1. Project:
Estimation of genetic variation among various
accessions of Shorea robusta plant using RAPD and
SCoT PCR primers
Name: Sarthak “Ankit” Patnaik
Dates of Project: 7/8/14 – 7/25/14
School of Life Sciences, Sambalpur University
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2. ACKNOWLEDGEMENT
I would like to express my deepest gratitude to Dr. Jogeshwar Panigrahi, School of Life
Sciences for allowing me to conduct research in his laboratory for the past six weeks. With his
guidance and teaching, I have learned several new lab techniques, which will undoubtedly prove
crucial to my lab work in the years to come. I am thankful to the head of the School of Life
Sciences for allowing me to work in the school.
It would also have been impossible to conduct the research here this summer without the
help of the research scholars: Sujit Kumar Mishra, Sen Seth, Alok Ranjan Sahu, Ramya Ranjan
Mishra, and Sobha Chandra Rath. Thank you for answering my many questions this summer and
teaching me more about plant genetics. The time you all spent outside of your own work to assist
me was invaluable.
Also, thanks to my family members for everything.
Jyoti Bihar, Sambalpur University
July 25, 2014 Sarthak “Ankit” Patnaik
University of North Carolina – Chapel Hill
Department of Arts & Sciences
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3. Introduction and Objectives
Shorea robusta, which is commonly known as śāl, is a common tree plant native to the
Indian subcontinent (Oudhia and Ganguali, 1998). Sal forests are prevalent in the foothills of the
Himalyas, and are distributed throughout Nepal, Bangladesh, and India, with approximately 10
million hectares located in India (Tewari, 1995). Sal trees have been shown to grow as evergreen
to deciduous trees in wet and dry areas, respectively (Devoe and Gautam, 2006). Although sal
trees were previously harvested for timber, it was later determined that several parts of the plant
can be utilized for various uses. These uses include production of livestock fodder (Panday,
1982), creation of leaf plates (Rajan, 1995), oil from seeds for use in cooking (Verma and
Sharma, 1978), as well as cow feed derived from seeds (Rai and Shukla, 1977), resin or latex
derived from heartwood (FRIB, 1947), and tannin and gum from the tree bark (Karnik and
Sharma, 1968). In addition to these more practical uses, sal has also found its way into several
Hindu scriptures and is also used in rituals and for medicinal purposes (Ahmad Wani et. al,
2012).
In scientific terms, the sal plant is known as Shorea robusta, with genus Shorea and
species robusta. This tree is a member of the Dipterocarpaceae family and the order Malvales.
According to the IUCN (International Union for Conservation of Nature), the plant is very
prevalent with no visible threat to its survival; the Shorea robusta conservation status is listed as
the lowest threat level or least concern (Ashton, 1998).
As we have seen, the Shorea robusta plant proves to be readily accessible in India, as
well as versatile, which makes it a good candidate for further genetic study. The objective of this
experiment was to better understand the genetic variation amongst obtained wild accessions of
Shorea robusta leaves. The project intended that I obtain 10 accessions of the plant so that DNA
could be isolated and purified from the explant samples. The DNA would then be amplified
using 10 different RAPD (Random Amplified Polymorphic DNA) and 10 different SCoT (Start
Codon Targeted) primers. After examining the amplified DNA using gel electrophoresis and UV
light analysis, the similarities and differences between band patterns could be noted, thus
revealing somewhat the genetic similarities and differences between the accessions. After
analysis, the data could be run through FreeTree software using Nei’s method for genetic
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4. distance. Finally, this data could be viewed using TreeView software to create a cluster tree
depicting the accessions.
Previously, Shorea robusta had not been worked with in this lab, so this project hoped to
accomplish a few things. Firstly, the project would allow for the estimation of genetic variation
amongst different accessions of this plant, so that we could see how the plant compares in
different environments around Burla. Secondly, the project will prove beneficial for future
projects as it helps standardize a protocol for DNA isolation and purification of a previously
unknown plant species. Lastly, the project allowed me to practice and apply techniques that I
learned throughout my stay in this laboratory, which includes DNA isolation and purification,
gel electrophoresis, PCR amplification, and use of FreeTree and TreeView software for genetic
analysis.
