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

Anthony Brusa, Eric L. Patterson, Todd A. Gaines, Kevin Dorn, Philip Westra, Crystal D. Sparks, Don Wyse

Research output: Contribution to journalArticlepeer-review


BACKGROUND: Amaranthus palmeri is an aggressive and prolific weed species with major impact on agricultural yield and is a prohibited noxious weed across the Midwest. Morphological identification of A. palmeri from other Amaranthus species is extremely difficult in seeds, which has led to genetic testing for seed identification in commercial seed lots. RESULTS: We created an inexpensive and reliable genetic test based on novel, species-specific, single nucleotide polymorphisms (SNPs) from GBS (Genotyping by Sequencing) data. We report three SNP-based genetic tests for identifying A. palmeri alone or in a mixed pool of Amaranthus spp. Sensitivity ranged from 99.8 to 100%, specificity from 99.59 to 100%. Accuracy for all three tests is > 99.7%. All three are capable of reliably detecting one A. palmeri seed in a pool of 200 Amaranthus spp. seeds. The test was validated across 20 populations of A. palmeri, along with eight other Amaranthus species, the largest and most genetically diverse panel of Amaranthus samples to date. CONCLUSION: Our work represents a marked improvement over existing commercial assays resulting in an identification assay that is (i) accurate, (ii) robust, (iii) easy to interpret and (iv) applicable to both leaf tissue and pools of up to 200 seeds. Included is a data transformation method for calling of closely grouped competitive fluorescence assays. We also present a comprehensive GBS dataset from the largest geographic panel of Amaranthus populations sequenced. Our approach serves as a model for developing markers for other difficult to identify species.

Original languageEnglish (US)
JournalPest management science
StateAccepted/In press - 2021

Bibliographical note

Funding Information:
The authors would like to thank Drs Adrian Steck, Jim Anderson, Rob Stupar and David Marks for their advice during the course of this work. The authors would also like to thank Denise Thiede and Anthony Cortilet at the Minnesota Department of Agriculture for their insights regarding seed regulatory testing. Funding for this work was provided by the Minnesota Invasive Terrestrial Plants and Pests Center (MITPPC) through the Minnesota Environment and Natural Resources Trust Fund. Graphical abstract was created using BioRender.com


  • Amaranthus palmeri
  • genotyping
  • Palmer amaranth
  • seed genetic testing
  • species identification
  • weed identification

PubMed: MeSH publication types

  • Journal Article

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