From seed to harvest — protecting food crops with a single platform

Climate change is impacting global agriculture, threatening the future of crop production and food security for the growing population¹. With extreme weather events, changing growing conditions, and altered plant pathogen dynamics, there is a demand for more resilient crops combined with effective disease monitoring programmes. Researchers are now demonstrating how Oxford Nanopore sequencing can deliver genomic data that is crucial in maintaining crop production in the face of these diverse pressures.

Breeding resilient crops with safety and precision

Precision breeding via careful transgene insertion enables important agricultural traits, such as high crop yields and disease resistance, to be introduced from one plant into another. In a study by Liu et al.², researchers used this technique to introduce a gene that enhances stress tolerance in low-nitrogen growing conditions into maize (Zea mays), resulting in two transgenic lines.

The researchers then needed to confirm that the transgene had integrated correctly into the host genome and that no unwanted edits had occurred. Molecular evidence of this is of the utmost importance for the safety and reliability of new plant breeds². The authors described how PCR is commonly used for this verification, but the short amplicons provide limited resolution, and complex rearrangements can impact primer sequences. Meanwhile, the size, ploidy, and often highly repetitive nature of plant genomes make them challenging to resolve with legacy short-read sequencing technologies, hindering thorough genomic analysis and, potentially, thorough screening.

To tackle this problem, the team chose Oxford Nanopore sequencing to characterise their transgenic maize. Whereas short-read technologies struggle to span complex regions, Oxford Nanopore technology sequences native DNA and produces reads of unrestricted length, delivering the coverage needed to resolve areas of plant genomes that legacy methods miss³.

The researchers performed PCR-free, whole-genome nanopore sequencing of the transgenic maize lines and a wildtype control to 10x depth of coverage. They confirmed the presence of the inserted gene in each transgenic line, and the long nanopore reads revealed the host genome sequences flanking each insertion. This allowed the team to design primers for both sites, generate amplicons, and perform targeted sequencing.

The nanopore sequencing data ‘unequivocally’ confirmed that whilst one transgene resulted in a 75 bp deletion from the host genome, neither insertion impacted protein-coding regions. Having used the Oxford Nanopore platform to confidently confirm accurate transgene insertion without unwanted effects on the maize genome, the group concluded that this technique establishes ‘a precise method for detecting newly created transgenic maize events, which will contribute to subsequent safety assessments’.

Pinpointing the genetics of useful crop traits

For other crops, breeding new varieties is not necessarily the goal. Max Schmidt (Geisenheim University, Germany) presented how the breeding of new grape vine (Vitis vinifera) varieties is uncommon, due to the demand for traditional wine grape varieties with known characteristics⁴. However, their propagation via cuttings over extended periods has resulted in considerable phenotypic variation, even within a specific variety. The traditional method of selecting for desirable traits from amongst this variation is a labour-intensive process with uncertain outcomes, taking as long as 30 years.

Max’s team is applying genomics to accelerate the search for resilient traits in Riesling grape vines^4,5. Using whole-genome nanopore sequencing on MinION and GridION devices, they assembled a reference genome for the variety. This produced an approximately 930 Mb phased diploid assembly, with the nanopore reads of unrestricted length enabling most chromosomes to be assembled in fewer than five contigs.

Max explained that as climate change results in warmer summers with higher rainfall in Germany, grapes that grow in denser clusters are at greater risk of mould, limiting crop yields⁴. To identify genetic drivers of mould resistance, the team investigated a Riesling grape vine clone with a looser cluster phenotype.

Using previous phenotypic and short-read data, the group identified eight loci of interest but found none mapped within coding regions of a pre-existing Pinot Noir reference genome. In contrast, when they mapped the loci to their Oxford Nanopore-generated diploid Riesling reference genome, they identified one variant of interest in a single haplotype — in a region that did not exist in the Pinot Noir reference. This was predicted to impact a transcription factor involved in berry development, making it a promising candidate for targeted selection of the pathogen-resistant trait. The team’s research highlights the potential of nanopore sequencing to reduce the selection process by years or decades for this economically important crop.

Guarding against the growing threat of plant pathogens

Even once crops are established, circulating pathogens threaten their success. To combat this, researchers are using nanopore sequencing to take plant pathogen detection into the field. Savva et al. described the significant and increasing threat that fungal plant diseases pose to agriculture and food stability, as well as biodiversity⁶. The ‘notorious’ wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) can devastate wheat crops and frequently overcomes pathogen-resistant varieties. Effective management of Pgt therefore requires a fast test deployed at the point of sampling, enabling farmers to take rapid action.

Using the portable Oxford Nanopore MinION, the team developed the first point-of-care Pgt genotyping workflow⁶. Their mobile and real-time plant disease (MARPLE) pathology platform combines targeted PCR with rapid nanopore library preparation, MinION sequencing, and automated analysis to characterise 276 genes in the fungal pathogen.

In resource-limited settings in Kenya and Ethiopia, the fully field-based workflow provided precise Pgt strain typing and fungicide sensitivity information from infected wheat leaves within 48 hours, providing an ‘early warning system’ for changes in circulating strains and resistance. The researchers also performed a phylogenetic analysis of the samples alongside previous Pgt isolates, highlighting the relatedness between them (Figure 1). This timely information will enable rapid action to reduce the crop losses caused by this destructive pathogen, helping to protect food security.

Figure 1. Phylogenetic analysis of Pgt samples from Kenya and Ethiopia revealed close relationships between the field-sequenced samples and representative isolates previously identified in East Africa. Figure from Savva et al.⁶ and available under Creative Commons license (creativecommons.org/licenses/by/4.0/)

From improving and selecting optimal plant varieties to protecting precious food crops in the field, these studies demonstrate how researchers are utilising Oxford Nanopore sequencing to address the pressing need to maintain food security in increasingly challenging environmental conditions.

Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.