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I. Title: Discussion and Analysis of Genetic Diversity Through the Use of Genotyping to

Determine Mode of Reproduction in Hemerocallis fulva

II. Introduction:

Genotyping makes use of laboratory techniques to determine differences in DNA

sequences between organisms. This technique is especially useful when studying the

three types of biodiversity, which are ecosystem diversity, species diversity, and genetic

diversity. By studying model organisms such as plants and fruit flies, we are led to a

greater understanding of the structure and organization of entire ecosystems. Specifically,

studying the differences in genetic composition among individuals within a species

(genetic diversity) allows for the analysis of population dynamics. This area of research

studies vigor, persistence, and extinction of species in a population to gain knowledge on

biodiversity, which includes the whole variety of life on earth (Brzyski 2020). Since

biodiversity is especially important when considering patterns of natural selection and

global climate change, performing individual genotyping experiments is a safe and

effective way to understand how species and populations are being affected in differing

conditions on earth.

Experimental data not only provides information that can be used in population

genetics, but can also be useful in identifying differences in DNA between individual

organisms. For example, genotyping certain model plants allows for the determination of

mode of reproduction, which can be either sexual or asexual. This is important because

the mode of reproduction can be directly related to the genetic diversity of populations by

studying certain species of model organisms. Sexual reproduction often yields more

genetically diverse populations because of recombination (Brzyski 2020). It is often very

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difficult to distinguish whether some plants are reproducing sexually or clonally without

doing research in this area. In a study from 2007, an invasive plant species named

Fallopia japonica was studied by a team of British researchers. F. japonica has invaded

Europe and North America and serves as a model organism for studying modes of

reproduction. The researchers studied simple sequence repeats (SSR’s) in the DNA of

these plants. In Britain, it was thought that these plants only reproduce clonally through

vegetative growth. However, a study of the invasive species in the United States revealed

that F. japonica may also reproduce sexually with others of its kind. Of the three

Massachusetts populations that were sampled, it was determined that 26 different

genotypes existed, with one of them being identical to the species in Europe (Grimsby et

al. 2007). With this study, it is evident that members of the same species can reproduce

differently in varying environments, leading to genetic diversity among organisms and

ultimately populations.

Another example where organisms behaved differently based on their

environments comes from a 2009 study on the woodland herb Anemone nemorosa. In this

study, A. nemorosa individuals were sampled from 12 different sites in Switzerland. Half

of the sites were areas of suburban forest and the other half were highly-trafficked areas

of recreational use. It was found that the organisms likely trampled on by humans altered

their size and production of flowers. An excess of aborted seeds in this area was also

found, indicating that the sexual reproductive potential of the population was reduced

(Rusterholz & Baur 2009). With a reduction in sexual reproductive potential comes a

decrease in genetic diversity, which often has a negative effect on population overall.

This study also demonstrates how the environment has a large effect on genetic diversity
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of populations from different areas. In these studies, the environment also had a direct

effect on the mode of reproduction in organisms.

The two studies previously mentioned both found evidence that the location and

nature of the environment that organisms are in have a direct effect on their mode of

reproduction. However, the studies investigate the genetic diversity in a population to

solely understand how a specific difference in environment affects their reproduction. In

reality, there are likely multiple environmental effects that cause populations to reproduce

in different ways. In order to better understand the effect that the environment has on the

species as a whole, we must first genotype individuals of these populations to see how

they are similar to each other. It is imperative that not only individuals living in the same

area are compared, but also populations across the world. This allows for better

understanding of genetic diversity in both individual populations and the species as a

whole, which is important when considering natural selection and climate change.

Our experiment aims to achieve both of these goals by studying genetic diversity

within populations and within the species as a whole through the use of genotyping. By

determining the mode of reproduction present in Hemerocallis fulva, an orange daylily,

we are able to study various areas of population dynamics and biodiversity as a whole

through the revealed genetic differences of individuals. We hypothesize that there will be

low genetic diversity among all populations of H. fulva because it is an organism that

reproduces clonally, indicating that the environment should not have an effect on mode of

reproduction.

