*3.3. Detection of EST-SSR Markers and Designing of Primers*

SSR loci in the transcriptome were identified using the microsatellite identification tool MISA (http://misaweb.ipk-gatersleben.de/) (accessed on 3 December 2020) [53]. The parameters of identifying the mono-, di-, tri-, tetra-, penta-, and hexanucleotide motifs were set to minimum numbers of 10, 6, 5, 5, 5, and 5 repeat units, respectively. Primer pairs were designed using Primer 3.0 software [54], according to the following parameters: the length range of the optimal primer sizes was 18–23 bp, the optimal annealing temperature was from 55–65 ◦C, the PCR product size range was 80–300 bp, and the GC content was 40–60%.
