STUDENT’S GUIDE
Case Study
Who’s the daddy?
James Clarkson
Polyploidy in plant evolution
Dean Madden [Ed.]
Royal Botanic Gardens, Kew
NCBE, University of Reading
Version 1.1
Case Stu
Case Stud
who’s the daddy? polyploidy in plant evolution
Polypoidy in plant
evolution
Multiple sets of chromosomes
David Monniaux, Wikimedia Commons.
Humans, like nearly all animals, have two sets of chromosomes, one from
each parent. We are diploid organisms, arising from the fusion of a haploid
sperm and a haploid egg. Polyploid organisms, in contrast, have three or
more sets of chromosomes. Such duplicate sets of chromosomes are more
than are required for the plant to function, but they can bring advantages.
Polyploids have larger cells to accommodate their extra DNA and therefore
the plants are often larger than their diploid parents. The regulation
and expression of genes in polyploids, often in new combinations, also
contributes to greater variety.
Damien Boilley, Wikimedia Commons.
Polyploidy has occurred independently many times in plant evolution,
and is an important mechanism in the production of new species. Recent
genomic studies have revealed ancient whole genome duplications in
plants. This implies that most, if not all, ‘diploid’ plants are actually
paleopolyploids (ancient polyploids). Therefore it might be more accurate to
think of most plants as polyploids with some having undergone additional
rounds of polyploidy more recently. Some estimates suggest that between
30 and 80% of living plant species are polyploid.
Ploidy
No. of sets of
chromosomes
Apple, banana, citrus,
ginger, seedless
watermelon
Triploid
3
Apple, durum wheat,
cotton, potato, cabbage,
leek, tobacco, peanut
Tetraploid
4
Bread wheat, triticale,
oat, kiwifruit
Hexaploid
6
Strawberry, sugarcane
Octaploid
8
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iStock photo.
Crops
iStock photo.
Their importance to agriculture and therefore the economic value of
polyploids cannot be understated. The four most widely-cultivated crop
plants in the world (wheat, rice, soybeans and maize) are all polyploids. In
fact, 15 of the 21 most important crops, as measured by area of cultivation,
are polyploids.
Wheat, rice, soya and maize, the
four most widely-cultivated crop
plants, are all polyploid.
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who’s the daddy? polyploidy in plant evolution
IMAGE AFTER: Wikimedia Commons.
Parent
Gametes
Faulty
meiosis
Zygote
Diploid
Tetraploid
Diploid
One of the ways in which polyploidy
can arise in plants.
Diploid
How polyploids are formed
Markus Hagenlocher, Wikimedia Commons.
There are two sorts of polyploid plants: autopolyploids and allopolyploids.
Autopolyploids have multiple sets of chromosomes from the same species.
They can result from the fusion of gametes formed after an error in meiosis
(see diagram above). They can also be formed by spontaneous doubling
of the chromosomes. Both processes can occur naturally. Autopolyploids,
such as bananas, are often infertile so such species have to be propagated
from vegetative tissue.
Allopolyploids have sets of chromosomes derived from different species.
For example, the common brassicas Brassica oleracea, B. rapa and B. nigra
hybridise to form three allotetraploids: B. napus, B. juncea and B. carinata.
The relationships between these plants are often represented in a diagram:
Triticale, a cereal crop, has six sets
of chromosomes: four from wheat
and two from rye.
B. nigra
n=8
IMAGE AFTER: Wikimedia Commons.
B.carinata
n=17
B. juncea
n=18
B. oleracea
n=9
B.rapa
n=10
B. napus
n=19
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The ‘Triangle of U’, showing the
relationships between three
common brassicas and hybrid
allotetraploid species.
B. nigra = Black mustard
B. juncea = Indian mustard
B. rapa = Field mustard (turnip)
B. napus = Oilseed rape
B. oleracea = Wild cabbage
B. carinata = Ethiopian mustard
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who’s the daddy? polyploidy in plant evolution
Nicotiana — a model plant
Ken Hammond, US Department of Agriculture / Wikimedia Commons.
