Source: NORTH CAROLINA STATE UNIV submitted to
DEVELOPMENT AND USE OF GENOMIC TOOLS TO IMPROVE FIRS FOR USE AS CHRISTMAS TREES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0229728
Grant No.
2012-51181-19940
Project No.
NCZ09476
Proposal No.
2012-01547
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2012
Project End Date
Aug 31, 2017
Grant Year
2012
Project Director
Frampton, J.
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Forestry and Environmental Resources
Non Technical Summary
Next-generation DNA sequencing technology will be employed to accelerate the development and use of genetic information to improve firs for use as Christmas trees, an important specialty crop for which American consumers spend over $1 billion annually. The initial focus will be on improving postharvest needle retention, because surveys indicate that messiness is a major concern of real Christmas tree consumers. A two-step process will be used to identify single nucleotide polymorphic markers (SNPs) with predictive power: 1) candidate genes will be identified via RNA sequencing and 2) SNPs in candidate genes will be screened for association with phenotypes by targeted sequencing of genomic DNA. Additional traits of interest addressed are resistance to Phytophthora root rot, a disease that reduces productivity and increases costs, and adaptability of two promising exotic species that will be evaluated across fir production regions in a related ongoing collaborative project. Consumer surveys and grower/retailer focus groups are included in this proposal to help identify their preferences and hence, traits to improve in the future. An integrated outreach program will educate growers about producing and using genetically improved planting stock as well as consumer preferences.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010670108080%
2010670116010%
2120670108010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0670 - Short rotation woody crops, including holiday trees;

Field Of Science
1080 - Genetics; 1160 - Pathology;
Goals / Objectives
The long-term goal of this project is to improve the sustainability and profitability of the U.S. Christmas tree industry by developing and using genomic tools to produce low cost and high quality Christmas trees with properties desired by consumers. Specifically, we will: 1.Identify single nucleotide polymorphic markers (SNPs) with predictive power for desirable traits in true firs through a two-step process: a.Identify candidate genes by RNA sequencing (RNA Seq) b.Screen SNPs in candidate genes for association with phenotypes using targeted sequencing of genomic DNA. 2.Use SNP markers to accelerate production of trees with superior postharvest needle retention and resistance to Phytophthora root rot, and leverage marker-informed breeding with data from a separate industry-supported study of adaptability to climate change. 3. Conduct surveys and focus groups to increase our understanding of consumer Christmas tree preferences and improve the ability of growers to respond. 4.Educate and advise growers in genetic improvement techniques, seed orchard establishment, and using improved genotypes to produce high quality Christmas trees desired by consumers. Numerous outcomes are expected from this project including: * Genetic marker systems to select traits of importance to the Christmas tree industry * A catalog of fir genotypes from regional applied tree improvement efforts * Expanded use of genetically improved planting stock in the Christmas tree industry * Increased understanding of consumer preferences relating to Christmas trees * Growers more knowledgeable about consumer preferences and how to use improved genetic material and cultural practices to better meet their needs Collectively, these outcomes will increase the sustainability and profitability of the U.S. Christmas tree industry and result in more satisfied Christmas tree consumers. Widespread use of Phytophthora resistant planting stock will reduce chemical inputs into the environment. Moreover, additional consumers will purchase real Christmas trees which have a smaller carbon footprint as well as less environmental impact during production and disposal than artificial trees.
Project Methods
Postharvest needle retention will be the first trait addressed in this project, because of its prominent role in the Christmas tree industry's competitiveness with artificial trees. We will identify candidate genes via RNA sequencing (RNA-Seq) of tissue samples from eastern fir trees with extreme values for this trait based on existing phenotypic data. Candidate genes will be identified based on differential expression between pools of trees with different phenotypes, on the presence of different alleles in the pools, or on similarity to genes known to be involved in abscission zone formation in model plants. Using capture libraries of fir candidate genes, SNP markers with predictive power for this trait will be identified and verified on fir trees of other species with existing phenotypic data. To better understand Phytophthora root rot we will 1) describe the variability of Phytophthora species from different production regions, 2) investigate interactions between fir species and Phytophthora species important across Christmas tree production regions, 3) conduct histopathological studies of the infection and colonization process for susceptible and resistant firs to Phytophthora species, and 4) investigate the genetic control of resistance and develop SNP markers for resistant loci. In preparation for the future need of firs that are adapted to climate change, we will leverage efforts of the concurrent industry-supported Collaborative Fir Germplasm Evaluation (CoFirGE) Project by installing weather stations at each field site and applying techniques developed to select trees from these trials. We will conduct a national survey to better understand consumer preferences in real Christmas trees, and then implement regional focus groups of Christmas tree producers and retailers to help ascertain how the industry can best use these results. This information will feed into the project outreach program. Throughout the project, integrated outreach efforts will be ongoing to help growers better understand their customers and how to use genetically improved material. These efforts will include producing materials summarizing the information generated from the consumer survey and grower focus groups, development of a Genetic Improvement of Christmas Trees website, and regional meetings on establishing and managing genetically improved seed orchards. Results from all aspects of this project will be published in peer-reviewed journals for use among the scientific community. Grower-oriented material will be developed and disseminated through the outreach portion of this project. The SNP markers will be used to evaluate material in regional tree improvement efforts allowing new seed orchards to be developed or existing seed orchards to be rogued. Results from the national survey and regional grower/retailer focus groups will guide growers and breeders toward producing and marketing high quality Christmas trees valued by the buyers.

