Identification of Hawaiian and Polynesian Plants along with an Investigation of their Phylogenetics...

PLANTS DNA EXTRACTION AND BARCODING 1

Identification of Hawaiian and Polynesian Plants along with an

Investigation of their Phylogenetics by means of DNA Extraction

and Barcoding

Helmi Merkhi

BIOL 2053

April 20th, 2015


Identification of Hawaiian and Polynesian Plants along with an

Investigation of their Phylogenetics by means of DNA Extraction

and Barcoding

As humans expanded, their need to distinguish the

multitude of organisms with which they interact increased. The

method of taxonomy started during the classical era. The ancient

Greeks and Romans began classifying and naming organisms.

Particularly those that were useful to them like medical plants

and certain beneficial animals. They made names for the animals

they hunted and those that were dangerous to them, as well as,

plants that helped make healing remedies to cure the sick (Knapp,

2007). More recently however, in 1753, Carl Linnaeus, a Swedish

natural scientist implemented a logical system of taxonomic

classification. In the traditional Linnaean system of

classification, which is still widely used today, the kingdoms of

life are subdivided, in a hierarchic manner, into successively


narrower categories. These categories are phylum, class, order,

family, genus, and species (Skinner & Murck, 2011). The

conventional organisms’ binomial nomenclature used today is

derived from this taxonomic system.

Certain processes like mutations, speciation, genetic drift

and natural selection lead to today’s spectacular biodiversity.

Yet on the other hand, urbanization, logging, climate change, and

the increasing numbers of human-introduced invasive species lead

to what is called the 6th mass extinction biologists currently

monitor. Consequently, the taxonomy field became very useful for

organizing and keeping track of the various species along with

detecting and managing invasive alien ones. Hawaii sets a good

example with its numerous exotic species on the verge of

extinction. The forest structure no longer resembles its native

past with 50% of its plant taxa at risk of endangerment and 8.33%

declared extinct (Sakai et al., 2002). With globalization and

tourism as a main industry, taxonomy is needed more than ever

before in order to distinguish the native Hawaiian species from

the invasive alien ones. Identifying alien species is a first


step towards taking the right measures to preserve the native

species.

In the same taxonomy context, new technologies have been

implemented to enable the identification of the numerous

organisms with higher precision. A relatively new method of DNA

extraction and barcoding is now used worldwide. DNA extraction

serves to obtain and purify the DNA from its surrounding cellular

environment through a certain biochemical procedure. DNA

barcoding serves a different purpose; it is designed to use a

specific DNA sequence to serve as a unique species identifier.

The two procedures work together as the DNA obtained from the

extraction is analyzed and sequenced in the DNA barcoding

procedure. The two methods are fundamental to the experiment

carried in this lab. From a holistic perspective, the goal of

this lab is to identify a number of native Hawaiian and

Polynesian plants by means of DNA extraction and barcoding.

A phylogenetic tree depicting the evolutionary relationships

of the plant species studied is the ultimate result of the lab.

However, to reach such result multiple steps are taken. The task


starts with DNA extraction. The obtained DNA is then amplified

and enhanced through a process called PCR (Polymerase chain

reaction). Gel electrophoresis is a sub-step that allows the

monitoring of the quality of the amplified DNA, before it is

barcoded. The sequencing and identification of the species relies

on certain computer programs which are DNA subway and BLAST

(basic local alignment search tool). Once identified, the

different plant species are organized into a phylogenetic tree

using another program named PHYLIP. The plants were pre-

identified in order to test the efficiency of the entire process

by comparing the results to the true plants’ species.

Three hypotheses were tested throughout this lab. The first

was related to the DNA extraction, PCR amplification and gel

electrophoresis. It was hypothesized that correctly following the

procedure for DNA extraction and PCR amplification will result in

a successful DNA extract that is humanely visible. It was

predicted that if the DNA extraction and PCR are carried out

correctly, then DNA bands will be visible in the gel. The second

hypothesis pertained to the efficiency of DNA barcoding with rbcL


(RuBisCO) gene. For this section it was hypothesized that the

identified plant species from the DNA BLASTN program would be

identical to the true identity of the plants. It was predicted

that if rbcL gene holds enough genetic information then the

BLASTN program will precisely identify the different species of

the plants. The third hypothesis is related to the phylogenetic

section of the experiment. It was hypothesized that the resulting

Neighbor Joining (NJ) phylogenetic tree would depict the

evolutionary relationships of plant species in a correct and

logical manner. It was predicted that if the phylogenetic tree

depicts the proper evolutionary relationships of the different

plants than the rbcL gene is suitable for this task.