Nicotiana is a plant genus which contains of 75 species, including tobacco
(N. tabacum) and its wild relatives. The main centre of diversity for
the genus is South America although species of the genus are native to
Australia, Africa and North/Central America. Although N. tabacum is only
a single species it receives a disproportionately large amount of attention
from scientists. This is because N. tabacum has become a model organism
for genetic studies. Model organisms are studied to understand particular
biological phenomena, with the expectation that discoveries made will
provide an insight into the workings of other organisms. In 1986 it was the
first flowering plant to have its entire chloroplast genome sequenced (the
nuclear genome is currently being sequenced). Tobacco is also the most
widely-cultivated non-food crop in the world.
Left: Willie Greeninge checks
his tobacco plants at his farm in
Chatham, Virginia, USA.
Right: Botanical illustration of
the flowers and seeds of Nicotiana
tabacum.
Nicotiana polyploids
About half of the species of Nicotiana are diploid and half are polyploid. All
of the polyploids are allotetraploids (polyploids with chromosomes from two
species). They have four sets of chromosomes: two sets from their maternal
parent and two from their paternal parent. They can therefore be thought
of as polyploid hybrids containing twice as much DNA as is required for a
functional organism.
The allotetraploids in Nicotiana range from relatively recently-formed
species (thousands of years old) to ancient species (millions of years old).
The base (diploid) chromosome number in Nicotiana is n=12 and therefore
most allotetraploids are n=24.
Both the tobacco plant, N. tabacum, and its wild relative N. clevelandii
(‘Cleveland’s tobacco’) are allotetraploids.
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Bi-parental vs maternal inheritance
The cell nucleus contains the chromosomes that house the nuclear genes.
Nuclear genes undergo recombination (re-shuffling at meiosis) and are
bi-parentally inherited (that is, one set of chromosomes is inherited from
each parent).
Chloroplasts are cellular organelles that perform photosynthesis. They
have their own genome, a small circular chromosome which resembles
a bacterial genome. In contrast to the nuclear genome, the chloroplast
genome is non-recombining and in almost all flowering plants it is inherited
as a unit from ‘mother’ to offspring. This difference in inheritance pattern
between nuclear and chloroplast genomes will be used in the following
exercise to determine the ‘parents’ of two allotetraploid species of Nicotiana.
Outer
membrane
Inner
membrane
Stroma
Lipid
globule
Stroma
lamellae
Starch granule
Structure of a chloroplast, showing
the location of the DNA.
Granum
Mitochondrial Eve
ATP synthase particles
Mitochondria, the organelles that perform respiration, are
also maternally inherited. They have been used by researchers
to trace the maternal lineage in various animal groups.
The ‘Mitochondrial Eve’ story was popularised by Richard
Dawkins in his book ‘River out of Eden’. In that case,
mitochondrial evolution was used to pinpoint the maternal
ancestor of all living humans.
River out of Eden: A Darwinian view of life by Richard Dawkins
(2001) Phoenix. ISBN: 978 1857994056.
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Intermembrane
space Matrix
Cristae
Ribosome
Granules
DNA
Inner membrane
Outer membrane
Structure of a mitochondrion, showing
the location of the DNA.
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DRAWING BY: Mariana Ruiz Villarreal, Wikimedia Commons.
DRAWING BY: Dean Maden.
DNA within the
chloroplast
who’s the daddy? polyploidy in plant evolution
Who’s the daddy?
In the following exercise, you will first be given a tree diagram generated
using DNA sequence data from one of the nuclear genes from the two
allotetraploids, N. tabacum and N. clevelandii, and several related haploid
species that could be ‘parents’ of these two polyploids.
