Licor 4300 sequencer

The Mu-TD protocol was performed following methods described in Ramekar et al. (2018) (link). The specific primer sequences used are shown in Table 1. Amplification products were visualized using the gel system on a LI-COR 4300 sequencer according to the manufacturer’s protocol (LI-COR Biotech, Lincoln, USA). Fluorescently-labeled DNA (50–700 bp; 50–700 sizing standard LI-COR) served as molecular weight markers. Polymorphic parental lines were used to genotype the RIL population using 34 transposon insertion-SCARs previously developed by Roy et al. (2017) (link). The PCR mixture (20 μl) contained 20 ng of template DNA, 10× PCR buffer, 0.2 mM of each dNTP, 0.5 μm of the forward and reverse primers, and 0.025 U of i-Star Taq DNA polymerase (Intron Biotechnology, Korea). DNA was amplified using the following protocol: pre-denaturation at 95°C for 5 min; then 20 cycles of denaturation at 95°C for 30 s, then primer annealing at 56°C for 30 s, extension at 72°C for 1 min, and final extension at 72°C for 5 min to ensure complete extension of the PCR products. Amplicons were analyzed via gel electrophoresis with a 1.0% agarose gel. DNA fragments were visualized by ethidium bromide staining under UV light. Because both marker systems are dominant, they were manually scored as a binary response variable, with 1 (presence) and 0 (absence), respectively.