*2.2. Genome SSR Identification and Development in Cucurbita Genomes*

The genome information of watermelon, melon, cucumber, and pumpkin was downloaded from http://cucurbitgenomics.org/ (2020). To develop a set of higher polymorphic SSR primers for the future study, the criteria used for microsatellite identification in this study was from 2 to 8 bp, and mononucleotides were not considered due to the difficulty in distinguishing bona fide microsatellites from sequencing or assembly error. The microsatellite identification tool (MISA) was used to identify and analyze SSR markers including perfect and compound microsatellites. The specific screening details were as follows: repeats with a minimum length of 18 bp (for di- and tetra-nucleotides), 20 bp (for penta-nucleotides), 24 bp (for hexa-nucleotides), 21 bp (for hepta-nucleotides), and 24 bp (for octa-nucleotides). The oligonucleotide primers for these SSRs were designed according to the flanking genomic sequence using Primer3 software (v.1.1.4). Primers were designed to generate amplicons of 100–300 bp in length with the following minimum, optimum, and maximum values for Primer3 parameters: primer length (bp): 18–20–24; Tm (◦C): 50–55–60. Other parameters used the default program values.
