A genome-enabled, high-throughput, and multiplexed fingerprinting platform for strawberry (Fragaria L.)

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.