**1. Introduction**

The *Cucurbita* genus (2n = 2x = 40), belonging to the Cucurbitaceae family, contains more than 13 species [1]. Most *Cucurbita* species are wild resources, and only three domesticated species, *Cucurbita maxima*, *Cucurbita moschata*, and *Cucurbita pepo*, are widely cultivated and have become important food crops globally [2]. At present, Asia has the largest pumpkin cultivation area, and China is the main producer of pumpkins. In 2012, the planting area of pumpkin was approximately 3.8 × 104 Hm<sup>2</sup> in China, and the total output reached 7.0 × <sup>10</sup><sup>6</sup> tons (http://www.fao.org/faostat/zh/#data/QC/visualize, 2020). Due to the fact of their long history of cultivation and domestication, *Cucurbita* species show a greater diversity in fruit shape, size, and color than other Cucurbitaceae species [3]. Furthermore, *Cucurbita* species have strong roots and exhibit good adaption to different biotic and abiotic stresses, such as cold, viruses, and salinity, and so they are widely used as rootstocks in grafting [4,5]. Although they are a common global crop, fundamental

**Citation:** Zhu, L.; Zhu, H.; Li, Y.; Wang, Y.; Wu, X.; Li, J.; Zhang, Z.; Wang, Y.; Hu, J.; Yang, S.; et al. Genome Wide Characterization, Comparative and Genetic Diversity Analysis of Simple Sequence Repeats in Cucurbita Species. *Horticulturae* **2021**, *7*, 143. https://doi.org/ 10.3390/horticulturae7060143

Academic Editor: Yuyang Zhang

Received: 19 May 2021 Accepted: 5 June 2021 Published: 8 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

genetic research on *Cucurbita* is lacking, and few studies have been conducted to improve the cultivation and breeding of this genus.

Simple sequence repeats (SSRs) are widely used in genetic mapping constructions, genetic diversity analyses, and genome-wide association studies due to the fact of their relative abundance, multi-allelism, co-dominance, and low cost [6,7]. In the Cucurbitaceae family, the whole-genome sequencing of *Cucumis sativus*, *Cucumis melon*, and *Citrullus lanatus* has been completed [8–10], and genome-wide SSR markers have been characterized and developed in these crops, which has greatly promoted their application in gene and quantitative trait locus (QTL) mapping as well as in comparative genomics [11–13]. A rough syntenic relationship between melon (2n = 2x = 24) and cucumber (2n = 2x = 14) chromosomes was revealed by comparative mapping using 199 SSR markers developed from cucumber [14]. Later, Yang et al. (2014) developed a higher density map of *Cucumis hystrix* containing 416 SSR markers, and 151 and 50 markers were derived from cucumber and melon, respectively. With these shared markers among the three *Cucumis* species, the chromosome-level syntenic relationships were well established, which was further confirmed by fluorescence in situ hybridization (FISH) [15]. Ninety-one syntenic blocks were divided between cucumber and melon, and 53 syntenic blocks were identified between cucumber and *Cucumis hystrix*. Furthermore, the genome-wide SSR markers developed from melon and watermelon have made it possible to more clearly define chromosomal syntenic relationships, and the complicated mosaic patterns of chromosome synteny between melon, watermelon, and cucumber have been well established based on cross-species SSR markers [12,15]. However, the syntenic relationships and chromosomal rearrangements between *Cucurbita* species and other Cucurbitaceae crops are still largely fragmented and incomplete.

Based on the conserved sequences among species or genera, some amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and SSR markers were developed in previous studies [16–20]. However, these restricted markers are insufficient for research on genetic diversity, genetic mapping, and comparative genomics. Esteras et al. (2012) constructed the first genetic map in pumpkin using 304 single nucleotide polymorphisms (SNPs) and 11 SSR markers and found that the linkage groups of pumpkin were partially homoeologous to cucumber chromosomes. The applications of these expressed sequence tag (EST)-SNP markers are still greatly limited due to the small numbers of markers, the high cost of enzymes, and the complicated operating procedure [21]. Due to the lack of genome-wide coverage and polymorphic markers, in-depth application and comparative analysis still cannot be conducted. With the development of high-throughput sequencing technology, there has been an increase in studies on *Cucurbita*, and the whole-genome sequences of three important cucurbit crops have become available. Based on these SNPs' data, the whole-genome synteny analysis indicated that both the *C. maxima* and *C. moschata* genomes underwent a whole-genome duplication (WGD) event and that pairs of *C. maxima* (or *C. moschata*) homoeologous regions are shared between chromosomes corresponding to the two sub-genomes [22]. Montero et al. also identified that the covered regions in most of the *C. pepo* genome had experienced a WGD event [23]. Furthermore, some transcriptomes of *Cucurbita* species have become available, and EST-SSRs were developed from them [24–28]. To date, the development of SSR markers in *Cucurbita* species is still limited.

The whole-genome sequences of *C. moschata*, *C. maxima*, and *C. pepo* have been completely assembled, which will greatly promote the large-scale development of SSR markers, allowing for the construction of high-resolution maps, gene mapping, and genome-wide association studies (GWAS). In this study, we conducted a genome-wide identification of SSR motifs in three *Cucurbita* species, analyzed the distribution and frequency of different repeat types, identified cross-species transferable SSR markers by in silico PCR analysis, and studied the chromosome synteny of *C. pepo* with other Cucurbitaceae crops. In addition, 66 core SSR markers were identified in *Cucurbita* genomes and used to evaluate the genetic diversity and population structure of 61 *C. pepo* germplasms. Our study will

be useful for research on the population structure, genetic diversity, molecular-assisted selection, and map-based cloning in *Cucurbita* species.

## **2. Methods**
