DOES SOURCE CLIMATE OR GENOTYPE BETTER PREDICT EARLY PINE SEEDLING PERFORMANCE IN RESTORATION PROJECTS?

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022080
• Grant No.: 2020-67014-30873
• Cumulative Award Amt.: $200,000.00
• Proposal No.: 2019-05480
• Project Start Date: Jun 1, 2020
• Project End Date: Jan 31, 2026
• Grant Year: 2020
• Program Code: [A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
UNIVERSITY OF CALIFORNIA AT MERCED
5200 N LAKE RD
MERCED,CA 953435001

Performing Department
Life & Environmental Sciences

Non Technical Summary
Selecting appropriate seed sources for replanting after large fires or other disturbances is an important part of maintaining healthy forests. However, as temperature and rainfall patterns shift, local tree populations may not grow or survive as well as they once did - so how can managers identify other seed sources that might do better under future conditions? One approach could be to select seed from areas that, in the past, had a climate that was similar to that of the planting site now (or in the near future). Another approach could be to use genetics to identify individual trees whose offspring would likely perform well under the current or future conditions at the planting site. In this project, we will test the ability of source climate versus genotype to predict the growth and survival of ponderosa pine seedlings in a post-fire restoration planting. The seedlings in this restoration planting were sourced from an orchard that contains ponderosa pine genotypes from different climates and locations across the Sierra Nevada. Twelve seed lots were selected from the local breeding zone, 4 from each of the 3 elevation bands- low, mid and high. We can calculate the difference in climate between the source and the planting site. We have also sequenced the DNA of 223 of the trees in the seed orchard, and identified places in the genome that vary depending on what climate the tree is from. In this study, we will test whether the number of genetic variants predicted to do well in the planting site predicts growth and survival of seedlings better than the difference in climate between the source and planting site. The results will help develop tools managers may use to select seed sources for restoration or tree breeding efforts. Because this project focuses on a working seed orchard, custom seed mixes could be developed for different planting sites based on the genotypes of the trees in the orchard and deployed immediately at almost no additional cost.

Animal Health Component

50%

Research Effort Categories

Basic

25%

Applied

50%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	0612	1080	70%
123	0612	1080	30%

Knowledge Area
202 - Plant Genetic Resources; 123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
0612 - Conifer forests of the West;

Field Of Science
1080 - Genetics;

Keywords

pines

forestry

seed

source

climate

snp

Goals / Objectives
Goal: To test the ability of genotype vs. source climate to predict seedling performance under operational reforestation conditions in order to advance germplasm selection procedures. Objective 1: Identify SNP markers associated with variation in home climate in ponderosa pine (climate associated SNPs).Objective 2: Genotype seedlings planted in a post-fire restoration project at the climate associated SNPs, and assess the extent to which growth and survival of individuals and seedlots is related to the number of putatively locally advantageous alleles (variants statistically associated with the planting area's climate).Objective 3: Conduct a similar analysis to objective 2 based on source seed zone + elevation and assess whether using climate associated SNPs would improve the ability to select appropriate seed sources for a planting site.Objective 4: If the genetic information does improve prediction of performance, begin developing an early-stage tool as well as collecting additional data that would enable seed orchards to use genotype to create customized seed lots and/or select individuals for controlled crosses.

