Abstract

1

PCR Analysis as a Possible Means for Sex
Determination of Eastern Redcedar (Juniperus
virginiana) Jaclyn Knibbe, Dr. Elizabeth
Heeg-Truesdell, Dr. Todd Tracy Biology
Department, Northwestern College, Orange City
Iowa
Female Red Cedar
Male Red Cedar
Abstract Although native to northern U.S., the
eastern red cedar (Juniperus virginiana) has
quickly become invasive in areas of the Midwest
where natural regeneration of planted trees is
occurring. Because of the negative impact of red
cedar on pastureland and prairies, ecologists
have begun finding ways to control its spread.
The best known methods include controlled burns
and cutting the trunk at its base. However, adult
eastern red cedars, once burned, can still cast
large shadows on nearby plants needing sunlight. 
Additionally, the dense, prickly foliage of the
tree make it difficult to cut the trunk. We
propose an alternative method via sex
determination of seedlings by means of PCR
analysis.  That is, female eastern red
cedars could be identified via PCR and either
not planted or removed as seedlings in a more
efficient manner than if they were allowed to
grow to maturity.  Farmers would still benefit
from the eastern red cedars maintenance-free
windbreaks, but without the hassle of their
eventual invasion into pastureland and prairie.
 Our research investigated a gene (Accession
AF151429) that codes for a pollen protein, and we
tested whether female red cedars lack this gene
and can thus be identified and removed before
maturity.
Introduction The invasive nature of the eastern
red cedar has become a significant problem with
farmers in the Midwest and surrounding areas. The
current methods for mass removal of the juniper
involve controlled burns or cutting the tree at
the base of its trunk. Controlled burns, however,
leave larger adult trees still standing. These
large and dense foliaged adults can still cast
great shade upon the surrounding prairie life.
Cutting the tree at its base can become a
difficult procedure as well. The foliage is very
thick and spiky and requires thick gloves to
prevent cuts. The eastern red cedar is a rare
plant species (among 4) in that it has separate
male and female individuals. If we can find a
gene that is only found in one sex, we can select
against it. In Papaya plants, for instance, once
the gene for a male was found, they amplified
that gene with PCR and noted that only males
contained a band in the gel electrophoresis.
Those seedlings containing the male band could
then be removed. Similarly, our research began
by finding the sex-specific gene located in the
eastern red cedar. A pollen gene was found with
nucleotide BLAST on PubMed The gene was then
examined if it was a sex-specific gene or not.
(Figure 2)
Figure 2 PCR steps 1) Denaturation Heat
briefly to separate DNA strands 2) Annealing
cool to allow primers to form hydrogen bonds with
ends of target sequence. 3) Extension DNA
polymerase adds nucleotides to the 3 end of each
primer.
Figure 1 The red cedar was originally
concentrated in Wisconsin and northern Minnesota.
Fire suppression allowed for the red cedar to
spread through the Great Plains and eastern.
Discussion After making changes to
the protocol and changing PCR temperatures and
duration time periods, we were able to extract
enough DNA for a successful PCR reaction. (Figure
4) This gave bands in the 500bp region, which
indicated that the pollen gene (520bp) was
located and copied. Female 2 did not show a band,
however. Because primer-dimers did show up, it
was determined that the solution was at least
loaded. Two conclusions could then be made.
Either no DNA was extracted from female 2
(considering the difficulty in DNA extraction
with conifers), or females 1 and 3 were false
positives. Contamination could have occurred
where male DNA got into the female DNA. With
proper lab techniques used, however, this
possibility was unlikely. Gloves were used
throughout the entire procedure and filter tips
were used for transferring solutions. To
make a definite conclusion, a positive control
was needed. Photosystem II primers from the
BioRad (2008) primer kit were used as a positive
control (Figure 5). Photosystem II is the first
protein complex in light-dependent
photosynthesis. Every plant that photosynthesizes
contains the gene for photosystem II at a length
of 455bp. The eastern red cedar photosynthesizes,
and therefore contains the gene for photosystem
II. Figure 5 showed that both males and females
contained a band around 500bp. This concluded
that the photosystem II primers found DNA and
were able to make copies in the PCR reaction.
Since female 2 did not show a band for
photosystem II, it must have not contained any
extracted DNA to begin with. Therefore, females 1
and 3 were not false positives and contained the
pollen gene. We conclude then, that female
Juniperus virginiana contains the gene sequence
for pollen formation. This is not entirely
uncommon, however. Most organisms that separate
into male and female during development contain
most of the same genetic information. It is the
transcription factors (normally located on the Y
chromosome in males) that make the physical,
expressed differences between males and females
(Gilbert 2003). In the case of the eastern red
cedar, females have the pollen gene but lack the
expression factors necessary to produce pollen
protein. Conversely, eastern red cedar females
contain expression factors for berry production
while males do not.
Figure 2 Gene sequence of the pollen gene. For
better PCR accuracy, only half of the gene was
framed by primers (circled in red). The band
length around 520 bp should also show up after
gel electrophoresis to confirm the success of the
primers.
Red Cedar Benefits Dense foliage makes great
windbreaks Low maintenance grows
quickly Heartwood is good for furniture and for
warp-sensitive musical instruments.
Red Cedar Disadvantages Reproduces
quickly Shades out and steals nutrients from
native prairie life Has become highly invasive
in pasturelands
Figure 3 Graphical representation of
hypothesized PCR product bands. If females dont
contain the pollen gene, they will not contain a
PCR product band.
Results

• Figure 4
• Bands show up around 500 bp, indicating the
  pollen gene (520bp) was successfully detected by
  the primers and copied. Female 2 does not contain
  a band giving two possible conclusions
• Females 1 and 3 are false positives for the
  pollen gene
• Because DNA extraction was not always 100
  successful, Female 2 did not contain any
  extracted DNA initially.

