Transcript Verifying and Documenting a New Mutation

Verifying and Documenting
a New Mutation
A presentation for The Angelfish Society
August 16, 2009 Meeting
By Tamar Stephens
Introduction
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Imagine that you are raising a spawn of angelfish, and as
they develop you spot one that is different from anything
you’ve seen before.
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You wonder, is this an uncommon variation of one of the
phenotypes you expected, or is it something altogether
new?
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You find yourself asking on the forums, and studying
photographs of angelfish, trying to figure out what it is.
The more it grows, the more convinced you are that this
one little fish has a new trait – and it’s in your tank!
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You want to find out for sure if this is something new –
but what do you do?
Identifying a new trait
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If you breed angelfish, you have no doubt spent
a lot of time learning how to identify the different
varieties, and to predict the outcomes of the
crosses that you do with your angelfish.
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The first challenge when you observe a new trait
is to try to determine whether it really is a new,
previously-unidentified inheritable trait.
Is it really a new mutation?
Make sure it really is a new trait!
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Study the TAS phenotype library to see if you
find a phenotype that it resembles.
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Ask on the forums, post photos, and ask if
anyone has seen anything like it before.
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Ask your friends
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Send photos to the Standards Committee
Rule Out Known Factors
You will need to rule out whether it is:
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an unexpected occurrence of a hidden recessive,
expression of a gene that is masked through epistasis
in the parent fish,
an uncommon expression of a known trait,
a trait that has partial penetrance and did not express
in the parents
or a result of an environmental condition.
Let’s look at these factors
Is it a hidden recessive?
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Any variety of angelfish can
carry a single dose of albino
(a), gold (g), pearlscale (p)
and/or halfblack (h) without
these traits expressing.
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Some varieties, such as
marbles and gold marbles, can
carry a single dose of
stripeless (S) without any
obvious effect on the
phenotype.
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When you cross two angelfish,
you may have no idea what
recessives are hidden in their
genotypes.
This silver angelfish could be
carrying a single dose of gold,
pearlscale, albino, or halfblack.
Is it a trait that was masked by
epistasis?
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Epistasis results when a
gene at one locus alters
or masks the expression
of a gene at another
locus.
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For example, gold is a
recessive trait, but when
it is present in double
dose, it will mask the
expression of zebra (Z),
smokey (Sm) and/or
halfblack.
This gold angelfish could be carrying a
single dose of smokey, zebra or a double
dose of half-black. The double dose of gold
suppresses the expression of these genes.
Is it an uncommon expression of a
known trait?
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Silver or zebra can have
broken or incomplete bars.
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Gold marble shows a lot of
variation in the expression
within a spawn. Gold
marble has random black
markings.
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Some will have a lot of
black, and some will have
only a speck of black.
Some will appear to have
partial stripes.
Some will appear to mimic
half-black.
The amount and location of
black coloration is highly
variable in gold marble.
Is it the result of an environmental
condition?
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Even transient mild water quality
problems or a brief bacterial
bloom on the bottom of the tank
can damage the fins of young fry
and small juveniles resulting in
bent or deformed fins and tails.
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Continuous light can prevent
expression of stripes in silver
angels.
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Some phenotypes are very
sensitive to environmental
conditions: orange in koi;
expression of half-black;
expression of pearlscale; combtail.
The expression of orange in koi
angelfish can be influenced by
environmental conditions.
You are convinced it is a new trait.
Now what?
What do you do now?
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Pamper it! Take really good care of this little
angelfish! Protect it and make sure you
don’t accidentally bag it up and sell it!
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Document the appearance of this fish. Take
photos of it at various stages as it grows.
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Record the phenotypes and anything you
know about the genotypes of the parents.
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When it reaches maturity, cross it with
multiple mates to create a larger gene pool
of individuals with the allele.
Questions to answer about a
suspected new mutation
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How, specifically, does it alter the phenotype?
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Is it on a previously unidentified locus, or is it an allele of
a known gene?
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Is it dominant, partially dominant, or recessive?
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How does it interact with other alleles at same locus?
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How does it interact with genes/alleles at other loci?