By looking at which species are grouped together on the tree, you will be
able to see which species the ‘parents’ are. What you won’t be able to tell is
which species is the ‘mother’ and which is the ‘father’. In other words, you
won’t know which set of chromosomes came from the pollen and which
from the ovules.
Because the chloroplast genome is transferred in the ‘maternal’ line only,
however (that is, only in the ovules) if you compare DNA sequences from
the plants’ chloroplast genomes, you should be able to see which species
was the ‘female’ donor. The second part of the exercise therefore is to
perform an analysis using DNA sequences from the chloroplast genome.
Once you know who the ‘mummy’ is, a process of deduction will reveal the
identity of the ‘daddy’!
PART 1: Analysing the nuclear DNA
Glutamine synthetase is an enzyme involved in nitrogen metabolism. It is
encoded by a single nuclear gene, and all the species under investigation
have a copy of it. Part of the DNA sequence of this gene (a fragment about 900
base-pairs long) was used to construct the tree diagram on the following
page. Symonanthus aromaticus is an Australian relative of Nicotiana.
Questions
Examine the tree diagram on the following page.
a. Which species has been used as an outgroup?
b. Which species are haploid?
c. Which species are allotetraploid?
d. Describe in general terms how the species are grouped on the tree.
e. Why do N. tabacum and N. clevelandii occur twice on the tree?
f. Which species appear to have crossed to form
i. N. clevelandii and
ii. N. tabacum?
Hint: similar gene sequences will be placed closer together on the tree.
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who’s the daddy? polyploidy in plant evolution
A tree based on nuclear
DNA sequences of part of
the glutamine synthetase
gene.
N. attenuata n=12
N. clevelandii n=24
N. undulata n=12
N. sylvestris n=12
N. tabacum n=24
N. obtusifolia n=12
N. clevelandii n=24
N. tormentosiformis n=12
N. tabacum n=24
Symonanthus
aromaticus
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PART 2: Analysing chloroplast DNA
The second part of the analysis involves the use of part of a chloroplast
gene for nicotinamide dehydrogenase (ndhF), which is an enzyme
involved in photosynthesis.
1. Double-click on the document Nicotiana.geneious. This will start up
Geneious and load the data into the programme. This file contains five
DNA sequences from diploid Nicotiana species plus a sequence from
Symonanthus aromaticus, which will be used as the ‘outgroup’.
2. If you zoom in on the sequences, you will see that they are already
aligned to each other so that the tree can be constructed.
Zoom buttons
These dashes show
where the sequences
have been aligned.
3. The next stage is to import the sequences of the two allotetraploid
species, N. tabacum and N. clevelandii. If you have a connection to the
internet, you can download them. Alternatively, you may have been
provided with the files locally. If you have an internet connection
carry on; if not, go to Step 10 on page 11.
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If you have an internet connection
4. Click on the Nucleotide name in the left-hand panel:
A ‘Search’ box will
appear at the top,
here.
Click
‘Nucleotide’
here.
5. Type the accession code of the Nicotiana clevelandii DNA sequence into
the box — AJ585925 —and click the Search button:
6. The sequence will then download from GenBank:
GenBank
GenBank is a DNA sequence database that is publicly available on the
web. In 2005, GenBank stored one hundred billion (100,000,000,000)
bases of sequence data. This number is slightly less than the number of
stars in the Milky Way and GenBank has grown further since 2005. The
database contains sequence data from organisms as diverse as humans,
earthworms, apple trees, mushrooms and bacteria with 165,000
organisms in total (in the 2005 count). It includes many entire genomes
such as the human genome. Over 13,000 DNA sequence records exist in
GenBank for the model organism Nicotiana tabacum.
