For RAD sequencing, a double‐digestion method was applied. Six accessions were selected as representatives for each cultivar, while three accessions—each one for the Polygonatum odoratum wild samples, P. cyrtonema, and P. humile. A total of 100 ng of genomic DNA were extracted from each accession and digested using 5 U each of two restriction enzymes, namely MluCI and SphI (New England Biolabs, USA) at 37°C for 5 hr. The restriction–ligation reactions were heat‐inactivated at 65°C for 20 min with T4 DNA ligase (New England Biolabs, USA), ATP (New England Biolabs, USA), MluCI adapter, and SphI adapter. Subsequently, the mixture was incubated at 12°C for 12 hr. A 40‐µl preamplification reaction was conducted using 5 µl of the diluted restriction–ligation mixture using a pair of single‐selective‐nucleotide primers (EcoRI and MseI adapters). PCR amplification was conducted on a T100™ thermal cycler (Bio‐Rad, USA) with thermal settings programmed as follows: 20 cycles of 94°C for 30 s, 56°C for 60 s, and 72°C for 60 s. Prior to the PCR purification step, PCR products were pooled and incubated at 37°C with MluCI, SphI, T4 DNA ligase, ATP, and EcoRI+MseI adapters (Illumina, USA). The pooled samples were further purified using a E.Z.N.A.® Cycle Pure Kit (Omega Bio‐Tek, USA) and separated on a 2% agarose gel via electrophoresis. Paired‐end sequencing was performed on purified fragments extracted from the agarose gel ranging from 350 to 500 bp (with indexes and adaptors) in size via Illumina NovaSeq platform (Illumina, USA).
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