*2.3. In Silico PCR and Synteny Analysis of Cross-Species SSR Markers*

Using the SSR markers from pumpkin (*C. pepo* MU-CU-16) genome as a reference, we comparatively analyzed the genome SSR information of cucumber (Gy14), melon (DH92), watermelon (97103), *C. moschata* cv. Rifu, and *C. maxima* cv. Rimu. This was performed with a custom Perl script that used the NCBI BLASTN program as a search engine with an expected value of 10 and filtering. We allowed up to five nucleotide mismatches at the 5'-end of the primer, no mismatches at the 3'-end, and a minimum of 90% overall match homology. To establish the syntenic relationships of chromosomes between *C. pepo* with *C. sativus*, *C. lanatus*, *C. melo*, *C. maxima*, and *C. moschata*, we discarded these SSR markers with multiply physical locations in the same genome, only retaining the SSR markers in the genomes which had a single in silico PCR product. In addition, these shared SSR markers located on the unanchored scaffolds of the chromosome were further filtered. The SSR marker-based syntenic relationships were finally visualized with visualization blocks in Circos software v.0.55 [29].
