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Genetic Variability in Relation to Plant Rarity

Lauren Kosslow

Seton Hill University

13 December 2019
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Introduction:

The overall goal of this project funded by the National Science Foundation is to

determine why some plant species are rare and why other plant species are common to improve

conservation efforts for rare species. Environmental changes serve as a threat to plant species,

particularly rare species. A plant adapting to an environmental change is a lengthy process,

which may be inhibited by the low genetic diversity of rare plant species (2). However, recent

studies on a plant’s plasticity, or its ability to adapt to environmental changes, and genetic

diversity have shown that plasticity could help explain differences in the distribution of location

when genetic diversity could not (3). Our study expands upon the idea of studying both plasticity

and genetic diversity to determine why some plant species are rare and why some plant species

are common to ultimately aid in conservation. Our part of the study focuses on genetic diversity.

We studied genetic diversity by using microsatellite, or simple sequence repeat, markers to

quantify genetic variation and genotype individuals. Neutral markers, or non coding regions, do

not have a predisposition to being selected for, unlike adaptive markers, or coding regions.

Therefore, we used neutral markers to investigate genotype identification.

Testing for DNA amplification was the first step toward testing for polymorphisms, or

differences in DNA. It first had to be determined if the chosen primers worked, which was

shown through DNA amplification on a 1% agarose gel. If the DNA amplified, the samples and

primers underwent further testing to determine if polymorphisms were present.

Polymorphisms indicate the presence of different alleles, and alleles indicate a more

genetically variable species. Polymorphisms were shown through different amounts of DNA

bands on a 4% agarose gel.

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Eleven primers originally designed for use on another echinacea species, Echinacea

angustifolia (1), were first tested for reasons of cost effectiveness. The primers designed for

Echinacea angustifolia were Ech03, Ech07, Ech05, Ech11, Ech13, Ech13Z, Ech15, Ech28,

Ech36, Ech37, and Ech47 (1). The primers were tested on one common echinacea species,

Echinacea purpurea, and one rare echinacea species, Echinacea tennesseensis. One common

echinacea species and one rare echinacea species were tested to determine if one species was

more genetically variable. More genetic variability could indicate a better ability to adapt to

environmental changes. Echinacea purpurea DNA samples included populations from Cuivre

River State Park (CRSP), and enterprise south (ES). Echinacea tennesseensis DNA samples

included populations from vesta (VES), lane farm (LF), couchville (CO), and mount view (MV).

After initial testing with primers designed for Echinacea angustifolia, primers were

designed specifically for Echinacea purpurea and Echinacea tennesseensis. The primers

designed for use on Echinacea purpurea were 97687, 9640, 15024, and 66750, and the primers

designed for use on Echinacea tennesseensis were 1044, 8553, 19374, 195974, 252258, and

340701. All ten of the primers designed for both Echinacea purpurea and Echinacea

tennesseensis were tested on both Echinacea purpurea and Echinacea tennesseensis DNA

samples to test for cross amplification and polymorphisms.

We hypothesize why some species are rare and other are common depends on an

organism’s ability to adapt to new environments, which depends on genetic variability, and,

therefore, a more common species is more genetically variable, allowing it to better adapt to new

environments.
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Materials and Methods:

DNA Extraction. The CTAB buffer was prepared by adding approximately 15 uL of 2-

mercaptoethanol to 7.5 mL of 2X CTAB per seven samples. The buffer was incubated in a

previously prepared hot water bath at approximately 60 °C for 20-30 mins. A small amount of

dry tissue, approximately half of a leaf to a whole leaf, was added to a mortar. A small amount of

sterilized sand and an approximately equal amount of PVP were then added to the mortar. After

incubation, 500 uL of CTAB buffer was added to the mortar, and the tissue was ground

thoroughly with the pestle. Another 500 uL of CTAB buffer was added to the tissue, for a final

volume of 1000 uL CTAB buffer, and ground thoroughly again. The tissue, in a mostly liquid

form, was poured into a labeled 1.5 mL microcentrifuge tube. The samples were then incubated

for 1 hr in a previously prepared hot water bath at approximately 65-70 °C. After incubation,

approximately 700 uL of chloroform : isoamyl alcohol (24:1) was added to each sample in the

hood. The samples were then spun at 13,000 rpm for 20 mins, or until a clear liquid layer

appeared above the samples. The top aqueous layers of each sample were transferred to

respective, labeled microcentrifuge tubes, and the waste was discarded. 540 uL of cold

isopropanol was added to each sample and inverted four times. The samples were then placed in

the freezer at -20 °C for overnight incubation. After 24 hr incubation, the samples were spun for

