Sequence assessment of comigrating AFLPTM bands in Echinacea — implications for comparative biological studies

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Abstract
The extent of sequence identity among clones derived from monomorphic and polymorphic AFLPTM polymorphism bands was quantified. A total of 79 fragments from a monomorphic band of 273 bp and 48 fragments from a polymorphic band of 159 bp, isolated from individuals belonging to different populations, varieties, and species of Echinacea, were cloned and sequenced. The monomorphic fragments exhibited above 90% sequence identity among clones within samples. Sequence identity within variety ranged from 82.78% to 94.87% and within species from 75.82% to 98.9% and was 57.97% in the genus. The polymorphic fragments exhibited much less sequence identity. In some instances, even two clones from the same fragment were different in their size and sequence. Within sample, clone sequence identity ranged from 100% to 51.57%, within variety from 33.33% to 100% in one variety, and from 23.66% to 45% within species and was as low as 1.25% within the genus. In addition, sequences of the same size were aligned to verify the nature of their sequence dissimilarity/similarity. Within each size group, identical sequences were found across species and varieties. In general, comigrating bands cannot be considered homologous. Thus, the use of AFLPTM band data for comparative studies is appropriate only if the results emanating from such analyses are considered as approximations and are interpreted as phenotypic but not genotypic.