Amplicon Cloning and Sequencing

All PCR fragments with frequencies higher than 5% in a panel of parental lines were cloned. PCR reactions were purified by using the High Pure PCR Product Purification Kit (Roche, Basel, Switzerland). Amplification was checked in 2% agarose TAE gel, and the amplified fragments were ligated into the Promega pGEM®-T Easy vector and transformed with JM109 Escherichia coli cells according to the kit instructions. Colonies were screened by direct colony-PCR using MYB10F2/MYB10NR2 genotyping and capillary electrophoresis. For colonies carrying one fragment of interest, colony-PCR was repeated with common vector T7 and SP6 primers, using the same PCR reaction and thermocycler conditions. PCR products were purified, checked on agarose gel, and sequenced with T7 and SP6 primers by the fluorescent dye termination detection in the ABI 3730 DNA Analyzer.