The 2011 ASHS Annual Conference

Spigelia marilandica (L.) L., an herbaceous perennial native to the Southeastern United States, is used as an ornamental and for pharmacological purposes. Plant conservationists and breeders, therefore, are interested in determining its population genetic diversity.  Amplified fragment length polymorphism (ALFP) fingerprints, which require upwards of 100ng of DNA, can effectively measure this diversity across the species’ range.  Since DNA concentration is linked to AFLP results, an optimum protocol was developed to extract DNA from leaf material of wild-growing plants. Young leaf material was collected from 10 populations throughout Georgia, Florida, and South Carolina and stored at -80°C prior to DNA extraction. A modified Omega Bio-Tek protocol was used to extract DNA.  A second protocol, which added 0.9 µL carrier RNA in the column step, was used in an attempt to increase DNA yields.  Yields of DNA with and without the addition of carrier RNA were checked for quantity and quality using a 1.5% agarose gel. The majority of DNA extracted from collections which used the addition of carrier RNA showed some variation as follows. The concentration of DNA was increased from 66.6% to 830% when carrier RNA was added to samples collected in Dalton, GA.  DNA extraction from 30 collections in Kathleen, GA showed variability. Of the 30 individuals collected, 13 had visible DNA only using carrier RNA, three did not generate DNA using either protocol, and of those plants that generated DNA without carrier RNA, the addition of carrier RNA increased the quantity of DNA from 40% to 500%.  Additionally, a second Kathleen, GA population only produced DNA when the carrier RNA treatment was used. No DNA concentration difference resulted in extractions with or without carrier RNA from populations in Lithonia, GA and Marianna, FL. A Wakulla Springs, FL population had similar DNA quantities in 23 of 30 samples, although 7 of 30 samples collected only generated DNA when carrier RNA was used.  Of 25 collections from Bonaire, GA, 19 samples resulted in DNA only when carrier RNA was used.  While the remaining four samples did not produce DNA using either treatment, two produced DNA without the carrier RNA.  The carrier RNA-modified Omega Bio-Tek protocol has since been effective in extracting DNA from Spigelia gentianoides, an endangered congener of S. marilandica, collected in Sneads, FL.  The use of carrier RNA with the Omega Bio-Tek protocol was generally effective in getting or increasing DNA concentration in Spigelia species.