III. Methods:

Day 1: Sample Preparation, Grinding, Extraction, and Isolation of DNA

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The CTAB buffer, a detergent used to disrupt cell membranes, was prepared by the

instructor and incubated at 60˚C for 30 minutes prior to use. About a dime or nickel size amount

of three different leaf samples were obtained by each group member. Each group member

labeled two microcentrifuge tubes with their initials and the name of their sample. The three

samples included Clop1, Shu12, Wal3. A minimal amount of sand and PVP was estimated and

added, along with the leaf sample, to a mortar and pestle to help break through the abundance of

cellulose in the plant. The sand was used as an abrasive material and the PVP was used to rid the

sample of phenolic compounds. 500 uL of CTAB buffer was added and the mixture was grinded.

After forming a paste, an additional 500 uL of CTAB buffer was added. The liquid mixture was

poured into a labeled microcentrifuge tube and incubated for 1 hour at 65-70˚C. The mortar and

pestle were cleaned. After the incubation period, 700 uL of chloroform: isoamyl alcohol was

added, in order to separate the DNA from other substances (like proteins). The microcentrifuge

tube was centrifuged at 13,000 rpm for 20 minutes. The top aqueous layer was transferred into

another labeled microcentrifuge tube. 540 uL of cold isopropanol was added, to precipitate the

DNA, and the microcentrifuge tube was inverted 2-4 times. The samples were stored at -20˚C for

future analysis.

Day 2: Isolation of the Pellet and Final Cleaning and Resuspension of DNA

The sample was spun in a centrifuge for 3 minutes at 13,000 rpm. If the sample contained

a clear pellet, the supernatant was discarded. If the sample contained gelatinous material above

the pellet, all but a thin band of isopropanol was removed with a micropipette. The pellet was

washed with 500 uL 75% EtOH to remove impurities, and the tube was inverted several times.

The sample was spun for 3 minutes. The supernatant was removed and the pellet was air-dried

for 30 minutes until it looked dry and did not smell of alcohol. The pellet was resuspended in 80
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uL 1X TE, to make sure the DNA was at the proper pH, with a 20-200 uL pipette. The pipette

was set to 50 uL and gently pumped to break up the pellet. 8 uL of 7.5 M ammonium acetate was

added to the tube in order to remove any excess DNTPs. 180 uL of 100% EtOH was also added.

The contents were mixed and stored at -20˚C.

Day 3: PCR and Electropherograms

The sample was spun for 3 minutes at 13,000 rpm and the supernatant was then decanted

afterwards. The sample was air-dried for about 30 minutes and resuspended in 1X TAE to make

sure the DNA was at the proper pH. For 15-30 minutes, the sample was incubated in a water bath

at 37-40°C to ensure the DNA was suspended in solution. An agarose gel then confirmed the

presence of DNA. The sample was stored at -20°C while a small gel rig was created. 1 g of

agarose was weighed out and placed in a flask. 50 mL of 1X TAE was added to the flask and

swirled to incorporate. The solution was microwaved for 2.5 minutes until the solution was

bubbling and completely clear. The solution was cooled slightly and 10 uL of GelRed was added

for every 100 uL of solution. Once again, the solution was swirled to incorporate then poured

into the prepared gel tray. The flask was washed immediately and the gel was allowed to cool

completely before loading.

Day 4: GenAlEx Data

GenAlEx was used to obtain multiple statistical analyses of the data set. This included

descriptive statistics, alleles per locus, observed heterozygosity, expected heterozygosity, and

genotypes per population.

IV. Results:
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Table 1. The mean and standard error values of the individual loci for each population.

Population Measure Number of Observed Expected

Alleles Heterozygosity Heterozygosity

Mean 1.750 0.750 0.375

PCR Standard Error 0.164 0.164 0.082

Mean 1.750 0.750 0.375

PER Standard Error 0.164 0.164 0.082

Mean 4.250 0.745 0.494

SHU Standard Error 0.453 0.155 0.057

Mean 3.250 0.768 0.506

WAL Standard Error 0.313 0.153 0.058

Mean 2.250 0.750 0.413

WC Standard Error 0.491 0.164 0.092

Table 2. The mean and standard error values of the populations for each locus.

Number of Alleles

Locus Number Mean Standard Error

1 3.600 0.748
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2 2.000 0.000

3 3.000 0.632

4 1.800 0.735

5 1.600 0.400

6 3.000 0.447

7 3.400 0.600

8 2.800 0.583

Table 3. The number of organisms and genotypes per population.