Methods

1. DNA extraction, PCR amplification and gel electrophoresis:

The first step was to choose 2 plant leaves from the 20

specimens available. The objective of the first section of the

experiment was to isolate the DNA (Deoxyribonucleic acid) from

the plant tissue. The larger end of a 1000 µL pipette was pressed

against the plant leaves to collect a small disk of the plant’s


tissue. The Larger leaves were saved as voucher specimens. To

prevent cross-contamination, a new pipette tip was used for each

plant specimen. It is also important to note that labeling was a

critical part throughout the entire identification procedure. The

specimens were then placed into 1.5 mL tubes followed by 100 µL

of nuclei lysis solution. This solution is used to disintegrate

the organelles surrounding the DNA like the chloroplasts and

mitochondria.

The next step was inserting a plastic pestle in the inner

surface of the 1.5 mL tube to grind up the rigid plant material

such as the cell walls. 500 mL of nuclei lysis were added to the

test tubes which were then incubated for 10 minutes in a 65 °C

water bath. The next step was adding 3 mL of RNase to degrade the

RNA as it is not the target DNA for this experiment. The tubes

were then incubated again for 15 minutes in a 37 °C water bath.

200 µL of protein precipitation solution was added and the test

tubes were then placed in the vortex machine for 5 seconds. The

next step was to incubate the test tubes in an ice bath for 5

minutes.


The next step is fundamental to the DNA extraction

procedure. The test tubes are placed in a micro-centrifuge for 4

minutes at maximum speed in order to separate the pellet and the

cell debris from the DNA. 600 µL of the clear supernatant

solution was then transferred to a new test tube were it was

mixed with another 600 µL solution of isopropanol. The mixture

was micro-centrifuged again for 1 minute. Differently from the

previous step however, it is the pellet that contained the DNA

and not the supernatant solution. The isolated pellet was then

washed with a 70% ethanol solution and micro-centrifuged again

for 1 minute. The DNA pellet was once separated from its ethanol

solvent solution and left to air dry for 15 minutes.

Once the ethanol solution completely evaporated, 100 µL of

DNA rehydration solution was added in which the final DNA pellet

dissolved. Finally the test tubes were incubated overnight at 4°C

and stored at -20 °C. 2.5 µL from each solution was extracted for

the Polymerase chain reaction (PCR). The main PCR steps are:

first, heating at 94 °C to denature DNA strands. Second, cooling

at 55°C to anneal primers to template. Lastly, warming at 72 °C


to activate the polymerase, which extends primers and replicates

DNA. The three steps cycle is repeated 35 times in order to

amplify the DNA sequence.

To check the quality of the resulting DNA sequence from the

PCR, a method of gel electrophoresis is employed. In general

terms, the DNA sequences are loaded into separate wells in a gel.

The left most well of the gel is filled with a DNA marker. The

gel is placed in an electrophoresis chamber with the wells close

to the black (negative) electrode. On a voltage of 130V the gel

is left to electrophorese for 20 to 30 minutes. Because DNA

nucleotides have different sizes and charges, they will migrate

towards the red (positive) electrode which indicates the success

of the DNA extraction and PCR. The gels are stained with

CaliforniaBLU stain which allows the visibility of the DNA bands.

2. Barcoding with the rbcL Gene:

The DNA samples pertaining to successful gel

electrophoresis were selected and sent to certain laboratories to

be analyzed and transformed to computer DNA trace files. These

trace files are then uploaded to an online computer program


called DNA subway. The program electronically turns the trace

files into a barcode and allows the user to perform certain tasks

such as locating sequences of god quality and trimming out

sequences of mediocre quality. The program also allows the user

to pair up the forward and reverse sequences to locate possible

mutations or mismatches. The finalized DNA sequences are then

copied into another program called BLAST. This program compares

the DNA sequences to the ones previously saved into an online

database. The data base is implemented by the National Center of

Biotechnology Information (NCBI). The program matches the

sequences and yields the name of the organism to which the DNA

sequence belongs.

3. Phylogenetics using the rbcL gene:

The last part of the experiment was creating a

phylogenetic tree that represents the evolutionary relationships

between the identified plant species. The neighbor joining (NJ)

tree is a final depiction of the evolutionary closeness of the

studied plant species based on their rbcL genetic information. A


subprogram of the DNA subway called PHYLIP NJ is used to generate

the phylogenetic tree.

Results

1. DNA extraction, PCR amplification and gel electrophoresis:

For this part of the experiment, the effectiveness of the

DNA extraction technique along with the PCR amplification are

tested. It was hypothesized that carefully following the standard

procedure for DNA extraction and PCR would successfully yield a

DNA extract. It was predicted that if the DNA extraction and PCR

were successful than DNA bands will be humanely visible through

the gel electrophoresis. Since the process of isolating the

plant’s DNA from its tissue is very long and intricate, the

margin of error is quite large. A good method to verify the

quality of the extracted and amplified plant rbcL DNA, before

moving into the DNA barcoding process, is gel electrophoresis.

