Identification and validation of Amaranthus species-specific snps within the ITS region: Applications in quantitative species identification

The Amaranthus genus consists of as many as 70 species, many with similar morphology. The development and validation of a DNA barcode specific to key amaranths would aid in plant identification. These barcodes can be used to develop assays for single-species identification, critical for species surveillance and contaminant screening. A reference panel of 75 internal transcribed spacer (ITS) sequences from the National Center for Biotechnology Information (NCBI) across 11 Amaranthus spp. was analyzed for species-specific single-nucleotide polymorphisms (SNPs). Identified SNPs were validated using 92 accessions of an Amaranthus spp. diversity panel. Of the 75 investigated ITS sequences from NCBI, 13 were identified as potentially mislabeled. Phylogenetic analysis of ITS from the reference panel distinguished 9 of the 11 investigated species. Nine SNPs were validated as species specific. Five species were distinguished with single SNPs. To illustrate the utility of the ITS SNP analysis, a quantitative assay for Amaranthus tuberculatus (Moq.) Sauer identification was constructed targeting SNP 73G and validated using simulated population samples. Results from this research will aid in population screening for A. tuberculatus, and in the development of other quantitative detection assays for Amaranthus.