PCR PER SHU WAL WC

Number

Of 6 13 43 7 7

Organisms in the

Sample

Number

Of 1 2 8 2 4

Genotypes
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Figure 1. The relationship showing genetic similarities and differences between the populations
PCR, PER, SHU, WAL, and WC.

Table 1 shows the mean and standard error values for the number of alleles, the observed

heterozygosity, and the expected heterozygosity for each population in this experiment. As seen,

the SHU population had the highest number of alleles at 4.250, while PCR and PER had the

lowest number of alleles with 1.75 each. The observed heterozygosity is generally very high

across all populations, however, the expected heterozygosity is relatively low. Table 2 shows the

number of alleles present per loci. This data shows the number of alleles at each of the eight loci

that were present in this population, helping display the bigger picture of overall genetic

diversity. Table 3 shows the number of organisms present in each sample as well as the number

of genotypes that are present for each population. This gives a numerical representation of the

genetic diversity of the five populations. Figure 1 shows the genotypes of all five populations

present in this experiment. Looking at the graph, a large cluster can be seen in the upper left

quadrant, and the rest of the populations can be seen in the upper and lower right quadrants.
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V. Discussion:

Multiple populations of H. fulva were studied through a genotyping experiment to relate

methods of reproduction to genetic diversity. As mentioned, evidence of sexual reproduction

often yields higher genetic diversity because of recombination and independent assortment.

Asexual reproduction yields offspring that are genetically identical to the parent organism. Since

the orange daylily typically reproduces clonally and does not experience genetic recombination,

we hypothesized that overall genetic diversity among the populations would be low and the

environment should not have an effect on mode of reproduction.

As previously mentioned, Table 1 includes the number of alleles, the observed

heterozygosity, and the expected heterozygosity of the five populations that were examined in

this experiment. A higher number of alleles indicates more genetic variability in a population. As

seen in the collected data, the SHU population had the most alleles, 4.25, and the PCR and PER

populations had the least number of alleles each at 1.75. This means that the SHU population

would be considered the most genetically variable of the five populations. The observed

heterozygosity was also present in Table 1. The observed heterozygosity shows the number of

heterozygotes present in a given population by locus. In general, the higher the observed

heterozygosity, the more diverse a population is and vise versa. All five of the populations show

a relatively high observed heterozygosity, the average being 0.75, classifying them as fairly

diverse. The expected heterozygosity is also seen in Table 1, and refers to what the

heterozygosity should be under Hardy-Weinburg equilibrium. In this case, the expected

heterozygosity is lower than the observed heterozygosity in every population. This means that

there is a possibility that populations that were previously isolated are now intermixing. Overall,

the data in Table 1 shows that the number of alleles per population is relatively low. However,
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the observed and expected heterozygosity indicate a large number of heterozygotes from

previously isolated locations are now intermixing.

The importance of Table 2 is centered around the mean number of alleles present at each

locus, all of which appear to be rather low. The highest value shown is 3.600, which is

comparatively low to other organisms. By determining the number of alleles at each locus, the

mode of reproduction for this organism can be identified as clonal reproduction. In order for an

organism to reproduce sexually, the organism must mate with another organism and their alleles

experience recombination. This process creates a new genetic combination in the offspring,

differing from both parents. Since there are so few alleles in the organism studied here, the

possibility of having different combinations from the parental organism is very low. Imagine

having genes A and B: those genes can only be distributed in the current combination or

separately and there is no other way for them to combine and be distributed. Furthermore, if

there are three genes, A, B, and C, there are only three possible ways to recombine those genes

and distribute them. That being said, the differences between the parental organism and the

offspring would be minimal. Thus, the hypothesis formulated indicating low genetic diversity

can be accepted and supported through not only Table 2, but all of the data collected.

Understanding this in terms of the number of alleles seen in Table 2 is key to supporting the

mode of reproduction as clonal. This will become even more apparent through the discussion of

Table 3.

The data present in Table 3 shows the number of organisms in each sample and the

number of genotypes present for each of the five populations. For example, in the PCR

population, there were six organisms, all with the same genotype. These organisms can then be
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explained as genetically identical clones. For the ease of discussion, these populations will be

explained in order of increasing genetic diversity.

In comparison to the PCR population, the PER population is slightly more diverse. The

PCR population only contains six organisms all sharing the same genotype, whereas the PER

population consists of thirteen organisms split between two genotypes.

