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Fall 2012 Newsletter

Barcoding Lilacs in Highland Park
Summer 2012 RWC Faculty Research Grant Report

By Dr. David Roll


Forty-seven different lilac plants were selected and studied this summer with the help of the Highland Park horticulturist Kent Millham. Three different visits to the park were made to collect leaf samples. Each time a plant was selected, we recorded the name of the plant by genus and species and identifier number, location of the plant with GPS coordinates, took digital pictures of the plants and the individual leaf morphology and collected two leaves to keep for DNA analysis in the laboratory. Once the leaves were collected in individual paper envelopes, they were transported back to the lab. The following day a paper punch was used to punch out a sample leaf for DNA analysis. Each sample was saved in an individual plastic sterile microfuge tube and then processed for DNA isolation using the procedure described in the lab manual developed by Cold Spring Harbor laboratory. The remaining leaves were then placed between sheets of Whatman #2 filter paper and pressed to dryness for a two to four week period. The plant names, identifier numbers and GPS locations are collected on an Excel spreadsheet and the digital pictures are stored on computer in a folder. The leaf samples have now been placed in plastic sheets and stored in a three-ring notebook for future reference.

The DNA isolation was completed for each plant using the Promega kit and samples were frozen at -20oC in microfuge tubes. This required the homogenization of the leaf fragment, lysis of the plant cells, and precipitation of the DNA by alcohol. PCR primers were selected and ordered from IDT to use in the PCR amplification protocol. The first round of analyses was done by analyzing the selected sequence of the Rubisco gene which is the standard gene used in DNA barcoding. DNA samples were then taken for PCR analysis using the amplification protocol listed in the Cold Spring Harbor protocol. Some 15 to 16 plants were analyzed by PCR after each collection. After running the PCR amplification, each amplified DNA plant sample was analyzed by agarose gel electrophoresis to ensure that the correct DNA sequences were amplified. After confirming the correct amplification of one major band, the original PCR reaction was taken and submitted to GENEWIZ, INC. for DNA sequencing. Two DNA sequences are collected for each lilac and then submitted to the DNA Subway web site where the data can be compared and analyzed using bioinformatics software. The DNA sequence data is stored as individual data files on computer.

A username and password were set up for submitting DNA sequence data to the DNA Subway program. The DNA sequence data has been collected and compared. To date, the sequence data for the Rubisco gene is close to 99% identical for all the lilacs analyzed to date. Additional genes have also been initially looked at this summer and that data is now being collected and analyzed. This includes the matK gene and the trnH-psbA gene sequences found in all plants. We are interested in finding additional DNA sequence data that may be able to distinguish genetic differences in the individual plants. The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and this may also be a useful gene to sequence in lilacs. A new universal primer set was chosen to amplify this gene in lilacs and that has been successfully done in two of the lilacs. The non-coding trnH-psbA intergenic spacer region found in plastids was successfully amplified in two lilac plants. These amplified sequences could be observed on agarose gels. These amplified sequences are now waiting to be sequenced by GENEWIZ, INC. These results will be added to the results with the Rubisco gene sequences. There are previously published reports to suggest that the trnH-psbA sequence combined with the Rubisco gene sequence can be used to correctly identify and discriminate between related species of land plants.

Initial results indicate that the Rubisco gene (rbcL) is not a good sequence to distinguish between different species or variants of the lilac plant but the rbcL gene sequence is useful in distinguishing between lilac and other land plants such as maple, oak, and pine trees. The other genes Matk and trnH-psbA may provide more differentiation between lilac species and variants. After sequencing the MatK and trnH-psbA gene sequences, we may be able to better distinguish between lilac species and variants.

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