Principal investigators: Aziz Ebrahimi, postdoctoral research associate, Forestry and Natural Resources, Purdue University (aebrahi@purdue.edu); Douglass Jacobs, Fred M. van Eck Professor of Forest Biology, Forestry and Natural Resources, Purdue University (djacobs@purdue.edu); Anna Conrad, research plant pathologist, USDA Forest Service; Carolyn Pike, regeneration specialist, USDA Forest Service, Eastern Region — State, Private and Tribal Forestry; John Couture, associate professor, Entomology and Forestry and Natural Resources, Purdue University.
Co-authors: Mojtaba Zamani Faradonbeh, postdoctoral research associate, Forestry and Natural Resources, Purdue University; Elisabeth G. Joll, PhD student, Forestry and Natural Resources, Purdue University; James Warren, biological scientist, USDA Forest Service.
Researchers at the HTIRC are harnessing innovative technology to protect one of North America’s most threatened trees. Butternut (Juglans cinerea) faces an existential threat due to butternut canker disease (BCD) and are threatened across much of their range. With dwindling natural populations and limited regeneration, the time to act is now.
Butternut trees are critical to forest ecosystems, offering habitat and food for wildlife while contributing to the biodiversity and sustainability of forests. They are essential species for indigenous tribes that use them for food and medicine resources. Their loss poses significant risks to the ecological balance of North American hardwood forests. Furthermore, butternut’s genetic diversity holds the potential for understanding disease resistance, which could benefit other threatened species. Protecting this iconic tree is not just about conserving a species but preserving a vital piece of our natural and cultural heritage.
Genotyping has become a crucial and efficient tool in efforts to identify genetic diversity and the genetic background within HTIRC’s extensive collection, laying the groundwork for future genome-wide association studies. Researchers analyzed genetic material from two main butternut plots planted at Martell Forest using advanced DNA markers. These studies have allowed the team to identify hybrids and pure butternuts and the overall genetic diversity in the plots. They also have also discovered hybrids and a few pure butternuts less susceptible to BCD.
In addition to genotyping, researchers used chemotyping (chemical phenotyping) to analyze disease severity and environmental interactions. Spectral data obtained from near-infrared (NIR) spectroscopy have proven to be a noninvasive and effective method for identifying pure butternut from its hybrid. This method also highlighted spectral shifts linked to disease severity, validating the potential for NIR as a rapid diagnostic tool. However, more data should be collected to prove this method in the future.
“This work integrates traditional morphology with state-of-the-art genomics and digital forestry techniques, supporting chemotyping for forest conservation,” co-principal investigator Aziz Ebrahimi said. “It provides a scalable framework for identifying resistant genes for butternut canker disease and could be a model for conserving other endangered hardwood species.”
In the future, HTIRC’s innovative strategies will guide reforestation efforts and conservation programs. By identifying disease-resistant trees and preserving the genetic integrity of butternut, researchers are setting the stage for long-term restoration. Future plans include using drone images (RGB, multispectral and hyperspectral images), genotyping additional trees and conducting genotype-phenotype association studies to deepen the understanding of resistance mechanisms. HTIRC’s work not only aims to restore butternut but also sets a precedent for the conservation of other threatened species, demonstrating how science and technology can work together to preserve natural heritage for future generations.
Figure 1: Hybridization process between pure butternut (Juglans cinerea) and Japanese walnut (Juglans ailantifolia). The initial cross produces a 50:50 hybrid offspring (JC: JA), displaying mixed traits of both species. Further backcrossing increases the proportion of one parent species (>75% JC or JA), advancing toward disease resistance and maintaining desired genetic characteristics.
Goals:
The primary goal of this research is to leverage existing butternut research plantings established over the past two decades to support conservation and resistance breeding efforts. The team aimed to integrate morphological, genotypic and chemotypic methods to distinguish pure butternut trees from hybrids, assess their genetic pedigrees and identify disease resistant hybrids.
Anticipated outcomes included the identification of hybrids with Japanese walnut genetics that exhibit resistance to butternut cancer disease. Researchers also sought to develop scalable tools for detection and disease screening using spectroscopy and advanced genotyping techniques.
Methods:
- Morphology: Conducted detailed monitoring of bud break progression in butternut trees using manual observations and biweekly drone imagery from mid-April to mid-May. Monthly drone-based RGB imaging extended data collection into the summer, capturing foliage development and leaf senescence in the fall.
- Genotyping: Used genomic single nucleotide polymorphism (SNP) panels and genotyping-by-sequencing (GBS) to analyze the genetic diversity and hybridity of ~1,500 trees.
Chemotyping: Leveraged near-infrared spectroscopy (NIR) to analyze foliar spectral data collected seasonally from northern and southern Indiana sites to determine if hybrid and pure butternut could be distinguished and to assess disease incidence and severity. Complementary chlorophyll content and leaf weight measurements provided additional insights into tree health.
Key Findings in 2024:
- Genotyping revealed clear distinctions among pure butternut, hybrids and Japanese walnut accessions, enabling the identification of promising disease resistant hybrids or pure butternut that are less susceptible.
- Chemotyping highlighted spectral shifts linked to species, validating the potential for NIR as a rapid field-based tool.
- Disease incidence and severity were correlated with genetic backgrounds, with some hybrids showing significant resistance to BCD.
- Drone imagery demonstrated a high potential for identifying phenological patterns, further supporting hybrid differentiation.
Future Research:
- Genotype-phenotype association analysis for identifying genes related to BCD resistance and adaptive traits
- Refining breeding strategies to enhance disease resistance while preserving genetic diversity
- Conducting long-term monitoring of families (both hybrid and pure butternut) that are less susceptible to BCD and planting them in provenance trials to evaluate disease resistance under climate change scenarios.
- Scale up drone-based imaging and spectroscopy tools for broader applications in forest health
- Improve finding use multi-omics approaches and transcriptome-wide association analysis
Key Collaborators/Partners:
- Keith Woeste (Washington office of the USDA Forest Service); Nicholas LaBonte (USFS National Forest System); Songlin Fei and Joseph Hupy (Purdue Institute for Digital Forestry); Martin Williams and Nathalie Isabel (Canadian Forest Service)