Isolation and Sequencing of Cassava MeFRK Genes

Full-length cDNAs of the MeFRK genes were isolated via RT-PCR, using a set of gene-specific primers (Table 2), which were designed based on BLAST analysis of the cassava genome database (Available online: http://www.phytozome.net/cassava) using seven published sequences of the AtFRK1–7 in A. thaliana [11 (link)]. The PCR reaction was carried out at a final volume of 50 μL, containing 1 μL of cDNA from different tissues, following the manufacturer’s instructions from the Ex Taq DNA polymerase kit (Takara, Japan). The PCR cycling conditions were as follows: 3 min at 94 °C, followed by 30 cycles of 94 °C for 30 s, a range of annealing temperatures for different MeFRKs from 57 to 63 °C for 30 s, 72 °C for 2 min, and a final extension of 10 min at 72 °C. The PCR products were separated on 1% agarose gel and purified by Axygen Purification kit (Axygen, Union, CA, USA), cloned into pMD18-T vector (Takara, Dalian, China), and sequenced (Shanghai Sangon Biological Engineering Technology and Services Co., Ltd, Shanghai, China).