DATWYLER, SHANNON L.* AND ANDREA D. WOLFE. Department of Evolution, Ecology and Organismal Biology, Ohio State University, Columbus, OH 43210-1293. - Assessment of ISSR band homology by southern hybridization with implications for data analysis and microsatellite development.
Despite numerous recent studies using inter-simple sequence repeat
(ISSR) markers, there have been no comprehensive studies examining
homology among ISSR fragments. In this study, we are assessing
homology among ISSR fragments by southern hybridization. Thirty two
taxa, representing a wide sampling among the Lamiales, with a focus on
Penstemon subg. Dasanthera were chosen for study. Three
ISSR primers representing different repeat motifs have been used for
DNA amplification. Southern blots were performed for all three
primers of interest. Probes for southern hybridization were generated
from three taxa (Penstemon cardwellii, Hyobanche
glabrata, and Scrophularia scopoli) for all three primers
by PCR cloning. Each ISSR band was hybridized separately to the
appropriate primer blot and visualized using the ECL non-radioactive
labeling kit. Preliminary data suggest that comigrating fragments
represent homologous fragments among closely-related taxa, but
homology assessment is more complicated among distantly-related taxa.
In addition, some ISSR bands have been demonstrated to hybridize with
multiple bands generated by the same primer, especially among
closely-related taxa. This could be the result of tandemly-repeated
SSRs of the same di- or trinucleotide repeat. Comparisons are being
made for genetic diversity statistics estimated including all bands
visualized using ethidium bromide-stained agarose gels and only bands
that are determined to be independent based on southern hybridization.
In addition, a new strategy for developing microsatellite loci from
ISSR fragments will be discussed.
Key words: ISSR band homology, microsatellite development, Scrophulariaceae, southern hybridization