Advantage 2 polymerase mix kit

All polymerase chain reaction (PCR) amplifications were performed using the Advantage 2 Polymerase Mix kit (Clontech) following the manufacturer instructions, in some cases including 4% of GC Rich solution (Roche Applied Science) in the master mix. Extra-long PCR (XL-PCR) amplifications were performed using the Expanded Long Template PCR System (Roche Applied Science), following the manufacturer instructions. Reverse transcription PCR (RT-PCR) were performed using either the ThermosTable rTh Reverse Transcriptase RNA PCR kit from Perking Elmer or the One^®Step RT-PCR kit (QIAGEN) following the manufacturer’s instructions. The PCR, XL-PCR or RT-PCR products were purified using the Qiaquick PCR purification kit from QIAGEN and cloned either into pSTBlue-1 Vector Vector using the Perfectly Blunt Cloning kit (Novagen) or into the pCR^®II Vector using the TA Cloning^® Kit Dual Promoter (pCR^®II) with One^®Shot INVαF’ kit from Invitrogen, as recommended by the manufacturers. Plasmids were isolated and purified using the QIAprep Spin Miniprep Kit (QIAGEN) and sequenced in both strands using the ABI 310 automated DNA sequencer and dye terminator chemistry (Big Dye V3 105 Dye Terminator Sequencing Kit, Applied Biosystems).

Genomic DNA was isolated with the DNeasy Plant Mini kit (QIAGEN, Gaithersburg, MD, USA) following the manufacture’s protocol, and used as the template for the PCR. PCR reactions were performed with Advantage 2 Polymerase Mix kit (Clontech, Mountain view, CA, USA). The primer pair VF397-VF399 was used to amplify FLS2-1, and VF395-VF396 to amplify FLS2-2 (Supplementary Table 2). The PCR products were purified using either a QIAquick PCR purification kit or QIAquick gen extraction kit (QIAGEN) and subsequently cloned into pGEM-T Easy vectors (Promega, Madison, WI, USA) and sequenced.
For the amplification of FLS2 candidates using rapid amplification of complementary DNA (cDNA) ends (RACE), total RNA was extracted using TriZol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions followed by DNase treatment and cleanup with the RNeasy Plant Mini Kit (QIAGEN). The cDNA was synthesized with the SuperScript III Reverse Transcriptase kit (Invitrogen) with Oligo dT primers.