Pinpointing Plates
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Your big chance has arrived
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1
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Great news! News of your studies into the location of the plates gene have reached the royal geneticists, and they are very impressed. They have given you a scholarship to study further, and you have been honored with an apprenticeship to the great dragon geneticist Nantal.
As if that weren't enough, Nantal wants to send you to a scientific conference far across the island to learn more about the latest techniques. Before you can go there, however, the geneticists want you to look further into the interesting question that brought you to their attention. As Nantal's apprentice, you know you will be in great hands. But of course, he wants you to keep thinking about your plates problem right away…
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Thinking it over
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2
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You have already learned one way to narrow down the location of a
hidden gene by examining other traits that are linked to it.
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Experimenting
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3
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Because the gene for horns is on Chromosome 1, we can guess that the hidden gene for plates probably is located there also. We will concentrate on Chromosome 1 in our next experiments (though you can peek at the others if you wish).
Below, you see a dragon and two views of its chromosomes. One of these views is familiar, and the other one is new. Try playing with the lower view and comparing what you see to the upper view. Can you figure out what is being shown here?
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Zooming In
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4
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Tracking Chromosomes
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5
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Continue to the next page to breed some dragons.
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Breeding Experiments
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6
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Making a back cross
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7
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Finding the plates gene
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8
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Finding the plates gene
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9
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Make another back cross, and use the shift key to select all the dragons that have plates. Do you notice any locations they have in common? It may help to breed several sets of dragons. (The chromosomes are smaller now, to make it easier to see.)
Try selecting some that don't have plates as well. Do these dragons have the same locations in common? Where do you think the plates gene is located?
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Narrowing it down
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10
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Narrowing it down
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11
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Narrowing it down
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12
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Running the numbers
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13
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In the chart below, you can choose two options for the locations A-E. If both a dragon's chromosomes are red at a location, we'll call it AA . If one is red and one is blue, we'll call it AB .
The first three rows have already been completed for you as an example. Use the image and the dragons shown to complete the rest of the rows for Dragons 4-9 correctly. As you complete the chart, pay attention to the graph below.
(*Dragons with plates)
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Running the numbers
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14
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Seeing it in action
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15
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Great detective work! Now you're finally deemed ready to go to the conference. You can hardly wait, because the legendary scientist VonTenz will be discussing a groundbreaking analysis technique there. You pack your bags and settle into the carriage seat for the treacherous ride over the Tomborg Mountain pass. Fortunately, the seat is comfortable and your driver is experienced, so you feel safe enough to look over an article that Nantar assigned you to read before the conference.
The article is by Dr. DePran, and describes early research into the technique you will hear about at the conference. You pull out the scroll you checked out of the Great Library and begin to read. Journal articles are always so difficult, but you have a feeling that this technique will come in handy sometime…
The first talk
The trip to the conference went quickly. Your first conference!
A chance to make friends with other researchers, and the great food!
A chance to listen to talks on the latest techniques, and the great food!
Discussions late into the night about possible new methods, (And did we mention the great food?!)
The first big talk is just about to start. It turns out that the famous researcher Professor DePran will be at the conference – the same one whose paper you read on the trip over. He will be describing his team’s discovery of the gene that is responsible for Dragon horns and the methods he helped develop for using Drakes as a model for Dragon genetics.
Professor DePran:
As we embarked on the search for the gene that is responsible for
horns in the Dragon, we were confronted by the time it would take to
perform our experiments. We need pure lines, inbred for at least 20
to 40 generations and with the Royal Dragon’s slow reproduction rate
(once every 500 years) it would take far too long. Fortunately there
is a very close model to the Dragon, the Drake. Drakes are small
common creatures in the same line as the Dragon but they reproduce 4
times in a year and weigh only an ounce or two. Just as the common
mouse has 98% the same genes as a human, the Drakes are 99% the same
as the Royal Dragon. Additionally, the Drake lines have been
isolated by geography for thousands of years, so the 6 common
strains of Drake breed true.
Professor DePran:
We picked
one strain with horns and one strain without horns
to breed and test. We have recently sequenced the genome of all the
common strains of Drake, but it would be far too expensive to do this
for all of our test animals. Instead we used “markers," sometimes
called SNPs or Single Nucleotide Polymorphisms. SNPs are a change
in a single base pair that are different between two strains of
Drake. By testing for these we can deduce which strain the region
around the SNPs came from. Here is a simple example for three
drakes. The red dot is a chromosome that we normally cannot see.
Each generation there is recombination or “crossover.” I have
colored the markers of one strain in white and the other in blue. If
this crossing over occurs once for each generation and the
distribution along the genome is random then we would expect the
markers closest to the gene of interest to more often accompany the
trait. And this is true.
Professor DePran:
The frequency of recombination between two locations is a measure of
the distance between two alleles. We can use this to find the
markers that are closest to the genes of interest by observing the
phenotype (horns) and genotyping the markers. Here is an example in
tabular form:
Professor DePran:
Looking at the patterns we can see that Marker 3 is a perfect match
for the pattern we see in the horn phenotype. We can simply count up
the matches and graph them to more easily see the results:
Of course when you have hundreds of Drakes and hundreds of markers it is much easier to have a statistical test that gives a number for each marker. We call this a LOD score. LOD is the Log (base 10) of the Odds of finding the gene at a particular location. The higher the LOD score the more likely we are to find the Gene in that location. This also gives us the ability to quantify thresholds and confidence intervals. Thresholds tell us how high a LOD score we need for a given probability. For example, we may want a threshold line that represents a 1 in 100 probability that we would see a LOD score that high by pure chance. Confidence intervals allow us to determine the extent of the region on the chromosome. For example, we can specify that the gene in question has a 95% chance of being between 400,000 and 450,000 base pairs on chromosome 1.
