Genotyping-by-sequencing for grassPea

GBS sequencing was conducted according to the method of Elshire et al., 2011 [29 (link)], with minor improvements. Briefly, the gDNA from 154 grasspea plants (2 parents and 152 F₂ progeny) was first digested by MseI (5’-T!TAA-3’) (New England Biolabs, ‘NEB’, Ipswich, MA, USA) at 37 °C, then subjected to restriction-ligation by T4 DNA ligase (NEB) and ATP (NEB) with a MseI Y-adapter N-containing barcode at 65 °C, followed by a second digestion with HaeIII (5’-GG!CC-3’) (NEB) at 37 °C. The digested fragments were purified using Agencourt AMPure XP (Beckman Coulter, ‘Beckman’, Brea, CA, USA) and used for PCR amplification, utilizing Phusion Master Mix (NEB) to add universal and index primers, as well as i5 and i7 index sequences, to the digested fragments. The purified PCR fragments (425–490 bp including indexes and adaptors) were screened and extracted using a 2% agarose gel with the Gel Extraction Kit (Qiagen, Valencia, CA, USA). The amplification products were purified using Agencourt AMPure XP (Beckman) and diluted before sequencing. Finally, an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used to perform 150-bp paired-end sequencing by Tianjin Novogene Technology Co., Ltd. (Tianjin, China).