Project Methods
- We have conducted Genotyping by Sequencing (GBS) on 223 ponderosa pine genotypes in the Chico Seed Orchard (CSO) that were sourced from across the Sierra Nevada mountains, CA. We have identified over 2 million Single Nucleotide Polymorphisms (SNPs).- We are currently conducting genotype-environment association analyses using these SNPs to identify loci for which genotype is linked to environmental conditions where the trees originated. These analyses will be conducted using both 1981-2010 climate for the source populations and 1921-1950 climate, as we hypothesize that climate-driven selection may be stronger early in a tree's life and thus the historical climate associations may be more accurate. We have also conducted a greenhouse experiment using the offspring of 50 of the genotyped trees, and will be conducting genotype-phenotype association analyses of drought-resistance traits.- For loci associated to environmental conditions or drought response, we will design primers for targeted sequencing. - Ponderosa pine seedlings sourced from known breeding zone + elevation blocks within the CSO have been planted as part of the restoration efforts following the King fire on the Eldorado National Forest. Usually only the local breeding zone + elevation (if available) is used in restoration plantings. This experiment 12 total seed lots from the local breeding zone, four each from low, middle, and high elevation band. Seedlings were planted in three different sites within the burn established in 2017, 2018, and 2019. Temperatures have increased in the Sierra Nevada since the early 20th century, so seed sources from lower elevations could potentially perform better. - We will calculate climate transfer distances between source and planting site in two ways: CD1) source mean 1981-2010 vs. planting site mean 2017-2021 and CD2) source mean 1921-1950 vs. planting site mean 2017-2021. If the predictive ability of CD2 is greater than that of CD1, this would suggest that observed climate shifts over the 20th century have already created adaptation lags.- The ability of SNP associations with environment or phenotype to predict seedling performance has rarely been tested, especially under field conditions as opposed to more controlled environments. We will target sequence a stratified random sample of the seedlings in the restoration plantings and test whether the frequency of alleles associated with climate conditions similar to the planting site, and therefore expected to be advantageous, is significantly associated with growth or survival.- Similar to the climate distance calculations, we will identify potential locally-favored SNPs in two ways: LG1) Associations of genotype with source tree 1981-2010 climate, with "local climate" for planting site defined as 2017-2021 average and LG2) Associations of genotype with source tree 1921-1950 climate, "local climate" 2017-2021 average. If L2 outperforms LG1, this would suggest that trees are adapted to early20th century conditions rather than current conditions. - We will model seed source survival and growth from planting until 2021 (year 2-4, depending on site) as a function of seedling age, planting site, and either a climate distance measure or a locally-favored allele frequency measure. We will also model individual seedling survival and growth as a function of these factors, except that individual genotype rather than seed source allele frequency will be used for the genetic models. In all cases, we will use generalized linear models, with a logit link in the case of survival. AIC values will be used for model selection, and residuals between predicted and observed performance will also be used to assess fit.- While breeding zone/elevation categories do differ in mean climate conditions, these areas are heterogeneous, with overlap between categories in the home climates of individual mother trees. Because of this, we hypothesize that the frequency of locally-favored SNPs may have better predictive ability than seed source average climate.- Results will be made available to scientists and managers through presentations, scientific publications, data publication, and reports as described under "products".- The four undergraduate student assistants (2 per summer) will be recruited from UC Merced and will gain experience in fieldwork (collecting needles from seedlings and measuring their height and survival) and DNA extraction. The Moran lab has previously provided research experiences for 22 undergraduate students, of whom 67% were female and 53% under-represented minorities.- The PhD student working on the project will be recruited to start in Fall 2021, and the support provided will give them a head start in developing a thesis project related to this dataset.- In our meetings and conversations with scientists and managers involved in tree breeding and restoration, we will assess the degree to which our results are applicable to their projects. We will use this information and the data collected to shape future research efforts to ensure that outputs can be turned into useful tools for breeding and seed selection.

Progress 06/01/24 to 05/31/25

Outputs
Target Audience:The ultimate target audiences includes foresters and forest ecologists. One talk discussing preliminary results was given by the PI at the Ecological Society of America meeting in August 2024 (this was listed in prior report, because registration occurred earlier, but actually occurred in this reporting period) Changes/Problems:The only new delay that arose in the last year was the requirement for the graduate student to return to China during the summer to renew her visa. However, she was able to complete the analyses by the beginning of 2025, and we are currently working on the manuscript. What opportunities for training and professional development has the project provided?One Asian woman PhD student worked on the project in the past year, gaining skills in genetic analysis, R model coding, and manuscript preparation. How have the results been disseminated to communities of interest?The manuscript that will report the main results of this project is currently being written, and the PI presented on preliminary results as part of a conference presentation: E.V. Moran, J. Wright, S. Ostoja, J. Stewart, J. Thorne, D. Young 2024, "Increasing publicly available tools for climate-smart seed sourcing and forest restoration" Ecological Society of America, Long Beach CA - oral presentation Two manuscripts related to the project but not directly funded by it have been published and will be cited within the MS currently in prep: - M. Shu & E.V. Moran. 2023. Identifying genetic variation associated with environmental variation and drought-tolerance phenotypes in ponderosa pine. Ecology & Evolution. 13:e10620. [The genotype-environment association study on which the genetic predictions for this study were based] - E.V. Moran, R. DeSilva, C. Canning. & J.W. Wright 2024. Testing source elevation versus genotype as predictors of sugar pine performance in a post-fire restoration planting Ecosphere 15:e70010 [Uses similar approach for sugar pine seedlings planted at the same sites] What do you plan to do during the next reporting period to accomplish the goals?The graduate student will finish writing the paper and submit it by summer 2025. Results useful to the forestry community will be shared at management-related conferences by the PI and/or graduate student in summer or fall 2025.

