*Article* **Genome Wide Characterization, Comparative and Genetic Diversity Analysis of Simple Sequence Repeats in Cucurbita Species**

**Lei Zhu 1,2, Huayu Zhu 1, Yanman Li 1, Yong Wang 1, Xiangbin Wu 2, Jintao Li 2, Zhenli Zhang 2, Yanjiao Wang 2, Jianbin Hu 1,2, Sen Yang 1, Luming Yang 1,2,\* and Shouru Sun 1,2,\***


**Abstract:** Simple sequence repeats (SSRs) are widely used in mapping constructions and comparative and genetic diversity analyses. Here, 103,056 SSR loci were found in *Cucurbita* species by in silico PCR. In general, the frequency of these SSRs decreased with the increase in the motif length, and di-nucleotide motifs were the most common type. For the same repeat types, the SSR frequency decreased sharply with the increase in the repeat number. The majority of the SSR loci were suitable for marker development (84.75% in *Cucurbita moschata*, 94.53% in *Cucurbita maxima*, and 95.09% in *Cucurbita pepo*). Using these markers, the cross-species transferable SSR markers between *C. pepo* and other Cucurbitaceae species were developed, and the complicated mosaic relationships among them were analyzed. Especially, the main syntenic relationships between *C. pepo* and *C. moschata* or *C. maxima* indicated that the chromosomes in the *Cucurbita* genomes were highly conserved during evolution. Furthermore, 66 core SSR markers were selected to measure the genetic diversity in 61 *C. pepo* germplasms, and they were divided into two groups by structure and unweighted pair group method with arithmetic analysis. These results will promote the utilization of SSRs in basic and applied research of *Cucurbita* species.

**Keywords:** pumpkin; simple sequence repeat (SSR); syntenic relationship; cross-species markers; population structure
