GBS of Pisum Sativum (pea): thre RIL crosses
Green tissue was collected from bulked stipules of four F 6 plants per line, flash frozen in liquid nitrogen, stored at 80C, and then ground for genomic DNA isolation. DNA was extracted using 400 mg of tissue using a CTAB method described by Rogers and Bendich (1985) and then checked on 1% agarose gel to assess yield and quality. The DNA was quantified with a Quant-iT PicoGreen dsDNA assay kit (Life Technologies). We used the proto- col by Elshire et al. (2011) with modifications. Each DNA sample (100 ng) was digested with ApeKI (NEB) and then ligated to a unique barcoded adapter plus a common adapter. Equal volumes of the ligated product were pooled and cleaned up with QIAquick PCR Purification Kit (QIA- GEN) for subsequent amplification. In the polymerase chain reaction, 50 ng template DNA was mixed with two primers and KAPA Library Amplification Readymix (KAPA Biosystems). Amplification was performed on a thermocycler for 10 cycles with 10 sec of denaturation at 98C followed by 30 sec of annealing at 65C and a 30-sec extension at 72C. Each library was sequenced in two lanes on Illumina HiSeq 2000 at the Genomic Sequencing and Analysis Facility at the University of Texas at Austin.