Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Ecosystem Science & Management
Non Technical Summary
The decimation of eastern hardwood forests in response to Emerald Ash Borer and its continued expansion west emphasizes the imminent threat facing forest-based ecosystems and economies in the United States. Consequently, there is a need for proactive conservation of genetic resources and development of genomic resources to pair with future breeding programs in Fraxinus. While there is growing emphasis on the importance of genetics in conservation and substantial progress has been made in developing whole-genome sequences for F. excelsior and F. pennsylvanica, there remains a substantial gap in our understanding of intraspecific variation, or the raw material upon which natural selection acts, for ash species. Thus, to safeguard biodiversity for species a risk, conservation and restoration policies need to include genomic screening of seed and living collections and quantify genetic variation captured within and across populations. This proposal is for 2 years of funding to establish a genomic screening program for ex situ and living collections of ash, focused currently on Oregon Ash. We will create a genomic passport for Oregon ash maintained in collections for future use in genecology, seed orchard establishment, and breeding program development. Assessment of genomic variation and differentiation alongside gene-environment associations will advance our understanding of neutral and adaptive processes influencing the species distribution. These data will be key to establishing seed zones and seed-transfer guidelines for restoration purposes. Finally, the genomic resources established here will provide a foundation for future comparative work directly applicable to state and national forest health initiatives and will better situate The Schatz Center for future external funding competitions.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Given the expanding infestation risk of EAB to F. latifolia and the growing need to develop genomic resources for use in gene conservation alongside a developing EAB-resistant breeding program we (i) propose to pair genomic monitoring with range wide ex situ collections of F. latifolia to assess range-wide population genetic variation and differentiation, (ii) quantify range-wide gene-environment associations, and (iii) quantify genetic variation of families maintained in living collections for future use in genecology and a developing EAB- resistance breeding program. Filling these knowledge gaps will provide baseline knowledge essential to proactive conservation, restoration, and developing breeding programs for species preservation and enable future comparative genomics research in Fraxinus.
Project Methods
This proposal complements ongoing efforts to establish range-wide ex situ seed collections for Oregon Ash and living collections for genecology, seed orchard development, and EAB- resistance breeding program. With the inevitable movement of EAB westward, IUCN has identified Oregon Ash as 'Near Threatened' (Barstow et al, 2018). This study takes a proactive approach to gene conservation and develops genomic resources for F. latifolia within both ex situ and living collections. This study will advance our understanding of the distribution of neutral and adaptive genetic variation in a species with no genetic or genecology resources, andprovide a 'genomic passport' for seeds and seedlings for future research. In addition, this proposal leverages newly available reference genomes in closely related F. pennsylvanica (Carlson et al. In Prep) to facilitate sequence alignment within bioinformatics pipelines.1. Genomic screening for ex situ and living tissue collections in Oregon ash. We will extract genomic DNA from foliage dried on silica provided by the Huntington Botanic Garden, Oregon Department of Forestry, and USDA - Forest Service. For this project, we will generate population genomic data using a GBS approach commonly used in forest trees (Di Santo et al, 2021a; Uckele et al, 2021). This is a ddRADseq protocol that reduces the genome complexity by digesting DNA with restriction enzymes and subsequently selecting a subset of the fragment distribution for high-throughput sequencing on the Illumina platform. Briefly, genomic DNA is cut with restriction enzymes, EcoRI and MseI, and Illumina based adaptors are ligated to fragments from each individual. Each EcoRI adaptor consists of the cut site bases, a unique eight to ten base DNA barcode, and an Illumina adaptor; the MseI adaptors consist solely of the endonuclease cut site and the opposite Illumina adaptor. Uniquely barcoded ligation products from all individuals are pooled and PCR-amplified using standard Illumina primers. In total, 756 uniquely barcoded adaptors are available and therefore we can pool large numbers of samples on individual sequencing runs. We will perform size selection for a region between 350-450 bases using a Pippin Prep quantitative electrophoresis device (Sage Science, Inc.) in order to reduce the fraction of the genome targeted for sequencing. Each library will contain 450-600 uniquely barcoded individuals and libraries will be sequenced with the highest output Illumina NovaSeq chemistry at the facility that is currently most cost effective (each lane of NovaSeq: ~2 billion short DNA reads).We will use computational methods for sequence assembly and variant calling, together with a well-established pipeline of Perl, Python and Unix tools, to identify Single Nucleotide Polymorphisms (SNPs) across samples. These analyses are routinely utilized and will be run on the local lab computing resources (Braasch et al, 2021; Parchman et al, 2016). These methods offer a cost-effective method for generating population genomic data that can accurately quantify genome-wide levels of genetic diversity, as well as potential genetic differentiation among natural and restored populations of virtually any plant taxon.2. Range-wide assessment of population genomic structure and differentiation. Using populations maintained ex situ, we will evaluate range-wide genomic structure using a principal components analysis implemented in ADEGENET (Jombart, 2008; Jombart and Ahmed, 2011). We will use these data to evaluate the likely number of genetic clusters preserved within ex situ collections for F. latifolia. To evaluate contemporary standing genetic variation preserved within collections we will calculate expected heterozygosity (HE), inbreeding coefficients (FIS), linkage disequilibrium effective population size (LD-Ne), and coancestry coefficients (!) based on approaches outlined in Braasch et al. 2021. We will calculate pairwise genetic differences between populations using Weir and Cockerham's unbiased estimate of FST (Weir and Cockerham, 1984). Following this, to test for patterns of neutral evolution produced by isolation-by-distance (IBD) we will test for correlations between genetic differentiation (FST) and pairwise spatial distance.3. Landscape genomics to understand genotype-environment associations. For widely distributed species, understanding the scale over which abiotic factors influence the distribution of genetic variation will be important to establishing seed transfer recommendations (Yoko et al, 2020). To compare patterns of neutral evolution with those that may reflect adaptation to local environments we will perform genome scans to identify loci putatively under selection using differentiation and GEA approaches. We will use differentiation approaches that do not include environmental data, BAYESCAN version 2.1 (Foll and Gaggiotti, 2008) and OUTFLANK version 0.2 (Whitlock and Lotterhos, 2014) with those that do: BayENV2 (Coop et al, 2010) and RDA (Forester et al, 2018). We will leverage environmental datasets associated with provenance origin to identify loci or regions of the genome associated with environmental gradients. We will use both univariate BayENV2 (Coop et al, 2010) and multivariate redundancy analysis (RDA, (Legendre and Legendre, 2012) to perform genotype-environment associations (Forester et al, 2016; Forester et al, 2018). Combining approaches reduces false-positive or false-negative signals associated with adjustments that might be made due population genetic structure (Flanagan et al, 2018). Using loci derived from these analyses we will develop a spatial framework of adaptive genetic variation across the landscape that may be used in establishing seed-transfer recommendations (Shryock et al, 2020; Thomassen et al, 2010).4. Demonstrate the utility of genomic monitoring in ex situ and living collections to gene conservation, restoration, and plant breeding. We will work with colleagues to create a genomic passport for ex situ and living collections of ash screened within this project. In addition, these data will be paired with phenotypic data on growth and resistance in collaboration with the USDA Forest Service. Longer-term, the goal of this program will be to establish a genomic database characterizing intraspecific genomic variation within populations across different Fraxinus species. This will facilitate comparative research across ash species and provide a foundation necessary for seed orchard and breeding program development.