Michael Marchizza Poster

Genetic Variation In Genus Magnolia Using Chloroplast Gene Spacer Sequences

Abstract

The Family Magnoliacae are woody trees and shrubs comprising 12 genera and about 220 species. There are about 75 species in the Magnolia genus. Eight of these are native to North America. Some of these species were introduced to England in the 18th century for ornamental purposes. Many of the natural and heritage cultivars are still maintained both here in the United States and in England.

It would be of interest to study the genetic diversity in these cultivars relative to the diversity in natural populations. Though the word Magnolia has come to be associated with the Southern United States, there are some species of Magnolia that thrive throughout the Eastern United States. Two of the native American species, Magnolia virginiana (the sweet bay magnolia) and Magnolia grandiflora (the bull bay or Southern Magnolia) will be used for an initial investigation into genetic variation both within each species and between the two species by comparing a region of chloroplast DNA.

The sweet bay magnolia is a graceful evergreen (southern habitats) to semi-evergreen (northern habitats) that performs well in a wide range of soil conditions from wet to slightly dry. It often grows as a multi-trunked clump and can be seen as shrub-like in the northern part of its range and taller and more tree form as you move farther south. Because of its form and fragrant ivory, lemon-scented flowers it is often used in landscaping.

Southern magnolia is a large, broad-leafed evergreen. It typically has a straight and erect trunk with spreading branches. Its large, white fragrant flowers make it ideal as a landscape tree.

DNA was extracted from individuals of both species. The DNA was isolated and a set of primers were used for PCR (polymerase chain reaction) in order to amplify the chloroplast regions under study. Chloroplast genomes resemble large bacterial plasmids or small chromosomes. Their sizes range from 110,000 to 160,000 base pairs. They were the first plant genomes to be characterized, and because of their small size and limited number of repeated elements, they are excellent candidates for use in genetic variation studies in plants.Once the chloroplast genomic regions were amplified, they were then sequenced to compare the genetic variation of the individuals within that region.

Eventually, additional specimens representing more of the populations from all over the Eastern United States will be collected and compared in order to produce genealogies.

Finally, other primers will be used to amplify and sequence other regions of the chloroplast DNA and also regions of the nuclear ribosomal DNA.

Introduction

The individual plants chosen are all representative of their respective species. All four of the plants studied were found on the grounds of the Museum Support Center in Suitland, Maryland.

Magnolia virginiana is characterized by a small multi-stemmed trunk, bright green oval foliage, and small creamy white flowers. The aggregate elongated fruit changes from green to red as it matures and ripens in August.

Magnolia grandiflora is characterized by a straight trunk with spreading branches, large dark glossy evergreen leaves, and large white flowers. The fruits are reddish-brown cone-like structures that fully mature in the autumn.

Materials and Methods

Leaves were collected from two individuals of each species. The leaves were frozen using liquid nitrogen, then crushed with a mortar and pestle. DNA was isolated from the leaf tissues using the Qiagen Plant Tissue Dneasy Plant Mini Kit. The leaves were ground under liquid nitrogen to a fine powder to disrupt the plant material. Buffers and enzymes were added and the mixture was incubated in order to lyse the cells. After a series of steps involving other buffers and centrifugations, the cell debris was discarded and the DNA was eluted with 200 ul of water into a microcentrifuge tube. Next, an agarose gel was run with a molecular marker ladder in order to see if DNA was present and to estimate its concentration in ng/ul.

