Conserving a threatened North American walnut: a chromosome-scale reference genome for butternut (Julgans cinera)

The conservation of native species is critical for maintaining biodiversity and healthy ecosystems in the face of increasing environmental threats. A team of students and faculty at the University of Connecticut recently demonstrated how DNA sequencing can play a pivotal role in the conservation of a key plant species.

Among the threatened native species in North America is the butternut tree (Juglans cinerea), a variety of walnut that has made it onto the endangered status list due to factors including climate change and an invasive fungus, called Ophiognomonia clavigignenti-juglandacearum (Oc-j).

This fungus causes butternut canker, a lethal disease characterised by the growth of visible sores on the tree’s trunk, depriving it of essential nutrients. Fortunately, a dedicated research and training initiative at the University of Connecticut (UConn) is determined to safeguard the species. The team, led by Jill Wegrzyn, Rachel O’Neill, and Nicole Pauloski, among others, recently achieved a significant milestone in their research: creating the world’s first comprehensive chromosome-scale reference genome for the butternut, using Oxford Nanopore’s sequencing technology.

Project overview

The butternut’s ecological and cultural significance in North America drove the decision to focus on the tree, coupled with its local presence in Connecticut and relative genetic simplicity compared to other tree species. High-quality sample extraction was performed by Natural Resources Canada, before sequencing at the university, on a PromethION 24, delivering 100x depth of coverage in a single run. The first year of the programme saw a team of five undergraduate students, mentored by three graduate students and one postdoctoral researcher, actively involved in tasks such as quality control, running multiple assemblies, removing contaminants, and structurally annotating gene locations, concluding with the first scientific publication for the training program. But the work hasn’t stopped with the paper – the students are feeding their findings back to government bodies as well.

‘All the first co-authors of this paper are undergraduates -- after the sample was extracted and sequenced, they did everything,’ said Jill Wegrzyn, an associate professor at UConn and the Computational Biology Core Director. ‘The students are also well connected with the forest service in Canada and the US, getting feedback on what’s important in the conservation framework. The goal is to have an industry, government, and academic collaboration to achieve this.’

Having established a comprehensive chromosome-scale reference genome, the team has laid the foundation for advancing genomic research and informing future conservation efforts. While natural resistance to butternut canker is rare, preliminary indications can now be investigated further through trait mapping and studying mechanisms of resistance. This in turn has the potential to inform breeding strategies and bioengineering interventions to preserve this endangered species.

‘One way the reference genome helps is if you have a large collection of individuals and you want to identify which genes are associated with beneficial traits, you’ll typically sequence individuals and map those sequences against a reference genome,’ said Karl Fetter, a UConn postdoc. ‘Without a reference genome you have a much smaller collection of genetic data to work with.’

Challenges

But the path to publication was not without its challenges. Tree genomes are inherently long and complex, riddled with highly repetitive DNA segments that are difficult to decipher. The unique ability of Oxford Nanopore’s technology to generate ultra long-reads and effectively manage highly repetitive content played a crucial role in the accurate construction of the genome.

Another significant challenge was cultivating the computational skills and knowledge to perform de novo genome assembly, structural and functional annotation, phylogenetic, and gene family evolution analysis. In its first year, students successfully learned these skills as part of the course’s curriculum. Wegrzn and O’Neil’s program is now in its second year of development, with four students from the previous year mentoring the new cohort of eight students in these essential skills. In addition to successfully creating the world’s first reference genome, the team have established a structured framework for training and development, with the aim of growing the programme and generating (at least) one new reference genome each year with goals for future publications.

‘A significant challenge is having the people with the computational skills to do the genome assembly, analysis and annotations,’ said Michelle Neitzey, a PhD student and program mentor. ‘One of the things Jill has achieved here is not only a framework to do the genome assembly and annotations, but also train the next generation in those skills. It’s a huge challenge.’

Training the next generation

The resounding success of the programme has led to an overwhelming response from students with over 90 new applicants looking for a spot in the next cohort. The team are focussed on developing solutions and are actively working with ORG.one to identify new species worthy of reference genomes. Pumpkin ash, deep sea zigzag coral, and the red-vented cockatoo are also being thoroughly sequenced by the Biodiversity and Conservation Genomics team at UConn’s Institute for Systems Genomics. Their vision extends to scaling up to population genomics and making information widely accessible and useful for breeding programmes and conservation initiatives.

‘Looking forward we are aiming for at least a new species every year,’ Wegrzyn said. ‘Right now, we have two teams working simultaneously, so hopefully we’ll have two new genomes by the end of this year. We’re not only focussed on plants either, corals, birds, and a range of mammals are in the pipeline also.’

She added that she has a clear goal for what she wants this program to achieve: ‘My interests lie in developing solutions. Being a computational biologist, I am interested in developing reliable software that helps us answer these questions faster and more reliably, be that detecting genes more accurately or assembling genomes at a higher quality. We want to put these genomes in the hands of those who can immediately use them, enabling real change on the ground. We also want to take these genomes and scale to populations and make that information available.’

Through transformative research and training programs such as this, classrooms around the world can evolve into hubs of scientific discovery, propelling global conservation initiatives, and empowering the next generation to harness nanopore sequencing for a more sustainable future. To support this movement, Oxford Nanopore recently launched Education Beta, a pilot programme providing education-based pricing for a range of products to amplify the efforts of educators in the existing nanopore sequencing community. For more information regarding Education Beta, please click here. To learn more about ORG.one, click here.