Micro fast track kit

Because the short style is controlled by the S allele (see the Introduction), thrum plants of ‘HTC’ were used for cloning. Genomic DNA was isolated from young leaves with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Total RNA was isolated from buds, mRNA was isolated from total RNA with a Micro-Fast Track Kit (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesised with the Universal Ribo Clone cDNA synthesis system (Promega, Madison, WI, USA). Based on the N-terminal and internal partial amino acid sequences, degenerate primers were designed and used to obtain partial cDNA and genomic DNA fragments. Thermocycling conditions were as follows: initial denaturation at 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 42 °C for 30 s, 72 °C for 1 min; and final extension at 72 °C for 5 min. Amplified DNA fragments were cloned with the TA Cloning Kit (Invitrogen) and sequenced on an ABI3100 sequencer (Applied Biosystems, Waltham, MA, USA). We performed 3ʹ RACE with the 3ʹ-Full RACE Core Set (Takara, Otsu, Japan) to determine the sequence of the 3ʹ region of the gene, and then conducted genome walking with the GeneRacer Kit (Invitrogen) to determine the sequence of the 5ʹ region of the gene. Primers are listed in Supplementary Table S2.