The DNA was then amplified using PCR. In order to amplify the chloroplast trnL genomic region, two primers were used: trnL-c and trnL-f. PCR ready to go beads were used because these already contain the necessary buffers, salts, polymerase and nucleotides needed for the reaction. Samples of DNA from each of the four individual Magnolia plants, in addition to pea plant DNA (positive control) were prepared for PCR, along with a negative control in separate tubes containing PCR ready to go beads. The tubes were then placed in a thermal cycler. The thermal cycler was programmed to run at 94 deg C for 3 minutes, and then to run 35 cycles of 94 deg C for 30 sec, 56 deg C for 30 sec and 72deg C for 1 minute. This cycle allowed the DNA to melt, the primers to anneal, and the strand to lengthen, copying the trnL template region repeatedly. Each cycle increased the number of strands produced. After the final cycle, the cycler heated the mixtures to 72 deg C for 7 minutes, then kept them at a constant 4 deg C until needed for the next step. The PCR products were then loaded into agarose gels in order to see if the amplification had occurred.

In the gel lane 1 contained the negative control, lane 6 contained the molecular ladder, and lanes 11 and 12 contained the pea control. Lanes 2-5 contained the M. virginiana DNA samples and lanes 7-10 contained the M. grandiflora DNA samples. The gel was then visualized using UV light. The results show that the trnL chloroplast region was successfully amplified for at least one of the plants from each species. The PCR bands for the Magnolia samples are seen at mid-gel and appear to be in the range of 800-900 base pairs. The bands for the pea appear to be in the range of 700 base pairs. The primers can be seen at the lower end of the gel.

Next, the PCR product was cleaned using GeneClean II. By mixing the PCR product with NaI and glassmilk, the extraneous materials were washed away leaving only the chloroplast genomic trnL region. The PCR product produced after the cleaning was then loaded into an agarose gel and visualized using UV light. After determining PCR product was present, the DNA in the remaining solution was ready to be measured by fluoremetry in order to ascertain the DNA concentration in ng/ul. The concentrations were found to be 72 ng/ul for M. virginiana 1, 68 ng/ul for M. virginiana 2, 67 ng/ul for M. grandiflora 1 and 66 ng/ul for M. grandiflora 2. The DNA was then ready for the sequencing reaction and clean-up for submission to the sequencer program using the ABI3100.

Results and Conclusion

The DNA was then prepared for the sequencing reaction Each DNA sample was mixed with each of four different trnL primers (trnl-c, trnL-d, trnL-e, and trnl_f). The tubes were then placed in the Thermal Cycler. After completion of the sequencing reactions, the sequencing products were cleaned and purified using sephadex. They were then submitted on a submission template to the ABI 3100 sequencer. The sequencer read the DNA for each of the four Magnolia samples and produced a visualization of the sequence of bases in the region submitted.

The example to the right shows how as each base is read in the DNA sample by the sequencer, a different color of light is produced depending on the dideoxynucleotide detected: Red Thymine, Blue Cytosine, Green Adenine, and Yellow Guanine.

A chromatogram is then produced so that the base sequence can be read and analyzed. The sequenced regions of the Magnolia trees are then aligned. For the region that was sequenced and compared, there were no differences found in base sequence for the individuals involved. Not only were the sequences identical for the two Magnolia grandiflora individuals, but they also matched the sequence for the Magnolia virginiana individual. At this location, no variation in the genetic sequence was seen for the approximately 300 base pairs sequenced. However, this only included a small region of the total base pairs and could have included a region that was conserved in this Genus.

Acknowledgements

I would sincerely like to thank my Supervising Scientist, Dr. Elizabeth Zimmer, along with Carrie McCracken, Lee Weight and the rest of the staff at the Laboratory for Molecular Science in Suitland, Maryland for all of their help, knowledge and time given to me. Also a special thanks to Mary Sangrey, the Smithsonian National Museum of Natural History and the National Science Foundation for giving me the opportunity to have this wonderful experience.

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The information presented here represents preliminary research as the result of ten-weeks of investigation in-residence at the National Museum of Natural History. This is not an official publication of the information.

As preliminary information, results and/or findings should not be cited as part of conclusive work. Please contact the authors first if you wish to utilize the information presented here.


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Research Training Program VIRTUAL POSTER SESSION 2003

Research Training Program
VIRTUAL POSTER SESSION
2003