*2.2. Source of Genic Sequences, SSR Identification and Primer Design*

In this study, the wild pepper 'Xiaomijiao' (*C. frutescens*) was sequenced at Beijing Nuohe Zhiyuan Technology Co., Ltd. The genome sequencing of 'Xiaomijiao' (*C. frutescens*) was performed with Illumina HiSeq4000 (300x coverage) and PacBio Sequel (30x coverage). The assembled sequences, which totaled 2.95 G bases, were used in this study to characterize the distribution of microsatellites in the pepper genome. The completeness of the 2.95 Gbp assembly is supported by the mapping of over 99% of ~3 million EST reads (generated using HiSeq4000 technology) from 'Xiaomijiao' (*C. frutescens*) leaf, stem and root tissues. A MISA (MIcroSAtellite) SSR identification tool program was employed for the sequence identification [35]. It was confirmed that 2 to 6 nucleotide motifs could be considered for identifying the presence of microsatellites. The minimum repeating units for the dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides were defined as 6, 5, 4, 4, and 4, respectively. We allowed up to 5 nucleotide mismatches at the 5' end of the primer, but no mismatches at the 3' end, and a minimum of 80% overall match homology. For a given primer pair, we considered that a specific amplicon was generated if both forward and reverse primers were mapped to the same chromosomes/scaffold. Then, based on the MISA results, Primer 5 software was employed for designing the SSR primers. They generated amplicon sizes of 100 to 300 bp with the following criteria: 22 to 25 bp lengths with 40 to 70% GC content levels; 45 to 65 ◦C melting temperature (Tm); and the remaining parameters used the program's default values. Eight pepper cultivars, referred to as GRIF 9194, GRIF 9316, PI 439309, PI 439489, PI 631142, VI029462, VI062180, and LJ091, respectively, were selected for validating the primers via PCR and electrophoretic techniques. A total number of 240 SSR markers, which were distributed among 12 linkage

groups, were screened. Finally, 33 markers were identified as being evenly distributed along the linkage groups, which produced clear bands with high polymorphism. These markers were further used to analyze all of the accessions, as detailed in Table 1.


