*4.2. Chromosome Synteny Analysis between C. pepo and Other Cucurbitaceae Species*

Chromosome synteny analysis has been conducted in many species, such as cucumber, watermelon, and melon, but few studies have been conducted on the chromosome synteny among different *Cucurbita* species or between *Cucurbita* species and other Cucurbitaceae crops. In this study, the genome-wide SSR development from the three *Cucurbita* genomes provided the possibility to identify their syntenic relationships at a high-resolution level via in silico PCR analysis. Though the sizes of the pumpkin genomes are similar to that of other sequenced Cucurbitaceae species, the number of cross-species SSR markers in the *Cucurbita* genus is much higher. Compared to hundreds of shared markers in previous studies [14], we identified many more cross-species transferable SSR markers in the *Cucurbita* genus that were used for chromosome synteny analysis. The WGD event in *Cucurbita*, which has not been observed in other sequenced Cucurbitaceae species, such as cucumber [8], melon [10], and watermelon [9], may be a possible reason leading to the high abundance of SSR markers.

According to the cross-species transferable SSR markers, 52, 61, and 89 syntenic blocks distributed on all chromosomes were identified between *C. pepo* with cucumber, melon, and watermelon, respectively (Figure 3). Similar homoeologous blocks were detected by whole-genome comparison [22], suggesting that the cross-species transferable SSR markers are useful and reliable in genome comparisons and chromosome synteny analyses. In most cases, there were multiple synteny blocks detected between *C. pepo* and other Cucurbitaceae species due to the fact of chromosome fission. The most complicated syntenic pattern existed on chromosome Cpe1 of *C. pepo*, which was syntenic to seven watermelon chromosomes, indicating that complicated structural changes occurred after their divergence from a common ancestor. The ratio of collinear blocks to inversion blocks was nearly 1:1 in *Cucurbita*, and the reason for this may be that genome duplication and inter-chromosomal exchanges occurred randomly during chromosome evolution.

Based on the cross-species transferable SSR markers, we identified more highly conserved syntenic blocks among *Cucurbita* species than melon, cucumber, or watermelon. We found that each block among three *Cucurbita* species of the same genus contained many more shared common SSR markers, and these homoeologous chromosomes were much conserved, which further confirmed their close evolutionary relationships in the Cucurbitaceae family. For example, the *C. pepo* syntenic block contained more markers than that in melon [12]. Due to the WGD during chromosome evolution and speciation, the number of the chromosomes and cross-markers increased. However, those blocks were highly conserved during chromosome evolution among different Cucurbitaceae species. The chromosomal pair analysis by cross-species SSR markers showed that there were eight large-scale inversions on different chromosomes between *C. pepo* and *C. moschata* or between *C. pepo* and *C. maxima*, indicating that *C. pepo* experienced more complex evolutionary processes (Figure 4). Interestingly, Chr4 contained a mosaic region among *Cucurbita* species. The reason might be due to the fact of genome duplication, large-scale inter-chromosomal exchanges, or long-term evolutionary forces. Whether the partial inversion of chromosome 4 in *C. pepo* will affect the mapping, cloning, and study of some traits is worth exploring in the future.