3 mins at 13,000 rpm, resulting in a visible pellet. The supernatant was then decanted, followed

by the addition of 500 uL of 75% EtOH to each sample. The samples were spun again for 3 mins

at 13,000 rpm. The supernatant was decanted again, and the tubes were allowed to dry at room

temperature for approximately 20-30 mins. The pellets were resuspended by adding 80 uL of 1X

TE with filter tips, followed by pumping to break up the pellets. 8 uL of 7.5 M ammonium

acetate was added to the side of each of the tubes, followed by 180 uL of 100% (or 95%) EtOH.
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The tubes were then inverted and placed in the freezer at -20 °C for overnight incubation. After

24 hr incubation, the samples were spun for 3 mins at 13,000 rpm. The supernatant was

decanted, and the tubes were allowed to dry at room temperature for approximately 20-30 mins.

The pellets were resuspended by adding 1 uL of 1X TE with filter tips, followed by pumping to

break up the pellets. The samples were incubated in a hot water bath at approximately 37-40 °C

for 15-40 mins. Following incubation, the samples were spun briefly to bring down the sample.

The presence of DNA was verified by a 1% agarose gel. Samples were stored for future use at

-20 °C.

Testing Primers for DNA Amplification. The polymerase chain reaction (PCR) cocktail was

prepared by adding 12.5 uL of Master Mix, 1 uL of Primer Mix, and 11 uL of dH₂O per sample.

24.5 uL of the PCR cocktail was added to labeled microcentrifuge tubes, followed by the

addition of 0.5 uL of each respective diluted DNA sample, for a final volume of 25 uL. The

diluted DNA was prepared by adding 22.5 uL of dH₂O to 7.5 uL of DNA, for a final volume of

30 uL. The samples were then placed in the thermocycler for PCR for approximately 2 hrs, set at

94 °C for 3 min for the initial denaturation. The thermocycler was set at 35 cycles of

denaturation (94 °C for 30 sec), annealing (temperature specific for each primer for 30 sec) , and

elongation (72 °C for 60 sec). The thermocycler was set for final elongation at 72 °C for 10

mins. DNA amplification was verified by 1% agarose gel.

Testing Echinacea angustifolia Primers for Polymorphism. If a primer showed DNA

amplification, the primer was then used to test for polymorphism. The polymerase chain reaction

(PCR) cocktail was prepared by adding 12.5 uL of Master Mix, 1 uL of Primer Mix, and 11 uL
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of dH₂O per sample. 24.5 uL of the PCR cocktail was added to labeled microcentrifuge tubes,

followed by the addition of 0.5 uL of each respective diluted DNA sample, for a final volume of

25 uL. The diluted DNA was prepared by adding 22.5 uL of dH₂O to 7.5 uL of DNA, for a final

volume of 30 uL. The samples were then placed in the thermocycler for PCR for approximately

2 hrs, set at 94 °C for 3 min for the initial denaturation. The thermocycler was set at 35 cycles of

denaturation (94 °C for 30 sec), annealing (temperature specific for each primer for 30 sec) , and

elongation (72 °C for 60 sec). The thermocycler was set for final elongation at 72 °C for 10

mins. DNA polymorphisms were verified by 4% agarose gel. Polymorphisms were indicated by

height differences between bands or by more than one band.

Testing Echinacea purpurea and Echinacea tennesseensis Primers for Cross Amplification.

The polymerase chain reaction (PCR) cocktail was prepared by adding 12.5 uL of Master Mix, 1

uL of Primer Mix, and 11 uL of dH₂O per sample. 24.5 uL of the PCR cocktail was added to

labeled microcentrifuge tubes, followed by the addition of 0.5 uL of each respective diluted

DNA sample, for a final volume of 25 uL. The diluted DNA was prepared by adding 22.5 uL of

dH₂O to 7.5 uL of DNA, for a final volume of 30 uL. The samples were then placed in the

thermocycler for PCR for approximately 2 hrs, set at 94 °C for 3 min for the initial denaturation.

