The genomic sequence of the rice OsFlo2 gene (NC_008397) was used as a query for BLAST searches against the wheat sequences database in the URGI (https://urgi.versailles.inra.fr/). All wheat scaffold sequences with high similarity to the rice OsFlo2 sequence were assembled to construct a putative TaFlo2 gene using DNAMAN (http://www.lynnon.com). Based on the scaffold sequences, six conserved primer pairs were used to specifically amplify TaFlo2 coding and promoter sequences from the three wheat sub-genomes: A, B and D (Table 1). The TaFlo2 mRNA of 4902 bp was cloned in Chinese Spring using three primer pairs designed from the predicted mRNA sequence (Table 1). Genomic DNA was extracted from young seedlings of each line using the CTAB method . A 20-μl reaction volume comprising 0.5 μl (5 μM) of each primer, 2× Taq mix (GenStar, Beijing, China) and 100 ng of DNA was used for PCR amplification that consisted of a cycle profile of 5 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 60 °C and 4 min at 72 °C; and a final extension of 10 min at 72 °C. The PCR products were detected by electrophoresis in 1% agarose gels with nucleic acid dye (Tiangen, Beijing, China), and gel images were captured using a UV spectrometer (BioRad, Hercules, CA, USA). The targeted PCR products were obtained from the agarose gels and purified using the TIANgel MIDI Purification Kit (Tiangen, Beijing, China). The purified PCR products were then ligated into the pGEM-T Easy cloning vector (TransGen Biotech, Beijing, China). The ligation product was transformed to 50 μl of Trans1-T1 competent cells by the heat shock method (Tiangen, Beijing, China). Positive clones from each transformation were selected based on positive PCR tests and were sequenced (Beijing Genomics Institute). The sequences were analyzed using DNAMAN software (http://www.lynnon.com).Table 1Primer sequences used in this studyPrimer namePrimer sequence (5′-3′)Position on scaffold sequenceAnnealing temperature (°C)PCR product sizeFunctionFlo2-1FTGTGCTGGAATCACCCACTC793–812601061cloning TaFlo2 /polymorphism detectionFlo2-1RGCGCGGCGAAAACTAATCAT1853–1844Flo2-2FGTGCCGTCCATAATCGTTGC1546–1565601781cloning TaFlo2 /polymorphism detectionFlo2-2RCATGTGCGGCAAAAGACACA3326–3307Flo2-3FAACGGGCATGTGTCTTTTGC3299–3318603025cloning TaFlo2 /polymorphism detectionFlo2-3RCGACGCAGCTCTGAAAATCG6332–6313Flo2-4FCGCTTAGCAGTGGATTTGCC5719–5738603948cloning TaFlo2Flo2-4RATCCAACAAACAGGTGCCCA9667–9647Flo2-5FTTGCGGAAGCCCATCATTCT8387–8406603836cloning TaFlo2Flo2-5RTGACCTTCTGCGGATGCTTT1222–12,203Flo2-6FCAGAACAGGGCCGGTACAAT11,368–11,387602600cloning TaFlo2Flo2-6RCGCTCATCTGGATAGGGCAA13,967–13,948TaFlo2-InDel8FACCCCTCCTCCGTTATCGTC1337–135660145/1538-bp InDel polymorphism in TaFlo2-A1 TaFlo2-InDel8RCCTCCTTCTTCTTGCGGTCG1470–1489Flo2-A1FGTGCTCCGATCCGATGTGCAGTTAT5387–5411585872A specificFlo2-A1RGTGCACAACCAAGTAAAAGG5973–5954Flo2-B1FGTC ATC ACTAGAGGA ATTTTCC6851–6872589022B specificFlo2-B1RCTCTCAGAACTGTGGAT7752–7736Flo2-D1FCTGTATCTGTAATTTGTTCCG5378–5398583262D specificFlo2-D1RCTTCCGAAAAATGTGGGG5704–5687mFlo2-1FTAACGGTGGTGCACTTGTGT–581868Cloning mRNAmFlo2-1RTCAGCCGCAAGTTATGCTCA–mFlo2-2FTGCGGACGAGATGGAAAACA–581809Cloning mRNAmFlo2-2RAGCAGTCAGCCGATGGTATG–mFlo2-3FATGCGTACTCCCTAAGCGTG–581889Cloning mRNAmFlo2-3RCACGAAGTGCTGCTTGCTTT–eTaFlo2FCCATTCGGCTTTCGTGCAAA–55134Expression analysiseTaFlo2RTGTTTTCCATCTCGTCCGCA–ActinFAGCCATACTGTGCCAATC–55134Internal controlActinRGCAGTGGTGGTGAAGGAGTAA–