Table 4.

PCR conditions and primer sequences used for LsVe1L and LsVe4L multiplex assays

Target locusa	Forward primerb	Reverse primerb	Annealing temperaturec	Amplicon sized
LsVe1L	5′-CAA GGG CTC TAT GTC ATT CCT CC	5′-GAC CCA TGG AAG CTG TTG GAT CT	60 °C	569 bp
LsVe4L	5′-CTT GTC CCA GAT AGA GTT GTC CAC C	5′-CAG ACC CTG GAA ATC TTT GGT TTG A	57 °C	505 bp
HPPD 1	5′-TCC CAA CTC CTC ACA CTC CTT AAT C	5′-GTA CGG AAC AAA GAG GAA GAG CC	57 °C or 60 °C	244 bp

^aThe lettuce HPPD was targeted as a DNA quality control in both the LsVe1L and LsVe4L multiplex assays

^bEach 25 μL PCR reaction contained 1.25 μL of each of the four primers at 10 μM each to amplify HPPD plus LsVe1L or HPPD plus LsVe4L, 12.5 μL 2x GoTaq Colorless Master Mix (Promega Corp., Madison, WI), and 7.5 μL DNA template from a 1 ng/μL stock

^cThe PCR program consisted of an initial denaturation at 94 °C for 2 min, followed by 32 cycles of denaturation at 94 °C for 10 s, 20 s at the assay-specific annealing temperature, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. PCRs were set up on ice under sterile conditions and the thermocycler was preheated to 94 °C before adding the reactions

^dPCR products (8 μL each) were run on a 1% agarose gel