Progress 09/01/12 to 08/31/17

Outputs
Target Audience:Science-based knowledge was delivered to Christmas tree growers in various U.S. regions including the Pacific Northwest, the Lakes States region, the South, and Pennsylvania/New York. Information including a description of this project and/or related Christmas tree genetics topics was disseminated via presentations at grower meetings and publications in trade journals. Focus groups of Christmas tree growers were held in three production regions (PNW, Lake States and NC) to discuss survey results of consumer perferences. A five-course webinar series on Christmas Tree Genetics and Tree Improvement was offered to growers. Information was also delivered to fellow scientists and extension professionals at conferences in the U.S. and abroad. Other target audiences included nursery and green industry personnel, and students. These target audiences as well as the general public were also reached by the project web site (http://www.christmastreegenetics.org). Changes/Problems:The original proposal was to use hybrid-capture sequencing to follow up on discovery of genes differentially expressed in relation toPhytophthora root rot resistance or genetic propensity for good or poor needle retention. By the time the differential gene expression results were obtained, many other conifer genomics research groups had tested hybrid-capture sequencing methods and reported that the resulting data were extremely difficult to analyze, due to the presence of repetitive sequences in conifer genomes that are closely related to functional genes but are not functional. This feature of conifer genome structure was not well understood at the time the proposal was written. We chose to pursue quantitative reverse-transcription-PCR (qRT-PCR) as an alternative approach to investigate the association between differential gene expression in the trees sampled for RNA-seq analysis and other trees with known phenotypes for needle retention. This technique requires identification of a set of control genes whose expression does not change, for use as a reference against which differences in gene expression of the target genes can be measured. Unfortunately, the same problem of repetitive sequences closely related to functional genes in conifer genomes arose in these experiments as well, leaving our measurements of differential gene expression by DNA sequencing as the most reliable measures available. The cost of DNA sequencing exceeded the available budget for the projected scale of validation experiments, but we were able to obtain DNA sequence data from balsam fir and Canaan fir samples with known phenotypes for needle retention, and compare patterns of differential gene expression in those closely-related fir species with the patterns observed in Fraser fir. The candidate genes identified as differentially expressed across all three species are not yet conclusively demonstrated to be causally associated with differences in needle retention, but they are validated in three independent samples of well-replicated gene expression analyses. These genes will be suitable candidates for further investigation in future research on the mechanisms that control differences in needle retention in firs. What opportunities for training and professional development has the project provided?Seminars, workshops, and lectures focusing on the topics of genetic improvement, seed orchard development, needle retention improvement, grafting and fir species evaluation were made available to Christmas tree growers and other industry stakeholders. Work opportunities have been provided for undergraduate students providing technical training in many areas including bioinformatics, histology, plant pathology, greenhouse culture, cone and seed processing, postharvest evaluations and related areas. Graduate students and post-docs have been provided opportunities to participate in trade and scientific conferences. Post-doc Lilian Matallana won the best oral presentation award at the 2014 IUFRO Forest Tree Breeding Conference in Prague, Czech Republic, and at a NCSU campus-wide research symposium. How have the results been disseminated to communities of interest?Project results have been shared with communities of interest via the www.christmastreegenetics.org website, a five-part webinar series, at scientific conferences, trade associations meetings, publications in trade and scientific journals, other webinars, media interviews, small focus groups and individual meetings. (See Target Audience and Products sections for specifics.) Raw sequence reads from RNA-seq studies for four fir taxa (balsam, Canaan, Fraser and Trojan fir) have been submitted to GenBank (NCBI SRA Accessions PRJNA387306 and PRJNA357112) and are currently available for use by the scientific community. Assembled transcriptomes of these four fir taxa have also been submitted to the NCBI Transcriptome Shotgun Assembly (TSA) database and will be made public after publication of project results. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Christmas trees are an important specialty crop for which American consumers spend over $1 billion annually. This project employed genetic approaches to help ameliorate two major problems identified by the industry: needle shed (consumer issue) and Phytophthora root rot disease (producer issue). We identified candidate genes associated with needle loss in Fraser, Canaan and balsam fir, as well as Phytophthora root rot resistance in Trojan fir. Raw sequence reads and transcriptome libraries of all four of these fir species are archived at GenBank and TreeGenes and will be available for continued research. We identified the relative aggressiveness of key Phytophthora species on all major fir species produced in the U.S. We also helped Christmas tree growers understand tree improvement activities, evaluate clones in current seed orchards for needle retention, cull existing orchards and establish new orchards to produce improved planting stock for the industry. Objective 1 - Post-harvest needle retention RNA was extracted from the needle abscission zone of three fir taxa, balsam fir, Canaan fir and Fraser fir)),to examine gene expression at extremes of needle retention (phenotyped as good and poor). Libraries derived from 83 samples were sequenced via Illumina HiSeq 2500 (100bp PE) and post-QC and de novo assembly (Trinity v.2.2.0) resulted in 2,276,379 balsam fir, 1,180,304 Fraser fir, and 1,869,548 Canaan fir transcripts. Frame selected transcripts (GeneMarkS-T) were filtered using enTAP to remove contaminants (bacterial, fungal, insect) and subsequently clustered into a reference transcriptome for each taxa: balsam fir (410,662 transcripts), Fraser fir (232,832 transcripts), and Canaan fir (494,637 transcripts). Reads from each library were aligned (Bowtie2) to their respective reference transcriptome and used to generate expression estimates (DESeq2). 134 differentially expressed genes were identified in balsam fir (63 up-regulated and 68 down-regulated), 474 in Canaan fir (323 up-regulated and 151 down-regulated), and 177 in Fraser fir (89 up-regulated and 88 down-regulated). Up-regulated were more expressed in 'good' retention phenotypes while down-regulated genes were less expressed.After functional annotation, genes were clustered (NCBI's DAVID) to highlight differences in gene expression. Strong up-regulation (>4-fold) was observed in balsam fir with proteins such as TMV resistance protein, Calmodulin-like protein, Beclin-1 protein, and methylenetetra-hydrofolate reductase. Several were down-regulated strongly (>4-fold), including: anthocyanidin reductase, two-component response regulator-like APRR2, and peptidyl-prolyl cis-trans isomerase (PASTICCINO1). Network analysis identified that concanavalin A-like lectin/glucanase domain containing genes were frequently up-regulated in balsam fir and are associated with abscission control in Arabidopsis. In Canaan fir, CRK2, a cystine-rich receptor-like kinase involved in growth regulation and stress adaptation and KAO2, a gibberellin biosynthetic gene, and GLR3.3 were all strongly upregulated. Fewer genes were strongly downregulated in Canaan fir including the protease, FACE-2, and 7 LRR receptor-like serine/threonine-protein kinases. BPM4 was the only strongly upregulated (>4-fold) gene in Fraser fir, and an uncharacterized oxidoreductase encoding gene was the only one highly downregulated. Annotation clustering revealed downregulation in defense response and upregulation of integral membrane components. Protein serine/threonine kinase activity was highly enriched in both upregulated and downregulated genes. These results offer new genes and pathways to consider that potentially contribute to variation observed in Abies needle abscission and also connect to mechanisms in other systems. Objective 2 - Phytophthora Root Rot Resistance A survey of Phytophthora species present in fir production regions was completed and the relative aggressiveness of key Phytophthora species on seven Abies species under two temperature regimes elucidated. Additionally, RNA was extracted from Trojan fir (Abies equi-trojani)roots to capture gene expression at the extremes of susceptibility to Phytophthora cinnamomi. 10 pooled libraries of 10 samples each were sequenced on an Illumina HiSeq 2500 (100bp SE) to produce a total of 22,4497,907 reads following quality control. Susceptible, resistant and uninoculated control libraries were assembled independently and de novo (Trinity v.2.2.0) to generate 180,530 transcripts. Frame selection (GeneMARKS-T) was performed and transcripts clustered (Usearch)to generate a single reference (39,538 sequences) after contaminants were removed via EnTAP (0.3% and 13.1% of assembled transcripts in each library). Reads from each library were aligned to the reference transcriptome (Bowtie2) to generate gene expression estimates (DESeq2). After functional annotation (EnTAP), transcripts were clustered (NCBI's DAVID) to highlight related differences in the gene expression. 6298 differentially expressed (DE) genes were identified between resistant and susceptible groups (343 upregulated in susceptible, 5955 in resistant), including SAUR72 (small auxin upregulated RNA), and HSP90-1 (heat-shock protein), which were significantly upregulated in susceptible trees, and five OMP16 (outer membrane lipoprotein)homologs greatly upregulated (4.7 to 9.8-fold) in resistant trees. 1545 genes were differentially expressed between resistant and control groups (1219 upregulated in control, 326 in resistant) and comparison between susceptible and control groups yielded 4837 DE genes (115 upregulated in control, 4722 in susceptible). Overall, expression of xanthine and alcohol dehydrogenases increased in resistant trees relative to controls. 38 annotated genes were downregulated in susceptible trees and upregulated in resistant trees relative to controls including YSl5 (YELLOW STRIPE like 5), cspA (cold-shock protein A) and several ribosomal proteins. Apoptosis related genes were upregulated in resistant plants across comparisons, including XCP1, a cysteine peptidase which is involved in tracheary element autolysis. Objective 3 - Surveys & Focus Groups As a team member, the National Christmas Tree Association added questions to their annual national consumer surveys to improve industry understanding of factors that are keeping consumers from buying real Christmas trees. Additionally, a national online Qualtrics Panel Survey was conducted to better understand factors influencing real and artificial Christmas tree consumers. Some industry recommendations from this survey include: 1) capture first time buyers, 2) reduce mess and increase convenience, 3) sell the smell, 4) appeal to children and 5) recognize that artificial tree buyers love Christmas, too. These results were presented to growers in three regions (PNW, Lake States, NC) accompanied by focus group discussions on how to take advantage of this knowledge. Objective 4 - Education & Extension Numerous presentations at state, regional, and national Christmas grower association meetings were made and articles published in trade journals. Advice and help managing regional seed orchards was provided to growers including performing needle retention assessments of candidate and existing orchard clones and grafting select material for future seed orchard establishment. Seven Turkish and Trojan fir field trials in NC, MI, OR, PA and WA were managed as part of the Collaborative Fir Germplasm Evaluation (CoFirGE) Project. We presented a five-part webinar series on Christmas Tree Genetics and Tree Improvement. 205 growers from 21 states and 2 Canadian provinces participated live and hundreds have viewed the recorded presentations. 96% of participants replying to a post-webinar survey improved their understanding of the topics presented while 74% indicated that they would make a change in their business as a result of the series.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kohlway, W., R. Whetten, D.M. Benson, A.M. Braham and J. Frampton. 2017. Response of Turkish and Trojan Fir Seedlings to Phytophthora cinnamomi and P. cryptogea. Scandinavian Journal of Forest Research. 32(5): 406-411.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pettersson, J.M., J. Frampton, J. R�nnberg, D. Shew, D.M. Benson and M.A. Cubeta. 2016. Increased number of Phytophthora species found in Fraser fir Christmas tree plantations in the Southern Appalachians. Scandinavian Journal of Forest Research. 32(5):412-420.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Petterson, M. J. Frampton and J. Sidebottom. 2017. Influence of Phytophthora root rot on planting trends of Fraser fir Christmas trees in the Southern Appalachian Mountains. Tree Planters Notes. 60(1): 4-11
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: McKeever, K. and G. Chastagner. 2017. Section 36. Phytophthora Root Rot, Stem Canker, and Shoot Blight in Christmas Trees. In: Hansen, E.M., K.J. Lewis, and G.A. Chastagner (Eds) Compendium of Conifer Diseases. Revised Second Edition. APS Press.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Crain, B. A., & Cregg, B.M. 2017. Regulation and management of cone induction in temperate conifers. Forest Science.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Crain, B. A., & Cregg, B.M. 2017. Using irrigation and mulch to control cone production in Fraser fir (Abies fraseri). Scandinavian Journal of Forest Research, 32(5) 384-390.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Crain, B. A., & Cregg, B.M. 2017. Gibberellic acid inhibitors control height growth and cone production in Abies fraseri. Scandinavian Journal of Forest Research, 32(5) 391-396.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Anonymous. 2017. Consumer Poll Figures and Trends. American Christmas Tree Journal, Spring-Summer 2017: 14-16.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Hinesley, L. E. and G. A. Chastagner. 2016. Christmas tree keepability. Pp. 650-658. In: Gross, Kenneth C., Chien Yi Wang, Mikal Saltveit and Gene Lester (Eds.). The Commercial Storage of Fruits, Vegetables, and Florist and Nursery Crops. USDA Agriculture Handbook 66. Revised Feb. 2016. USDA, Agricultural Research Service, Beltsville, MD. 780p.
  • Type: Websites Status: Published Year Published: 2017 Citation: NCTA. 2017. Consumer survey results. http://www.realchristmastrees.org/dnn/News-Media/Industry-Statistics/Consumer-Survey
  • Type: Websites Status: Published Year Published: 2017 Citation: Christmas Tree Genetics web site. 2017. Updates including relevant research, extension and trade publications.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: McKeever. K.M. 2016. Assessing Status of and Resistance to Phytophthora Root Rot on True Fir (Abies spp.). PhD Dissertation, Washington State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Curry, A. 2017. Assessing Abies nordmanniana and Abies bornmuelleriana seed quality factors. August. MS thesis. Washington State University.
  • Type: Theses/Dissertations Status: Under Review Year Published: 2018 Citation: Kohlway, W. 2018. Insights into Phytophthora root rot resistance in Trojan fir. PhD Disseration, N.C. State University. Raleigh.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Cregg, B.M., Ellison, D.S. & ODonnell, J. 2017. Coning and shoot growth response of Fraser fir trees to paclobutrazol application. 13th International Christmas Tree Research and Extension Conference. September 4-8, 2017, Akureyri, Iceland. (abstract and presentation)
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Chastagner, G.A., K. Riley, K. P. Coats, H. Eikemo and V. Talg�. 2017. Delphinella abietis and Grovesiella abieticola on Abies lasiocarpa in Western USA. Scandinavian J. Forest Research. 32(5): 432-437.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Landgren, C., U.B. Nielsen, and G. Chastagner. 2017. Comparison of noble fir progeny from U.S. Pacific Northwest collection regions and Denmark for Christmas tree traits. Scandinavian J. Forest Research 32(5): 366-375.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Cregg, B.M., & Ellison, D.S. 2017. Seasonal development of cold hardiness and needle retention in Christmas tree plantations in Michigan. 13th International Christmas Tree Research and Extension Conference. September 4-8, 2017, Akureyri, Iceland. (abstract and presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Ellison, D.S., Cregg, B.M., & ODonnell, J. 2017. Evaluation of cone control options in Fraser fir plantations. 13th International Christmas Tree Research and Extension Conference. September 4-8, 2017, Akureyri, Iceland. (abstract and presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Matallana, L. K. Goode, M. Rojas-Pierce, K. Coats, G. Chastagner, J. Frampton and R. Whetten. 2017. Christmas tree needle separation: a marriage that ends on the carpet. 13th International Christmas Tree Research and Extension Conference, 4-8 Sept. Akureyri, Iceland. (abstract and presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Frampton, J. M. Pettersson and A.M. Braham. 2017. Family variation in resistance to Phytophthora root rot in Eastern white pine. 13th International Christmas Tree Research and Extension Conference, 4-8 Sept. Akureyri, Iceland. (abstract and presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: McKeever, K.M. and G. Chastagner. 2017. Interactions between Root Rotting Phytophthora, Abies and Environment. 13th International Christmas Tree Research and Extension Conference, 4-8 Sept. Akureyri, Iceland. (abstract and presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Matallana, L. 2017. How genomics is shaping the future of Christmas trees. 2017 Marcus Wallenberg Prize. October 25th, 2017. Stockholm, Sweden. (Presentation ranked in the top five best.)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Goode, K. 2017. Biological Divorce: How Christmas Tree Needles Go Their Own Way. 26th Annual spring undergraduate research symposium at NCSU. April 12th, 2017. Raleigh, NC (USA). (poster and abstract)