Materials and Methodology
The entire process of obtaining Shorea robusta explant samples and progressing toward
estimation of genetic variation required four distinct steps: DNA isolation and purification,
equilibration of isolated DNA, PCR amplification of DNA using RAPD (Random Amplified
Polymorphic DNA) and SCoT (Start Codon Targeted) primers, and finally analysis of genetic
variation using FreeTree and TreeView software. The procedural section will therefore be split
into different subsections for clearer organization.
I. DNA Isolation and Purification
Trial 1 (DNA Isolation)
DNA isolation and purification proved to be a very troublesome and time-consuming step
in the project, and caused a delay in the overall procedure. Previously, DNA had never been
isolated from the Shorea robusta plant in this lab, so the process had to be standardized before
we could proceed further. Because of this, several trials were undergone before a consistent
method for isolating and purifying DNA could be determined. Originally, we attempted to isolate
DNA from 10 accessions of Shorea robusta plant using the protocol for Cajanus cajan (pigeon
pea), standardized by Shivaramkrishnan et al. in 1997. This procedure involved first, obtaining
fresh leaf samples of Shorea robusta plant with masses ranging from 2 – 4 g. The masses are
listed in the following table:
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5. Explant # Mass (g)
1 3.30
2 2.45
3 2.63
4 1.95
5 2.17
6 2.00
7 2.24
8 2.38
9 2.40
10 2.88
Figure 1: Table listing masses of explant samples for DNA Isolation (Trial 1)
The samples were grinded into a fine powder using -198°C liquid nitrogen, to freeze and
preserve the DNA, with mortar and pestle. Each sample was placed into a respective, labelled
Oakridge tube, containing heated 20 mL extraction buffer (2% CTAB, 200 mM Tris-HCl at pH
8.0, 1.4 M NaCl, 20 mM EDTA, and 2% β-mercaptoethanol – buffer for Cajanus Cajan
protocol). The ten Oakridge tubes containing samples and extraction buffer were placed in a 65°
C water bath for 1 hour. After an hour, the tubes were allowed to cool to room temperature by
placing in an ice bucket, before an equal volume of chloroform:isoamyl alcohol (24:1 v/v) was
added to each solution; the contents were mixed by inversion. The tubes were placed in a
centrifuge for 15 minutes at 4° C and 8000 rpm in order to precipitate degraded proteins and
plant waste products. This way, DNA, located in the supernatant, could be separated from
unwanted compounds. The addition of chloroform:isoamyl alcohol and subsequent
centrifugation was repeated in order to further precipitate and remove degraded proteins and
waste products. After this, an equal volume of isopropanol was added to the supernatant in order
to precipitate out DNA and the solutions were centrifuged for 20 minutes at 4° C and 6500 rpm.
At this point, we expected to see DNA floating about in the solution but none was visible, a
problem compounded by the fact that much of the plant pigment had remained in the solution,
causing the color to remain a murky brown color. All solution was removed to leave behind
DNA, which was washed twice with 2 mL of 70% ethanol. After air-drying, the DNA was
dissolved in 2 mL T50E10 buffer.
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6. Trial 1 (DNA Purification)
Although it was not possible to determine if DNA had been successfully extracted from
the explant samples due to the dark brown color of the solution, we proceeded to the purification
steps of the protocol. This began with the addition of 10 μL of RNAse–A (10 mg/mL) to each
test tube, before placing the tubes in a water bath set at 37° C for 1 hour. After removing, 15 μL
of proteinase-K (20 mg/mL) was added to each tube before placing in 37° C and 65° C water
baths for 30 minutes and 10 minutes, respectively. An equal volume of
Phenol:choloroform:isoamyl alcohol (25:24:1 v/v) was added to each solution to further separate
DNA from proteins. The tubes were placed in a centrifuge for 10 minutes at 4° C and 5000 rpm.
At this point, however, the procedure faced its biggest obstacle, which ultimately grinded the
DNA extraction process to a halt. After removing from the centrifuge, the supernatant layers,
containing DNA, had solidified and could not be removed from the test tubes. Because the DNA
could not be separated from the other compounds, this trial could not continue.