Impacts
What was accomplished under these goals? Sequencing of ponderosa pine seedlings, SNP calling, and identification of putative locally adapted alleles was completed by Fall 2024 (Objectives 1 & 2 complete) Models including the genotype data have been run. The manuscript is being written, and is expected to be submitted by summer 2025 (Objective 3). Because the genotype did not turn out to be a very strong predictor, we will not be proceeding with objective 4 as planned, but instead will be focusing on other projects that use past climate as a predictor of climate-adapted seedling sources.

Publications

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The ultimate target audiences includes foresters and forest ecologists. One talk discussing preliminary results was given by the PI at the Ecological Society of America meeting in August 2024. Changes/Problems:Not having DNA sequence completion overlap with the availability of a paid expert in bioinformatics on this project (Drs. Shu & DeSilva) slowed progress. However, new PhD Yinyue Du was able to learn the DNA sequence analysis techniques with occasional pro-bono help from Dr. DeSilva and R modeling techiques from PI Moran, and is close to completing the planned project analyses and manuscript. What opportunities for training and professional development has the project provided?One Asian woman PhD student worked on the project in the past year, gaining skills in genetic analysis and R model coding. How have the results been disseminated to communities of interest?The manuscript that will report the main results of this project is currently being written, and the PI presented on preliminary results as part of a conference presentation: E.V. Moran, J. Wright, S. Ostoja, J. Stewart, J. Thorne, D. Young 2024, "Increasing publicly available tools for climate-smart seed sourcing and forest restoration" Ecological Society of America, Long Beach CA - oral presentation Two manuscripts related to the project but not directly funded by it have been published and will be cited within the MS currently in prep: - M. Shu & E.V. Moran. 2023. Identifying genetic variation associated with environmental variation and drought-tolerance phenotypes in ponderosa pine. Ecology & Evolution. 13:e10620. [The genotype-environment association study on which the genetic predictions for this study were based] - E.V. Moran, R. DeSilva, C. Canning. & J.W. Wright 2024. Testing source elevation versus genotype as predictors of sugar pine performance in a post-fire restoration planting Ecosphere 15:e70010 [Uses similar approach for sugar pine seedlings planted at the same sites] What do you plan to do during the next reporting period to accomplish the goals?We plan to submit the manuscript in spring 2025. If an extension to the grant is approved, the graduate student will carry out any requested revisions in summer 2025 and resubmit the paper for publication. Results useful to the forestry community will be shared at management-related conferences by the PI and/or graduate student in summer 2025.

Impacts
What was accomplished under these goals? Sequencing of ponderosa pine seedlings, SNP calling, and identification of putative locally adapted alleles was completed by Fall 2024 (Objectives 1 & 2 complete) Models including the genotype data have been run and results are currently being compared and the manuscript written (Objective 3).

Publications

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The ultimate target audiences includes foresters and forest ecologists. Three talks discussing preliminary results have been given, one at the Placerville Nursery client meeting (managers/practitioners), one at an international university (graduate and undergraduate students), and one at a large national scientific meeting. Changes/Problems:Problems with probe specificity and delays in processing at the Davis Genome Center slowed the genetic analyses considerably. Fortunately, reductions in costs of GBS analyses meant that, even though the probes turned out to be useless, we could still afford to sequence all seedlings. Not having reliable sequence availability overlap with the availability of a paid expert in bioinformatics on this project (Drs. Shu & DeSilva) has also slowed progress. However, both these former employees remain interested in seeing things through to publication, and Dr. DeSilva is helping to train a new graduate student to complete the analyses. What opportunities for training and professional development has the project provided?Dr. Rainbow DeSilva was hired as a research specialist to help advance this project and a partner project analyzing data for sugar pine at the same sites. She is currently a lecturer at another institution, but has continued to be involved in the project. She is currently starting to train a new female PhD student to carry on the analyses, now that the first batch of good sequence data has been received. Two female undergraduates were hired in summer 2021 to conduct the field measurements and needle collection, giving them valuable experience in fieldwork and standards of data collection. How have the results been disseminated to communities of interest?Two manuscripts related to the project have been submitted to scientific journals, but neither has yet been published. Another manuscript is in progress. Presentations including preliminary results for how elevation transfer affected ponderosa pine survival and growth in the post-fire planting included: Invited talk, Universidad Michoacana de San Nicolás de Hidalgo: EV. Moran, Nov 23, 2022, "Tree genetics and assisted migration experiments in California's Sierra Nevada Mountains", Morelia, Mexico Invited talk, Placerville Nursery Client Meeting: EV. Moran, Oct 18, 2022, "Assisted migration experiments in post-fire and intact forest landscapes" E.V. Moran, R. DeSilva, M. Shu^, J. Wright, C. Canning, August 2022, "Can association genetics help guide seed selection for restoration plantings in a warming climate?" Ecological Society of America, Montreal - oral presentation What do you plan to do during the next reporting period to accomplish the goals?Currently in progress/Summer 2023: - Genotyping of planted seedlings at SNPs associated with climate gradients or drought responsiveness. - Writing code to calculate whether an individually has the "locally advantageous" genotype at a given SNP. Fall 2023: - Test whether the frequency of alleles associated with climate conditions similar to the planting site, and therefore expected to be advantageous, is significantly associated with growth or survival. - Model individual and seed source survival and growth from planting until 2021 (year 2-4, depending on site) as a function of seedling age, planting site, and either a climate distance measure or a locally-favored allele frequency measure. - Disseminate results in publications and presentations.