All crosses should be carried
forward for two generations.
Do a P1 Cross (P = parental. This is a cross
between two non-sibling angelfish.)
1.
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Observe the offspring (F1’s) and record what you
see.
If more than one phenotype is present, count the
number of each phenotype.
Do an F1 Cross (F = filial, a cross between two
siblings from the first cross)
2.
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Observe the offspring (F2’s) and record what you
see.
If more than one phenotype is present, count the
number of each phenotype.
Ideally: Cross with a Wild Type
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This is the best and most direct way to obtain
some angelfish that exhibit only the new trait.
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To do this, you need to be sure you have a wild
type (silver) that comes from a line that has not
been interbred with any other mutations.
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Wild types (silver) that come from lines that have
been interbred with other color varieties can carry
single doses of hidden recessive traits, including
gold, albino, pearlscale, or half-black.
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If you don’t know that your silver angelfish is
carrying a recessive trait, you may reach
erroneous conclusions because part of the effect
may be caused by one of these recessive alleles.
Example – “Platinum” mutation
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Based on crosses performed by Ken Kennedy and others
following his example, the platinum phenotype appears to
be a result of a double dose of a new recessive mutation
in combination with a double dose of gold, also a
recessive mutation. This means that gold and the new
mutation are at different gene loci.
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Let’s take a look at what happens when we cross a
platinum with a wild type. For the sake of this exercise, we
are going to label the allele for this new mutation with “u”
for unknown. A platinum then would have the genotype:
g/g–u/u
(note: This new mutation has not yet been fully documented
through offspring counts. This is a hypothetical example
based on how the new mutation seems to work.)
Wild x Platinum P1 Cross
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First we do a P1 cross:
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Wild (+/+-+/+) x Platinum (g/g-u/u)
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If our hypothesis is valid, then all of the gametes (egg or
sperm) will either be + - + from the wild type or g - u
from the platinum.
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The resulting F1 offspring then will all have the
genotype: +/g - +/u
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We observe that 100% of the F1 offspring look like
ordinary silver wild types. This confirms that both gold
and the new mutation are recessive to the wild type
alleles.
F1 Cross
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Next we do an F1 cross: +/g-+/u x +/g-+/u
 Now if our hypothesis is correct, the possible
gametes (eggs or sperm) will have one of these
four combinations:
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+-+
+-u
g-+
g–u
Let’s put these into a punnet square and see
what we will get for offspring.
Punnet Square – F1 Cross
+-+
+-u
g-+
g-u
+-+
+/+-+/+
silver
+/+-+/u
silver
+/g-+/+
silver
+/g-+/u
silver
+-u
+/+-+/u
silver
+/+-u/u
*new*
+/g-+/u
silver
+/g-u/u
*new*
g-+
+/g-+/+
silver
+/g-+/u
silver
g/g-+/+
gold
g/g-+/u
gold
g-u
+/g-+/u
silver
+/g-u/u
*new”
g/g-+/u
gold
g/g-u/u
*platinum*
The Punnet square shows all the possible outcomes of this
hypothetical cross.
F1 Cross – Hypothetical Results
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By doing a P1 cross with our original platinum we were
able to separate the new mutation out from the gold in the
F2 generation. This allows us to see the effects of this
new mutation without the confounding effects of the gold.
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From the Punnet square, we can see that we will
hypothetically get four phenotypes in the F2 offspring in
these ratios:
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9/16 silver
3/16 gold
3/16 new trait (which has been reported to look like a “blue” silver)
1/16 platinum
Anytime you get this 9:3:3:1 ratio on offspring, you know
there are two separate gene loci involved.
Do offspring counts
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Let’s suppose that we do see four phenotypes in the fry.
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This cross confirms that the platinum trait is the result of
a new recessive mutation that modifies gold to produce
the platinum phenotype. But to understand the
inheritance, we need to do offspring counts.
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Suppose there are a total of 369 fry in the spawn, and
the numbers of each phenotype are as follows:
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211 silver
68 “blue” silver
72 gold
18 platinum
Calculate percent of each type
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A ratio of 9:3:3:1, in percents, would yield 56%, 19%,
19%, 6%.
