Project: Estimation of genetic variation among various

accessions of Shorea robusta plant using RAPD and SCoT PCR primers

Name: Sarthak “Ankit” PatnaikDates of Project: 7/8/14 – 7/25/14

School of Life Sciences, Sambalpur University

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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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Introduction and ObjectivesShorea 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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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 MethodologyThe 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

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fresh leaf samples of Shorea robusta plant with masses ranging from 2 – 4 g. The masses are

listed in the following table:

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

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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.

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,

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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.

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).

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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

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 mMβ-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

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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,

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 9

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)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:

Sample # 1 2 3 4 5 6 7 8

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Factor of

Dilution8x 8x 26x 20x 15x 20x 8x 8x

Figure 6: Chart of dilution factors required for each sample in order to reach same band intensity

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

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Total Volume = 20 μLFigure 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

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.

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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 InterpretationUnfortunately, 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,

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

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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 ProceduresIn 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

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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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in experimental animals. Indian J Pharmacol. 44(4), 493 – 499.

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online May 12, 2006. www.iucnredlist.org.

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robusta Gaertn. f.) forest and prospects for multiple-product forest management – a review.

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