Progress 09/01/15 to 08/31/16

Outputs
Target Audience:Science-based knowledge was delivered to Christmas tree growers in various U.S. regions including the Pacific Northwest, the Lakes States region, North Carolina, and Pennsylvania. Information including a description of this project and/or related Christmas tree genetics topics was disseminated via presentations at grower meetings and publications in trade journals. This information was also delivered to fellow scientist and extension professionals at conferences in the U.S. and abroad. Other target audiences included nursery and green industry personnel, and students. These target audiences as well as the general public will also be reached by the project web site (http://www.christmastreegenetics.org). Changes/Problems:The transcriptome assembly presented two minor challenges. The primary challenge resulted from frame selection approaches that introduced some error into the final annotation and expression results. As a result, two different approaches were used to revise the assemblies and DE analysis. The combined approach is preferred for DE analysis but was not amenable to computational approaches to generate an acceptable assembly. These experiments delayed results to the project but the current approach has improved these to ensure that the final deliverables will be available before the end of the year. After RNA-seq and gene expression analyses, it is important to determine if the expression of selected candidate genes correlated with needle abscission in trees with good or poor needle retention phenotypes. RT-qPCR is a method of choice for rapid quantification of mRNA transcription. To determine the exactly amount of amplifiable template nucleic acid present in the reaction, it is necessary to compare the amplification of those genes with the expression of endogenous controls or "housekeeping" genes. The reliability and accuracy of the method remains influenced by many experimental variations, but the selection of housekeeping genes is probably the most crucial aspect to assure biological meaningful data. Those genes need to be transcribed on the tissue of interest, among all samples and in a constant rate under specific experimental conditions. We have been testing the expression of common housekeeping genes reported in model plant species and some conifers. Nevertheless, we have been unable to identify appropriate housekeeping genes that allow us the validation of our candidate genes for needle retention studies. Therefore, we decided to search in our RNA-seq data for a set of housekeeping candidate genes. We are currently evaluating differences in expression of those genes using some statistical algorithms. Our aim is to validate a number of housekeeping genes for their use as internal standards in transcriptome analysis of needle retention in Fraser fir. The histopathology study of infection and colonization of susceptible and resistant Abies spp. by Phytophthora spp. at WSU has been delayed and likely will not be completed during this project. This was largely due to the one-year delay in starting the host:pathogen:environment interactions studies due to problems associated with the production of the 20,630 seedlings for use in the initial greenhouse study. The data from these studies were going to be the basis for developing the protocol for the histopathology study. While the final data from the second year of these studies were collected this past summer, data analysis will not be completed until fall 2016. The graduate student who was scheduled to conduct the histopathology studies will be graduating in December, and given the complexity of the histopathology studies, it is unlikely they studies can be completed prior to the termination of this project in 2017. What opportunities for training and professional development has the project provided?Seminars, workshops, and lectures focusing on the topics of genetic improvement, seed orchard development, needle retention improvement, grafting and fir species evaluation were made available to Christmas tree growers and other industry stakeholders. Work opportunities have been provided for undergraduate students providing technical training in many areas including bioinformatics, histology, greenhouse culture, cone and seed processing, postharvest evaluations and related areas. Graduate students and post-docs have been provided opportunities to participate in trade and scientific conferences. Post-doc Lilian Matallana was won the best presentation award at the NCSU campus-wide research symposium for her presentation, "Decoding Needle Abscission: The Genome behind your Christmas Tree". How have the results been disseminated to communities of interest?Project results have been shared with communities of interest via the www.christmastreegenetics.org website, at scientific conferences, trade associations meetings, publications in trade and scientific journals, webinars, media interviews, small groups and individual meetings. (See Target and Products sections for specifics.) What do you plan to do during the next reporting period to accomplish the goals?Major activities for the final year of this project include: * Complete the genomics aspects of the project including: - sequence rna from the second rep of Fraser fir needles abscission zone samples (to balance with other two species) - repeat bioinformatics analyses with new sequences - conduct gene expression analyses using RT-qPCR on candidate needle retention genes - archive data with Genbank and Tree Genes - write publication of results * Complete Phytophthora root rot studies including: - graduate two Ph.D. students, Katie McKeever (WSU) and Will Kohlway (NCSU) - write publication of results * Develop and carry out a five-part webinar for Christmas tree growers on Christmas tree genetics and tree improvement. * Assess four-year height and budbreak in Turkish and Trojan fir field trials of the CoFirGE Project in MI, NC, OR, PA and WA. Postharvest needle retention assessments will also be made for tress at the WA site. Analyze and write publication of results. * Continue to educate and advise Christmas tree growers about tree improvement activities

