Evidence for low genetic variation in interrupted fern, and implications for clonal growth By Rebecca Dalley Abstract An investigation of the possibility of ancient genets in interrupted fern, through use of molecular techniques and measurements. RAPD-­‐PCR was used with 39 different primers, in order to try and find variation in this species at three separate locations at Wintergreen Ski Resort in Virginia. Almost no variation was found, only one primer produced variation, and only in four of the one hundred and thirty samples run. This result allows us to hypothesis that this particular species of fern must have a very low rate of genetic variation. It also allows for the possibility that many of these samples could be genetically identical and very old. Introduction Clonal growth in plants is a form of asexual reproduction. Clonal growth consists of genets and ramets. Genets consist of all plants derived from a single zygote and are genetically identical to other seemingly independent plants. Ramets are potentially or actually physiologically independent units of a genet and have all functions common to the physical individual of non-­‐clonal organisms (Pan 2001). Clonal growth in plants can be used to approximate the age of the ramets, by measuring the distance between known ramets of a genet when the growth rate of the plant is known. When physical connections are visible then it is easy to calculate the age. If the connections between ramets have rotted, it is still possible to age the plants by using their DNA and examining common markers to see if they are identical. Studies on other plants such as Picea mariana found genets containing multiple stems that were estimated to be around 1800 years old (Laberge et al., 2000). With improving methods for genetic analysis, longer genet life spans have been reported in shrubs, herbs and grasses, 1 indicating that they are several hundreds to thousands of years old (Escaravage et al., 1998; Stehlik et al., 2000; Wesche et al., 2005; de Witte et al., 2010). Research on clonal growth in Populus tremuloides, a species of common Aspen, resulted in an approximated age range of 169 to 1998 years old (Ally et al. 2008). RAPDs (Random Amplification of Polymorphic DNA) are simple primers that are ten nucleotides in length. They are randomly assigned in PCR reactions to an unknown DNA sample in order to discover possible genomic divergence in the DNA. It cannot be used to determine actual genetic sequences, but is useful in making simple conclusions about whether the specific organism is identical to other units or different from another. They have a wide range of applications in gene mapping, population genetics, molecular evolutionary genetics and plant and animal breeding (Bardakci 2000). RAPDs are a low cost method of genetic analysis, though there is still some debate on the reproducibility of RAPDs. Interrupted fern or Osmunda claytoniana L. is a fern that grows in the Eastern US from the great lakes to Mississippi, and also in most of Eastern Canada.1 Ferns and many other plants are known for using horizontal stems, called rhizomes, for asexual reproduction and clonal growth. These rhizomes creep along just above or below the soil, take root, and form ramets. Interrupted fern is known to grow approximately 0.6 cm per year (J. Steven pers. comm.). Using this information, we can measure the distances between known ramets in a genet and calculate the approximate age of the fern genet. A similar plant commonly called the bracken fern was studied using a similar method and the results estimated that the different genets ranged from 429 years to over 1,000 years old (Werth et al. 1993). 1
