*3.2. GBS Analysis*

Next, in order to conduct a genotyping-by-sequencing (GBS) analysis, all 96 plants were collected, and a construction of 96-plex GBS library was generated. As such, a total of approximately 108.0 Gbp of DNA sequences (715,257,004 reads) were obtained from single-lane sequencing using Illumina Hiseq X ten platform (Table S1). The GBS raw data ere de-multiplexed according to the 96 barcode sequences. The de-multiplexed sequences of the 96 samples were trimmed by eliminating the sequences of the barcode and adaptor and removing the low-quality information. Finally, the average number and total length of trimmed reads were 6,046,776 and 696 Mbp, respectively. In addition, the average length of trimmed reads (bp) and the total trimmed raw data were 119.99 bp and 94.45%, respectively (Table S1). The trimmed data were further mapped to the reference genome: *Capsicum. annuum* cv. CM334 ver. 1.55, sourced by Sol Genomics Network (http://www.sgn.cornell.edu/ (accessed on 19 November 2019). The average numbers of mapped reads and mapped regions were 5,216,671 and 125,718, respectively (Table S1). The average depth and length of the mapped regions were 14.75 and 233.34 bp, which covered 0.56% of the reference genome. To further mine SNPs from the sequence data, in-house GBS analysis pipeline was applied with filtering criteria. A total of 628,437 raw SNPs was identified in 94 F2 lines (Table S2). The SNPs were filtered to identify putative markers using the criteria of 30% missing values across the genotyped individual and MAF ≥ 25%, which yielded a total of 146,217 SNPs. Moreover, 11,020 SNPs were filtered using both missing <30% and MAF > 25% condition. Furthermore, 4387 homozygous SNPs in KC352 were selected (Table S2). After generating a map of the genotyping and SNP selections using 500 kb as the window size, 1643 SNP markers were identified, and a total of 1639 SNPs were produced with polymorphic SNP between KC352 and 14F6002-14 as the parents (Table S2).
