**4. Discussion**

The genome wide analysis of SSRs could provide the opportunity to decipher the optimal functions of these repeats in the regulation and organization of a genome. Also, the potential uses of these markers, such as diversity and population analyses, evolutionary history, and genome and comparative mapping, are currently being explored [19–21,40–42]. In previous studies, based on the results of sequencing analyses, assessments of the highquality genome sequences of *C. frutescens* were made possible, thereby accrediting the opportunity to develop suitable SSR primers. In the present study, it was observed that

the dinucleotide and trinucleotide motif repeats were the most abundant, accounting for 85.74%. The remainder of the repeats (14.26%) were contributed by the tetranucleotides, pentanucleotides, and hexanucleotides, as illustrated in Figure 1E. This phenomenon has also been reported in other plants, such as *Radix codonopsis*, *Anthuriumand raeanum*, and *Camellia sinensis*, respectively [43–45]. Nevertheless, a few reports have found high abundance of tetranucleotide repeats in some plants, such as *Cucumis sativus*, *Medicago truncatula*, and *Vitis vinifera* [46–48]. The differences in the previous finding may have been due to the dissimilarities in paradigms adopted for the SSR identifications. Moreover, the examination of the SSR motif frequency manifested that the scatterings of the dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides repeats were generally skewed toward fewer numbers of repeats. These findings indicated that there were predominantly fewer repeats along the pepper genome. The results obtained in this study indicated that higher repeats were found in dinucleotide and trinucleotide SSRs. However, repeats were fewer in number or absent among the tetranucleotide, pentanucleotide, and hexanucleotide SSRs (Figure 1A). Similarly, in other plants, such as citrus, watermelon, and tea, the same trends were observed [18,19,45]. This may be due to the obvious differences in the frequencies and types of SSR motifs. In the pepper genome, this study found AT/TA was the most common, while the CG/GC motif was very rare in the dinucleotide repeats (Figure 1B). These findings were consistent with the motif frequencies found among cucumber, strawberry, maize, *Radix codonopsis*, potato, plum, watermelon, and horseradish [19,43,48–53]. However, our results greatly differed from the motif frequencies observed in rice, citrus, onion, and *Atremisia frigida* [18,54–56], where AG/CT has been found to be the most dominant type. Similarly, TTG, AAT, and ATT were the prevailing motifs of the trinucleotide in this study, with CCG and AGG being the predominant motifs in the monocotyledons, such as barley, rice, and corn. It was found that the number of SSRs, along with their structure and repeat motifs, will greatly differ compared with those in plant species.

It has been found that SSR markers are very much beneficial in population genetics and molecular breeding. However, their effectiveness mainly relies on the marker quality and the accuracy of the experimentation [25]. In the present study, 240 selected SSR loci markers were scrutinized, resulting in 33 unique markers. It was found that when evaluating 147 pepper cultivars, these markers demonstrated remarkable and unambiguous amplification bands (Table 1). The screened SSR polymorphism primers accounted for 13.8% of the total. Previously, Li et al. obtained 17 pairs of SSR polymorphism primers with clear bands and high polymorphism from 152 pairs of SSR primers covering 12 chromosomes [33]. These accounted for 11.2% of the total number of SSR primers, which was slightly lower than that obtained in this result. Liu et al. evaluated 85 pairs of SSR polymorphic primers and 12 pairs were scrutinized [57]. These accounted for 14.1% of the total, which was similar to this study's research results. Wu et al. used three different peppers as templates to select 65 pairs of SSR polymorphism primers from 153 pairs of SSR primers [42]. These accounted for 42.5% of the total number, which was substantially higher than that obtained in this study. Therefore, it was determined that the proportion of SSR polymorphism primers screened in this study was relatively low, which may have been attributed to the small differences existing in these peppers However, although the proportion of polymorphic primers was low, the results could still be used to analyze the genetic diversity of the pepper population. In regard to pepper, many SSR markers have been developed and mapped to linkage groups [58–60], which provide a key basis for analyzing pepper genetic diversity. However, such factors as the number, size, and types of SSR markers, frequencies of the SSR motifs, as well as the various sampling schemes, apparently result in differences in genetic diversity [25,61].

It is of major significance for the collection and efficient utilization of germplasm resources to continue to carry out genetic diversity evaluations. Highly polymorphic, as well as stable markers, are the prerequisites for studying genetic relationships and diversity. Nevertheless, it was found that the SSR loci showed less diversity, as evidenced by low

polymorphism information index and gene diversity when compared to earlier reports. For example, in the study conducted by Nicolaï [62], a PIC of 0.67 and a gene diversity of 0.7 were reported, which were 0.38% and 0.37% higher than those of the current study (PIC: 0.29; Gene Diversity: 0.33). However, this may have been due to the volume and types of test sampling. In the aforementioned study, 1352 accessions from 89 countries were utilized, including 11 species of *Capsicum*. However, this study only examined a single species, *C. frutescens*. Therefore, compared with Li's report [33], the amount of accessions used was approximate. The PIC was approximate (slightly higher than this study), but the number of markers was different. Therefore, it was considered that the hypothesis that the difference in genetic diversity was influenced by the number of SSR markers had been confirmed.

Previously, researchers reported that genetic diversity of some peppers (*Capsicum* spp.) accessions, including *Capsicum chinense*, *Capsicum annuum*. In 2016, 71 *C. chinense* accessions from different Brazilian geographic regions, using fruit morphological descriptors and AFLP molecular markers, were analyzed [63]. The results found no association between the morphological descriptors and AFLP markers [63]. In the same year, the researchers investigated patterns of molecular diversity using a transcriptome-based 48 single nucleotide polymorphisms (SNPs) in a large germplasm collection comprising 3821 accessions. Among the 11 species examined, *Capsicum annuum* showed the highest genetic diversity (HE = 0.44, I = 0.69), whereas the wild species *C. galapagoense* showed the lowest genetic diversity (HE = 0.06, I = 0.07). The *Capsicum* germplasm collection was divided into 10 clusters (cluster 1 to 10) based on population structure analysis, and five groups (group A to E) based on phylogenetic analysis [64]. The dendrogram constructed in this study from 147 pepper accessions using NJ methods, indicated that the genetic relatedness of the pepper cultivars clustered in the majority of the groups were in good agreement with their geographic origins. This study's analysis results were also consistent with the previous findings reported by Luo et al. and Jia et al. [7,65]. Moreover, the geographical sources of the Group III and Group V materials were found to be diverse, and not only attributed to Asian and African countries, but also to Latin American countries. It was observed that the pepper plants of the same geographical origin were not strictly divided into the same groups. For example, the eight pepper materials from China were grouped into Group I and Group III. These findings suggested that numerous complex migrations had occurred in the pepper genotype as the result of human migration, which had led to their adoption, acclimatization, and local selection.