Plants.usda.gov 2 Interrupted fern growth is slow. We suspect that if clonal growth is as extensive as all the fern groups appear in the forest floor, then these genets could be very old indeed. It is unknown how old the ferns are because the physical connections rot away and no visible connection remains in plants that are farther away. Without using genetics to compare the possible ramets, there is currently no other way to determine their age. I investigated what the sizes of genets were, so that I could discover how old the genets were by measuring the distance between ramets and multiplying their length by the known growth rate of the interrupted fern. The ferns we studied were collected in three locations2 at the Wintergreen Resort, Nelson County, Virginia: Fern Place (the front yard of a house off of Laurel Springs Drive), Cedar Drive (the biggest area and mostly forest) and on Blackwood Drive (behind the dumpster). If all or most of the ferns are from the same genet, or if they contain plants from many different little genets, then we can calculate the approximate age of the fern genet(s). If these ferns are discovered to be very old, then they need to be protected from harm. The more knowledgeable we are about the interrupted fern and clonal growth, the more we can investigate clonal growth as a method of asexual reproduction. It will help in developing an understanding of the genetic patterns and traits exhibited by ramets in a genet, not just of the interrupted fern, but of all plants that use clonal growth as a means of reproduction. It will elucidate the history of the current forest understory, and provide a possible age for the current state of the forest. 2
GPS coordinates: Fern Place ± 17ft N 37.92615°, W 078.95555° [elevation 3,412ft] Cedar Drive ± 17ft N 37.91665°, W 078.96222° [elevation 3,564ft] Blackrock Drive ± 16ft N 37.91116°, W 078.93894° [elevation 3,312ft] 3 Methods The ferns we are sampling from are located in northern central Virginia in the temperate region. They grow on land that has been part of Wintergreen Resort since the 1970’s. The forest is primarily deciduous. It contains oak, hickory and maple trees at low and middle elevations, and at high elevations the forest is mainly coniferous. The forests used to be primarily chestnut trees, but after the blight of the chestnut3, it is almost impossible to find any surviving mature specimens. The forests in this area were possibly logged, but they were never farmed or plowed. The ferns grow in partial to full shade under the tree canopy. The forest floor is populated with evergreen shrubs typical of the mesophytic forest and a dense, nutrient-­‐
demanding flora (Weakley et al. 2012). At the Fern Place location, an x -­‐ y coordinate plane of 15 m by 10 m was plotted with measuring tapes and flags, and then samples were taken every meter with a possible deviation of 40cm. A small 4 m x 4 m plot inside the larger plot was intensely sampled and every plant was recorded (see figure 1). The Cedar Drive location had an x, y coordinate plane of 50 m by 50 m, and samples were taken and recorded every 5 meters with a deviation of 2.4 m, with a central, intensely sampled area of 10 m by 10 m where samples were taken every meter. Using sterilized scissors, the plant tissue was collected when the new growth reached approximately six to twelve inches above ground. One or two fiddle-­‐heads and some new leaves were removed, placed into individual plastic bags, and instantly frozen on dry ice at each location. All 3
The Chestnut Blight started by the introduction of an Asian species of chestnut tree in NY in 1904 and after about forty years the American chestnut was almost extinct. American Chestnut: The Life, Death and Rebirth of a Perfect Tree (Freinkel). 4 samples were collected in late April or early May of 2013, as fiddle heads were approximately 6 to 12 inches high (see figure 2). Seventy percent ethanol was used to sanitize all the surfaces used, including the mortars and pestles. (This also assists with limiting the possibility of DNA contamination.) The DNA was extracted by grinding the plant tissue under liquid Nitrogen using a mortar and pestle, then a DNeasy® Plant Mini Kit (QIAGEN™, Germany) was used. The Quick-­‐Start protocol was followed and step 11 was repeated once, which resulted in 200 µl of liquid containing the DNA. The DNA solution was then quantified using the Qubit™ fluorometer. The protocol was followed and 10 µl of each DNA sample was tested. The TC-­‐5000 gradient thermal cycler (TECHNE) was used for the PCR reactions. The DNA was diluted to have 60 ng of DNA per reaction and each reaction was 25 µl total volume. We used AmpliTaq Gold® 360 Master Mix (invitrogen™), as well as PCR grade