Verifying and Documenting Verifying and Documenting a New Mutationa New Mutation

A presentation for The Angelfish SocietyA presentation for The Angelfish Society

August 16, 2009 MeetingAugust 16, 2009 Meeting

By Tamar StephensBy Tamar Stephens

IntroductionIntroduction Imagine that you are raising a spawn of angelfish, and as Imagine that you are raising a spawn of angelfish, and as

they develop you spot one that is different from anything they develop you spot one that is different from anything you’ve seen before. you’ve seen before.

You wonder, is this an uncommon variation of one of the You wonder, is this an uncommon variation of one of the phenotypes you expected, or is it something altogether phenotypes you expected, or is it something altogether new? new?

You find yourself asking on the forums, and studying You find yourself asking on the forums, and studying photographs of angelfish, trying to figure out what it is. photographs of angelfish, trying to figure out what it is. The more it grows, the more convinced you are that this The more it grows, the more convinced you are that this one little fish has a new trait – and it’s in your tank! one little fish has a new trait – and it’s in your tank!

You want to find out for sure if this is something new – You want to find out for sure if this is something new – but what do you do?but what do you do?

Identifying a new traitIdentifying a new trait

If you breed angelfish, you have no doubt spent If you breed angelfish, you have no doubt spent a lot of time learning how to identify the different a lot of time learning how to identify the different varieties, and to predict the outcomes of the varieties, and to predict the outcomes of the crosses that you do with your angelfish. crosses that you do with your angelfish.

The first challenge when you observe a new trait The first challenge when you observe a new trait is to try to determine whether it really is a new, is to try to determine whether it really is a new, previously-unidentified inheritable trait. previously-unidentified inheritable trait.

Is it really a new mutation?Is it really a new mutation?

Make sure it really is a new trait!Make sure it really is a new trait!

Study the TAS phenotype library to see if you Study the TAS phenotype library to see if you find a phenotype that it resembles.find a phenotype that it resembles.

Ask on the forums, post photos, and ask if Ask on the forums, post photos, and ask if anyone has seen anything like it before.anyone has seen anything like it before.

Ask your friendsAsk your friends

Send photos to the Standards CommitteeSend photos to the Standards Committee

Rule Out Known Factors Rule Out Known Factors

You will need to rule out whether it is:You will need to rule out whether it is: an unexpected occurrence of a hidden recessive, an unexpected occurrence of a hidden recessive, expression of a gene that is masked through epistasis expression of a gene that is masked through epistasis

in the parent fish, in the parent fish, an uncommon expression of a known trait, an uncommon expression of a known trait, a trait that has partial penetrance and did not express a trait that has partial penetrance and did not express

in the parentsin the parents or a result of an environmental condition. or a result of an environmental condition.

Let’s look at these factorsLet’s look at these factors

Is it a hidden recessive?Is it a hidden recessive? Any variety of angelfish can Any variety of angelfish can

carry a single dose of albino carry a single dose of albino (a), gold (g), pearlscale (p) (a), gold (g), pearlscale (p) and/or halfblack (h) without and/or halfblack (h) without these traits expressing. these traits expressing.

Some varieties, such as Some varieties, such as marbles and gold marbles, can marbles and gold marbles, can carry a single dose of carry a single dose of stripeless (S) without any stripeless (S) without any obvious effect on the obvious effect on the phenotype. phenotype.

When you cross two angelfish, When you cross two angelfish,

you may have no idea what you may have no idea what recessives are hidden in their recessives are hidden in their genotypes. genotypes.

This silver angelfish could be carrying a single dose of gold, pearlscale, albino, or half-black.

Is it a trait that was masked by Is it a trait that was masked by epistasis?epistasis?

Epistasis results when a Epistasis results when a gene at one locus alters gene at one locus alters or masks the expression or masks the expression of a gene at another of a gene at another locus. locus.

For example, gold is a For example, gold is a

recessive trait, but when recessive trait, but when it is present in double it is present in double dose, it will mask the dose, it will mask the expression of zebra (Z), expression of zebra (Z), smokey (Sm) and/or smokey (Sm) and/or halfblack. 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 Is it an uncommon expression of a known trait?known trait?

Silver or zebra can have Silver or zebra can have broken or incomplete bars.broken or incomplete bars.

Gold marble shows a lot of Gold marble shows a lot of variation in the expression variation in the expression within a spawn. Gold within a spawn. Gold marble has random black marble has random black markings. markings.

Some will have a lot of Some will have a lot of black, and some will have black, and some will have only a speck of black. only a speck of black.

Some will appear to have Some will appear to have partial stripes. partial stripes.

Some will appear to mimic Some will appear to mimic half-black. half-black.

The amount and location of black coloration is highly variable in gold marble.

Is it the result of an environmental Is it the result of an environmental condition?condition?

Even transient mild water quality Even transient mild water quality problems or a brief bacterial problems or a brief bacterial bloom on the bottom of the tank bloom on the bottom of the tank can damage the fins of young fry can damage the fins of young fry and small juveniles resulting in and small juveniles resulting in bent or deformed fins and tails.bent or deformed fins and tails.

Continuous light can prevent Continuous light can prevent expression of stripes in silver expression of stripes in silver angels.angels.

Some phenotypes are very Some phenotypes are very sensitive to environmental sensitive to environmental conditions: orange in koi; conditions: orange in koi; expression of half-black; expression of half-black; expression of pearlscale; comb-expression of pearlscale; comb-tail. tail.

The expression of orange in koi angelfish can be influenced by environmental conditions.

You are convinced it is a new trait.You are convinced it is a new trait.Now what?Now what?

What do you do now?What do you do now?

Pamper it! Take really good care of this little Pamper it! Take really good care of this little angelfish! Protect it and make sure you angelfish! Protect it and make sure you don’t accidentally bag it up and sell it!don’t accidentally bag it up and sell it!