The next population in increasing order of genetic diversity would be SHU. SHU is more

diverse than both PCR and PER with eight genotypes overall. However, with forty-three

organisms sharing eight genotypes, we see a similar ratio in comparison to the previously

discussed populations. Most of the organisms present in the SHU population are thus genetically

identical.

In the WAL population, there are seven organisms present, which is less than some of

the previous populations. Nevertheless, the WAL population has two genotypes which is the

same number as the PER population. This means that the organisms represented by each

genotype are genetically identical clones. Even so, compared to one another, the two genotypes

are genetically different. Therefore, the WAL population shows more genetic diversity than the

previous populations because less organisms share the same genotype.

The most genetically diverse population out of the five would then be WC. The WC

population only has seven organisms, although it has four genotypes, which means there aren’t

as many organisms sharing the same genotype. This means that there are three genetically unique

organisms and four genetically identical clones. Out of all of the populations discussed, this is

the most genetically diverse but still contains genetically identical clones within the population.

By looking at Figure 1, which is a graphical representation of Table 3, the genetic

similarities and differences between the five populations can be observed. As previously
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discussed, the PCR population has six organisms sharing the same genotype. This can be seen in

the upper left quadrant of the graph where the PCR population, represented by a blue diamond,

all appear in the same spot. This indicates that all organisms of the PCR population are

genetically identical clones. The PER population has thirteen individuals with two genotypes.

The PER population, represented by a pink square, appears in two clusters seen in the upper left

quadrant and the lower right quadrant. The two different groupings of the PER population is

indicative of the two genotypes. The WAL population, represented by a purple X, also has two

genotypes, and therefore appears in two different locations in Figure 1. WAL appears in the

clusters in the upper left and upper right quadrants. The SHU population was the largest of the

five observed and had eight unique genotypes. SHU, visualized by a green triangle, appears in

clusters in both upper quadrants as well as the lower right quadrant. The WC population,

represented by a blue X, has four unique genotypes, and appears only in the lower right quadrant.

Looking at Figure 1 as a whole, a trend can be noticed. This trend is that almost every

population (with the exception of WC) all appear in the cluster seen in the upper left quadrant of

the graph. This reveals that some organisms within the populations PER, PCR, WAL, and SHU

are all identical clones. Although the cluster in the upper left quadrant accounts for 57 of the 76

organisms sampled, other trends can also be noticed. The area in the lower right quadrant shows

organisms from PER, SHU, and WC, meaning that these samples are not clones but are rather

genetically similar to one another. A similar case can be seen in the top right quadrant where

organisms of SHU and WAL are found, displaying the same trend as seen in the lower right

quadrant.

Overall, the results of this experiment allowed us to accept our hypothesis that genetic

diversity is low among all populations of H. fulva. However, some evidence of sexual
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reproduction among organisms was observed, as shown in Table 1. Data from Table 1 also

shows that a few organisms from each population are genetically different from one another.

Thus, supporting the idea that sexual reproduction is occurring, but not frequently. The highest

average number of alleles per locus for all of the loci studied was 3.600. Since this number is

very low in comparison to other organisms that reproduce sexually, the data supports the idea

that sexual reproduction is not the most common mode of reproduction in H. fulva.

Although the populations reveal similar data about mode of reproduction, the WC

population reveals a higher number of genetic diversity, and possibly, a higher amount of sexual

reproduction. Table 3 shows very few different genotypes among all populations except WC. In

general, about seven organisms from the same population share one genotype. This is not the

case with the WC population, which reveals four different genotypes among the seven

organisms. Figure 1 shows this genetic diversity within the WC population because of the larger

spread of the points on the graph. The data from Table 1 shows that WC has a higher standard

error of alleles per population than the other groups and supports this idea that there is more

genetic diversity present in this group.

Sexual reproduction is evident among these organisms, but asexual reproduction is more

prevalent as supported by the data from this experiment. From analyzing the data in Table 1, it is

possible that some populations were previously isolated heterozygotes and are now intermixed

within the same population. Overall, this supports the idea that evidence of sexual reproduction

may actually be data supporting asexual reproduction that ocurred before these experimented

populations were classified together. The data shows that sexual reproduction is still infrequently

occurring within the populations, indicating that there should be genetic differences between

populations. This idea is supported by Figure 1, which shows a general trend of genomic
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similarities among members of the same population, such as SHU and WC. However, the reason

these trends do not show strong correlations between members of the same population is because

asexual reproduction is the more prominent mode of reproduction, thus yielding offspring that

are genetically identical to the parent organism and not influenced by location.