water when diluting. Thirty-­‐five primers (Eurofins MWG Operon) (see Table 1) were tested on four different DNA samples taken from the Fern Place location. The primers were re-­‐suspended with 1xTE at a pH of 7. Two DNA samples were known ramets because of physical connections, and one was behind the Fern Place plot, and the other across the street from the Fern Place plot. The PCR program used was an initial denaturation of 5 minutes at 95°C. Then forty cycles of: Denaturation -­‐ 30 seconds at 95°C, Annealing -­‐ 45 seconds at the maximum temperature for the primer (Tm), Extension -­‐ 2 minutes at 72°C. The final step was an extension of 7 minutes at 72°C, then hold at 4°C until used in electrophoresis. 5 Electrophoresis gels of 1.5% agarose were run at 150 volts, and used to discover possible bands. The DNA was stained with EZ-­‐Vision one (Amresco®), and we used a 2-­‐Log DNA ladder (New England BioLabs®) then changed to a more accurate TrackIt™ 100 bp DNA Ladder (invitrogen™ by Life Technologies™). The agarose gels were imaged with a gel documentation system (Bio Rad™) and the images were quantified using Quantity One™ software (Bio Rad™). Results: The DNA extractions were all successful, and all of the DNA was quantifiable. The DNA concentrations ranged between 8 µg/ml to 100 µg/ml. The average concentration for the DNA was approximately 35 µg/ml. The PCR reactions were successful, and only 2 primers did not amplify the fern genomic DNA at all. In our samples from the Fern Place location, one of the DNA samples was from behind the house at Fern Place, one sample was from across the street from Fern Place and the other two were known ramets in Fern Place, identifiable because of a visible physical connection. The electrophoresis gels showed that there were no obvious differences in the four DNA extractions that we ran with all of the primers, except for one primer (OPBF-­‐13). This primer was then run on 130 of the 163 total extractions from Fern Place. Only four DNA samples showed any variation in all of these reactions (See figures 3 & 4). In Figure 5 we can see that the area of these different plans is located randomly, but with lots of room in between each. This could be because they are different through sexual reproduction and the zygote landed in that specific location, or because of a mutation in the individual fern unit. Some of the DNA samples would not amplify during PCR, and approximately 30% never show up on the gels. 6 The Cedar Drive fern DNA never amplified in PCR reactions. Eight tissue samples were extracted and run with multiple primers that worked with the Fern Place DNA and no bands were seen. 12 10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 Figure 1: A plot map of the fern samples taken at Fern Place (± 17ft N 37.92615°, W 078.95555° [elevation 3,412ft]). Samples were taken every one meter in a 15 by 10 meter square. A 4 by 4 meter square was intensely sampled and a tissue sample was taken of every fern in that location. 7 Figure 2: An image of an interrupted fern at the location called Fern Place, on the day of tissue sample collection. (May 2013) Figure 3: UV ray illuminated DNA from PCR using OPBF-­‐13 primer. The bands are in 1.5% agarose gels that have run for approximately 1.5 hours at 150 volts. The ladder used in well 1 and 12 is TrackIt™ 100bp DNA Ladder (invitrogen™ by Life Technologies™). The left side is the original image and the right is with the analysis software set at 4% error for matching bands. Most are quite similar, but there are four bands in the last visible sample DNA lane (number 10). 8 Figure 4: Electrophoresis gels that were run at 150 volts, for 1.5 hours. Primer OPBF-­‐13 was used. Both gels show a slight variation of band lengths on well number 9 when counting from left to right. 12 10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 Figure 5: A graph plotting all samples taken at Fern Place. The four red marks are the four DNA samples that had different bands from all of the others that were amplified. 