*3.3. The Genetic Diversity and Population Structure Analysis of the C. pepo Germplasm*

In our preliminary study, approximately 400 SSR markers were screened using 61 accessions of *C. pepo* germplasm. Finally, a total of 66 core SSR markers were selected based on the allelic number, the genomic coverage, and the efficiency of PCR amplification (Table S12). These markers exhibited clear band spectrums and were evenly distributed on the chromosomes. In this study, 276 alleles were detected by the 66 SSR markers in the 61 *C. pepo* accessions with an average of 4.18 loci per SSR marker. The number of Na ranged from two to nine. The highest number of Na was nine, which was detected by SSR010246, SSR026560, SSR026918, SSR027656, and SSR026980, followed by SSR011546, SSR003315, and SSR026797 with eight alleles. The number of Ne varied from 1.03 to 6.07 with an average of 2.31. The SI ranged from 0.083 to 1.96 with an average of 0.83. The PIC value ranged from 0.03 to 0.83 with an average of 0.43 (Table S13).

We further used a model-based approach for population structure analysis of the 61 *C. pepo* accessions. According to the results of the structural operation, when K = 2, ΔK showed a significant peak value, indicating that the 61 accessions used in this study could be obviously divided into two groups (Figure S2), named group I and group II. The five *C. pepo* subsp. *ovifer* accessions (2, 29, 30, 31, and 45) were clustered into group I (8.20%), and all of them were wild materials. Most of the *C. pepo* subsp. *pepo* accessions were clustered into group II (91.80%), which were all cultivated materials (Figure 5A). This indicated that the SSR markers we used could clearly distinguish the cultivated materials from the wild materials. The backgrounds of the cultivated accessions were narrow, except for accession 45 in group I, which should have a complex genetic background, similar to accession 14 and 16 in group II. The UPGMA analysis revealed that the 61 *C. pepo* accessions were divided into two clusters (Figure 5B), which was consistent with their population structure. The five *C. pepo* subsp. *ovifer* accessions were clustered together at the base of the phylogenetic tree, which further supported our population structure analysis.

**Figure 5.** The genetic diversity of the 61 accessions based on SSR markers. (**A**) Population structure of 61 accessions in *C. pepo* by the model-based analysis. The scale of the *y*-axis represents the percentage of genetic components, and the *x*-axis represents the different materials. (**B**) Phylogenetic tree of 61 accessions in the *C. pepo* by UPGMA analysis; Group I (red makers) and Group II correspond to the structure analysis.
