Total RNA was isolated from leave and stem of plants subjected to the various treatments described above. Contaminating DNA was removed by treating with RNase-free DNase I and the first cDNA strand was synthesized from 1 μg total RNA using PrimeScript® Reverse Transcriptase (Takara, Dalian, China) and an oligo (dT) primer, according to the manufacturer’s instructions. qPCRs were performed in an Eppendorf Real Time PCR System (Mastercycler®ep realplex, Germany) using a SYBR Premix Ex Taq™ Kit (Takara), according to the manufacturer’s protocol. Gene-specific primers were designed using Primer5 software (sequences given in Table 7). Each 20 μL qPCR contained 5 μL diluted cDNA, 100 nM of each primer, and 10 μl SYBR Green PCR master mix, and was exposed to an initial denaturation (95°C/2 min), followed by 40 cycles of 95°C/15 s, 60°C/15 s, 72°C/15 s. After amplication, all results were screened to verify a single peak melting curve for the specificity of the amplifications. Three biological replicates were performed for each sample. Relative transcript abundance was obtained by including the C. nankingense EF1α gene as the reference, and was based on the 2-ΔΔCT method [73 (link)].

Primers of quantitative reverse transcription-polymerase chain reaction for validation of RNA-Seq data

GeneIDPrimer F (5′-3′)Primer R (5′-3′)Blast nr
CL695.Contig1_S2_3GTGACGAGTTGGTGATGGTGGTTACCACCTACGAAGGCCAWRKY transcription factor 4
Unigene75748_S2_1CGGGTGAAATGCTCTCAAATTGCCAAATGGTTCTAAAGGGWRKY transcription factor 1
Unigene83309_S2_1ATTTAAACACGCGGATCGACCCAGAGTGTGGCTTGGTACADREBa
Unigene73473_S2_1TAAAGGTGGGCCAGAAAATGATCATACGCCAGAGCAGCTTDREB2 transcription factor
Unigene27271_S2_3ACAACATCCCCTTGGATGAAGGGTGACAGCATTTGAAGGTAP2 transcription factor
Unigene93511_S2_3TGTGCCGCTGTTATCCATTACCACACTATCACAGCCCCTTEthylene-responsive transcription factor
Unigene37079_S2_1TCTTCTTTCCCTTTCTGCGATGGATCTCCCTCATGACTCCbHLH128-like
Unigene56969_S2_3GCATTTGCAGCTGATTCTGAGCTATCACCGTTGACCCACTDOF domain class transcription factor
Unigene13900_S2_3ATCGTGTCGCCGGTATTTAGGTTGTAGACAAAGCGTCGCALEA protein 1
CL2985.Contig1_S2_1CATCCCCATATTGGTTCCAGGAACACGAAGCAAGAGGGTCdehydrin
CL4257.Contig4_S2_1CTTCTTGCACACTGGTCGAAGGGGCTTGCTAGGGATAAAGDEAD-box ATP-dependent RNA helicase 56-like
Unigene6176_S2_3TGTTTGGCTTGTCAAACTGGTCCGTGTTATTCCTTTTGCCDEAD-box ATP-dependent RNA helicase 32-like
Unigene84542_S2_3CCAGGTTTCGTTTTCGTCATGCCTTGAATGCTTTCCACATGibberellin-regulated protein
Unigene85549_S2_3ACCTCTGTCGGTCCATCAACTCGGAACGAGCTCATCTTTTjasmonate ZIM-domain protein 2
Unigene48078_S2_3TTTCAGCCGATGGTGATGTAGTCGTGCCCCACAAGATACTSerine/threonine-protein kinase
Free full text: Click here