Pollen Gene The pollen gene (accession
AF151429) was initially located to find
differences between several allergenic conifers.
Messenger RNA (mRNA) from pollen protein was
taken and converted into cDNA in order to
determine its sequence (Figure 2). Because the
pollen gene was converted back to cDNA from mRNA,
it is possible to use the sequence for seedlings.
If the gene sequence was pure mRNA, seedlings
would not likely be producing pollen and
therefore would not contain mRNA. It was
hypothesized that only male red cedars would
contain the pollen gene and would only contain a
band from the PCR product. Since females wouldnt
contain the gene, the primers would not locate
the specific sequence and would not make copies.
(Figure 3)
Because two conclusions were a possible from the
result of the gel from Figure 4, another PCR
reaction was performed with a positive control
using photosystem II primers. Photosystem II is
the first protein complex in light-dependent
photosynthesis. Every plant that
photosynthesizes (including the eastern red
cedar) contains the gene for photosystem II at a
length of 455bp. Therefore, both male and female
plants should contain a band for photosystem II
if DNA is present.
Future Directions Since the pollen gene is
not located on the Y-chromosome, it can not be
used as a sex-determinate gene. However, a
northern blot could be performed to screen for
the presence of mRNA. Since only males produce
pollen, they would only be producing mRNA to make
the pollen protein. Similarly, a western blot
could be performed to screen for the presence of
the pollen protein. Yet, the goal of our research
is to determine the sex of an individual when it
has not yet matured. Seedlings would not likely
produce pollen until they reached reproductive
maturity. Therefore, it might be difficult to
find any pollen mRNA or protein in seedlings.
A second option is to find the transcription
factors that initiate the expression of the
pollen gene. Females contain the pollen gene, but
since pollen is not produced, they must lack the
expression factors needed. This would require
more research in that these expression factors
have not yet been found and decoded. Only a
select number of genes have been decoded for the
eastern red cedar. If transcription
factors become too difficult to locate, we could
start from scratch and locate a completely new
gene hypothesized to be on the Y-chromosome. From
past research, the genes located on the
Y-chromosome have been found in dioecious plants.
Research by Ling, I. et al. (2003) indentified a
sex-associated gene in the tree, Ginkgo Biloba.
Of the 8,372 RAPD bands, only a 682 bp RAPD
maker generated by a primer (S1478) of random
decamer squence, named S1478-682, was found to be
associated with the male plants (Ling 2003). To
find a specific RAPD marker takes a lot of
research filled with many trials and errors. We
can search in the same region that the Ginkgo
sex-specific marker is to find a possible homolog
for the eastern red cedar sex-determinate gene.
Once again, this will require further decoding of
Juniperus virginiana genes. As more research is
done on the eastern red cedar, more decoded genes
will be available for sex-determination. In the
meantime, the best way to remove invasive cedars
remains with controlled burns and cutting at the
trunk.
Methods The DNA extraction protocol was
adapted from C.S. Kim et al., (Oxford Journal,
1997). Red cedar samples were collected using
the lid of a 1.5 ml microcentrifuge tube to clip
off pieces of the needles, which prevented any
contamination. Needles were ground up with a
mortar and pestle in one drop of 1
2-mercaptoethanol and 300 mul of extraction
buffer (250 mM NaCl, 25 mM EDTA, 0.5 SDS, 200 mM
Tris-HCl pH 8.0). After incubating at room
temperature for one hour, freshly prepared PVP
(10,000 MW PVP, Sigma, 6 of final volume) and ½
volume of 7.5 M ammonium acetate were added
individually. The solution was incubated on ice
for 30 minutes and centrifuged for 10 minutes
(10,000 g). The supernatant was then transferred
to a new tube and 1 vol isopropanol is added and
then incubated at -20º C overnight. The mixture
was centrifuged at 10,000 g and the supernatant
was discarded. The DNA pellet was dried by
blotting on a paper towel. DNA pellet was
resuspended in 500 mul of TE buffer (10 mM
Tris-HCl ph 8.0, 0.1 mM EDTA pH 8.0). The mixture
was placed in a boiling water bath for 5 minutes.
One vol chloroform-isoamyl alcohol (241) was
added and emulsified by inverted shaking. Another
vol of chloroform-isoamyl alcohol (241) was
added and emulsified. Once centrifuged (10,000 g)
for 5 minutes, the supernatant was transferred to
a new tube and 1 vol of isopropanol was added and
incubated at -20º C for 10 minutes. After
centrifuging (10,000 g) for 10 minutes, the
pellet was dried by blotting on a paper towel and
then resuspended in 30 mul of TE buffer.
Best PCR procedure for primers Denature
94C.2 minutes Loop (x40) Denature 94C.1
minute Anneal 59C.1
minute Extend 72C....2
minutes Final Extension 72C.10 minutes Hold
4C
Figure 5 The positive control, photosystem II,
shows that Female 2 did not contain successfully
extracted DNA. Pollen gene bands show to be
around 500bp and photosystem II bands show up
around 500bp as well. This also concludes that
both male and female eastern red cedars may
contain the gene for pollen.
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Acknowledgements Funding for this project was
provided by a Northwestern College Scholarship
Grant and a grant from the Iowa Science
Foundation.