Genetic Screening of Wheat Cultivars

All cultivars were screened for known vernalization (VNR1, VRN2, and VRN3) and photoperiod (PPD1) genes. The genotyping included the recessive and dominant alleles of VRN-A1(vrn-A1, Vrn-A1a, Vrn-A1b, Vrn-A1c) (Yan et al. 2004a (link)), VRN-B1 (vrn-B1, Vrn-B1), null alleles ZCCT-A1, ZCCT-B1, and ZCCT-D1 (Zhu et al. 2011 (link)), and functional alleles ZCCT-A2, ZCCT-B2 and ZCCT-D2 of VRN2 (Distelfeld et al. 2009 (link); Kippes et al. 2016 (link)), photoperiod-insensitive alleles Ppd-A1a, Ppd-B1a, Ppd-D1a and sensitive alleles Ppd-A1b, Ppd-B1b and Ppd-D1b of Ppd1 (Beales et al. 2007 (link); Nishida et al. 2013 (link)). The primers and the protocols used to amplify the target fragments are summarized in Table S2. DNA extraction was conducted following the protocol of DNAeasy Plant Mini Kit (Qiagen, Hilden, Germany). The polymerase chain reactions (PCR) were performed in a 25 μL reaction volume containing 100 ng of genomic DNA, 1 × Taq DNA polymerase reaction buffer, 10 μM of each forward and reverse primer, 0.2 mM of dNTP, and 0.5 unit of Taq DNA polymerase (NEB, Frankfurt, Germany). The PCRs were conducted in the thermocycler Flex cycler (Analytik GmbH, Jena, Germany). PCR profiles were visualized by electrophoresis on a 1% agarose gel stained with 0.04 μl/mL peqGreen (VWR, Darmstadt, Germany).