Impacts
What was accomplished under these goals? Impact Christmas trees are an important specialty crop for which American consumers spend over $1 billion annually. This project seeks to ameliorate two major problems identified by the industry: needle shed (consumer issue) and Phytophthora root rot disease (producer issue). We have identified candidate genes associated with needle loss in Fraser fir. We recently obtained dna sequence data for balsam and Canaan fir and are conducting analyses across all three species to find sequences associated with needle loss. We have also worked to help Christmas tree growers understand the importance of genetics toward improving needle retention and have helped them rate clones in current seed orchards and plantations for this trait and have helped cull existing orchards and establish new orchards. We have identified the relative aggressiveness of key species of the pathogen on all the major fir species produced in the U.S. We have also identified dna sequences associated with Phytophthora resistance in Trojan fir and are collecting more sequence information to improve our confidence in these results. Objectives 1 & 2 RNA-Seq libraries were generated from three different fir taxa from tissue at the needle abscission zone. Individual RNA-Seq libraries were generated for individuals from two different phenotypic classes in Fraser fir, Canaan fir, and balsam fir. The libraries were multiplexed and sequenced on the Illumina HiSeq 2500 (100bp PE). The resulting libraries were assembled both independently and combined. The first analysis was performed using individual assemblies of each library using Trinity, performing frame selection with GeneMarkS-T, and clustering the resulting assemblies with Usearch/UClust to generate a reference for each of the three sets. The assemblies here ranged in size from 45K to 75K transcripts with an average N50 exceeding 750bp in length. Differential expression was performed on all three sets via DESeq2 and DE genes were identified and annotated across all three. Full functional annotation of the resulting assembly was performed. The largest fold change genes ranged from 20 to over 50 across the three analysis runs. Evaluation of the frame selection approach generated some erroneous frame starts and shifts in the final transcripts that were evident from the annotation/expression results. Also, the combined clustering approach introduced some bias in the expression analysis. To determine if a fully combined assembly approach would be viable to contend with some of these issues, two different k-mer runs (a standard k-mer level of 1 and a k-mer depth of 5) were run with Trinity. The latter parameter is intended to contend with assembly fragmentation. Test runs of both k-mer levels were performed on both the Canaan fir and balsam fir assemblies. Both assemblies resulted in final transcript counts of > 500,000 transcripts and relatively short N50 (transcript lengths) of 600-650bp. It was determined that the individuals were too diverse to be amenable to this approach even with the additional k-mer filter. Returning to the original assemblies, we have generated a new orthologous gene set across all three species working with the original individual assemblies and a modified approach (primarily relying on database comparisons) for frame selection. Phytophthora research focused on completion of the second large-scale greenhouse host:pathogen:environment interaction screening trial. Three isolates of four pathogen species (P. cambivora, P. cinnamomi, P. kelmania, and P. pini) were used to inoculate seven host species (Fraser, noble, white, Canaan, balsam Turkish and Nordmann firs) under two temperature conditions (cool, 15 - 21°C and warm, 27 - 32°C). Disease progress curves representing mortality over time indicate that mortality was generally lower in the cooler greenhouse than under warmer ambient temperatures. Phytophthora cinnamomi generally caused disease on a greater number of species than other Phytophthora treatments. Phytophthora kelmania and P. pini had little effect on Nordmann and Turkish firs, but were moderately-to-highly virulent on the other hosts, particularly at warmer temperatures. Phytophthora cambivora generally only damaged noble and Fraser firs, with some moderate effect on white fir. Observational comparison of root rot severity due to Phytophthora treatment for each host also suggested a trend of greater damage on host roots infected by P. cinnamomi, followed by P. kelmania, P. pini, and P. cambivora. These trends were not absolute; for example, P. pini was quite virulent on the more susceptible host species, and statistical analysis indicated that significant differences among mean root rot severity caused by P. cinnamomi, P. kelmania, and P. pini were generally perceptible only on resistant hosts. On more resistant hosts (Turkish and Nordmann firs), root rot severity caused by P. kelmania, P. pini, and P. cambivora rarely deviated statistically from root rot ratings assessed on control seedlings. Phytophthora cambivora was arguably the least damaging Phytophthora, differing significantly from the other three treatments in nearly every fir species except for noble fir. Objective 3 - Surveys + Focus Groups Following the 2015 sales season, a new question was added to the annual national poll conducted by the National Christmas Tree Association (NCTA) aimed at understanding which convenience factors are keeping consumers from buying a real Christmas tree. Thirty percent of those surveyed who were not using a Christmas tree (real or artificial), indicated that there was something that would convince them to buy a tree. This market segment represents over 13 million household and could sustainably expand the real tree market. About 18% of those surveyed and not currently displaying a tree would consider using one if watering was more convenient. Because devices to easily water trees are available, the industry and manufacturers should work together to educate the public on how these devices address this major convenience issue. About three quarters of those surveyed lived in metro areas and were mainly inhibited by transportation problems and proximity to sales lots. Objective 4 - Education & Extension Numerous presentations at grower association meetings were made and articles published in trade journals. (See other sections of this report.) Advice and help managing regional seed orchards was provided to growers including performing needle retention assessments of candidate and existing orchard clones and grafting select material for future seed orchard establishment. Eight Turkish and Trojan fir field trials in NC, MI, OR, PA and WA were managed as part of the Collaborative Fir Germplasm Evaluation (CoFirGE) Project.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Crain, B. A. 2016. Environmental and chemical control of cone formation in plantation-grown Abies fraseri (Doctoral dissertation, Michigan State University).
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Kohlway, W.H. 2015. Mapping Phytophthora root rot resistance in fir species through genotyping by sequencing. MS Thesis, N.C. State University. 52p.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Grinwis, R. and Cregg, B. 2016. What fir? Diversifying landscapes with exotic firs. The Michigan Landscape 59(1):32-35.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cregg, B. 2016. What do conifer enthusiasts need to know about mycorrhizae? Conifer Quarterly 33(2):5-8.
  • Type: Other Status: Published Year Published: 2015 Citation: Cregg, B. and ODonnell, J. 2015. Why is my Christmas tree beginning to grow? MSU Extension News Dec. 2, 2015.
  • Type: Other Status: Published Year Published: 2015 Citation: Cregg, B. and ODonnell, J. 2015. Choosing the right Christmas tree. MSU Extension News November 23, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cregg, B. 2015. Living Christmas trees: Another real tree option. MSU Extension News November 24, 2015.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: McKeever, K., and G. A Chastagner. 2016. A survey of Phytophthora species associated with Abies in U.S. Christmas tree farms. Plant Disease. 100: 1161-1169.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Matallana, L. 2015. Christmas tree researcher profile: Dr, Lilian Matallana from Colombia. Limbs &Needles. 42(1):22-23.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Pettersson, J.M., J. Frampton, J. R�nnberg, D. Shew, D.M. Benson and M.A. Cubeta. 2016. Increased number of Phytophthora species found in Fraser fir Christmas tree plantations in the Southern Appalachians. Scandinavian Journal of Forest Research. Published online: 05 Dec 2016. (in press)
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Kohlway, W., R. Whetten, D.M. Benson, A.M. Braham and J. Frampton. 2016. Response of Turkish and Trojan Fir Seedlings to Phytophthora cinnamomi and P. cryptogea. (Scandinavian Journal of Forest Research. (in press)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kurt, Y., J. Frampton, F. Isik, C. Landgren and G. Chastagner. 2016. Variation in needle and cone characteristics and seed germination ability of Abies bornmuelleriana and Abies equi-trojani populations from Turkey. Turkish Journal of Agriculture and Forestry. 40:169-176).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: McKeever and Chastagner. 2015. Screening Abies for resistance to Phytophthora root rot. 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. P. 30. (Presentation & Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chastagner, G., Y. Kurt, J. Frampton, F. Isik, and C. Landgren. 2015. Variation in postharvest needle retention characteristics of Turkish and Trojan fir populations from Turkey. 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. p. 54. (Presentation & Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kurt, K., C. Landgren, G. Chastagner, B. Cregg, P. Nzokou, J. ODonnell, R. Bates, U.B. Nielsen, R. Cowles, F. Isik and J. Frampton. 2015. Variation in survival and bud break of Turkish and Trojan fir in the United States. 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. P. 40. Presentation & Abstract
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Matallana, L. K. Coats, G. Chastagner, J. Frampton, and R. Whetten. 2015. Inferring gene networks regulating needle abscission in Fraser fir through RNA-seq data. 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. P. 46. Presentation & Abstract
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Bucholz, E. D. Tilotta, R. Jetton, L. Lucia and J. Frampton. 2015. Effect of bornyl acetate on reproduction of the green peach aphid and balsam wholly adelgid. 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. P. 23. (Presentation & Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pettersson, J.M., J. Frampton, J. Ronnberg, D. Shew, D.M. Benson and M.A. Cubeta. 2015. Increased number of Phytophthora species found in Fraser fir Chrsitmas tree plantations in the Southern Appalachians. 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. P. 27. (Presentation & Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kohlway, W., R. Whetten, D.M. Benson, A.M. Braham and J. Frampton. 2015. Response of Turkish and Trojan fir seedlings to Phytophthora cinnamomi and P. cryptogea. P2 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. P. 29. (Presentation & Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Whetten, R. 2015. Genomic technologies and applied tree breeding. 12th International Christmas Tree Research and Extension Conference. NIBIO Book 1 NR 01 2005. September 6-11, 2015. Honne, Norway. P. 21. (Presentation & Abstract)
  • Type: Other Status: Other Year Published: 2015 Citation: Matallana, L., J. Frampton and R. Whetten. 2015. Transcriptomic analyses of gymnosperm species. Postgraduate seminar invitation. Scuola Superiore Sant. 14 Spet. Pisa, Italy.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Matallana, L. K. Coats, G. Chastagner, J. Frampton and R. Whetten. 2016. Falling needles: clues from your Christmas tree genome. 30th Annual Plant Molecular Biology Retreat. 23 Sept. Wrightsville Beach, NC.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Matallana, L. J. Frampton and R. Whetten. 2016. Decoding needle abscission: the genome behind your Christmas tree. NCSU Postdoctoral Research Symposium. 17 May. Raleigh, NC.  Awarded best poster presentation.


