The group collected plant samples from three locations on Mount Obama using a quadrat sampling method to identify species through DNA barcoding. They were able to extract DNA from 11 samples and sequence DNA from 8 plants. By inputting the sequences into databases, the group identified three species: Emilia coccinea, Solanum, and Passiflora suberosa. There was no overlap between the species found at the peak and those previously collected at the base. The experiment proved DNA barcoding can accurately identify biodiversity in Antigua.
1. DNA Barcoding: Spring 2019
Mount Obama PEÂK
Group 1: Sam Beyin, Joseph Mitchell, Ed Bauter, Adeelah Sayed, Precious Nwoko
2. DNA Barcoding
❏ A method of identifying species based on a common designated gene sequence.
❏ A region of chloroplast gene rbcL, RuBisCo large subunit, is used for plants.
❏ Advantages include minimal sample needed, applicable to all stages of life, and
can differentiate phenotypically similar species.
❏ Our goal in this project was to identify plant species on the peak of Mount
Obama, and compare them to those found at the base of the mountain.
❏ We collected 20 plants in total, taking only small pieces of the leaves to
minimize any disturbances.
3. Sampling locations
❏ We used the
quadrat sampling
method (1m:1m
▦) to choose and
collect our plants.
❏ Q1: PEAK
❏ Q2: initial
❏ Q3: further
descent
Mount Obama (Boggy Peak), Shekerley Mountains
ELEVATION -- 402m / 1319ft
4.
5. Methods
❏ DNA Extraction: We were limited to
testing 11 samples. Using in the DNeasy
Plant Handbook, we extracted DNA from
the samples.
❏ DNA Amplification: Following the
protocol, PCR was done using primers
rbcLaF (2µM) and rbcLaR (2µM)
6. Process
❏ Sequence Analysis: PCR product
was analyzed on 1% agarose gel
using gel electrophoresis to
confirm DNA had been
successfully extracted.
❏ Sample Sequencing: successful
samples were sent to Genewiz
for sequencing.
7. Analysis
❏ DNA Analysis: Following the
bioinformatics directive, we
collaborated and submitted our
sequences from DNA Subway to BOLD.
❏ We used the results from BOLD and
photographical evidence of the
specimens to positively identify the
plant genus/species we found.
8. Emilia coccinea
Common name:
Tassel Flower or Scarlet Magic
Medicinal Use:
Leaves can be crushed and used
topically to treat sores. They can
also be infused with other herbs
and used in eye drops.
10. Passiflora suberosa
Common Name:
Corky Passion Vine
Medicinal Use: Leaves can be
crushed and used topically to treat
hives and alleviate itch. Leaves
may also be incorporated into
mixture to alleviate indigestion.
11. Discussion
❏ 8 / 11 DNA samples were successfully amplified using PCR.
❏ We were able to sequence the DNA from these 8 plants, input our sequences
into the NCBI database, and identify the plants we found.
❏ Possible sources of error:
❏ Measurement (pipetting) errors
❏ Deviation from protocol in cycling/amplifying
❏ Transferring errors when changing test tubes
❏ Minimizing these errors in future experiments will increase the yield.
12. Discussion
❏ In fall 2018, a previous research group collected samples from the base of
Mount Obama.
❏ Our group collected plants from the peak to see if there was any species
overlap between the two locations.
❏ We did not note any species overlap with the previous group. This could be
due to the great amount of biodiversity in our rural environment and our
limited sample size.
Morinda pubescens, Mimosa pudica, Turnera ulmifolia
13. Conclusion
❏ The success of this experiment proves DNA barcoding can be an accurate
way to identify species on Antigua in order to contribute to a global
conservation effort.
❏ We hope that our contribution to the Barcode of Life Data System can help
further the knowledge we have on species diversity on the island.
14. Acknowledgements
❏ We would like to thank Dr. Joseph Cross, Dr. Karron James, and Dr.
Michael Friedman for helping us make this research possible.
❏ We would also like to recognize the AUA Research Grant and Dr. Richard
Millis for providing the necessary funding for this project.
❏ A special thanks to Marly Chaudry, Sandra Secondus, and Josephine
Sebagisha who allowed us to compare our findings at the peak of Mount
Obama in Spring 2019 with theirs at the base of the mountain in Fall 2018.
Editor's Notes
Stayed on same side, shaded area
Within species DNA polymorphism is typically low or absent in coding pDNA regions like rbcL and matK but can be frequent in non-coding regions like trnH-psbA.
Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification.
We verified and modified the alignment manually where inconsistencies were found, and translated the sequences into amino-acid sequences to guide the alignment. Forward and reverse sequences were trimmed at both ends of the alignment in order to avoid too many missing data at the ends.
Percentage identity >90% used with basic local alignment search tool BLAST to positively ID species or at least genus. Sequences availability in the database is a major limiting factor of DNA barcoding.
Farming is done near the base of the mountain, so previous land clearing could have also contributed to our lack of overlap.
Future direction - get more samples, better lab technique
The use of DNA sequences as barcodes to discriminate between species is based in part on the assumption that species bear unique barcode haplotypes. But large percentages of species were found to share haplotypes in several barcoding studies.
