*3.4. Polymorphism Analysis with SSR*

The results of polyacrylamide gel electrophoresis of several highly polymorphic SSR markers are shown in Figure 2C. In total, 91 alleles were obtained with the aforementioned 33 amplified SSR primers. Among those markers, the Number of Alleles (Na) per locus ranged from 2 (Chr1SSR8, Chr2SSR12, Chr2SSR14, Chr3SSR5, Chr3SSR6, Chr4SSR11, Chr5SSR13, Chr5SSR14, Chr6SSR12, Chr6SSR20, Chr7SSR2, Chr8SSR6, Chr9SSR16, Chr10SSR14, Chr11SSR14, and Chr11SSR16) to 6 (Chr4SSR20), with an observed average of 2.8 alleles. The Effective Number of Allele (Ne) per locus ranged from 1.0288 (Chr7SSR2) to 3.6226 (Chr7SSR15), with an observed average of 1.7055 alleles. The major allele frequency (MAF) ranged from a low of 0.3231 (Chr7SSR15) to a high reaching 0.9858 (Chr7SSR2), with an average of 0.7547. In addition, the Observed Heterozygosity (Ho) ranged from 0.000 (Chr1SSR8, Chr4SSR11, Chr6SSR17, Chr7SSR2, Chr10SSR12, Chr11SSR4, and Chr11SSR14) to 0.9863 (Chr5SSR16), with an average of 0.0989 observed. It was also determined that the Expected Heterozygosity (He) ranged from 0.0281 (Chr7SSR2) to 0.7264 (Chr7SSR15), with an average of 0.3313. Also, the Shannon information index (I) ranged between 0.0744 and 1.3273, with an average of 0.5758, and the polymorphic information content (PIC) ranged from 0.0276 to 0.6718, with a mean value of 0.2893. In the present study, the calculation of the mean gene diversity confirmed it to be 0.3300 for all of the 147 types of material, as shown in Table 2. It was observed that the different markers had displayed different polymorphism. For example, primer Chr7SSR15 was found to be the most informative (PIC value: 0.6718), whereas primer Chr7SSR2 was the least informative (PIC value: 0.0276). Therefore, this study concluded that when considered altogether, the performances of the selected SSR markers were very effective in detecting genetic diversity.

**Figure 2.** Regional distribution of the 147 pepper (*C. frutescens*) cultivars, and the detailed information material collected from these countries. (**A**) Regional distribution of the 147 pepper cultivars in this study, mainly collected from Central and South America, Africa, and Asia; (**B**) Labeling of 25 countries on the map, and information material collected from these countries (including an unidentified source); (**C**) Polyacrylamide gel electrophoresis analysis of 7 SSR markers with high polymorphism on samples 1 to 15.


**Table 2.** Polymorphism analysis of the 147 *C. frutescens* accessions with SSR primers.
