*4.3. The Genetic Diversity and Population Structure of C. pepo Germplasm*

Previously, because of the scarcity of genomic sequences, there were limited molecular markers available to study the genetic diversity and population structure of *Cucurbita* species. Though the genetic diversity of *Cucurbita* species has been evaluated using sequence-related amplified polymorphism (SRAP), AFLP, SSR, RAPD, and inter-simple sequence repeats (ISSRs), most of the markers used have high randomness, lack precise location information, and have low genomic coverage and poor polymorphism, which greatly limit their application [18,51,52]. With the draft genome available for three cucurbit crops, we developed 91,248 SSR markers with precise physical locations on chromosomes and evaluated the genetic diversity of 61 pumpkin accessions using 66 core SSR markers.

The population structure of 61 accessions revealed that the background of some materials was mixed between group I and group II, suggesting that these accessions may have undergone gene exchange between two subspecies. The materials were collected from different provinces in China, and they were obviously classified into two subspecies, subsp. *ovifer* (or subsp. *texana*) and subsp. *pepo*, which is consistent with previous studies [21,51]. However, the three subspecies of *C. pepo* classified by Decker are *C. pepo* subsp. *fraterna* (Bailey) Andres, *C. pepo* subsp. *texana* (Scheele) Filov, and *C. pepo* subsp. *pepo* [53]. The putative ancestor for *C. pepo*, namely, subsp. *fraterna* from northeastern Mexico, has been considered a wild gourd [54]. The population structure and UPGMA results indicated that these accessions of *C. pepo* in China come from the common ancestor. Thus, there have great prospects for germplasm improvement.

The *Cucurbita* genus contains several economically important crops, but its breeding has lagged behind the other Cucurbitaceous crops. Limited high-quality cultivars cannot meet the production requirements. Thus, different breeding programs can be facilitated using marker assisted selection. The whole-genome SSR markers detected in this study will promote the development and utilization in basic and applied research of *Cucurbita* species.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/horticulturae7060143/s1, Figure S1: The top five types of each SSR repeat motif and their frequencies in *C. moschata*, *C. maxima*, and *C. pepo*, Figure S2: The optimal K-values analysis by using Structure Harvester, Table S1: The list of the *C. pepo* introduction accessions, Table S2: The distribution of different nucleotide repeats in the genome of three *Cucurbita* species, Table S3: The distribution of SSR loci on different chromosomes in *C. moschata*, *C. maxima*, and *C. pepo*, Table S4: The identified SSR markers in *C. moschata*, Table S5: The identified SSR markers in *C. maxima*, Table S6: The identified SSR markers in *C. pepo*, Table S7: List of cross-species SSR markers between *C. pepo* and *C. sativus* identified by in silico PCR, Table S8: List of cross-species SSR markers between *C. pepo* and melon identified by in silico PCR, Table S9: List of cross-species SSR markers between *C. pepo* and watermelon identified by in silico PCR, Table S10: List of cross-species SSR markers between *C. pepo* and *C. maxima* identified by in silico PCR, Table S11: List of cross-species SSR markers between *C. pepo* and *C. moschata* identified by in silico PCR, Table S12: The total SSR markers in *C. pepo* genetic diversity and population structure analysis, Table S13: Polymorphism and allelic diversity of SSR markers in *C. pepo* materials.

**Author Contributions:** L.Z. and H.Z. performed the data analysis and wrote the manuscript. Y.L., Y.W. (Yong Wang), and X.W. conducted validation of SSR polymorphism, genetic diversity, and population structure analysis. J.L., Z.Z., and Y.W. (Yanjiao Wang). participated in the SSR genetic diversity analysis. J.H., S.Y., and Y.L. conducted the germplasm collection. S.S. and L.Y. designed the experiments and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Major Public Welfare Project of Henan Province (202102110202) and the Science and Technology Project of China National Tobacco Corporation (Henan Tobacco Company) (2018410000270095).

**Data Availability Statement:** All generated or analyzed data during this study are reflected in the present article.

**Acknowledgments:** The authors are highly grateful to Yufeng Wu of Nanjing Agricultural University with the Circos analysis. The *C. pepo* accessions used in this study were introduced from the National Crop Germplasm Resource Platform of China. We are also indebted to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for improvement.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