This method revolves around certain DNA characteristics; the

difference sizes of its composing nucleotides as well as their

different negative charges. The resulting gel after staining with

CaliforniaBLU is shown in figure 1.


Figure #1 photograph of the CaliforniaBLU stained gel.

The blue colored bands represent the DNA material after migratingaway from the wells due to the electrical potential to which theywere exerted. The numbers on top indicate the DNA samples to which the DNA bands belong. The first sets of bands on the left are marker indicators. The CaliforniaBLU stain along with the PCR

M 11, 12 29, 30 -- -- -- --


amplification largely contributed to making the DNA sequences visible.

The two bands that formed belong to four DNA samples. Since

each two samples came from the same plant species, it was

acceptable to combine them. The DNA band from samples 11 and 12

and the DNA band from samples 29 and 30 reacted to the

electrophoreses process by migrating towards the positive

electrode. And most significantly, they are clearly visible.

Therefore, both the hypothesis and the prediction for this

section were confirmed.

2. Barcoding with the rbcL Gene:

In this section, the capability of the rbcL gene to contain

enough genetic information for the correct species identification

is tested. In other words, the objective of this section is to

test whether or not, DNA from the rbcL gene is enough to identify

an unknown species. It was hypothesized that the identified plant

species from the DNA BLASTN program would be identical to the

true identity of the plants. It was predicted that if the rbcL


gene holds enough genetic information then the BLASTN program

will precisely identify the different species of the plants. The

results from this section are organized in the following table.

Table#1. List of the scientific names of the plants generated by

the blast program in comparison to the actual plants identity.

The results from the DNA sequence matching were not accurate. The

blast had only one direct hit.

No. Blast Hit (scientificname)

Actual Scientific name

Common name Location

Collection Date

2 Psilotum nudum

Psilotum nudum

moa, whisk fern yard 11/18/14

4 Vitex negundo Vitex rotundifolia

pohinahina, beachvitex

yard 11/18/14

6 Scaevola aemula

Scaevola taccada

naupaka yard 11/18/14

9 Ipomoea cordatotriloba

Ipomoea batatas

sweet potato, regular leaves

yard 11/18/14

11 Cordyline australis

Cordyline fruticosa

ti yard 11/18/14

12 Diplopeltis huegli

Dodonaea viscosa

a’ali’i, hopbush yard 11/18/14

14 Archidendron hirsutum

Acacia koa koa Aiea ridge trail

11/18/14

17 Leucobryum Junipersideum

Leucobryum sp. moss Aiea loop trail

11/18/14


Only one plant species out of eight DNA samples was

accurately identified. The Psilotum nudum, a native Hawaiian whisk

fern often referred to as moa or moa nehele in Hawaiian. The

identification of the other seven species wasn’t completely off,

yet, it was not correct. As a result, both the hypothesis and the

prediction were refuted for this section.

3. Phylogenetics using the rbcL gene:

This section of the experiment tests whether the phylogeny

from DNA subway matches the general understanding of plant

evolution. In different terms, whether or not the genetic

information from rbcL gene are suitable for phylogeny. It was

hypothesized that the resulting Neighbor Joining (NJ)

phylogenetic tree would depict the evolutionary relationships of

plant species in a correct and logical manner. It was predicted

that if the phylogenetic tree depicts the proper evolutionary

relationships of the different plants than the rbcL gene is

suitable for this task. At first, the program PHYLIP NJ generated

a non-logical phylogenetic tree. It placed the Psilotum nudum and

the Leucobryum sp. as sister species in the same clade, sharing a


close common ancestor. This connection seemed illogical since

Psilotum nudum is a whisk fern and Leucobryum sp. is a type of moss.

The program however, gives the option to select an outlier. When

the change is made and Leucobryum sp. is chosen as an outlier

because it’s a moss, the resulting neighbor joining phylogenetic

tree is represented by figure#2

Figure#2. Phylogenetic tree of the eight plant species identified. Sample number 17, Leucobryum sp. was selected to be an outlier. Thenumbers at each node represent the confidence of the pairings. Value of 100 are best and values less than 50 are extremely suspect.

The phylogenetic tree first suggested by the DNA subway was

different from the one in figure#2. The moss, having no recent


common ancestor with any other plant species is key to testing

the authenticity of the phylogeny automatically produced by the

program. Failure to generate a logical phylogenetic tree reflects

on the validity of the use of rbcL as a lone DNA source. The DNA

sequence taken from the rbcL gene is either incompatible or not

enough to create a phylogenetic tree. As a result, both the

hypothesis and the prediction for this section were refuted.

