Development and application of RAPD analysis for intra- and interspecific characterization within the genus Amelanchier

Abstract

The development and application of RAPD analysis for intra- and interspecific characterization within the genus Amelanchier was the main objective of this study. Distinguishing among saskatoon cultivars (A. alnifolia Nutt.) and among Amelanchier species is difficult based on morphology. The ability to clearly identify cultivars is important in terms of maintenance, distribution and identification of genetic variability. An understanding of the taxonomy of Amelanchier is important because it offers a system of nomenclature which allows for comparative references and is useful for the enhancement and development of germplasm. A protocol for the extraction of DNA suitable for amplification by the polymerase chain reaction (PCR) from Amelanchier leaves was established. This protocol consistently yielded suitable DNA, regardless of plant growing conditions or leaf age. A protocol for the reliable PCR-amplification of saskatoon DNA was established by optimizing the reaction components. The reproducibility of amplification products was affected by variation in any of the reaction components examined. Variation in the magnesium concentration by as little as 0.5 mM had a dramatic effect on the intensity and numbers of amplification products. RAPD analysis was used to distinguish among 16 cultivars of saskatoon. Eight 9-base primers generated reproducible polymorphic markers which uniquely characterized twelve cultivars and two pairs of 2 cultivars. Polymorphism was not detected among five sources of the cultivar Thiessen, whereas variability was found among seedlings from self-pollinated Thiessen. The cultivars Regent and Parkhill could be distinguished from one source, but were indistinguishable from another, suggesting the latter source had mislabeled these cultivars. RAPD analysis was also used to assess the genetic relationships among 56 individuals representing 16 species of Amelanchier. Amplification products were analyzed using cluster analysis, principal coordinates analysis, and Wagner parsimony. Based on these analyses, taxa were divided into these three groups: (1) A. arborea, A. laevis, A. canadensis, A. intermedia, and A. x grandiflora; (2) A. alnifolia, A. florida, A. cusickii, A. oxyodon, and A. gaspensis, and (3) A. spicata, A. sanguinea, and A. stolonifera. These results suggest the third group may have arisen through hybridization between the first two groups. Generally, the genetic relationships presented here can be supported by the literature.