*2.5. Sequencing, Alignment, and SNP Genotyping*

Raw reads were de-multiplexed, and the barcode sequences were trimmed using SEEDERS in-house python script as previously described in [49]. Reads were also trimmed using the Cutadapt (ver. 1.8.3) to eliminate the sequences of the common adapter. The demultiplexed reads were processed by the SolexaQA package ver.1.13 [41]. Next, bad-quality bases with low Phred quality score (Q = 20 or 0.05 probability of error) were trimmed using DynamicTrim in the SolexaQA package, and the read lengths lower than 25 bp with poorquality sequence were discarded, using Lengthsort program in the SolexaQA package [55]. The processed and cleaned reads were applied to align with *C. annuum* cv. CM334 reference genome (ver. 1.55, http://www.sgn.cornell.edu/ (accessed on 19 November 2019), and the read depth was counted by the number of aligned reads via the Burrows–Wheeler Aligner (BWA, 0.6.1-r104) program as described in [56]. The BWA was carried out with following default options: gap open penalty (−O) = 15, number of threads (−t) = 16, mismatch penalty (−M) = 6, maximum differences in the seed (−k) = 1, and gap extension penalty (−E) = 8, except for seed length (−l) = 30. The detection of raw SNPs and the consensus sequences were acquired from the resulting mapped reads with BAM format file using

SAMtool (v.0.1.16) utilities [57]. For SNP calling, the varFilter command in the SAMtool was utilized with default options as previously described in [17,49]. Finally, on the basis of ratio of SNP/InDel reads in the mapped reads, variant types of SNP were grouped with three categories: homozygous SNP/InDel for read rate ≥90%, heterozygous SNP/InDel for read rate ≥40% and ≤60%, and the rest defined as "etc." [49,58,59].

#### *2.6. Linkage Map Construction*

Genetic linkage maps of F2 segregating lines were illustrated using the JoinMap ver. 4.0 (Kyazma B.V., Wageningen, The Netherlands). A total of 1550 SNPs were grouped into 16 linkage groups (LGs) with a logarithm of the odds (LOD) threshold score ≥5.5, and a maximum distance of 30 centiMorgans (cM) were used. The genetic map distance of the SNP markers was converted to cM using the Kosambi's mapping function [60]. To remove the skewed SNP and the segregation distortion, the chi-square test (*p* < 0.001) was applied, and the SNP markers were filtered with identical segregation or missing rate ≥30%. Final genetic linkage maps of KC352 and 14F6002-14 with the 1550 SNPs were drawn using MapChart ver. 2.3 software [61].