Discussion

This experiment had three objectives: The first one was

testing the effectiveness of the standard DNA extraction and PCR

procedures. The second one was examining the capacity of the rbcL

gene to provide enough genetic information in order to identify

multiple plant samples. The third objective of this experiment is

to verify whether or not the rbcL gene alone is suitable for

phylogeny.

The first hypothesis was related to the DNA

extraction, PCR amplification and gel electrophoresis. It was

hypothesized that correctly following the procedure for DNA

extraction and PCR amplification will result in a successful DNA


extract that is humanely visible. This hypothesis was supported

by the results. As predicted, DNA bands appeared in the gel at

the end of the gel electrophoresis procedure. Two DNA bands,

pertaining to four plant samples, migrated from the negative

electrode to the positive electrode and were clearly visible

(figure#1).

This is important because not only it means that DNA

extraction and PCR amplification procedures were carried out

correctly, but it also gives credibility to the complex and

intricate procedure itself. In the big image, such procedure is

critical to the advancement of biology as a whole, especially

that DNA analysis is widely used today for a multitude of

purposes. Additionally, this procedure allows non experts in the

taxonomy field to identify any organism. A good example is the

story of the two eleven graders from New York who were able to

detect numerous market substitutions through DNA extraction and

barcoding.

The second hypothesis pertained to the efficiency of

DNA barcoding with rbcL (RuBisCO) gene. For this section it was


hypothesized that the identified plant species from the DNA

BLASTN program would be identical to the true identity of the

plants. It was predicted that if rbcL gene holds enough genetic

information then the BLASTN program will precisely identify the

different species of the plants. Neither the hypothesis not the

prediction were supported by the results. Only one of the eight

plant species was accurately identified (table#1). However, the

BLASTN program was 75% efficient in determining the plants’

genus. Six out of the eight plants had a matching genus.

Failure to identify the exact plant species could be

due errors. The intricate DNA extraction procedure comes with a

wide margin for error. For example the use of a pipette tip twice

by mistake or the mislabeling of one of the test tubes could lead

to contamination and ultimately to a misidentification of the

species. It is important to mention that due to contamination,

samples 8, 20, and 21 had a bad quality barcodes. Prevention of

such contamination could have slightly altered the results. But

in general, it is concluded that the genetic information from

rbcL might not be enough to precisely identify any unknown


species. Today, species are sometimes subdivided even further

into subspecies, varieties, or types (Skinner & Murck, 2011).

With the science continuously aiming for higher level of detail

and more precision, relying on the genetic information from rbcL

gene alone is not enough. Further research and programs have to

be developed to expand the DNA barcoding to larger sequences

including rbcL and COI and other genes. This would allow for a

higher precision in the identification of unknown species and

even subspecies. This would be important in numerous fields such

as biodiversity preservation and taxonomy.

The last section of this experiment was designed to

test the suitability of the rbcL gene for phylogenetic studies. A

phylogenetic tree was constructed through a subprogram of the

genetic subway using the genetic barcodes extracted from the rbcL

gene. It was hypothesized that the resulting Neighbor Joining

(NJ) phylogenetic tree would depict the evolutionary

relationships of plant species in a correct and logical manner.

It was predicted that if the phylogenetic tree depicts the proper

evolutionary relationships of the different plants than the rbcL


gene is suitable for this task. Both the hypothesis and the

prediction for this section were refuted by the results.

The generated phylogenetic tree had a moss and a whisk

fern in the same clade as if they had a close common ancestor.

This is not plausible and it is probably why the program had the

option to alter the phylogenetic by choosing the outlier.

Manually selecting the moss as an outlier (figure#2) resulting in

a more logical tree with higher confidence rates. Only one

pairing confidence was under 50. It is concluded from such

results the genetic information provided by the rbcL gene alone

might not be enough to form an accurate phylogenetic tree. This

could be due to the fact that this gene was picked by the

scientific community for barcoding and not for phylogenetic

analysis. It is more likely to get an accurate phylogenetic tree

if the genetic barcoding includes multiple gene. Additionally, to

further investigate the evolutionary relationships between

organisms, the genetic information has to be supplemented by the

morphological traits of the organisms.


Literature Cited

Knapp, S. (2007, March). What's in a name? A history of Taxonomy.

In Natural History

Museum. Retrieved April 18, 2015, from

http://www.nhm.ac.uk/nature-online/science-of-natural-

history/taxonomy-systematics/history-taxonomy/session1/

Sakai, A.K. et al. (2002) Patterns of endangerment in the Hawaiian flora. Systematic

Biology 52, 276-302

Skinner, B. & Murck, B. (2011). The blue planet: an introduction to earth system

science. Hoboken, NJ: Wiley

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