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www.ncbi.nlm.nih.gov
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who’s the daddy? polyploidy in plant evolution
7. With the sequence selected in the top window, click on the Text View
tab above the main Geneious window. This will display information
about the DNA sequence including details of the organism that it came
from, sequence features (such as the position of introns and exons) and
a reference for where and when it was first published:
8. You must now transfer the sequence you have downloaded into the
same folder as the six sequences you already have. Do this by clicking
on the name of the sequence in the top window, and dragging the file
to the folder in which the other sequences are stored (probably the
‘Sample Documents’ or ‘Local’ folder):
GenBank database
accession codes
The accession codes for the chloroplast
DNA sequences from the following
species are:
9. Repeat Steps 4–8 for the second DNA sequence — the one from Nicotiana
tabacum. Its accession code is: AJ585931. You should end up with three
documents in the same folder. Now go to Step 13 on the next page.
Copyright © James Clarkson and Dean Madden, 2011
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N. clevelandii
AJ585925
N. tabacum
AJ585931
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who’s the daddy? polyploidy in plant evolution
If you do not have an internet connection
10. Double click on the document named: AJ585925.geneious. This
contains the sequence data for Nicotiana clevelandii, which should now
be imported into Geneious.
11. Do the same for the document named: AJ585931.geneious. This
contains the sequence data for Nicotiana tabacum.
12. Ensure, if necessary, that both new DNA sequences are in the same
folder as the six sequences you already have. Do this by clicking on
the name of the sequence in the top window, and dragging the file
to the folder in which the other sequences are stored (probably the
‘Sample Documents’ or ‘Local’ folder):
13. You should now have a folder containing:
—
—
six ready-aligned chloroplast DNA sequences from diploid species (including an outgroup sequence);
two chloroplast DNA sequences from the two polyploid species,
N. clevelandii and N. tabacum.
14. It is useful to rename the two sequences that are currently identified
by codes, for ease of recognition. Click on each code in turn, wait for
the cursor to appear and type in the name of the species:
15. Select all three documents by holding down the Shift key as you click
on their names in the top window:
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who’s the daddy? polyploidy in plant evolution
16. Having selected the DNA sequences, you need to align them before
generating a phylogenetic tree. Click on the Alignment button at the
top of the Geneious window. A panel will appear. Click the OK button.
Alignment button
Ensure that you select ‘Create an
alignment of all sequences’ here.
17. The DNA sequences will now be aligned. When the process has
finished, you can check that they have been aligned by looking at the
data (remember, dashes indicate where alignment has taken place):
Tree button
Technical note
The Jukes-Cantor distance model
assumes that all base pair substitutions
(mutations) happen at the same rate (1
in 4 or 25%). Other mathematical models
assume that different bases mutate at
different rates.
18. Next, ensure that the newly-generated alignment is selected in the top
window, and click on the Tree button at the top of the Geneious window.
A panel will appear. Select Jukes-Cantor as the Genetic Distance Model,
the Tree build Method should be Neighbor-Joining and there should be
no outgroup. Click the OK button.
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The Neighbor-Joining method is a quick
and popular mathematical model
for calculating genetic distances and
drawing trees. Other methods will
produce slightly different results (and
take longer to do it).
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who’s the daddy? polyploidy in plant evolution
19. A tree will be generated. Ensure that the options selected in the panel
on the right are as shown below:
DO NOT select ‘Transform branches’
here. This will ensure that the
lengths of the branches on the tree
are proportional to the amount of
evolutionary change between the
different DNA sequences.
Questions
g. Examine the tree diagram. Which species are the two allotetraploids
(N. clevelandii and N. tabacum) grouped with? (These will be the
‘maternal’ parents of the two species.)
h. Look at the nuclear tree diagram again (page 7). For each of the two
species, which plant appears to be the ‘paternal’ parent? Copy and
complete the following table.
Tetraploid species
Male parent
Female parent
N. clevelandii
N. tabacum
i. Obtain a print-out of the chloroplast tree diagram. Using a ruler,
measure the lengths of the branches indicated in red above. The length
of each branch is proportional to the amount of evolutionary change.
What can you infer about relative evolutionary age of the two species,
N. clevelandii and N. tabacum?
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