M mlv reverse transcriptase kit

Total RNA for each sample was isolated using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (TIANGEN, Beijing, China) according to the manufacturer’s protocol. Total RNA was reversed to cDNA using an M-MLV Reverse Transcriptase Kit (Bioteke Corporation, Beijing, China). Quantitative Real-time PCR (qRT-PCR) was carried out on an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) in a 20 μL volume containing 100 ng of cDNA, 4 pM of each primer, and 10 μL SYBR Green PCR Master Mix system (TaKaRa). The PCR conditions and the calculation method of gene expression were the same as what had been described previously [68 (link)]. Information on the qRT-PCR primers for gene expression analysis and gene cloning used in this study was listed in S3 Table. The nucleotide sequences of GhMFT homoeologous genes marked with primer location for qRT-PCR were shown in S1 Fig. A cotton Ubiquitin7 (GhUBQ7, GenBank accession no. DQ116441) gene and an Arabidopsis Actin2 (AT3G18780) gene were used as internal controls, respectively. Three replicate assays were conducted with separately isolated RNA, and three technical triplicates were performed for each PCR reaction.