Genotyping-by-Sequencing Protocol for Genetic Analysis

The sequence data were generated using genotyping-by-sequencing (GBS) [20 (link),21 (link)]. The DNA was first digested using the methylation-sensitive restriction enzyme ApeKI (R0643L, New England Biolabs, Ipswich, MA, USA), and then the restriction fragments were ligated to a unique barcode adapter and universal adapter. The QIAquick PCR Purification Kit (28104, QIAGEN, Valencia, CA, USA) was used to purify an equal volume of pooled ligation products for PCR amplification. The amplicons were re-purified after amplification to generate clean PCR products. The generated library represented all 366 individual genotypes and was sequenced in each of the four lanes of an Illumina HiSequation PE150 instrument (Illumina, San Diego, CA, USA) to generate single-ended 100 bp reads.
The raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) in the GenBank database under accession number PRJNA565837.