TaqMan® assays for Ppd1_2B copy number determination
Significant anthesis and frost QTL were detected in the Ppd1_2Bregion. As variable copy number of the Ppd1_2B gene among parental lines was suspected, TaqMan assays were conducted to determine copy number in those lines. Based on a published protocol (Díaz et al., 2012), 20-µl PCR reactions were set up including 10 µl ddPCR Supermix for Probes (Bio-Rad), 0.4 µl probe plus forward and reverse primers (10 µM), 5 µl DNA (10 ng/µl), and 4.6 µl RNase/DNase-free water. The primer and probe sequences for Ppd1_2B gene and TaCO2 internal control were as those published by Díaz et al.(Díaz et al., 2012). PCR cycling parameters were 95°C for 15 min; 40 cycles of 95oC for 15 sec; and 60oC for 60 sec. Ppd1_2B copy number was analysed based on the ratios of absolute copy numbers against the TaCO2 control.