Trial 2 (DNA Isolation using activated charcoal, new extraction buffer, and potassium acetate)
For this trial, the same protocol was used to isolate DNA as in Trial 1, with a few
important changes. In this case, a spatula-full of activated charcoal was added to the samples (in
this case, two samples marked as Samples 1 and 2) and mixed in while grinding using mortar and
pestle. Activated charcoal has been shown to work effectively in various plant DNA extraction
protocols due to its absorption qualities; the charcoal was used to absorb the phenolic compounds
and plant pigments that had plagued the purification steps of the previous trial. Along with this
change, the extraction buffer composition differed (Shorea robusta Extraction Buffer 1),
especially due to the addition of PVP to assist in removing phenolic compounds (3.5 % CTAB,
200 mM Tris – HCl at pH 8.0, 1.5 M NaCl, 40 mM EDTA, 0.5% β-mercaptoethanol, and 2%
PVP). The third was that 4 mL of potassium acetate was also added to each solution before the
first addition of chloroform: isoamyl alcohol. The potassium acetate was intended to neutralize
the DNA in the nucleoplasm in order to assist in precipitation of degraded proteins out of the
nucleoplasm. Even with the addition of activated charcoal and potassium acetate, it was difficult
to see whether any DNA had been extracted, although much more pigmentation was absorbed as
the color lightened from a muddy brown to lighter yellow. It wasn’t until after the samples had
incubated at -20° C overnight that DNA was visible in the solution.
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7. Trial 3 (DNA Isolation using new extraction buffer and NO activated charcoal)
For this trial, two new samples were obtained which were marked as Sample 3 and 4.
Samples 3 and 4 underwent the same procedures that Samples 1 and 2 undertook, except
activated charcoal was not mixed with the explant when grinding. It is also important to note that
a new extraction buffer composition (Shorea robusta Extraction Buffer 2) was used with Sample
4 (3% CTAB, 200 mM Tris – HCl at pH 8.0, 1.5 M NaCl, 25 mM EDTA, 0.2% β-
mercaptoethanol, and 3% PVP). After completing the isolation protocol and storing at -20° C
overnight, DNA was visible in these samples as well.
Trial 4(DNA Isolation using new extraction buffers and NO activated charcoal)
Two more samples were obtained for this trial, which were labeled Sample 5 and 6. The
samples underwent very similar procedures for DNA isolation compared to that of Samples 3
and 4, with a few minor differences. Firstly, potassium acetate was not added to the solutions for
Samples 5 and 6, as it had been for Samples 1 – 4. Secondly, 2 mL of ammonium acetate and 5
mL chilled pure ethanol was added in addition to isopropanol for Sample 5, to remove cellular
and histone proteins, as well as precipitate out DNA. It is important to note that isopropanol was
not added to Sample 6. The third and major difference was in the compositions of the extraction
buffers for each sample (Sample 5 – Shorea robusta Extraction Buffer 4; Sample 6 – Shorea
robusta Extraction Buffer 3). Sample 5 utilized an extraction buffer very similar to that of the
Cajanus cajan protocol, but with a reduction in β-mercaptoethanol concentration (2% CTAB,
200 mM Tris – HCl at pH 8.0, 1.4 M NaCl, 20 mM EDTA, and 0.2% β-mercaptoethanol).
Sample 6 used an extraction buffer more attuned to extraction buffers 1 and 2 (2.5% CTAB, 200
mM Tris – HCl at pH 8.0, 1.5 M NaCl, 25 mM EDTA, 0.15% β-mercaptoethanol, and 1.5%
PVP). After incubating both samples at -20° C overnight, DNA was visible in the solutions.
Trial 5 (DNA Isolation using extraction buffer 3 and dicloromethane)
It is important to note that explant samples 1 – 6 all came from the accession of wild
Shorea robusta plant. For Samples 7 and 8, fresh, young leaves were obtained from a new
accession. It is believed to be easier to extract DNA from young leaves because of their lack of
protective organelle structures, namely the cell wall, so DNA should have been easily obtainable
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8. from these samples. Both samples underwent a very similar procedure to that of Sample 6 in that
both used Shorea robusta Extraction Buffer 3. However, ammonium acetate and chilled ethanol
was not added to either sample, nor was isopropanol. The main difference between the protocol
for Sample 7 and 8 was the use of dicloromethane in place of chloroform: isoamyl alcohol in
Sample 8; Sample 7 continued to use the normal addition of chloroform: isoamyl alcohol, as seen
in Samples 1 – 6. In either case, DNA was visible for each sample after an overnight incubation
period at -20° C.