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:Science-based knowledge was delivered to Christmas tree growers in various U.S. regions including the Pacific Northwest, the Lakes States region, North & South Carolina, and Pennsylvania. Information including a description of this project and/or related Christmas tree genetics topics was disseminated via presentations at grower meetings and publications in trade journals. This information was also delivered to fellow scientist and extension professionals at conferences in the U.S. as well as in Sweden. Other target audiences included nursery and green industry personnel, and students. These target audiences as well as the general public will also be reached by the project web site (http://www.christmastreegenetics.org). Changes/Problems:Determination of gene expression patterns relies heavily on the ability to identify specific mRNA transcripts in the pool of messenger RNA isolated from tissue samples such as abscission zones. A new technique has been developed for the Pacific Biosciences sequencing instrument that provides a more accurate and complete catalog of all the alternative splicing isoforms of mRNAs present in a particular RNA sample. We plan to take advantage of this technology to collect data to improve the existing RNA transcript dataset we have been using in our analysis of differential gene expression. The benefit of this effort will be realized through more accurate estimates of the abundance of different transcript splicing variants in both existing RNA-seq datasets and in new datasets that may be collected in the future. What opportunities for training and professional development has the project provided?Seminars, workshops, and lectures focusing on the topics of 'genetic improvement of Abies spp.', 'seed orchard development', 'needle retention improvement', 'grafting' and 'Abies species evaluation' were made available to Christmas tree growers and other industry stakeholders. Tours and temporary work opportunities have been provided for students. Graduate students and post-docs have been provided opportunities to participate in trade and scientific conferences. Field Days/Tours Co-organized Pacific Northwest Christmas Tree Association Short Course. March 2015. Wilsonville, OR Puget Sound Christmas Tree Association Annual Meeting, June 2015. Puyallup, WA Pacific Northwest Christmas Tree Association Summer Meeting. June 19, 2015. Rochester, WA Pacific Northwest Christmas Tree Association Summer Tour. June 20, 2015. Elma, WA How have the results been disseminated to communities of interest?Project results have been shared with communities of interest via the www.christmastreegenetics.org website, at scientific conferences, trade associations meetings, publications, media interview, small groups and individual meetings. See Target Audience, Products and Other Products sections for specifics. What do you plan to do during the next reporting period to accomplish the goals?Include other related species (Canaan and balsam fir) that exhibit high variation in needle retention to increase the number of RNA-seq libraries and improve the data set for further analysis. The transcription assembly from RNA-seq Fraser fir data without a reference genome is a continious learning process that needs improvement. Different software for RNA-seq analysis are available but validation of results will be required. The measurement of gene expression patterns by q-RT-PCR will continue as a means of testing the validity of the associations detected between the desirable needle-retention phenotype and gene expression patterns of specific gene networks active in abscission zones. In addition, histological examination of the abscission zone region will be carried out to assess differences in tissue differentiation patterns observed in trees with desirable versus undesirable needle retention characteristics. Repeat the greenhouse screening of Abies for resistance to Phytophthora root rot. Continue to educate and assist Christmas tree growers concerning the development and use of genetically improved planting stock.