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To determine the percent of each type, divide the
number of that phenotype by the total number in the
spawn, then multiply by 100.
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There were 211 silver in a spawn of 369. 211/369 x 100
= 57%.
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Now we do this calculation for all four phenotypes in the
F2 generation.
Percent of each phenotype
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When we calculate the percent of each phenotype and compare
with the predicted percent, we can see that we did get
approximately the predicted percent of each phenotype.
Phenotype:
No. in
spawn
Actual
percent
Predicted
percent
silver
211
57
56
“blue” silver
68
18
19
gold
72
20
19
platinum
18
5
6
We can tell just by looking at the results that actual offspring counts are
very close to the predicted outcome.
Why is it important to do offspring
counts?
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If you don’t get approximately the predicted ratio in each
spawn, then maybe the genetics is more complicated,
such as a gene linkage.
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Maybe a double dose of the new gene impairs the
immune system or makes the fry more vulnerable in
some way.
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It is beyond the scope of this presentation to go into all
the possibilities, but the bottom line is that without
offspring counts, you can’t confirm that you have the
genetics figured out correctly.
Further crosses
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To fully document the genetics of the new mutation,
crosses need to be made to determine:
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Whether it is an allele of a known gene
How it affects the phenotypic expression of alleles of other genes.
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You may need to do test crosses with the P1 parents to
confirm their genotypes.
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Just remember to do a P1 cross and an F1 cross and to
record what you observe in the offspring.
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Next let’s look at just what information you should record.
What should you record?
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Record all of your information in a computer file, in a
notebook, or both.
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P1 CROSS:
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All the information you have about the genotypes and phenotypes
of the parents. Include photographs of the parents.
Record the phenotypes you see in the F1 offspring – record the
numbers if you see more than one phenotype. Take photographs
of examples of each phenotype.
F1 CROSS:
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Phenotypes of the angelfish used for the F1 cross. Photograph
them.
Record the phenotypes you see in the F2 offspring – record the
numbers of each phenotype. Take photographs of examples of
each phenotype.
How do you get a new mutation
recognized by TAS?
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You need to submit your information to the Standards
Committee. The TAS bylaws say the committee must
use the following guidelines to evaluate a new mutation:
a. A color mutation must be determined to be a major or significant
mutation to earn a new gene name. In general, a slight
modification of an existing color variety would not qualify. The
mutation should produce statistically predictable phenotypes
and must be uniquely identifiable.
b. A major or significant mutation will be given the name used by
the person doing the documented gene research for that
mutation. This research must be reproduced and documented
by at least one other person and the research for this gene must
be accepted by at least 4 Committee members.
What should you submit?
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The following guidelines are on the TAS website under
“Standards”:
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1. Initial crosses should include test crosses to prove the
background genetics of the original parents.
2. Crosses should test for alleles with as many existing genes as
possible.
3. Dominant or recessive characteristics should be clearly shown.
4. Photographs labeling phenotypes should accompany the study.
5. Phenotypes should be uniquely identifiable and statistically
predictable.
6. Actual progeny counts should be included for all crosses, with
the statistical reasoning behind the conclusion given.
7. Included should be the manner in which a person can obtain
some of the same stock used in the study, in order to verify the
results.
What if you don’t have all this
information?
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The Standards Committee will accept incomplete
information, as long as it is sufficient to make a case for
the new mutation.
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The committee can evaluate the information you have
and give you advice on what to do next to fill in data
gaps.
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Just remember:
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Do P1 and F1 crosses,
Take photos, do offspring counts, and keep good records of all
crosses and the results.
Some mutations for which the genetics
have not been fully documented.
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Are you looking for a good project? The genetics of the
following phenotypic traits has not been fully
documented:
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Platinum
Combtail
Frank Grainer’s “sport”
Black “crossovers”
Hump on the heads of males
Suspected Genetic Defects for which genetics has not
been fully documented
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Missing ventrals in gold and gold marble
Missing ray extensions in tails
That’s all for now folks!