Before continuing on to the purification stage for Samples 1 – 8, tables have been
included detailing the compositions of the five different extraction buffers used, as well the
extraction buffer associated with each sample (Samples 1 – 8).
Extraction
Buffer
Component
Amount in
Cajanus Cajan
Extraction
Buffer
Amount in
Extraction
Buffer 1
Amount in
Extraction
Buffer 2
Amount in
Extraction
Buffer 3
Amount in
Extraction
Buffer 4
CTAB 2% 3.5% 3% 2.5% 2%
Tris – HCl 200 mM 200 mM 200 mM 200 mM 200 mM
NaCl 1.4 M 1.5 M 1.5 M 1.5 M 1.4 M
EDTA 20 mM 40 mM 25 mM 25 mM 20 mM
β-mercaptoethanol 2% 0.5% 0.2% 0.15% 0.2%
PVP - 2% 3% 1.5% -
Figure 2: Table displaying the compositions of all extraction buffers used in this experiment
Sample # Extraction Buffer Used/Protocol
1 & 2 1 with activated charcoal
3 1
4 2
5 4
6, 7, and 8 3
Figure 3: Table displaying the extraction buffers and protocols used with each sample
Note: Samples 1 – 6 were obtained from the same accession of Shorea robusta plant, while
Samples 7 & 8 were obtained from the same, secondary accession.
Trials 2 – 5 (DNA Purification of Samples 1 – 8)
Samples 1 – 8 underwent the same purification protocol as did the initial samples from
Trial 1, save for a few changes. For all samples, 10 μL RNAase-A was pipetted into each tube,
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9. mixed, and test tubes were placed in a 37° C water bath for 1 hour. Afterword, 15 μL proteinase-
K was mixed into each tube and tubes were placed in a 37° and 65° C water bath for 30 minutes
and 10 minutes, respectively. It is important to note that an equal volume of
Phenol:chloroform:isoamyl alcohol was then added only to Sample 7 and 8, while this step was
omitted for Samples 1 – 6. For all samples, an equal volume of chloroform:isoamyl alcohol was
then added to each solution and samples were centrifuged for 12 minutes at 4° C and 5000 rpm.
This step was repeated with the supernatant for all samples after which 1/10 volume of 3 M
ammonium acetate (pH 5.8) and 2x volume of chilled ethanol was added to the supernatant of all
samples. The DNA was hooked out for all samples, before being washed with 70% ethanol, air
dried, and dissolved in 500 μL T10E1 buffer. Purified DNA was much clearer than before,
although it is important to note that Samples 7 and 8 provided the most purified DNA, based on
appearance.
II. DNA Equilibration via Gel Electrophoresis
Initial equilibration of DNA samples
The newly purified samples needed to be diluted to the same concentration as the λ DNA
marker (20 ng/μL). Therefore, gel electrophoresis was required to determine by which factor to
dilute each sample with T10E1 buffer (50 ng/μL). A 0.8% agarose gel was prepared using 1X
TAE buffer (1 mL 50X TAE, 49 mL ddH2O, and 0.4 g agarose). The composition of 100 mL
50X TAE buffer is given in the table below:
Name of Component Amount added to buffer
1 M Tris 29.2 g
0.5 M EDTA 10.0 mL
Glacial Acetic Acid 5.7 mL
Total Volume = 100 mL (with added ddH2O)
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10. Figure 4: Table displaying the components of 50X TAE buffer, which is diluted 50 times and mixed with agarose to
form gel
Samples 1 – 8 were loaded in the wells, from right to left. Each well contained 3 μL total,
with 2 μL DNA sample, and 1 μL dark blue dye. Gel electrophoresis was run for approximately
1 hour at 50 V. Upon completion, the samples were examined under UV source to analyze each
sample’s band intensity.
Figure 5: Image of electrophoresed Samples 1 – 8 under UV light. NOTE: Samples 1 – 8 are read from right to left
Based on this, a dilution factor for Samples 1 – 8 was determined, and is given in the
table below:
Figure 6: Chart of dilution factors required for each sample in order to reach same band intensity
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Sample # 1 2 3 4 5 6 7 8
Factor of
Dilution
8x 8x 26x 20x 15x 20x 8x 8x

11. After dilution, the samples were stored at -20° C overnight in preparation for a gel run of
the equilibrated samples the next day. The next day, Samples 1 – 7 were run alongside the λ
DNA marker, to confirm correct dilution had been achieved.