The thermocycler was set at 35 cycles of denaturation (94 °C for 30 sec), annealing (temperature

specific for each primer for 30 sec) , and elongation (72 °C for 60 sec). The thermocycler was set

for final elongation at 72 °C for 10 mins. DNA amplification was verified by 1% agarose gel.

Cross amplification was indicated by DNA amplification in the species for which the primer was

not originally designed.

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Results:

Eleven primers originally designed for use on another echinacea species, Echinacea

angustifolia, were the primers first tested on Echinacea purpurea and Echinacea tennesseensis.

The PCR annealing temperatures specific for each primer were originally set as the temperature

recorded in the primer note for Echinacea angustifolia (1), shown in Table 1, but these

temperatures did not result in DNA amplification in the first few primers tested. Gradients were

run on a few primers to determine at what temperatures were best to conduct PCR. The

annealing temperatures for each primer were then changed to the temperatures calculated by the

ThermoFisher Annealing Temperature Calculator, also shown in Table 1, which resulted in DNA

amplification, shown in Table 2. One primer, Ech37 had no DNA amplification following a

gradient, and it was subsequently removed from the study, also shown in Table 2. Testing with

some Echinacea angustifolia primers resulted in polymorphisms, while others did not show

polymorphisms, shown in Table 2. Testing with some Echinacea purpurea and Echinacea

tennesseensis primers resulted in cross amplification, while others did not result in cross

amplification, shown in Table 3.

Table 1. Echinacea angustifolia Annealing Temperatures - Annealing temperatures for PCR

for the primers originally designed for use on Echinacea angustifolia. The annealing

temperatures from the Echinacea angustifolia primer note (1) and the annealing temperatures

from the ThermoFisher Annealing Temperature Calculator are shown.

Primer Primer Note Annealing ThermoFisher Annealing

Temperatures (1) (℃) Temperature Calculator
Annealing Temperatures (℃)
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Ech03 61 46.6

Ech07 64 48.4

Ech05 54 49.4

Ech11 57 50.9

Ech13 55 50.2

Ech13Z 55 50.2

Ech15 55 49.4

Ech28 59.8 53

Ech36 63 51

Ech37 55 48

Ech47 54 50

Table 2. Echinacea angustifolia Polymorphisms - Traits of the primers originally designed for

use on Echinacea angustifolia, including the primer name, if the 4% gel amplified DNA, the

annealing temperature during PCR, if the primer showed polymorphisms, and if yes, how the

samples were polymorphic.

Primer Did 4% Gel Annealing Polymorphic? How it was

Amplify DNA? Temperature Polymorphic
(℃)

Ech03 Yes 46.6 Yes Single band-

CO14, LF1,
MV2, CO2,
VES3
Two bands-
MV14, LF10,
CRSP23,
CRSP4,
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CRSP28,
CRSP20,
CRSP13, CRSP
17, CRSP1,
CRSP24

Ech07 Yes 48.4 Potentially Bands (CO14,

LF1, MV14,
CRSP28,
CRSP20,
CRSP13,
CRSP1) higher
than other bands
(MV2, VES11,
CRSP23,
CRSP4,
CRSP17,
CRSP24)

Ech05 Yes 49 Yes Tennesseensis

bands were
brighter than the
purpurea bands.

Ech11 Yes 51 Yes All samples

except CO14 had
2 bands, but the
wells were
messy.

Ech13 Yes 50.2 Potentially No Only 1 band in

all samples and
no height
differences, but
some bands were
longer than
others, some
wells were not
clean, and the
negative control
had a band,
which could be
from spillage.

Ech13z Yes 50.2 Yes All purpurea

samples had 1
clear band, and
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LF1 and VES11

bands may have
been higher than
the other
tennesseensis
bands. LF10 had
2 clear bands
and MV2 and
CO2 may have 2
bands. The
negative control
had a band,
which could be
from spillage.