Impacts
What was accomplished under these goals? Objectives 1 & 2 108 Fraser fir selections grafted in a clone bank at the N.C. Department of Agriculture & Consumer Services' Upper Mountain Research Station, Laurel Springs, N.C., were phenotyped for needle retention at three time points (10/13, 10/27 and 11/10) for the second consecutive year. Needle abscission zones samples were collected and stored for future RNA extractions. RNA-seq data from good and poor needle retaining Fraser fir selections were processed. After removing low quality reads, de novo assemblies were constructed from both individual and merged data. Following clustering, transcript abundances were quantified for both the individual and merged assemblies by two methods (Sailfish and Bowtie2 + RSEM) and differentially expressed genes identified. To validate associations with the needle retention phenotype and more accurately measure differential gene expression, select candidate genes are being evaluated using quantitative reverse-transcriptase polymerase chain reaction (q-RT-PCR). The identification of Phytophthora species from the survey of Fraser fir Christmas tree plantations in the Southern Appalachian Mountains was completed. In total, six species of Phytophthora were isolated from 82 sites in 13 counties. P. cinnamomi accounted for 71% of the isolates and P. cryptogea for 23%. Collectively, P. citrophthora, P. europaea, P. pini and P. sansomeana accounted for 6% of the isolates; these have not been identified in previously published Fraser fir surveys conducted in the region. While P. cinnamomi was still the predominant species isolated from infected Fraser fir roots, P. cryptogea appears to have become an important pathogen contributing to losses in the region. In a large-scale greenhouse Phytophthora-resistance screening study one-year-old seedlings of 7 species of fir were inoculated with 3 virulent isolates from each of 4 species of Phytophthora. The Phytophthora isolates were collected from fir roots during a nationwide sampling of tree plantations in 5 major U.S. Christmas tree production regions. The experiment was conducted simultaneously in two greenhouses set to two different temperatures. One greenhouse was maintained at 15 - 21°C to replicate prevailing conditions in temperate regions such as the Pacific Northwest while the other greenhouse was sustained at 26 - 32°C to simulate the southeastern U.S. and California. Throughout the course of the 20-week experiment, weekly ratings of plant condition (alive, failing, dead) were performed, and percent mortality was calculated over time. In general, noble and Fraser fir were extremely susceptible, while balsam, Canaan, and white fir were moderately susceptible. The ranking of balsam, white, and Canaan fir was dependent on Phytophthora species and isolate. Nordmann and Turkish fir were consistently quite resistant. Mortality was clearly greater under warmer ambient conditions than cooler temperatures. Interestingly, although the overall mortality differed quite markedly between the two temperatures, the ranking of the percentage of mortality caused by each Phytophthora species was very similar in each environment, with P. cinnamomi causing more mortality, followed by P. kelmania, pini, and cambivora. There were no statistical differences among isolates of the same Phytophthora species for Nordmann and Turkish firs since mortality was so low in these species, however, for other Abies-Phytophthora combinations, isolate did indeed have a bearing on disease progress. In June 2015, trees from this greenhouse study were destructively processed to collect additional data to compliment the observations for percent mortality. During processing, we took a final assessment of shoot conditions (alive, failing, dead), an estimation of the percentage of rot on the roots, and a determination of root biomass (oven-dry weight basis). Statistical analyses of the data collected in the first trial of this project are underway, and inoculation of the second trial is planned for December of 2015. Objective 3 A follow-up question about recycling was added to the annual Harris poll conducted by the National Christmas Tree Association (NCTA) to determine if tree recycling programs are strengthening sales. As in the previous year, even people who buy a real tree don't seem to be motivated by the success of tree recycling programs. And there's also a lack of evidence that recycling programs are helping to convince non-tree buyers to consider buying one. Disconcertingly, 63% of 18-34 year olds reported they either didn't know about tree recycling programs or know about them but don't know how to use and only 43% of all people who buy a tree know how to recycle it. The industry needs to do more to reinforce this message and be part of the awareness and education and maybe even implementation. Another question added to NCTA's Harris poll of consumers attempted to measure impact of variety and convenience factors on potential tree sales, i.e., are more variety in trees and "convenience" factors more important to no-tree households than to those who do buy a tree? Not surprisingly, those who already buy a tree are very picky about their aesthetics--height, width, fullness, color, etc. Further, for those already buying a real tree, convenience factors are not that important. However, while those not buying a real tree still rated aesthetics important, they also rated convenience factors as significantly more important. Though it's only one year of data, it's highly suggestive that the industry needs to improve convenience factors including recycling to target the 27% of households who do not buy a real tree. Objective 4 Eight Turkish and Trojan fir field trials in NC, NY, OR, PA and WA were managed as part of the national Collaborative Fir Germplasm Evaluation (CoFirGE) Project. Data for first year survival and second year budbreak were analyzed from nine sites (n=23,560 trees) and eight sites (n=22,518 trees), respectively. Budbreak at each site was measured on a scale from 0 (tight bud with no swelling) to 6 (shoot fully elongated). Overall, survival was excellent (>90%) except at two sites and no provenance differences were detected. However, the two Trojan fir provenances broke bud earlier than the three Turkish fir provenances. The overall heritability estimate for budbreak on an individual tree basis was low (0.06) but modest on a family means basis (0.59). Both varied considerably across regions (0.04-0.47 and 0.34-0.79, respectively). The latitude, longitude and elevation of the mother tree were significantly correlated with the survival (0.29. 0.30 and 0.30, respectively) and budbreak (-0.75, -0.78 and -0.78, respectively) of its progeny. While these early results are generally encouraging, some northern studies have encountered damages from extreme winter temperatures and/or late spring frosts. Assistance with genetically improved seed and seed orchards was provided to Christmas tree growers in several regions including: 1) collecting and processing cones from a Turkish fir seed orchard in New York as part of a seed quality project, 2) scouting provenances of Nordamann fir in the Republic of Georgia for future cone collections, 3) performing maintenance and assessing the third year of needle retention evaluations on clones in a Canaan fir seed orchard in PA, 4) establishing a new Fraser fir seed orchard at the Michigan State University Horticulture Teaching and Research Center using selections exhibiting delayed coning and excellent form, 5) grafting clones from the Penn State University Canaan fir seed orchard for establishing another orchard, 6) establishing a Virginia pine seed orchard in South Carolina, and 7) providing data on needle retention and other characteristics for clones of the NC Premium Fraser Fir Seed Coop's orchard.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: McKeever, K., and G. Chastagner. 2014. Regional Variation of Phytophthora Species from U.S. Christmas Tree Production Areas. 62nd Western International Forest Disease Work Conference (WIFDWC). September 9, 2014. Cedar City, Utah.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: McKeever and Chastagner. 2014. Community structures of root-rotting Phytophthora species affecting Abies in U.S. Christmas tree farms and screening true fir for resistance to Phytophthora root rot. IUFRO 7th Phytophthora in Forest and Natural Ecosystems Symposium. Nov. 9-14, 2014, Esquel, Argentina.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: McKeever and Chastagner. 2015. Field assessment of Turkish fir (Abies bornmuelleriana) tolerance to five rot Rotting Phytophthora species. 61st Annual Conference on Soilborne Plant Pathogens. March 2015, Riverside, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: McKeever and Chastagner. 2015. Regional variation of root-rotting Phytophthora species from U.S. Christmas tree production areas. American Phytopathological Society Annual Meeting, August 1-5, 2015, Pasadena, CA.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Kurt, Y., J. Frampton, F. Isik, C. Landgren and G. Chastagner. 2015. Variation in needle, cone and germination characteristics of Abies bornuelleriana and Abies equi-trojani populations from Turkey. Turkish Journal of Agriculture and Forestry.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cregg, B. 2015. Building a better Christmas Tree, Great Lakes Christmas Tree Journal, Winter volume 10 (1): 30-34.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: McKeever, Kathleen, and Gary Chastagner. 2014. Screening true fir resistance to Phytophthora root rot and structures of Phytophthora communities from Pacific Northwest Christmas tree farms. P. 26. In: MacDonald, M. (Ed). Proceedings IUFRO 11th International Christmas Tree Research and Extension Conference. 74 p. August 10-14, 2013. Truro, Nova Scotia.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: McKeever, K, and G. Chastagner. 2015. A survey of Phytophthora species causing root rot on Abies in U.S. Christmas tree farms. APS Abstract 134-O. http://www.apsnet.org/meetings/Documents/2015_meeting_abstracts/aps2015abO155.htm
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Landgren, Chal, Gary Chastagner, Ulrick Nielsen. 2014. Results from common source trials of Nordmann and Turkish fir in Denmark and the PNW, USA. P. 32-34. In: MacDonald, M. (Ed). Proceedings IUFRO 11th International Christmas Tree Research and Extension Conference. 74 p. August 10-14, 2013. Truro, Nova Scotia.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kurt Y., Frampton J. ve I??k F. 2015. Y?lba?? a?a�lar?na yeni adaylar: ABDde T�rkiye g�knarlar?. Bilim ve Gelecek 132: 84-87. (New Christmas tree candidates: fir species of Turkey in the United States. Science and Future. 132: 84-87.)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Frampton, J., R. Whetten, L. Matallana, Y. Kurt and W. Kohlway. 2014. DNA sequencing and Christmas tree research. Am. Christmas Tree J. 58(2): 24-26.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Frampton, J. and R. Whetten. 2014. DNA sequencing and Christmas tree research. Limbs & Needles 41(1): 20-23.


