*3.5. Genetic Diversity Analysis*

The genetic diversity and phylogenetic relationships were determined using 147 collected pepper cultivars from 26 different countries around the world (Figure 2 and Supplementary File S1). This study adopted Nei genetic distance and neighbor-joining methods, and a dendrogram was constructed based on the genotypes detected by the newly developed SSR markers (Figure 3). These were clustered into seven main groups (designated in this study as Groups I, II, III, IV, V, VI, and VII), which were comprised of 18, 37, 32, 20, 21, 5, and 14 members, respectively.

The dendrogram not only reflected the phylogenetic relationships of the cultivars, but was also consistent with their places of origin. Remarkably, the dendrogram revealed that Group I had become a unique branch. Furthermore, Group II consisted of 37 accessions, majority of which were from Latin America, with the exception of 3 accessions (PI 281,419 and PI 281,420 from the Philippines, and PI 281,347 from India). Of plant material collected from 15 countries, 32 types comprised Group III. All of the materials collected in Africa were found to be clustered in that group. In addition, 4 Chinese cultivars were also assigned to Group III. Group IV consisted of 20 accessions, among which 7 were from Guatemala; 7 were from South America; 3 were from United States; 2 were from Mexico; and 1 was from an unknown country, which this study speculated may have been of North American origin. Group V was composed of 21 accessions which were from various geographical

origins, such as North America, South America, and other Asian areas. The five members which were clustered in Group VI included four cultivars derived from Costa Rica, and one derived from El Salvador. In addition, 20 accessions derived from Latin America were clustered together in Group VII (Figure 3). These results suggested that the newly developed SSR markers were both stable and suitable for assessing the genetic relationships among *C. frutescens* cultivars.

**Figure 3.** Dendrogram was constructed based on the genotypes from 33 SSR markers using neighbor-joining methods, and the icons indicated the information of material source, which was shown in Figure 2B.
