|Chambers, Alan -|
|Carle, Scott -|
|Njuguna, Wambui -|
|Chamala, Srikar -|
|Whitaker, Vance -|
|Barbazuk, W. Brad -|
|Folta, Kevin -|
Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 26, 2012
Publication Date: January 6, 2013
Citation: Chambers, A., Carle, S., Njuguna, W., Chamala, S., Bassil, N.V., Whitaker, V.M., Barbazuk, W., Folta, K.M. 2013. A genome-enabled, high-throughput, and multiplexed fingerprinting platform for strawberry (Fragaria L.). Molecular Breeding. DOI:10.1007/s11032-012-9819-3. Interpretive Summary: We used a protocol for generating genetic markers composed of 3-9-base repeat unit patterns from the woodland strawberry reference DNA sequence. We devised a strategy of using a single label to generate fluorescently labeled products from eight of these DNA regions in one amplification reaction. This strategy was used to fingerprint 219 strawberries. The methods are comparable to DNA forensics in humans. The approach yielded reproducible, highly-variable, complex patterns. The technique was applied to detect closely-related individuals from different types of strawberries. The resulting fingerprints successfully differentiated the vast majority of individuals and found few individuals that were not true to type.
Technical Abstract: Strawberry (Fragaria L.) genotypes bear remarkable phenotypic similarity, even across ploidy levels. Additionally, breeding programs seek to introgress alleles from wild germplasm, so objective molecular description of genetic variation has great value. In this report, a high-throughput, robust protocol for generating highly-informative simple sequence repeat (SSR) patterns is presented to address these issues. The methods are comparable to SSR use in DNA typing in humans and are based on identification of high-number repeats composed of tetra- through nona-nucleotide repeat units found in the Fragaria vesca genome sequence. Individual SSR-containing regions were examined for variability over a range of 219 strawberry genotypes. A single-fluorophore secondary labeling strategy was devised that allows simultaneous amplification of eight SSR regions in a single PCR reaction. The approach yields reproducible, highly-variable, complex patterns (Shannon-Weaver Index 7.09–13.88). The technique may be applied to detect closely-related individuals across ploidy levels, including full sibling progeny in an inter-related octoploid pedigree. Genetic diversity among various cultivars and progenitor wild species in the United States Department of Agriculture-Agricultural Research Service Fragaria Supercore collection was also evaluated. The results build on known relationships, and also raise questions about accepted relationships between several genotypes.