*2.2. DNA Extraction*

A total of 96 plant samples from 9–10 leaf stage of 4-week-old plants were ground with the TissueLyser II (Qiagen, Hilden, Germany), and genomic DNA (gDNA) from the samples was extracted using cetyl trimethyl ammonium bromide (CTAB) method as previously described in [54].

## *2.3. Disease Assay and Resistance Index Scoring*

To evaluate resistance against *R. solanacearum,* resistance screening was conducted in 45–48 F2 offspring derived from a cross between parental lines of KC352 and 14F6002-14 with three independent replicates (Supplementary Figure S1). A total of 94 F2 lines were tested for the resistance screening and subsequent GBS analysis. To prepare the inoculum, the *R. solanacearum* of WR-1 strain isolates (race 1, biovar 3) were cultivated on NA medium and incubated at 28 ◦C for bacterial cell growth for 2–3 days. The plates were collected with distilled water to harvest the cells. The concentration was determined by measuring OD at 600 nm = 0.3, and the cell density was adjusted to approximately 107–108 cfu per mL before inoculation. Two parental lines and 94 F2 offspring were inoculated at the 6–7 leaf stage onto the plant roots with 5 mL of the bacterial suspension after wounding the plant roots by stabbing a scalpel along with two sides at a soil depth of 1–2 cm. The inoculated

plants were kept under vinyl-protected conditions at 25 to 30 ◦C, and disease resistance was continuously observed and recorded after inoculation. *C. annuum* KC352 was utilized as the resistant control, whereas *C. annuum* 14F6002-14 was utilized as the susceptible control to compare the severity of disease symptoms in F2-segregating populations. The disease symptoms and disease resistance index were evaluated on the basis with disease scale of 0–4 as previously described in [25], where 0 = no visible symptoms, 1 = 1 to 25% of wilted leaves, 2 = 26 to 50% of wilted leaves, 3 = 51 to 75% of wilted leaves, and 4 = 76 to 100% of wilted leaves.

#### *2.4. Preparation of Libraries for Genotype-by-Sequencing (GBS) Analysis*

A total of 96 individuals were subjected to GBS analysis. The quantity and quality of extracted gDNAs were validated using 1% agarose gel electrophoresis before running next-generation sequencing (NGS). The preparation of GBS libraries was conducted as provided by SEEDERS sequencing company (Daejeon, Korea). To construct GBS libraries, gDNAs were digested with *Ape*KI (New England Biolabs, Ipswitch, MA, USA) [41] with a minor modification. In detail, oligonucleotides for the top and bottom strands of each barcode adapter and a common adapter were separately diluted in 50 μM TE buffer and annealed in thermocycler conditions followed with 95 ◦C, 2 min (ramp down to 25 ◦C by 0.1 ◦C/s; 25 ◦C, 30 min; 4 ◦C hold). Barcodes and common adapters were diluted in 10× adapter buffer, including 500 mM NaCl and 100 mM Tris-Cl to 10 μM, and 2.4 μL of the mixture was applied into a 96-well PCR plate. Then, 100 ng/μL of DNA samples were added to individual adapter-containing wells and digested for overnight at 75 ◦C with 3.6 U ApeKI (New England Biolabs, Ipswitch, MA, USA) in 20-μL volumes. Adapters were then ligated to sticky ends by adding 30 μL of a mixture containing 10× ligase buffer and 200 unit of T4 DNA ligase (MG Med, Seoul, Korea) to individual wells. The samples were incubated at 22 ◦C for 2 h and heated to 65 ◦C for 20 min to remove the activity of the T4 DNA ligase. The 96 digested DNA samples possessing a different barcode adapter were combined with each 5 μL and were purified using a purification kit (QIAquick PCR Purification Kit; Qiagen, Valencia, CA, USA) following the manufacturer's instructions. Restriction fragments from each library were then amplified in 50-μL volumes containing 2 μL pooled DNA fragments, Herculase II Fusion DNA Polymerase (Agilent, Santa Clara, CA, USA), and 25 pmol with each of the primers in [41]. Polymerase chain reaction (PCR) was conducted with the following conditions: one cycle at 95 ◦C for 2 min, 16 cycles at 95 ◦C for 30 s, 62 ◦C for 30 s, 68 ◦C for 30 s, and stopped at 68 ◦C for 5 min. The amplified fragment size and library quality were assessed with Agilent Tape station with high-sensitivity DNA chip. Whole-genome sequences were conducted using Illumina Hiseq X ten platform (Illumina, San Diego, CA, USA).