Professor DePran:
We created a cross between Desert Drakes and Valley Drakes. Desert
Drakes always have horns and Valley Drakes never have horns. After
the first cross NONE of the Drakes had horns.
White is for drakes without horns, black is for drakes with horns. Round is Female, square is male. The result of NO drakes in the first generation having horns confirmed that horns are a recessive trait. If “H” is the allele for no horns and “h” is the allele for horns then: HH = no horns, Hh = no horns, and hh = horns.
For the Second generation we will cross a hh Male with a Hh Female:
Professor DePran:
For our QTL (Quantitative Trait Loci) analysis we took Females from
this generation of Heterozygote (mixed) Drakes that had horns and
crossed them to Desert drakes that did not have horns (as seen in
the second figure on the previous slide).
We created 418 Drakes for this cross and measured their phenotype. We genotyped the drakes at 200 markers and generated the LOD scores.
The threshold line shows that the LOD score is much higher than 1 chance in 100. The 95% Confidence Interval bars are from 310,000 to 320,000 base pairs on chromosome 1.
Professor DePran:
Over years of research we have accumulated vast knowledge about
different segments of the genome. Archivists and curators have
gathered this information and collected it in large data bases. One
of the ways we index this information is in the order it occurs in
the genome. These are the Genome Browsers. With the recent addition
of the full sequence on many organisms (including human, mouse,
dragon, and drake) we now have a way of looking at what features
occur in the segment between 310,000 and 320,000 base pairs on
chromosome 1.
When we go to the genome browser and look in this region we find only one gene!
And that one gene is Spint1 .
Knockouts (deactivation) of the Spint1 gene confirmed that this did prevent the formation of horns in the Desert Drake that would normally have horns.
Thank you for your time, I will now take questions……
Interlude
That was a very informative talk and covered many of the techniques
that are being used so much by researchers on the cutting edge. So much
information, but there is no time to do anything but jot down some
quick notes and get ready for the next talk….
You have been looking forward to meeting Professor VonTenz. He was DePran’s best student and has continued his work, extending it to the search for the cause of disease. Now we will get to see how these methods can be applied by one of the best...
Professor vonTenz
Up until 20 years ago, Allium cepa, the common onion , was not a common part of the dragon diet. After its introduction to Dragons we discovered two interesting facts:
Dragons really like onions (cooked, never raw), and
Some dragons get a very bad upset stomach after eating onions.
This allergic effect caused their heads to turn green (except for the green dragons, who turn purple) and the effect was severe enough that if exposed multiple time it can cause death in some dragons. The same effect was noted in the inbreed strains of Drakes and this allow us a way to investigate the genetic link to this allergy.
Professor vonTenz
The following graph shows the severity of the effect (as measured by
the pigment change in the face two hours after ingestion of a diet
containing 22% Allium cepa {in the form of Salsa, extra hot}:
(diamonds show the Mean value and the bars show two standard
deviations).
Professor vonTenz
We determined that the allergy was a recessive trait and that the
Swamp drake was homozygous for resistance (RR) and the Desert Drake
was homozygous for the Allergy (rr). We choose to cross the Swamp
drake with the Desert Drake to generate heterozygous offspring (Rr).
We then crossed these offspring to generate 506 second generation
offspring (RR. Rr. rr). These were tested for quantitative face
pigment change and genotyped.
Professor vonTenz
We expect 25% of the offspring to be RR, 50% to be Rh (for the Rh
and hR, we didn’t measure if the “R” came from dad or mom) and 25%
rr. We observed a strong allergy effect in about 25% of our second
generation drakes. Our QTL analysis used 30 markers on each
chromosome and showed a strong peak on Chromosome 2. The large LOD
score is indicative of a single gene. The Confidence Interval gave a
95% chance that the gene was between 54 and 58 centiMorgans (540,000
and 580,000 bp).
Professor vonTenz
Going to our Genome Browser and looking at this region of Chromosome
2 gives us five possible genes:
And we could predict that there were SNPs near enough to the gene to possibly be predictive markers for the allergy.
Professor vonTenz
We found the most predictive SNP to be SNP1156, giving an almost 99%
prediction of the alleles and predicting the allergic effect.
Through this work we will now be able to predict from a simple and
inexpensive test if a Dragon will be allergic to Allium cepa and
freeing our great friends to enjoy their Salsa without fear.
Additional work may lead to a way of correcting the mutation in the
Pomc1 gene in the future.
Thank you for your attention, I will now answer questions…….
Interlude
We had no idea of the massive amount of information that is
available in the archives. Thousands of researchers working for
decades to create and curate knowledge, all now at our fingertips.
So much and yet it covers less than 3% of the known genome. But the
power of the techniques gives us hope that our search is now
possible. We can go over our notes on the trip back to home, and
start to make research plans….
You can hardly wait to return to Gandwar and let all the dragons know about everything you learned. You have a feeling that this will all come in quite handy very soon.