Ech15 Yes 49.4 Potentially Most samples

showed 3 bands.
3 bands shows
polymorphism,
but it is
confusing
because only 2
alleles are
possible.

Ech28* Yes 51 Yes CRSP had 2

bands. CRSP23
and CRSP4
bands were
lower than
VES11 and
CRSP28 bands,
with height
differences
overall.

Ech36* Yes 51 Yes Height

differences
overall, but the
wells are messy.

Ech37 N/A N/A N/A 1% gel did not

amplify
following a
gradient, so a 4%
gel was not run.
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Ech47 Yes 50 Yes Purpurea

samples had
noticeable height
differences.
However, there
were clear,
bright bands in
an area with too
many bp.

Table 3. Echinacea purpurea and Echinacea tennesseensis Primers - Traits of the primers

designed specifically for use on Echinacea purpurea and Echinacea tennesseensis, including the

primer name, the species the primer was designed for, if the 1% gel amplified DNA, the

annealing temperature during PCR, if the primer showed polymorphisms and, if yes, how the

samples were polymorphic, and if there was cross amplification across both Echinacea purpurea

and Echinacea tennesseensis.

Primer Species Did 1% Annealing Polymorphic? How it was Cross

Gel Temperature Polymorphic Amplification
Amplify? (℃) ?

97687 Echinacea No 59 N/A N/A N/A

purpurea

9640 Echinacea Yes 60 No All 1 band at Yes

purpurea the same
height.

15024 Echinacea No 59 N/A N/A N/A

purpurea

66750 Echinacea Yes 60 No All 1 band at Yes

purpurea the same
height.

1044 Echinacea Yes 55 Yes Samples Yes

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tennesseensis drastically
brighter than
previous
samples, tall,
multiple
bands.

8553 Echinacea Yes 55 Maybe Slight band No

tennesseensis height
differences

19374 Echinacea Yes 55 Maybe Slight band Yes

tennesseensis height
differences

195974 Echinacea Yes 55? Yes Samples Yes

tennesseensis drastically
brighter than
previous
samples, tall,
multiple
bands.

252258 Echinacea Yes 60 No All 1 band at Yes

tennesseensis the same
height.

340701 Echinacea Yes 59 No All 1 band at Yes

tennesseensis the same
height.

11 12

13 14 15

16 17 18
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1 2 3 4 5 6 7 8 9 10

Figure 1. 4% Agarose Gel for Ech03 Primer. Examples of 1 band are shown in the bottom of

the gel in wells 2-5, and examples of 2 bands are shown in the bottom of the gel in wells 7 and 8

and in the top of the gel in wells 11, 12, 16, and 18.

Discussion:

Seven out of eleven primers designed for Echinacea angustifolia expressed usable

polymorphisms, indicating genetic variability. Two out of eleven primers designed for

Echinacea angustifolia expressed potential for polymorphisms, one out of eleven primers did not

express potential of polymorphisms, and one out of eleven primers did not amplify DNA in a 1%

gel, and was therefore not tested for expression of polymorphisms. Seven out of ten primers

designed for Echinacea purpurea and Echinacea tennesseensis showed cross amplification,

indicating genetic variability. Two out of ten primers designed for Echinacea purpurea and

Echinacea tennesseensis did not show DNA amplification and were, therefore, not eligible to

show cross amplification, and one out of ten primers showed DNA amplification but did not

show cross amplification.

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In the future, we will continue conducting PCR, 1% gels, and 4% gels to discover 20 or

more primers specifically designed for Echinacea purpurea and Echinacea tennesseensis that

express polymorphisms and cross amplification.

References:

1. Ison, J, Wagenius, S, Diedre, R, Ashley, MV (2013) Development and Evaluation of

Microsatellite Markers for a Native Prairie Perennial, Echinacea angustifolia

(Asteraceae) Applications in Plant Sciences 1(11): 1300049

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2. Leimu R, Fischer M (2008) A meta-analysis of local adaptation in plants. PLOS One 3:

e4010.

3. Lovell JT, McKay JK (2015) Ecological genetics of range size variation in Boechera spp.

(Brassicaceae) Ecology and Evolution 5: 4962-4975.