**1. Introduction**

Pepper is an indispensable spice, as well as an important vegetable crop which is cultivated around the globe. It originated in South America and belongs to the genus *Capsicum* (Solanaceae) [1–3]. This genus has many varieties of cultivated and wild species. However, only five of the species are commonly cited in the current literature as domesticated and culinary species. These include *C. annuum*, *C. chinense*, *C. frutescens*, *C. baccatum*, and *C. pubescens* [4]. Among those, *C. annuum* is considered to be the predominant species, which is comprised of many commercial varieties with major variations in the size, shape, color of the fruit, and, in particular, the pungency. However, after a long period of artificial selection, continuous cultivation, and domestication, the characteristic performance of pepper has tended to become diversified with narrowing genetic backgrounds. Consequently, the reduced genetic diversity index has engendered a straggle in the production of the piquant/hot pepper varieties. This has entailed searching and restoring potent traits

**Citation:** Zhong, Y.; Cheng, Y.; Ruan, M.; Ye, Q.; Wang, R.; Yao, Z.; Zhou, G.; Liu, J.; Yu, J.; Wan, H. High-Throughput SSR Marker Development and the Analysis of Genetic Diversity in *Capsicum frutescens*. *Horticulturae* **2021**, *7*, 187. https://doi.org/10.3390/ horticulturae7070187

Academic Editor: Yuyang Zhang

Received: 1 June 2021 Accepted: 3 July 2021 Published: 8 July 2021

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from related wild species/wild pepper varieties in order to improve the quality traits [5]. Furthermore, the enriched genetic diversity in the other four species, along with the rest of the wild species, has significantly rendered the possibility of improving *C. annuum* through various new technologies [6]. The genetic diversity analyses of the aforementioned accessions have provided excellent research resources, thereby utilizing their advantages and also the potential of reasonably allocating parent traits, which will definitely guarantee future breeding efficiency [7].

*C. frutescens* is a domesticated annual/perennial shrub crop bearing small erect, wheatshaped fruit, which are often strongly spicy in flavor. The unripe fruit are green to pale yellow, which turn red as ripening occurs. This species has many wild types, particularly one of the most commonly reported wild pepper species in China, which are mainly distributed in tropical areas such as Yunnan and Hainan provinces in China. A member of *C. frutescens* is locally known as 'Xiaomijiao' (Kunming Institute of Botany, Chinese Academy of Sciences, 1979) [8]. In addition, this plant species is known to be highly resistant to various biotic and abiotic stresses [8–11]. It also has the ability to withstand pathogenic attacks [9]. *C. frutescens* is characterized by high yield capacity, delicate flesh fragrance, and is also of ethnomedicinal importance. Therefore, investigating the genetic diversity of the germplasm resources of *C. frutescens* can potentially provide a basis for the improvement of existing cultivated pepper in China.

Simple sequence repeats (SSRs), or microsatellites, are a group of tandemly repeated DNA sequences comprised of one to six nucleotide units. These are ubiquitous in the genomes of prokaryotic and eukaryotic organisms [12,13]. SSRs are the most highly recognized genetic markers that are actively employed in plant breeding due to their distinguished traits of co-dominant inheritance, multi-allelic nature, extensive genome coverage, high abundance, and especially high reproducibility [12,14]. The polymorphism resulting from SSRs can be detected from amplifying genes by employing primers flanking the repeated motifs [15]. SSR markers are widely used when examining plants and animals from different aspects, such as analyzing population characteristics [16], functional diversity [17–19], constructing linkage maps [20,21], DNA fingerprinting [22–25], and assisted breeding techniques [26–28]. There are many SSR markers which have been developed and successfully used. However, it is imperative to develop a few with strong stability and highly polymorphic SSR markers in order to comply with such intricate studies as high-density genetic mapping, genome comparative mapping, and genome-wide association analysis.

It has been found that examining the diversity of the pepper germplasm via assessing the morphological characteristics is not only very stringent, but often results in misguidance. Furthermore, such attributes may be equally impacted by environmental conditions. However, molecular markers have been found to have several advantages in representing colossal amounts of information, as well as better stability and high analysis efficiency when used to study the genetic diversity of plant germplasm resources. Therefore, molecular markers are widely used in genetic diversity research [29]. For example, in recent years, molecular markers have been extensively employed for analyzing the genetic diversity of pepper germplasm resources. In addition, among the many types of molecular markers, SSRs are highly preferred for examining diversity due to their high polymorphism, ample repeatability, and co-dominance traits [30]. These characteristics have been supported by many earlier reports. For example, in the study conducted by Gu [31] regarding pepper diversity, 1904 pepper materials were accommodated into two categories by employing 29 pairs of polytropic SSR markers. Similarly, Zhang [32] used SSR labeling technology to analyze 372 pepper materials and then cluster them into three groups. The results were found to be consistent with the botanical characteristics. Chen [5], when comparing the efficiency of SRAP and SSR for the genetic diversity analyses of eight pepper germplasm, found that the SSRs were more efficient, with higher polymorphism and detection ability. In another related study, 14 pairs of SSR polymorphic primers were employed by Li [33], who also analyzed the genetic diversity of 169 pepper materials. It was observed that when

classifying them into seven groups and validating the findings, the results were in exact congruence with the basic classification of the pepper species.

Pepper germplasm resources are the prime material basis for the breeding and production of pepper crops. However, the genetic background of the pepper germplasm resources in China is relatively narrow. Therefore, it is indicated that an emphasis should be placed on gaining significant data resources with efficient utilization values from the collection and proper exploitation of wild germplasm resources. Among the available resources, 'Xiaomijiao' is the only wild *C. frutescens* plant found in China. As a result, this research focused on the objectives of studying the development of SSR molecular markers for *C. frutescens* on the basis of the 'Xiaomijiao' sequence data (unpublished), and then analyzed the genetic diversity of the germplasm resources collected from the genome levels through SSR markers. These new polymorphic microsatellite markers provide the basis for further population research. In this paper, the genetic diversity of 147 *C. frutescens* germplasm from 25 countries was analyzed to understand the genetic relationships and genetic composition of various accessions, so as to provide basis for more effective utilization of these germplasm resources in the future.