Figure 7: Image of electrophoresed diluted samples analyzed under UV light. NOTE: λ marker placed in right-most
well with Samples 1 – 7 read in order from right to left.
The above image shows that almost all samples were diluted correctly, although Sample
3 was diluted a further 2x. The samples were now ready for SCoT PCR analysis.
III. SCoT PCR Analysis of Shorea robusta samples
PCR amplification using SCoT primer (Samples 6 and 7)
SCoT PCR analysis was undertaken for samples 6 and 7 using the SCoT-1 DNA primer.
SCoT Master Mix was made using the components listed in the table below:
Component of Master Mix Amount added
Milli – q H2O 14.67 μL
10X assay buffer 2.5 μL
DNTB mix 1.0 μL
MgCl2 0.5 μL
Taq polymerase 0.33 μL
SCoT-1 primer 1 μL
Total Volume = 20 μL
Figure 8: Table displaying the compounds required for SCoT-1 Master Mix
For each sample prepared for PCR amplification, 3 μL of DNA sample was mixed with
20 μL of Master Mix. Therefore, 40 μL of total SCoT-1 master mix was prepared and aliquoted
into two 20 μL PCR tubes, whereby Samples 6 and 7 were each mixed into a tube. PCR
amplification was run using the eppendorf PCR machine and preprogrammed SCoT protocol
detailed below:
35x
Step Temperature Time
Initial Denaturation 94° C 5 minutes
Denaturation 94° C 1 minute
Annealing 50° C 1 minute
Elongation 72° C 2 minutes
Final Elongation 72° C 5 minutes
Figure 9: Table detailing steps undertaken in SCoT PCR amplification program. NOTE: 35 cycles were completed
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12. Analysis of amplified samples via gel electrophoresis (Samples 6 and 7)
Upon successful amplification of Samples 6 and 7 using the SCoT-1 primer, the DNA
could be analyzed using gel electrophoresis to determine if the products had indeed amplified. A
new 0.8% agarose gel was prepared and amplified Samples 6 and 7 were loaded into wells,
alongside a λ DNA marker of known base-pair length. After running gel electrophoresis, the gel
was analyzed under UV light whereupon it was determined that Sample 6 did not amplify, while
Sample 7 did amplify. It is therefore imperative to run more PCR primers with these samples to
see if the same results appear.
Figure 10: Image of amplified samples under UV light. One sample amplified using PCR primer (Sample 7), while
one did not (Sample 6)
Results and Interpretation
Unfortunately, there was not time to complete the PCR analysis of all samples. Therefore,
it is imperative to undergo PCR amplification with more SCoT primers, along with RAPD
primers, in order to better analyze the genetic variation amongst different accessions of Shorea
robusta plant. However, there was some success in this project in that the initial steps toward
standardization of DNA extraction for Shorea robusta occurred through multiple trials.
Trial 1 of DNA extraction was a failure, as no DNA was isolated and the procedure could
not even be completed. The major problem occurred after the addition of
Phenol:chloroform:isoamyl alcohol, which resulted in the solidification of the supernatant,
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13. rendering it impossible to isolate DNA from waste products. This was believed to be due to the
coagulation of the Phenol with excess lipid products that should have been removed, but had
remained in the solution. Shorea robusta leaves contain a large concentration of polysaccharides,
so it is essential to remove these compounds in the protocol. In response to this, steps were added
to the protocol of DNA isolation in order to better absorb the phenolic compounds, which
included the addition of activated charcoal to the grinding step, PVP to the extraction buffer, as
well as addition of potassium acetate to the solution. However, as we saw with Samples 1, 2, and
3, DNA was successfully isolated using Extraction Buffer 1 with or without activated charcoal.
This suggests that addition of activated charcoal is not necessary for DNA isolation of Shorea
robusta. It was also shown that the addition of potassium acetate, as well as the use of
dicloromethane was not necessary as well, as DNA was successfully extracted from samples that
did not use these compounds in their protocols. Therefore, it seems that the addition of PVP to
extraction buffer is the main change necessary for Shorea robusta DNA extraction.