Impacts
What was accomplished under these goals? Objective 1: completed - The genotype-environment association analysis using 223 ponderosa pine genotypes in the Chico Seed Orchard (CSO) sourced from across the Sierra Nevada mountains, was completed. We also conducted a genotype-phenotype association analysis using the offspring of 48 of these trees subjected to a greenhouse drought experiment. A manuscript detailing the results of this analysis has been submitted to BMC Plant Biology. Objective 2: In progress - We measured and censused the ponderosa pine seedlings planted in the three King Fire restoration areas in summer 2020, and collected needles from all live seedlings for genetic analysis. Seedlings were re-censused for height and survival in 2021. - Sequencing probes based on the SNPs associated with five environmental variables and several drought-response phenotypes were ordered from Tecan to genotype the planted ponderosa seedlings. However, these probes did not work because (unbeknownst to us) the company did not use the full Pinus taeda genome to test them, and thus they were insufficiently specific. - We decided to use Genotyping by Sequencing (GBS) instead, and then focus on the loci that had been found to be associated with climate in the prior analysis. - Due to delays at the Davis Genome Center, the first batch of sequences were only received in early May 2023. They are currently being analyzed to identify SNP genotypes at the target loci, while we wait for the second batch. Objective 3: In progress - We calculated climate transfer distances between seed source and planting sites based on long-term climate averages. Next year we will download the climate data needed to assess the transfer distance for the years since planting (2017-2021).

Publications

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The ultimate target audiences includes foresters and forest ecologists, but as the analysis has not been completed we have not conducted outreach to these audiences. Changes/Problems:We requested a year's extension based on the following challenges: - Covid-related rules on lab occupancy slowed labwork (DNA extractions). - The role of the Davis Genome Center in Covid sequencing slowed progress on other research projects, including ours. - When initially tested, DNA quality for the ponderosa pine samples was poor. However, this problem appears to have been resolved, and a test batch of sequencing using the first 96 samples yielded acceptable sequence quality. - We are currently experiencing some challenges with aligning the sequence fragments to call SNPs, but are working through this with assistance from Tecan customer service. What opportunities for training and professional development has the project provided?In the prior reporting period, graduating PhD student Mengjun Shu was hired as a postdoc to help advance the project. This has enabled her to submit two of her thesis chapter for publication (key to advancing her career), to interface with Tecan to design the probes for sequencing the ponderosa pine seedlings, and to advise a lab technician working with Dr. Wright on DNA extraction techniques for pine needles. Two female URM undergraduates were also hired in summer 2020 to conduct the field measurements and needle collection, giving them valuable experience in fieldwork and standards of data collection. In the current reporting period, the analyses were continued by Dr. Rainbow DeSilva, who was hired as a temporary staff scientist. This enabled her to continue developing the skills in tree genetics she had begun developing during her PhD. We also hired two more female undergraduates to do the follow-up field measurements of seedlings. How have the results been disseminated to communities of interest?Two manuscripts related to the project have been submitted to scientific journals, during the prior reporting period. However, both will need to be revised and submitted to different journals. What do you plan to do during the next reporting period to accomplish the goals?Summer 2022 - Finish genotyping planted seedlings at SNPs associated with climate gradients or drought responsiveness. - Test whether the frequency of alleles associated with climate conditions similar to the planting site, and therefore expected to be advantageous, is significantly associated with growth or survival. Fall 2022 & Spring 2023: - Finish modeling individual and seed source survival and growth from planting until 2021 (year 2-4, depending on site) as a function of seedling age, planting site, and either a climate distance measure or a locally-favored allele frequency measure. - Disseminate results in publications and presentations.

