Submitted to: Journal of Biomolecular Techniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2015
Publication Date: 1/18/2016
Citation: Alexander, L.W. 2016. Rapid, effective DNA isolation from Osmanthus via alkaline lysis. Journal of Biomolecular Techniques. 27:53-60.
Interpretive Summary: The genus Osmanthus has several popular horticultural varieties. Nursery growers seek Osmanthus varieties that are improved for flower size and color, flower fragrance, increased cold tolerance, and increased resistance to drought and disease. Molecular breeding approaches are needed to catalog genetic diversity of the genus, identify species and cultivars, and locate important traits on genetic maps. However, DNA isolation – a fundamental step in developing a molecular toolkit – has proven difficult for many species and cultivars of Osmanthus. This study demonstrated that an improved method of alkaline lysis is suitable for isolating abundant DNA from diverse Osmanthus species and cultivars. The procedure takes only 1.5 hours for 18 samples and costs as little as $0.74 per sample. This study shows that a single, cost-effective method can be used to extract Osmanthus DNA from a wide variety of leaf types. Plant breeders, geneticists, and plant biologists will benefit from this improved DNA isolation method.
Technical Abstract: The genus Osmanthus contains many popular ornamental species including fragrant tea-olive (O. fragrans) and Fortune’s tea-olive (Osmanthus x fortunei). Growers seek improved Osmanthus varieties; however, few molecular tools exist for this genus. Furthermore, the variability of leaf structure and presence of proteins and phenols in leaf tissue presents a challenge for reliable DNA extraction from all species and cultivars. The objective of this study was to develop a rapid, effective, and cost-efficient method of DNA isolation that could be used on all species and cultivars of Osmanthus. We used four different methods to isolate DNA from eight cultivars of Osmanthus representing four species. Absorbance spectra, DNA concentration, appearance on agarose gel, and performance in PCR were used to analyze quality, quantity, and integrity of isolated DNA. Method and cultivar (species) were significant sources of variation in DNA absorbance spectra and concentration, with method accounting for the largest share of variation for each analysis variable. The commercial kit DNA had the least contamination based on absorbance spectra, but had the lowest concentration, while alkaline lysis led to the most contamination, but highest concentration. The commercial kit and alkaline lysis resolved the most individuals the clearest on agarose gel, while the two CTAB methods had poorly resolved gels. All methods except CTAB lysis with phenol/chloroform extraction performed well in PCR. Additions to the alkaline lysis method including washing the DNA pellet and discarding the pellet after elution significantly decreased contamination without affecting yield. We recommend the improved alkaline lysis method as a rapid, effective, and cost-efficient method of isolating DNA from Osmanthus species.