9 Primer Used Number of Bands seen OPD-­‐01 OPD-­‐02 OPD-­‐03 OPD-­‐04 OPD-­‐05 OPD-­‐06 OPD-­‐07 OPD-­‐08 OPD-­‐09 OPD-­‐10 OPD-­‐11 OPD-­‐12 OPD-­‐13 OPD-­‐14 OPD-­‐15 OPD-­‐16 OPD-­‐17 OPD-­‐18 OPD-­‐19 OPD-­‐20 P-­‐A-­‐17 P-­‐B-­‐15 P-­‐C-­‐06 P-­‐D-­‐01 P-­‐D-­‐02 P-­‐D-­‐05 P-­‐D-­‐06 P-­‐D-­‐07 P-­‐F-­‐18 P-­‐G-­‐19 P-­‐A-­‐12 P-­‐A-­‐13 P-­‐B-­‐17 P-­‐C-­‐01 OPBF-­‐11 OPBF-­‐13 3 5 6 1 8 to 10 6 5 6 2 to 3 8 9 10 7 10 5 7 0 4 7 6 9 11 7 12 8 12 6 6 6 0 5 to 11 5 6 12 11 5 or 9 OPBF-­‐14 OPBF-­‐16 9 5 Noted Differences None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None Yes, there were four different banding patterns seen in different DNA samples. Not well amplified Not well amplified Sequence 5’ to 3’ ACCGCGAAGG GGACCCAACC GTCGCCGTCA TCTGGTGAGG TGAGCGGACA ACCTGAACGG TTGGCACGGG GTGTGCCCCA CTCTGGAGAC GGTCTACACC AGCGCCATTG CACCGTATCC GGGGTGACGA CTTCCCCAAG CATCCGTGCT AGGGCGTAAG TTTCCCACGG GAGAGCCAAC CTGGGGACTT ACCCGGTCAC CCTGGGCCTC AGGGGCGGGA CGTCTGCCCG GCTGTAGTGT CGCACCGCAC CTGGCGGCTG GCCTGGTTGC CGTGGGCAGG AGGCCGCTTA GTGGCCGCGC CCTGGGTCCA CCTGGGTGGA GAGGGCGAGC GCGGCTGGAG GACGACCGCA CCGCCGGTAA CCGCGTTGAG AGGGTCCGTG Table 1: A list of all the DNA primers used and the number of bands discovered after PCR amplification using the genomic fern DNA extracted from the collected samples. All primers were from Eurofins MWG Operon and resuspended in 1x TE at a pH of 7.0. Discussion The results from this research demonstrate that the ferns’ genome size must be immense for almost all the primers to successfully amplify in PCR. This is a very unusual thing to happen with RAPD-­‐PCR, as evidenced in other journals that used similar methods. In one similar 10 study with RAPDs, they tested 60 primers and only selected 7 primers that had clear and reproducible results (Chen et al., 2006). Another study found that, when using RAPD-­‐PCR all 25 primers amplified, but there were no polymorphic bands, and thus deduced that there was very low genetic diversity (Li et al., 2006). The interrupted fern possibly has a very large genome or chromosome number, which would explain why the primers all found DNA sections to amplify. The chromosome number found in other species of the genus Osmunda is n = 22 (Zhang et al. 2008). As all five of these species had n = 22, it would be reasonable to hypothesis that claytoniana L. also has 22 chromosomes. This is a fairly large number and, depending on the interrupted ferns chromosome size, could explain why everything amplified so well. An alternative method of discovering variation in the fern genome would be to use microsatellites. Microsatellites are perfect for discovering genetic divergence or similarities within many different populations. Microsatellites (also known as inner simple sequence repeats, ISSRs) have proven to be useful in population genetic studies, especially in detecting clonal diversity and fingerprinting closely related individuals (Zietkiewicz et al., 1994; Wolfe and Liston, 1998; Esselman et al., 1999). Microsatellites are small sections of a particular nucleic acid sequence containing small repeats of base pairs. The repeated sequence is normally no longer than 6 base pairs and is repeated multiple times in an allele. There is a possibility that multiple microsatellite repeats can be found in a genome. The length of these repeats can frequently be specific to a species or an individual. They may be highly polymorphic, especially if long and uninterrupted, and they are therefore useful genetic markers to determine how individuals are related (Goldstein 1999). 11 If we decide to use this other method, hopefully we can find more variation and be able to successfully deduce which units are identical to which. There is another alternative hypothesis – that all of the ferns in Fern Place are actually identical except for the four different ferns, but that seems unlikely. We cannot make a definite conclusion until further research is done. The DNA in Cedar Drive never amplified in any of the RAPD-­‐PCR. This could be because those particular ferns were not as reproductively active as the ferns located in Fern Place. The ferns found on Cedar Drive were also much smaller, more unfurled, and did not appear have many dark green leaflets containing the spores for reproduction. The ferns in Fern Place were very large and healthy in appearance. 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