Deep sequencing of High Plains wheat mosaic virus from sweet corn to guide seed health testing reveals multiple variants for all eight genome segments with limited reassortment

Authors: Wilson, Jennifer; Ohlson, Erik; Willie, Kristen; Khatri, Nitika; DU TOIT, LINDSEY - Washington State University

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 8/26/2026
Publication Date: 8/26/2026
Citation: Wilson, J.R., Ohlson, E.W., Willie, K.J., Khatri, N., Du Toit, L.J. 2026. Deep sequencing of High Plains wheat mosaic virus from sweet corn to guide seed health testing reveals multiple variants for all eight genome segments with limited reassortment. bioRxiv. 2026.08.25.746265. https://doi.org/10.64898/2026.08.25.746265.
DOI: https://doi.org/10.64898/2026.08.25.746265

Interpretive Summary: High Plains wheat mosaic virus (HPWMoV) infects major grain crops such as corn and wheat and has the potential to be moved by the seed trade. Sweet corn is particularly vulnerable to HPWMoV. Prior to import by some key trading partners, corn seed from the United States must be certified “free” of HPWMoV, usually through testing the seed for the presence of virus. However, current diagnostic assays are inconsistent and miss detection of 25% of infected samples. To improve diagnostic testing for HPWMoV, we sequenced 20 HPWMoV genomes obtained from sweet corn samples collected from the two major sweet corn seed production regions in the United States, more than tripling the amount of sequence information available for HPWMoV. Our sequencing results revealed there are two main types of HPWMoV in the United States, and that all previously sequenced HPWMoV samples from around the world can also be categorized into these two types. Using this sequence information, we developed two new diagnostic assays that recognize both HPWMoV types and can successfully detect HPWMoV in seed. These diagnostic assays can be used to certify seed “free” of HPWMoV, facilitating export to key trading partners and increasing profits for seed growers and American seed companies.

Technical Abstract: High Plains wheat mosaic virus (HPWMoV) is a wheat and maize-infecting virus of phytosanitary concern due to its potential for seed transmission. Recent phytosanitary restrictions have required sweet corn seed lots to test negative for HPWMoV prior to import into certain countries. To inform the design of more sensitive and broad-spectrum diagnostic primers for seed health testing and phytosanitary certification, we performed deep sequencing of HPWMoV-positive tissue collected from fields in two major sweet corn seed production regions in the Pacific Northwest, the Columbia Basin and Treasure Valley. Virus-like particle enrichment prior to Illumina sequencing facilitated near complete genome coverage (>95%) for the 21 HPWMoV isolates sequenced. De novo assembly of the eight viral genome segments revealed high levels of diversity for each segment, with at least two variants identified for each RNA and three variants for RNA3, RNA6, and RNA8. Within each sample, only one variant per RNA segment was usually present, with the notable exception of RNA3, sorting each isolate into what we designated type A and type B isolates. All but one previously sequenced HPWMoV isolate can be sorted into these two types. Two samples contained at least two variants for every RNA, totaling 17 genome segments, potentially representing a co-infection of type A and type B isolates. Despite this variability, we successfully designed two primer and probe sets for reverse transcription-quantitative polymerase chain reactions (RT-qPCR) that detected all 20 isolates tested in a duplex diagnostic assay, making the assay suitable for seed health testing for HPWMoV.