from the Graves source, and 139 B3 parents.

The GBS data had been further processed to declare whether or not 100-kb bins along the chromosomes were derived from Chinese or American chestnut.  Under the assumption that blight resistance was conferred by Chinese alleles, this transformation made the data easier to process and interpret.  I owe profound thanks to Jared Westbrook for assembling and leading the team of researchers who prepared these datasets.  Jared furthermore made further selections in B3-F2 progeny based on tests of their B3-F3 progeny, field traits of the B3-F2s themselves, and genomic selection based on SNPs identified by GBS.  Two-hundred, fifty were selected and 1275 rejected.

RESULTS

The largest Clapper family had 31 progeny, with the number of members of the second largest family declining to 20, the third to 14, with the tenth largest family having only 5 progeny.  Those numbers of family members were inadequate to test directly for homozygote deficiency, even though there were 825 Clapper progeny.  Thus, tests for heterozygous excess had to be conducted with aggregates of families

The location on a chromosome containing the largest number of Chinese chestnut alleles would be most associated with a resistance gene, assuming these all came from Chinese chestnut, other things being equal.  Taking the median position of the locus with the largest number of Chinese alleles was considered a good first approximation of loci associated with blight resistance.   The mean number of Chinese alleles varied considerably within and between chromosomes.  There were three chromosomes in Clapper progeny that had frequencies of Chinese alleles greater than 0.2, chromosomes 5, 10 & 12, and five chromosomes in Graves progeny, 1, 7, 9, 10 & 12.  These can be considered to be relatively major _chromosomes_ associated with resistance.  Interestingly, two chromosomes, 5 in Clapper and 10 in Graves, with high frequencies of Chinese alleles did not have a significant incease after the latest round of selection.  Although two chromosomes with high frequencies of Chinese alleles were shared between Clapper and Graves, the closest were 8 Megabases apart, so most likely did not contain homologous genes

If there had been no loss of resistance alleles during backcrossing, the B3 parents would have been exclusively heterozygous for resistance and it would have segregated 1:2:1 in B3F2s.  The expected segregation in B3-F2s was calculated from the allele frequencies in their B3 parents. The counting occurred at one locus per chromosome for the two sources of blight resistance.  There were massively fewer Chinese alleles, 67% less, than would have occurred under 1:2:1 segregation.  Thus, loss of alleles during backcrossing had a major influence on the decrease in blight resistance of B3-F2s compared to expectation.

Comparing the observed segregation of Chinese alleles in B3-F2s to that expected from allele frequencies in B3 parents would reveal the effect of homozygous deficiency on allele frequency.  It revealed only slight further erosion of the number of Chinese alleles.  But the frequency of loci homozygous for resistance declined 46% further.  It had already declined by 87% comparing expected to 1:2:1 segregation.  Recovery of progeny homozygous for resistance alleles would double their number compared to heterozygotes, and would be the means for increasing their number once most progeny were heterozygous.  Thus homozygous deficiency is likely an important factor limiting high levels of blight resistance. It is not an insurmountable problem however, since homozygotes were recovered.

VIRGINIA

Alex Sandercock, Virginia Tech

Landscape Genomics of American chestnut

Project falls under Objective 1 and 3:

Blight-resistant American chestnut trees have been developed from gene insertion methods and backcross breeding programs. These trees can reduce the impacts of C. parasitica, but they are not in great enough numbers to capture the genetic diversity present in the native population. Additionally, it is unknown which genes in the American chestnut genome are related to local adaptation. Introducing blight-resistant American chestnut trees that are locally adapted to their planting site will give them the best chance of survival. So, the development of a conservation and breeding strategy is necessary to instill blight-resistance into the American chestnut population while maintaining genomic diversity. This study seeks to re-sequence the genomes of ~500 American chestnut trees, describe the population structure and demographic history, and identify the genes related to local adaption. Thus far, 96 samples have been sequenced and used to develop a dataset consisting of ~47 million SNPs and INDELs, which shows evidence of a genetically diverse population. A DAPC and an ADMIXTURE analysis were performed to estimate population structure, and preliminary results show a two population and three population structure respectively. Though, both analyses are in agreement with an independent northeastern population beginning in New York and extending through Maine. Finally, an SMC++ analysis was used to estimate the demographic history of C. dentata. This showed a steep decline in the effective population size beginning ~2.7 million years ago followed by subsequent bottleneck events. The next steps will be to sequence the remaining American chestnut samples and complete a genotype-environment association analysis.