Cloning and Sequencing of Chrysanthemum Genes

For CmMAX1 degenerate primers were developed based on MAX1 amino acid sequences from Arabidopsis, tomato, poplar, soy, Medicago, vine and RicinusS1 Table. Furthermore, a BLAST search was done to the chrysanthemum transcriptome database of Xu et al. [62 (link)] using Arabidopsis genes known to be involved in branching (corresponding accession numbers are indicated in Table S.2). This resulted in orthologous sequences for CmMAX3, CmDRM1, CmSTM, CmRR1, CmHK3a, CmHK3b, CmAXR1, CmAXR2, CmIAA16, CmAXR6, CmIAA12, CmPIN1, CmTIR1 and CmTIR3. Reference genes were identified by BLAST against the NCBI database. These included CmACT2, CmATUB, CmUBQ10, CmUBC, CmEF1α, CmCACS, CmEXP5, CmEXP6, CmPGK, CmPSAA and CmHH3. Sequences of CmBTUB_AB608732 and CmMTP_AB542716.1. were available for Chrysanthemum. Primers S2 Table. were developed using Primer3 PLUS. Genes were cloned using a pGEM-T kit (Promega) and sequenced according to the protocol of the Big Dye Terminator Cycle Sequencing kit version 1.1 on an ABI Prism 3130 xl Genetic analyser (Applied Biosystems). BlastX [63 (link)] was used to validate isolated fragment identity.