Tomato spotted wilt virus (TSWV) is one of the most economically important viral pathogens in row crops, causing an estimated $1 billion in annual agricultural losses globally. TSWV infects peanut and more than 200 other host species. Despite advances in tolerant cultivars and agricultural management, genetic resistance remains a priority for breeding programs.
In the mid-1990s, a peanut line showing strong resistance to TSWV was identified. Although not agronomically elite, it has a highly valuable resistance phenotype. Over the next two decades, several mapping populations were developed and evaluated in an attempt to localize the underlying resistance locus, but traditional marker-based approaches and reference-based analyzes failed to identify causal variants.
The phenotype was reproducible in the field, but its genetic basis was undetectable.
Traditional trait-mapping approaches and single-reference analyzes could not unambiguously resolve the identity signal of the responsible genetic variant. The phenotype was there, but it was frustratingly out of reach for decades.
Changed with it. Hudson Alpha Institute for BiotechnologyOf fear Platform, and the power of pangenomes.
A known feature without a known cause
By then a team led by Hudson Alpha faculty investigator Josh Clevenger, Ph.Dstarted working on the problem, Breeders had already spent nearly 20 years trying to identify the causal locus.. Through the combined efforts and funding of the University of Georgia, USDA-ARS, and USDA NIFA, Dr. Clevenger’s team was able to solve the case and solve a decades-long mystery.
In viral resistance, structural variations and gene copy number may play important roles, but many standard genotyping approaches focus primarily on SNP markers and underrepresented genome sequences. If the gene for disease resistance was structural or dependent on transcribed gene cassettes, it could be easily missed.
To solve a problem that had resisted conventional methods for two decades, the team needed more than SNPs. They needed a deeper, more precise kind of insight.
The Khufu approach: accuracy through pangenomes
Hudson Alpha’s Khofo was designed to maximize the power of short-read, low-pass whole-genome sequencing. Paired with KhufuPAN, a Khufu add-on package for creating custom pangenome graphs, Khufu enables mapping within a broader genomic context that reflects true population diversity, rather than aligning short reads to a single reference genome, which can introduce reference bias and obscure structural differences.
Using sequencing scaled to thousands of individuals in separate populations, the team applied this approach to the TSWV resistance question. The result was transformational.
Instead of identifying a single SNP, the pangenome graph revealed A duplicated gene cassette containing four copies of the glutamate receptor gene. This copy number change proved to be key. Those carrying four copies put up stiff resistance. Those with low copies showed moderate resistance. Plants with zero copies were fully susceptible.
Conventional tools were not designed to detect or interpret this type of complex, copy number-dependent variation. Khufu’s ability to call and type structural variations within the pangenome framework made the difference.
From discovery to actionable insight
Variety recognition was only part of the solution. For breeders, insight must translate into action.
Because Khufu enables accurate identification of structural variation in large populations, the team can quickly incorporate this knowledge into the selection workflow. Rather than relying solely on phenotype observation under variable field pressure, breeders can now directly select for the optimal copy number sequence.
The team hypothesized that this locus may carry broader viral resistance beyond TSWV. The potential to extend conservation to additional geographies underscores the real-world impact of precise genomic insights.
Why it matters
The TSWV case is more than a technical success story. This represents a shift in how unresolved breeding challenges can be approached.
For 20 years, the point of resistance remained just out of reach. With the integration of Khofu’s low-pass sequencing and pangenome-guided variant detection, the team moved beyond approximate marker associations to accurate, actionable variant insights.
When breeders can see the full spectrum of genomic variation, including structural and copy number differences, they can solve problems that were once intractable.
Khufu didn’t just add more data. It provided clarity and turned a long-standing mystery into a workable solution, increasing disease resistance and enabling stronger breeding decisions for farmers and ranchers that have lost billions over decades.
Ready to uncover the variations your current workflow may be missing?
If you are working on complex traits that have not been resolved using traditional reference-based or SNP-based methods, Khufu and KhufuPAN can provide the clarity you need.
Schedule a free consultation with our team to discuss your species, population structure, and breeding goals, and discover how pangenome-guided diversity discovery can accelerate your program.
Book your consultation