Progress 09/01/13 to 08/31/14

Outputs
Target Audience:Science-based knowledge was delivered to Christmas tree growers in various U.S. regions including the Pacific Northwest, the Lakes States region, North & South Carolina, and Pennsylvania. Information including a description of this project and/or related Christmas tree genetics topics was disseminated via presentations at grower meetings and publications in trade journals. This information was also delivered to fellow scientist and extension professionals at conferences in the U.S. as well as on international visits to China and the Czech Republic. Other target audiences included nursery and green industry personnel, and students. These target audiences as well as the general public will also be reached by the project web site (http://www.christmastreegenetics.org). Changes/Problems:The project budget is not sufficient to allow collection of sufficient RNA-Seq data to conclusively test the hypothesis of association between the desirable needle retention phenotype and specific patterns of gene expression in identified gene networks. The proposed alternative was to use hybrid-capture DNA sequencing to recover specific fragments of genomic DNA from the coding sequences of mRNAs that show differential expression between trees with good needle retention and those with poor needle retention. Since the proposal was submitted, publication of draft genome sequence assemblies for Norway spruce and loblolly pine have demonstrated that conifer genomes are full of "processed pseudogenes" - short fragments of DNA derived from coding sequences of mRNAs that have been re-inserted into the nuclear genome. These processed pseudogenes make analysis of data from hybrid-capture experiments extremely difficult; many research groups around the world working with conifer genomes are facing the challenge of distinguishing between sequences derived from true functional genes and sequences derived from processed pseudogenes. The alternative approach chosen to validate associations between specific genes and the desirable needle retention phenotype is quantitative reverse-transcriptase polymerase chain reaction, or q-RT-PCR. This technique allows accurate measurement of differential gene expression in hundreds to thousands of RNA samples, for dozens of genes. Problems relating to the availability of seedlings resulted in the need to delay the start of the greenhouse host:pathogen studies by one year. What opportunities for training and professional development has the project provided?Seminars, workshops, and lectures focusing on the topics of 'genetic improvement of Abies sp.', 'seed orchard development', 'needle retention improvement', 'grafting' and 'Abies species evaluation' were made available to Christmas tree growers and other industry stakeholders. Tours and temporary work opportunities have been provided for students. Graduate students and post-docs have been provided opportunities to participate in trade and scientific conferences. Post-doc, Lilian Matallana was presented the "Best Oral Presentation" award at the IUFRO Forest Tree Breeding Conference in Prague for her talk on genome-wide SNP discovery and sequence variability in Fraser fir. How have the results been disseminated to communities of interest?Project results have been shared with communities of interest via the www.christmastreegenetics.org website, at scientific conferences, trade associations meetings, publications, media interviews, small groups and individual meetings. See Target and Products sections for specifics. What do you plan to do during the next reporting period to accomplish the goals?Additional needle retention phenotypic data and abscission zone tissue samples for RNA extraction will be collected from target fir species, to provide more opportunities to test the association of gene expression patterns and DNA sequence variation with the desirable needle retention phenotype. Complete ongoing studies: analysis of the CoFirGE Project data, the first greenhouse host:pathogen studies, the Phytophthora species survey, etc.

Impacts
What was accomplished under these goals? The project team met in St. Louis on July 15 to discuss progress to date and make plans for the next year. Objectives 1 & 2 In Washington, branches were harvested and samples of target tissue in the abscission zone between the needle and the stem were collected from 44 balsam fir, 30 Fraser fir, and 38 Canaan fir clones that were known to represent trees that exhibit either very good or very poor needle retention. Following the collection of the tissue samples, all of the branches were displayed in a temperature-controlled room and rates of needle loss and needle retention were monitored. Later, mRNA was isolated from specific branches based on needle loss data collected during these tests. In North Carolina, needle abscission zone (NAZ) samples were collected from 3 ramets of 30 clones at the N.C. Premium Fraser Fir Seed Orchard, Big Springs, N.C. and the needle retention of each sampled branch was phenotyped. NAZ samples were immediately placed on dry ice after collection and later stored at -80C for future RNA extractions. Efficient 96-well plate RNA and DNA extraction protocols for abscission zone samples of firs have been developed and will improve efficiency of future studies. The first RNA-seq libraries from Fraser fir abscission zones were created from selected clones that exhibit good and poor needle retention and the first RNA-seq analysis pipeline was established to address variation of transcriptome data from Fraser fir abscission zones. The usefulness of NIR in predicting needle retention and moisture content for Fraser fir was evaluated. Partial least squares regression models using NIR data were only capable of detecting a small portion of the variation (r2= 0.11) in post-harvest needle retention but showed potential for development to predict moisture content for monitoring freshness in the Christmas tree industry (r2=0.89). A survey of Phytophthora species causing root rot of Abies in U.S. Christmas tree farms was carried out. 32 Christmas tree farms were visited in 7 states representing 4 major production regions to isolate and identify Phytophthora species causing disease on true firs. Symptomatic tissues from lesion margins on roots and crowns were plated on PARPH-cV8 selective media to recover Phytophthora. Cultures were identified by DNA sequencing of the nuclear ITS and mitochondrial cox I regions. P. cambivora is the prominent PRR species in western WA and OR, while P. cinnamomi was prevalent in CA and NC. In the eastern US, the undescribed P. taxon 'kelmania' was isolated in great frequency and was found to be capable of killing fir species considered to be tolerant. Completion of Koch's Postulates confirmed pathogenicity of 12 unpublished Phytophthora-fir combinations. Another Phytophthora species survey focusing on Fraser fir Christmas tree plantations in the Southern Appalachian Mountains was initiated. Roots of three symptomatic trees from each of 103 sites were collected. Sites covered 14 counties in NC (n=8), VA (n=3) and TN (n=2). Overall isolation success was 54% but isolates were obtained from 80% of the sites sampled. Currently the Phytophthora species are being identified using morphology and ITS DNA sequencing. A field assessment of Turkish fir tolerance to five root-rotting Phytophthora species was undertaken. Thirty six seed sources of Turkish fir collected from 3 Turkish provenances, as well as 2 seed sources of Nordmann fir and 1 source each of noble and Fraser fir were included in the assessment. Inoculum consisted of a mix of 5 species of Phytophthora including P. cinnamomi, P. cambivora, P. cryptogea, P. pini, and P. cactorum. Results showed that while mortality of the noble and Fraser firs quickly reached 100%, the highest mortality of any Turkish fir seed source was less than 25%. One seed source of Nordmann fir suffered less than 20% mortality while the other was resistant during both years of the field trial. Mortality in all host species was clearly dominated by P. cryptogea, and P. cinnamomi was found more frequently on Turkish fir than on Fraser, noble, or Nordmann firs. Objective 3 As part of this project, a question was added to the annual Harris poll of Christmas tree consumers conducted by the National Christmas Tree Association (NCTA), specifically, to determine if the perception of consumers that Christmas trees are "too expensive" is inhibiting potential tree buyers. The results indicated that buyers do NOT perceive that trees are more expensive. The Harris statisticians recommended that we do not need to track this every year, maybe every 3-5 years. Another question added to the NCTA consumer survey in 2013 was designed to determine if tree-recycling programs are strengthening sales. A most disconcerting finding was that 50% of 18-34 year olds reported they didn't know about tree recycling (This should be the group that knows the MOST!) In addition, half of all people who buy a tree still don't know how to recycle it. We need to do more to reinforce this message and be part of the awareness, education and implementation. Two separate focus groups involving Washington State University students who displayed and did not display a real tree were conducted. Participants in the focus groups cited the following reasons for displaying a real tree: was an important part of a family (but not necessarily a holiday) tradition, offered valued time spent with family, finding and cutting down a tree was exciting as a kid, wanted to offer their children the same experience (tradition), and real trees smell good. Participants in the focus groups cited the following reasons for displaying an artificial tree: real trees are a hassle to get and maintain (i.e., they are messy), artificial trees more convenient, often with lights already on them, artificial trees more likely to be perfectly symmetrical, with a good top, and current life stage makes artificial tress more realistic, but may switch back when they have a family. A national sample of 290 U.S. residents recruited via Amazon's MTurk completed a survey assessing likelihood and reasons for displaying real vs. artificial trees. The sample had a mean age = 34.8, was 44% female and 77% Caucasian, with a median income between 30-35 thousand dollars per year. Overall, 58% planned to display an artificial tree, 28% planned to display a real tree, 3% planned to display both, and 11% planned to display no tree. Compared to the Harrison poll conducted that same year, the MTurk sample was more likely to display a real tree (28% vs. 18%) and less likely to not display a tree (11% vs. 29%). The MTurk sample was much more likely than the Harris poll respondents to get their tree from a farm (50% vs. 27%) and much less likely to get their tree from a lot (10% vs. 33%). One of the top five reasons for not displaying a real tree is that respondents already owned an artificial tree. Four additional reasons for not displaying a real tree all centered around inconvenience (of setting up, purchasing, maintaining, and disposing of real trees). Analysis of the open-ended comments further demonstrated that convenience was a major issue preventing people from displaying a real tree. The word "messy" was very common, as were the words "transportation," "space", and "pets." Objective 4 Numerous presentations at grower association meetings and publications in trade journals were performed. (See other sections of this report.) Advice and help managing regional seed orchards was provided to grower including performing needle retention assessments of orchard clones and grafting select material for future seed orchard establishment. Eight Turkish and Trojan fir field trials in NC, NY, OR, PA and WA were managed as part of the Collaborative Fir Germplasm Evaluation (CoFirGE) Project. Activities included data collection and weed management. First year survival and budbreak at the beginning of the second year were measured at each site.

