Statistical correlations of primer thermodynamic stability ΔG° for enhanced flax ISSR-PCR cultivar authentication

We revealed four statistically significant correlations related to inter-simple-sequence repeat (ISSR) patterns:  (1) between thermodynamic free energy ΔG° of ISSR primer sequence and PCR reamplification intensity (dAi), (2) between free energy ΔG° of ISSR primer sequence and PIC coefficient quantifying the polymorphism of ISSR patterns, (3) and (4) between free energy ΔG° of anchor sequence of primer and the number of total, and polymorphic bands in ISSR patterns, respectively. Methodological recommendations for effective ISSR primer design were inferred based on revealed correlations. In particular, free energy of ISSR primer sequence is recommended to be ΔG° > 160 kJ/mol of interaction and free energy of flanking anchor sequence in primer to be around ΔG° = 28 kJ/mol of interaction to produce ISSR patterns displaying maximum polymorphism of flax germplasm. Keywords: Cultivar authentication; flax germplasm; inter-microsatellite repeats; ISSR-PCR; thermodynamic free energy ΔG°