A needle in a seedstack: An improved method for detection of rare alleles in bulk seed testing through KASP

Authors: BRUSA, ANTHONY - University Of Minnesota; PATTERSON, ERIC - Michigan State University; GAINES, TODD - Colorado State University; Dorn, Kevin; WESTRA, PHILLIP - Colorado State University; KUEPPER, ANITA - Colorado State University; SPARKS, CRYSTAL - Colorado State University; WYSE, DON - University Of Minnesota

Submitted to: Pest Management Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/14/2021
Publication Date: 2/2/2021
Citation: Brusa, A., Patterson, E., Gaines, T., Dorn, K.M., Westra, P., Kuepper, A., Sparks, C., Wyse, D. 2021. A needle in a seedstack: An improved method for detection of rare alleles in bulk seed testing through KASP. Pest Management Science. 77(5):2477-2484. https://doi.org/10.1002/ps.6278.
DOI: https://doi.org/10.1002/ps.6278

Interpretive Summary: Palmer amaranth is an aggressive weedy species that can have major impacts on agricultural production. In recent years, Palmer amaranth has been spread through contaminated commercial seed lots. While Palmer amaranth is a prohibited, noxious weed species in many states, several other closely related Amaranthus species are naturalized to these areas. The seed of these non-weedy Amaranthus species are not easily distinguishable from Palmer amaranth. In this study, we describe a new genetic test that can identify Palmer amaranth seed contamination in a mixed seed lot containing both low levels of Palmer amaranth as well as other Amaranthus seed. This new test consists of 3 DNA markers, each of which were shown to have an accuracy of >99.7%. These DNA markers were shown to be effective across a wide genetic diversity of Palmer amaranth populations. This assay provides a valuable tool for both seed screening and identification of suspected Palmer samples collected in the field.

Technical Abstract: Finding rare alleles in mixed genetic pools of closely related DNA is a difficult task; however, there are situations where it is necessary, especially in diagnostics. For instance, Amaranthus palmeri is an aggressive and prolific weed species that has major impact on agricultural yield and has recently been declared a prohibited noxious weed in some US states while being a native species to other. Morphological identification that distinguishes A. palmeri from other Amaranthus species is difficult in seedling plants and nigh impossible for seeds, which has led to the need for genetic testing for Amaranthus species weed seed identification in commercial seed lots. In response we have developed a method for identifying novel (species-specific) single nucleotide polymorphisms (SNPs) from Genotyping by Sequencing (GBS) data and then created an inexpensive and reliable genetic test based on those SNPs. In this paper we describe three SNP-based genetic tests for identifying A. palmeri alone or in a mixed pool of Amaranthus spp. DNA. Accuracy for all three tests is >99.7%. Furthermore, we show that all three markers are capable of reliably detecting a single A. palmeri seed in a pool of 200 Amaranthus spp. seeds. The test was validated across 20 populations of A. palmeri, along with 8 other Amaranthus species, the largest and most genetically diverse panel of Amaranthus samples to date. This research represents a marked improvement over existing commercial assays resulting in a test that is 1) accurate, 2) robust, 3) easy to interpret and 4) applicable to both leaf tissue and pools of up to 200 seeds. This assay provides a valuable tool for both seed screening and identification of suspected Palmer samples collected in the field. Our approach serves as a model for developing such markers for other difficult to identify species.