Two types of multilocus markers—random amplified polymorphic DNA (RAPD) (Williams et al., 1990) and amplified fragment length polymorphism (AFLP) (Vos et al., 1995)—have been utilized in preliminary assessments of Echinacea diversity. Wolf et al. (1999) developed RAPD markers specific for the commercial species E. purpurea, E. angustifolia, and E. pallida and demonstrated the utility of these markers in distinguishing root mixtures of the latter two species. Kapteyn et al. (2002) also developed reproducible and diagnostic RAPD markers for the same species and for E. atrorubens, and further extended their analysis to an evaluation of genetic diversity. Similar levels of overall diversity for each of the commercial species were detected (Kapteyn et al., 2002). Notable was the analysis of molecular variance (AMOVA) result that 78.2%, 82.6%, and 98% of genetic variation occurred within E. angustifolia, E. pallida, and E. purpurea populations, respectively, rather than among them (Kapteyn et al., 2002). The high 98% within-population variance component signified no differences among the sampled E. purpurea accessions, a confounding result given their different commercial sources and history of selective breeding (Kapteyn et al., 2002). However, there were significant differences among some populations of the other two species, more so for E. angustifolia than for E. pallida, suggesting that certain accessions may be valuable breeding resources (Kapteyn et al., 2002). Also note that with only four individuals analyzed per accession (Kapteyn et al., 2002), differences among accessions may not have been fully resolved with the RAPD markers (Lynch and Milligan, 1994); see additional comments below.
Baum et al. (1999) were the first to utilize the AFLP technique (Vos et al., 1995) to assess diversity in Echinacea. Based on a preliminary study, they concluded that the cultivated E. purpurea of Trout Lake Farm, Trout Lake, Washington, had greater diversity than that of the wild species examined, which included E. angustifolia, E. pallida, E. paradoxa var. paradoxa, E. sanguinea, and E. simulata (Baum et al., 1999). This is unusual in that wild relatives are normally the reservoirs of greater genetic diversity (Chapman, 1989; Harlan, 1984). Clear interpretation of these data is hampered by the absence of wild E. purpurea in this study. Although the data show that commercial E. purpurea had the highest number of polymorphic sites, this number is dependent on sample size, which ranged from 2 individuals for E. paradoxa var. paradoxa to 55 for E. purpurea. Moreover, an intermediate (not maximum among the sampled species) value of average gene diversity over loci was calculated for E. purpurea (Baum et al., 1999); this is a more typical comparative measure. AFLP markers are particularly powerful (Mueller and Wolfenbarger, 1999) and the results of Baum et al. (1999) are certainly evidence of genetic diversity in Echinacea; however, more data are needed for comparisons among the species with these markers.
The expanded use of molecular markers in characterizing Echinacea will have great utility in diversity surveys; population, conservation, and evolutionary genetics; fingerprinting; genetic mapping; hybrid identification; and systematics (Karp et al., 1996; Milligan et al., 1994; Rieseberg and Ellstrand, 1993; Mueller and Wolfenbarger, 1999). All molecular markers have limitations, however, and appropriate applications, interpretations, and statistical analyses must be considered (Karp et al., 1996; Mueller and Wolfenbarger, 1999). This is particularly relevant to dominant markers such as RAPDs and AFLPs; given limited sample sizes, the above studies are preliminary and statistics can be biased even with data-pruning corrections (Lynch and Milligan, 1994). Additionally, levels of genetic variation are known to vary by marker (Russell et al., 1997), making the genetic diversity of Echinacea best assessed with multiple markers and techniques (Fritsch and Rieseberg, 1996).
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