qPCR oligonucleotides developed for this study to produce a genus-specific assay targeting a 100 bp section of the D-loop mtDNA region in bigheaded carp (genus Hypophthalmichthys: H. molitrix and H. nobilis).

Note that the two slightly different versions of the forward and reverse primers reflect three positions (shown in bold text) that vary within bigheaded carp, according to all available GenBank sequences. The “Hm” version of a primer matches all H. molitrix sequences while the “Hn” version matches all H. nobilis sequences in GenBank. Rather than ordering primers with degenerate bases, which would create unnecessary combinations of the two variable positions in the reverse primer, we simply ordered each version separately and combined them at equimolar concentration. The Hm and Hn variants do not provide discrimination between H. molitrix and H. nobilis. Rather, they are used in combination to maximize sensitivity within the genus while avoiding amplification from any non-Hypophthalmichthys species. 6FAM = fluorescein amidite reporter. MGB = minor groove binding moiety. NFQ = non-fluorescent quencher. BHQ1 = black hole quencher. +N = locked nucleic acid (LNA).qPCR oligonucleotides developed for this study to produce a genus-specific assay targeting a 100 bp section of the D-loop mtDNA region in bigheaded carp (genus Hypophthalmichthys: H. molitrix and H. nobilis).