The data from this genotyping experiment supports the conclusions drawn from similar

experiments in published literature. A 2007 article found low genetic diversity and high

heterozygosity in small populations of Sorbus torminalis, a European tree. The study suggested

that clonal reproduction and small population size were responsible for the findings. The

researchers also made inferences about a self-incompatibility system that maintained some level

of heterozygosity (Rasmussen & Kollmann 2007). The structure and results of this experiment

with small population sizes corresponded to the results of our experiment. However, we did not

study the orange daylily’s ability or inability to self-fertilize. In a future experiment, it may be

beneficial to research the presence of a self-incompatibility system for H. fulva to determine

whether the high percentages of observed heterozygosity were a result of this process.

Another article from 1994 showed an increase in genetic diversity in Phytophthora

infestans, a fungus that caused blight disease in potatoes for over 135 years. The increase in

genetic diversity was most notable in the last year discussed. Overall, from 1845 to 1980, the

fungus remained fairly genetically identical, but in 1980, a new mating type surfaced allowing

sexual reproduction in the organism. After a span of 135 years, the fungus, P. infestans, went

from having one genotype to having 134 genetically unique genotypes (Drenth, et al. 1994).

After seeing the results of this study, it would be interesting to continue to study H. fulva and

identify if it would have the capabilities of becoming a more sexually reproducing organism.
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Since we already see more genetic diversity in the WC population, it would be interesting to see

if after some period of time this genetic diversity becomes greater or not.

Further study of this organism could also potentially identify the conservation of asexual

organisms. It is extremely important to be able to conserve asexual organisms, since they are

clones because of the possibility of one unfavorable environmental factor wiping out an entire

population. Studying the conservation of these organisms would increase their chance of survival

in some circumstances.

Through this experiment and possible further studies involving H. fulva, we are able to

expand our understanding of genetic diversity, population dynamics, biodiversity, and in

essence, the world around us. By studying model organisms and comparing our experimental

results to those organisms, we can identify the genetic diversity of this population. Through

comparison and genotyping, identifying the mode of reproduction becomes even easier. The

mode of reproduction is therefore extremely important through the consideration of biodiversity

and the patterns through generations. These patterns are evident in natural selection, climate

change, etc. and are identified frequently through genotyping experiments. These experiments

allow us to see affected species and populations around the world and allow us to have a better

view of how the world is changing. These population dynamics are visualized through the

genetic composition of different organisms, and in order to view these genetic compositions, we

need genotyping. Genotyping is a huge asset to the field of genetics and allows for the study of

genetic composition, reproductive modes, population dynamics, and biodiversity, thus allowing

us to become closer to the world around us.

VI. References:

Drenth, A., Tas I. C. Q., & Govers, F. (1994) DNA fingerprinting uncovers a new sexually
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reproducing population of Phytophthora infestans in the Netherlands. European Journal

of Plant Pathology, 100, 97-107. https://link.springer.com/article/10.1007/BF01876244.

Grimsby, J. L., Tsirelson, D., Gammon, M. A., & Kesseli, R. (2007). Genetic diversity and

clonal

vs. sexual reproduction in Fallopia spp. American Journal of Botany, 94(6), 957-964.

https://bsapubs.onlinelibrary.wiley.com/doi/full/10.3732/ajb.94.6.957.

Rasmussen, K. K. & Kollmann, J. (2007). Low genetic diversity in small peripheral populations

of a rare European tree (Sorbus torminalis) dominated by clonal reproduction.

Conservation Genetics, 9, 1533-1539.

https://link.springer.com/content/pdf/10.1007/s10592-007-9492-y.pdf.

Rusterholz, H. & Baur, B. (2009). Disturbances by human tampling alter the performance, sexual

reproduction and genetic diversity in a clonal woodland herb. Perspectives in Plant

Ecology, Evolution and Systematics, 11(1), 17-29.

https://www.sciencedirect.com/science/article/abs/pii/S1433831908000656#!.