Among the various extraction buffers tested, Extraction Buffer 3 seems most effective for
DNA isolation, as evidenced by analysis of the gel runs. Sample 7, which used Extraction Buffer
3, had successfully extracted DNA, which was diluted and able to be amplified as well. Although
Sample 6 used the same extraction buffer and was unsuccessful in PCR amplification, this may
be due to a difference in the procedure for DNA purification. Because of the initial worry about
coagulation of phenol with excess lipid products present in solution, the addition of
phenol:chloroform:isoamyl alcohol was omitted from the protocol for Samples 1 – 6. Yet, DNA
was still successfully extracted so it seemed that the addition of phenol was not necessary to the
DNA purification protocol. However, phenol:chloroform:isoamyl alcohol was added to Samples
7 & 8, whereby Sample 7 successfully amplified using the SCoT-1 primer.
Phenol:chloroform:isoamyl alcohol has an important role in that it separates proteins attached to
extracted DNA. These bound proteins would otherwise prevent PCR primers from attaching to
DNA strands, thus negating amplification, which can be seen in Figure 10. Therefore, we have
determined a successful method for extracting DNA, which necessitates the use of
phenol:chloroform:isoamyl alcohol, and also shows success with the use of Extraction Buffer 3
and potassium acetate.
Conclusion and Future Procedures
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14. In conclusion, the project faced a major obstacle in the isolation of DNA from a plant
without a standardized protocol. Determining a method for extracting DNA took several trials
and days, which pushed back the entire project. Because of this, PCR amplification and analysis
of genetic variation using FreeTree could not be completed. However, the results collected
provide an important stepping-stone toward completion of the project in the future in that we
now know how to successfully extract, purify, and equilibrate DNA from Shorea robusta leaves.
In the days to come, 8 – 10 accessions of Shorea robusta will be collected from various
areas around Burla, for genetic analysis. Using the protocol standardized for Sample 7, DNA will
be extracted from each sample and amplified using various SCoT and RAPD primers. After
analyzing the bands between each sample and scoring as either present (1) or absent (0), the data
can be inputted into FreeTree software to calculate Nei’s estimation of genetic distance. Finally,
this data can be used to create a cluster analysis for these Shorea robusta samples.
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15. References
- Ahmad Wani, T. et. al 2012 Analgesic activity of the ethanolic extract of Shorea robusta resin
in experimental animals. Indian J Pharmacol. 44(4), 493 – 499.
- Ashton 1998. Shorea Robusta. 2006. IUCN Red List of Threatened Species. First published
online May 12, 2006. www.iucnredlist.org.
- Devoe, N. and Gautam, K. 2006 Ecological and anthropogenic niches of sal (Shorea
robusta Gaertn. f.) forest and prospects for multiple-product forest management – a review.
Forestry 79 (1): 81-101 first published online December 12, 2005 doi:10.1093/forestry/cpi063
- FRIB 1947 Experimental tapping of sal and blue pine. Forest Resource India Burma 1945–
6 1, 88–90.
- Ganguali, R.N. and Oudhia, P. 1998 Is Lantana camara responsible for Sal-borer infestation in
M.P.?. Insect Environment. 4 (1): 5.
- Karnik, M.G. and Sharma, O.P. 1968 Cellulose gums from sal (Shorea robusta) bark and
Bamboo (Dendrocalamus strictus). Indian Pulp Paper 22, 451–453.
- Panday, K.K. 1982 Fodder Trees and Tree Fodder in Nepal. Swiss Development Cooperation,
Berne.
- Rai, S.N. and Shukla, P.C. 1977 Influence of feeding deoiled sal seed meal with urea and
molassees on digestibility and balances of nitrogen, phosphorus and calcium in lactating
cows. Indian J. Anim. Sci. 47, 111–115.
- Rajan, R.P. 1995 Sal leaf plate processing and marketing in West Bengal. In Society and Non-
timber Forest Products in Tropical Asia. J. Fox (ed.). East-West Center, Honolulu, pp. 27–36.
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16. - Tewari, D.N. 1995 A Monograph on Sal (Shorea robusta Gaertn. f.). International Book
Distributors, Dehradun, India.
- Verma, V.P.S. and Sharma, B.K. 1978 Studies on production and collection of sal (Shorea
robusta Gaertn.) seeds. Indian For. 104, 414–420
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