SSR primer pair design

The SSR primer pairs were designed using Primer Premier 5.0 software (Tu et al., 2011) using the following standard parameters: target amplicon length of 80–300 bp, annealing temperature variation from 55 to 65°C, GC content from 50 to 70%, and primer size of 18–28 bp. Three primer pairs were designed for each SSR locus, and the primer pair producing DNA with the highest score was chosen for further use in SSR marker studies. Primers were synthesized by GENEWIZ, Inc. (Suzhou, China). In order to test the effectiveness of the SSR set for classification of flax varieties, 62 loci out of the total collection of novel SSR markers were selected randomly as sites for subsequent genotypic testing of 48 cultivars ultimately for use in genetic diversity analysis (Table ​(Table11).