Publications

  • Type: Websites Status: Published Year Published: 2014 Citation: Christmas Tree Genetics website. 2014. http://www.christmastreegenetics.org
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Frampton, J. G. Hodge and R. Jetton 2014. Evaluation of near infrared (NIR) spectroscopy to predict post-harvest needle retention in Fraser fir. IUFRO Forest Tree Breeding Conference, Book of Abstracts. Czech University of Life Sciences. August 25-29. Prague, Czech Republic. p25.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Matallana, L. J. Frampton and R. Whetten 2014. Genome-wide SNP discovery and sequence variability in Fraser fir. IUFRO Forest Tree Breeding Conference, Book of Abstracts. Czech University of Life Sciences. August 25-29. Prague, Czech Republic. p19.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Kohlway, W. J. Frampton and R. Whetten. 2014. Mapping Phytophthora root rot resistance in fir species through Genotyping by Sequencing. IUFRO Forest Tree Breeding Conference, Book of Abstracts. Czech University of Life Sciences. August 25-29. Prague, Czech Republic. p20.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Matallana, L. J. Frampton and R. Whetten. 2014. Understanding needle retention: an interesting Christmas tree lab story. Genomics Sciences Retreat. September 26-28. Carolina Beach. North Carolina.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Chastagner, G. 2014. Multi-year, Multi-institution Christmas Tree Research Project Gets Underway. Great Lakes Christmas Tree Journal 9(3): 25-28
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Matallana, L. J. Frampton and R. Whetten 2014. Identification of key molecular markers for needle retention in firs as Christmas trees. 2014 Genetics Retreat. (Poster presentation by L. Matallana). August 18. Chapel Hill. North Carolina.
  • Type: Websites Status: Published Year Published: 2014 Citation: NCSU Molecular Tree Breeding Lab. 2014. https://research.cnr.ncsu.edu/sites/moleculartreebreeding/
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Cregg. B. 2013. Looking at the Best of Both Worlds: Fir Hybridization. Great Lakes Christmas Tree Journal 8 (3): 28-33.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Cregg, B and J. ODonnell. 2013. Christmas tree Researchers meet in Nova Scotia, Great Lakes Christmas Tree Journal, 8 (4): 4-8.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Cregg, B. 2104. Species profile: Grand fir Abies grandis. Great Lakes Christmas Tree Journal 9 (1):30-32.


Progress 09/01/12 to 08/31/13

Outputs
Target Audience:Science-based knowledge was delivered to Christmas tree growers in various U.S. regions including Indiana, Michigan, North Carolina, Oregon, Pennsylvania, South Carolina, and Washington. Information including a description of this project and/or related Christmas tree genetics topics was disseminated via presentations at grower meetings and publications in trade journals. This information was also delivered to fellow scientist and extension professionals at conferences in the U.S. as well as internationally in Canada, the Czech Republic, and Turkey. These target audiences as well as the general public will also be reached by a recently initiated web site (http://www.artemisgroup.com/scri/index.htm). Changes/Problems:The balsam and fir samples stored in RNAlater yielded rna of unacceptably poor quality. All future samples for rna extraction will be immediately placed on dry ice in the field and later stored at -80 C. What opportunities for training and professional development has the project provided?Undergraduate students, graduate students and a post-doc were involved in this project and were mentored in research methods and ethics as well as trained to perform specific techniques. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Complete the national Phytophthora species survey and initiate a similar regional survey for Fraser fir Christmas tree plantations in the Southern Appalachian region (NC, VA & TN). Resample Fraser, balsam and Canaan fir abscission zones from good and poor needle retaining trees for phenotyping and rna extraction. Create rna-seq libraries from the Fraser fir abscission zones samples and develop a pipeline to analyze the transcriptome data. Add content to the Christmas Tree Genetics web site and make it available on the web. Begin surveying Christmas tree consumers.

Impacts
What was accomplished under these goals? During mid-October 2012, branches were collected and used to assess post-harvest needle retention for 30 Fraser fir, 10 balsam fir, and 10 Canaan fir trees. The trees selected had extremely good and extremely poor needle retention based on three prior years of assessment. All the branches were displayed in a temperature-controlled room and rates of needle loss and needle retention were monitored. For the above-mentioned branches, tissue from the needle abscission zones was also collected and stored at -80 C (Fraser fir) or in RNAlater (balsam and Canaan fir). Candidates were interviewed and in April 2012, a post-doctoral scientist, Lilian Matallan, was hired to spearhead tissue collection and RNA extraction for RNA-Seq experiments and and to collaborate with the project bioinformaticist in analyzing data for differential gene expression and SNP allele frequencies. Using the fir needle abscission zone tissue, techniques were refined to improve the quantity and quality of RNA extracted. Extracted RNA The project team met in St. Louis on July 30 to become acquainted and to report progress and plan future activities. A survey of Phytophthora species causing disease on fir species was initiated. Symptomatic roots and shoots from Christmas tree farms in 7 states representing four major Christmas tree producing regions were sampled. Phytophthora was isolated on selective media (PARPH-cV8) and the species of each culture is currently being identified by DNA sequencing of the nuclear ITS and mitochondrial cox I regions. Koch's Postulates is being carried out to verify pathogenicity of several Phytophthora-fir combinations. Discussions were undertaken to plan upcoming surveys and focus groups of Christmas tree consumers. A Christmas Tree Genetics website was drafted and temporarily posted at http://www.artemisgroup.com/scri/index.htm

Publications

  • Type: Websites Status: Published Year Published: 2012 Citation: Christmas Tree Genetics website 2012. http://www.artemisgroup.com/scri/index.htm
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Frampton, J. F. Isik, and M. Benson. 2012. Genetic variation of resistance to Phytophthora cinnamomi in Seedlings of two Turkish Abies taxa. 14th International Fir Symposium (IUFRO WP 1.01.19, 2.02.13 & 2.02.09). Kastamonu, Turkey. Sept. 12-14. (abstract and presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Whetten, R. and J. Frampton. 2012. New tools for genetic analysis of forest trees. 14th International Fir Symposium (IUFRO WP 1.01.19, 2.02.13 & 2.02.09). Kastamonu, Turkey. Sept. 12-14. (abstract and presentation)
  • Type: Other Status: Published Year Published: 2013 Citation: Whetten, R., F. Isik, S. McKeand and J. Frampton. 2013. The pine reference genome sequence and applied tree breeding. Plant & Animal Genome XXI. San Diego, CA., Jan 12-16. (abstract and presentation)
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: Frampton, J., A.M. Braham, L. Matallan and R. Whetten. 2013 Genetic control of post-harvest needle retention in Fraser fir. 11th International Christmas Tree Research and Extension Conference (IUFRO WP 2.02.09). Truro, Nova Scotia. August 13. (abstract and presentation)
  • Type: Other Status: Other Year Published: 2013 Citation: Cregg, B. 2013. Christmas tree production and selection research in the United States. Seminar for: Department of Dendrology and Forest Tree Breeding, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague. May 8, 2013.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: Chastagner, Gary, Andy McReynolds, and Kathy Riley. 2014. Mid-rotation growth and postharvest needle retention characteristics of balsam fir grown in western Washington. 11th International Christmas Tree Research and Extension Conference (IUFRO WP 2.02.09). Truro, Nova Scotia. August 13.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: Landgren, Chal, Gary Chastagner, Ulrik Nielsen. 2014. Results from common source trials of Nordmann and Turkish fir in Denmark and the PNW, USA. 11th International Christmas Tree Research and Extension Conference (IUFRO WP 2.02.09). Truro, Nova Scotia. August 13.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: McKeever, Kathleen, and Gary Chastagner. 2014. Screening true fir resistance to Phytophthora root rot and structures of Phytophthora communities from Pacific Northwest Christmas tree farms. 11th International Christmas Tree Research and Extension Conference (IUFRO WP 2.02.09). Truro, Nova Scotia. August 13.
  • Type: Other Status: Published Year Published: 2013 Citation: Bustard, J. and R. Bates. 2013. Mediterranean firs: a fad or the future? Pennsylvania Christmas Tree Growers Association Bulletin. 280:6-7.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Cregg, B. 2013. Fir hybridization. The Great Lakes Christmas Tree Journal 8(3):28-33.