Polymorphism in spruce assessed with RAPD and ISSR markers: Cloning and characterization of species-diagnostic sequences

The genus Picea comprises some 40–60 species widely distributed over the cooler regions of the northern hemisphere to the tropic of Cancer. White spruce (Picea glauca) and Engelmann spruce (P. engelmannii) are sister species, and thus very difficult to distinguish using morphological analyses. The main objective of this study was to identify and validate species-specific RAPD and ISSR PCR markers in the sister species P. glauca and P. engelmannii as well as in the closely related species, P. pungens var. glauca and P. rubens. Intra and inter-specific variations were analyzed in P. glauca and P. engelmannii . The level of polymorphism observed between the species using RAPD PCR was estimated to be 43.1%. RAPD primers 11 and 13 produced a combined intra-populational polymorphism level of 19.12%. The 41 RAPD primers screened in total generated 244 bands of which 61.1% were polymorphic. UBC-186 produced two species-diagnostic bands, one in P. glauca at 750 bp and the other in P. engelmannii at about 550 bp; primer E-2 produced a species-diagnostic band at about 2000 bp, found only in P. engelmannii; while primers E-7, UBC-402, and OPX-04 produced species diagnostic markers at 900 bp, 850 bp and 450 bp in P. rubens. None of the RAPD markers analyzed with the designed primers flanking the targeted regions was truly specific to one spruce species, and in fact, all showed amplification in most, if not all of the species studied. Like the RAPD primers, the ISSR primers used in this study generated very similar amounts of polymorphism (17.18%) in P. glauca and P. engelmannii populations. ISSR primer 827 produced a strong species-diagnostic marker in P. engelmannii at approximately 425 bp.