*3.2. Analysis of the SSR Repeat Motif Types and Frequencies*

As shown in Figure 1E, the distributions of the SSRs were also examined from the aspect of the number of repeat units. It was observed that for all the SSR types, the SSR frequencies decreased as the number of repeat units increased. Meanwhile, the change rates became more gradual for the dinucleotides when compared with the longer repeat motif types. The dominant numbers of repeats in the pepper SSR loci ranged between 4 and 10, with the exception of a few (more than 10). At the same time, the majority of the observed repeat times were 6, accounting for 26.20% (45,076). The dinucleotides were found to be the most abundant number of repeats, accounting for 55.16% (94,916), whereas the number of repeats for remaining trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide were determined to be 30.58% (52,614), 9.50% (16,351), 2.63% (4531), and 2.13% (3660), respectively, as shown in Figure 1E.

**Figure 1.** Total number, distribution, frequency, and distinct motif types of SSRs in the entire genome of *C. frutescens*: (**A**) Distribution of the SSR in the 'Xiaomijiao' genome; (**B**) Number of dinucleotide and trinucleotide repeat motifs in the genome; (**C**) Number of tetranucleotide repeat motifs in the genome; (**D**) Distribution and frequency of the SSR loci in the genome; (**E**) Relative frequency (%) of the SSR types with different repeat numbers in the 'Xiaomijiao' genome.

*3.3. Primer Design of the Pepper Plant Genomic SSR Markers*

For the designing of the primers in the current study, a total of 240 SSRs, which were distributed on different chromosomes, were selected. Then, the reliability was evaluated on eight pepper cultivars. It was determined that out of the total primer sets tested, 41 were successfully amplified showing full length polymorphisms. The remaining 199 primers were found to be either non-polymorphic, non-specific amplification with ambiguous bands, or not amplified, as evidenced from the gel results. Of the 41 amplified primer sets, only 33 (13.75%) were found to have generated both polymorphic and unambiguous bands on the gel. Therefore, those primer sets were selected for further analysis, as detailed in Table 1. Among the 33 SSR loci, 1 was observed to be dinucleotides, 11 were trinucleotides, 4 were tetranucleotides, 4 were pentanucleotides, and 13 were hexanucleotides, respectively.