Impacts
What was accomplished under these goals? Objective 1: Completed in prior reporting period. - The genotype-environment association analysis using 223 ponderosa pine genotypes in the Chico Seed Orchard (CSO) sourced from across the Sierra Nevada mountains, was completed. We also conducted a genotype-phenotype association analysis using the offspring of 48 of these trees subjected to a greenhouse drought experiment. A manuscript detailing the results of this analysis is currently in preparation for submission to a new journal. Objective 2: In progress - We measured and censused the ponderosa pine seedlings planted in the three King Fire restoration areas in summer 2020, and collected needles from all live seedlings for genetic analysis. DNA was extracted from all individuals and frozen. - Sequencing probes based on the SNPs associated with five environmental variables and several drought-response phenotypes were ordered from Tecan. - Covid-related issues slowed down lab work and processing of samples by Davis Genome Center. There were some concerns about DNA quality, but the first plate of 96 samples has been sequenced and the quality appears adequate for further analysis. - Statistical models of survival and growth that can incorporate genotype as well as other predictor variables are being constructed. Objective 3: In progress - We calculated climate transfer distances between seed source and planting sites based on long-term climate averages. We have recently download the climate data needed to assess the transfer distance for the years since planting (2017-2021).

Publications

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:The ultimate target audiences includes foresters and forest ecologists, but as the analysis has not been completed we have not conducted outreach to these audiences. Changes/Problems:We were not able to recruit a new graduate student to work on the project as COVID impacts resulted in a drop in graduate recruitment of almost 50% in our program. However, we were able to maintain former PhD student Mengjun Shu as a postdoc, and she has been invaluable in advancing the project goals. COVID-related rules on lab occupancy have also slowed labwork, but we should be on track to genotype all seedlings by the end of the summer. Hiring a postdoc used all of our initially planned graduate student support funds. However, savings achieved in other areas - notably probe cost - should enable us to hire the personnel needed to complete the analyses as planned. What opportunities for training and professional development has the project provided?Graduating PhD student Mengjun Shu was hired as a postdoc to help advance the project, as the genetic analysis was based on her thesis research, and the COVID pandemic made recruiting new graduate students and finding jobs for graduates more complicated. This has enabled her to submit two of her thesis chapter for publication (key to advancing her career), to interface with Tecan to design the probes for sequencing the ponderosa pine seedlings, and to advise a lab technician working with Dr. Wright on DNA extraction techniques for pine needles. Two female URM undergraduates were hired in summer 2020 to conduct the field measurements and needle collection, giving them valuable experience in fieldwork and standards of data collection. How have the results been disseminated to communities of interest?Two manuscripts related to the project have been submitted to scientific journals, but neither has yet been published. What do you plan to do during the next reporting period to accomplish the goals?Currently in progress: - Analyzing 2017-2020 survival and growth data for the seed lots from different elevations. - Hiring two female undergraduates to conduct the 2021 seedling measurements. - Extracting seedling DNA for sequencing with the probes (when delivered). - Writing code to calculate whether an individually has the "locally advantageous" genotype at a given SNP. Summer 2021 - Census the planted seedlings and re-collect needle samples if needed. - Genotype planted seedlings at SNPs associated with climate gradients or drought responsiveness. Fall 2021 & Spring 2022: - Test whether the frequency of alleles associated with climate conditions similar to the planting site, and therefore expected to be advantageous, is significantly associated with growth or survival. - Model individual and seed source survival and growth from planting until 2021 (year 2-4, depending on site) as a function of seedling age, planting site, and either a climate distance measure or a locally-favored allele frequency measure. - Disseminate results in publications and presentations.

Impacts
What was accomplished under these goals? Objective 1: completed - The genotype-environment association analysis using 223 ponderosa pine genotypes in the Chico Seed Orchard (CSO) sourced from across the Sierra Nevada mountains, was completed. We also conducted a genotype-phenotype association analysis using the offspring of 48 of these trees subjected to a greenhouse drought experiment. A manuscript detailing the results of this analysis has been submitted to BMC Plant Biology. Objective 2: In progress - We measured and censused the ponderosa pine seedlings planted in the three King Fire restoration areas in summer 2020, and collected needles from all live seedlings for genetic analysis. - Sequencing probes based on the SNPs associated with five environmental variables and several drought-response phenotypes have been ordered from Tecan, and will be used to genotype the planted ponderosa seedlings. Objective 3: In progress - We calculated climate transfer distances between seed source and planting sites based on long-term climate averages. Next year we will download the climate data needed to assess the transfer distance for the years since planting (2017-2021).

Publications