GBS Genotyping of F6 Plant Lines

DNA was extracted from green tissue that was collected from bulked stipules of four F₆ plants per line using a CTAB method [63 (link)], checking its quality on 1% agarose gel. We adopted the GBS protocol by Elshire et al. [27 (link)] with modifications. After quantification by a Quant-iT™ PicoGreen® dsDNA assay kit (Life Technologies, P7589), each DNA sample (100 ng) was digested with ApeKI (NEB, R0643L) and then ligated to a unique barcoded adapter plus a common adapter. Equal volume of the ligated product was pooled and cleaned up with QIAquick PCR purification kit (QIAGEN, 28104) for subsequent amplification. In PCR, 50 ng template DNA was mixed with two primers and KAPA Library Amplification Readymix (KAPA Biosystems KK2611). Amplification was carried out on a thermocycler for 10 cycles with 10 s of denaturation at 98 °C, followed by 30 s of annealing at 65 °C and 30 s extension at 72 °C. Each library was sequenced in two lanes on Illumina HiSeq 2000 at the Genomic Sequencing and Analysis Facility of the University of Texas, Austin, TX.