Application of ISSR, RAPD, and cytological markers to the certification of Picea mariana, P. glauca, and P. engelmannii trees, and their putative hybrids.

Picea glauca (white spruce) and P. engelmannii (Engelmann spruce) are so similar and integrated that it is impossible to distinguish between them and their hybrids using morphological characteristics. Although natural hybrids between P. glauca and P. mariana (black spruce) do not generally occur, even though the 2 species are sympatric in North America, a first-generation hybrid, called the Rosendahl spruce, has been reported in the literature. In this study, several inter-simple sequence repeat (ISSR) markers were developed, as were randomly amplified polymorphic DNA (RAPD) markers, to certify spruce trees and their hybrids. ISSR fingerprinting was more efficient than RAPD assay; it detected 70% polymorphic DNA markers among the spruce species analyzed, whereas RAPD fingerprinting detected only 53%. Species-diagnostic ISSR and RAPD markers differentiating P. glauca from P. engelmannii and P. mariana were cloned and sequenced. Molecular certification of the spruce samples analyzed confirmed that all the seeds from interior spruce populations were true hybrids of P. glauca and P. engelmannii. But the analysis of seeds derived from the putative Rosendahl spruce indicated that this tree is likely a pure P. glauca genotype, rather than a hybrid of P. glauca and P. mariana. These data were confirmed by cytological analyses. Further analysis, using a more sensitive DNA amplification method with designed primers flanking the species-diagnostic ISSR and RAPD markers, revealed that such sequences are not generally species-specific because they are present in other spruce species.