Document the appearance of this fish. Take Document the appearance of this fish. Take photos of it at various stages as it grows. photos of it at various stages as it grows.

Record the phenotypes and anything you Record the phenotypes and anything you

know about the genotypes of the parents. know about the genotypes of the parents.

When it reaches maturity, cross it with When it reaches maturity, cross it with multiple mates to create a larger gene pool multiple mates to create a larger gene pool of individuals with the allele. of individuals with the allele.

Questions to answer about a Questions to answer about a suspected new mutationsuspected new mutation

How, specifically, does it alter the phenotype?How, specifically, does it alter the phenotype?

Is it on a previously unidentified locus, or is it an allele of Is it on a previously unidentified locus, or is it an allele of a known gene?a known gene?

Is it dominant, partially dominant, or recessive?Is it dominant, partially dominant, or recessive?

How does it interact with other alleles at same locus?How does it interact with other alleles at same locus?

How does it interact with genes/alleles at other loci?How does it interact with genes/alleles at other loci?

All crosses should be carried All crosses should be carried forward for two generations.forward for two generations.

1.1. Do a P1 Cross (P = parental. This is a cross Do a P1 Cross (P = parental. This is a cross between two non-sibling angelfish.)between two non-sibling angelfish.)

Observe the offspring (F1’s) and record what you Observe the offspring (F1’s) and record what you see. see.

If more than one phenotype is present, count the If more than one phenotype is present, count the number of each phenotype.number of each phenotype.

2.2. Do an F1 Cross (F = filial, a cross between two Do an F1 Cross (F = filial, a cross between two siblings from the first cross)siblings from the first cross)

Observe the offspring (F2’s) and record what you Observe the offspring (F2’s) and record what you see. see.

If more than one phenotype is present, count the If more than one phenotype is present, count the number of each phenotype.number of each phenotype.

Ideally: Cross with a Wild TypeIdeally: Cross with a Wild Type This is the best and most direct way to obtain This is the best and most direct way to obtain

some angelfish that exhibit only the new trait.some angelfish that exhibit only the new trait.

To do this, you need to be sure you have a wild To do this, you need to be sure you have a wild type (silver) that comes from a line that has not type (silver) that comes from a line that has not been interbred with any other mutations. been interbred with any other mutations.

Wild types (silver) that come from lines that have Wild types (silver) that come from lines that have been interbred with other color varieties can carry been interbred with other color varieties can carry single doses of hidden recessive traits, including single doses of hidden recessive traits, including gold, albino, pearlscale, or half-black. gold, albino, pearlscale, or half-black.

If you don’t know that your silver angelfish is If you don’t know that your silver angelfish is carrying a recessive trait, you may reach carrying a recessive trait, you may reach erroneous conclusions because part of the effect erroneous conclusions because part of the effect may be caused by one of these recessive alleles. may be caused by one of these recessive alleles.

Example – “Platinum” mutationExample – “Platinum” mutation Based on crosses performed by Ken Kennedy and others Based on crosses performed by Ken Kennedy and others

following his example, the platinum phenotype appears to following his example, the platinum phenotype appears to be a result of a double dose of a new recessive mutation be a result of a double dose of a new recessive mutation in combination with a double dose of gold, also a in combination with a double dose of gold, also a recessive mutation. This means that gold and the new recessive mutation. This means that gold and the new mutation are at different gene loci.mutation are at different gene loci.

Let’s take a look at what happens when we cross a Let’s take a look at what happens when we cross a platinum with a wild type. For the sake of this exercise, we platinum with a wild type. For the sake of this exercise, we are going to label the allele for this new mutation with “u” are going to label the allele for this new mutation with “u” for unknown. A platinum then would have the genotype:for unknown. A platinum then would have the genotype:

g/g–u/ug/g–u/u

(note: This new mutation has not yet been fully documented (note: This new mutation has not yet been fully documented through offspring counts. This is a hypothetical example through offspring counts. This is a hypothetical example

based on how the new mutation seems to work.)based on how the new mutation seems to work.)

Wild x Platinum P1 CrossWild x Platinum P1 Cross First we do a P1 cross: First we do a P1 cross:

Wild (+/+-+/+) x Platinum (g/g-u/u)Wild (+/+-+/+) x Platinum (g/g-u/u)

If our hypothesis is valid, then all of the gametes (egg or If our hypothesis is valid, then all of the gametes (egg or sperm) will either be + - + from the wild type or g - u sperm) will either be + - + from the wild type or g - u from the platinum.from the platinum.

The resulting F1 offspring then will all have the The resulting F1 offspring then will all have the genotype: +/g - +/ugenotype: +/g - +/u

We observe that 100% of the F1 offspring look like We observe that 100% of the F1 offspring look like ordinary silver wild types. This confirms that both gold ordinary silver wild types. This confirms that both gold and the new mutation are recessive to the wild type and the new mutation are recessive to the wild type alleles.alleles.

F1 CrossF1 Cross

Next we do an F1 cross: +/g-+/u x +/g-+/uNext we do an F1 cross: +/g-+/u x +/g-+/u Now if our hypothesis is correct, the possible Now if our hypothesis is correct, the possible

gametes (eggs or sperm) will have one of these gametes (eggs or sperm) will have one of these four combinations:four combinations: + - ++ - + + - u+ - u g - +g - + g – ug – u

Let’s put these into a punnet square and see Let’s put these into a punnet square and see what we will get for offspring. what we will get for offspring.

Punnet Square – F1 CrossPunnet Square – F1 Cross

+ - ++ - + + - u+ - u g - +g - + g - ug - u

+ - ++ - + +/+-+/++/+-+/+

silversilver

+/+-+/u+/+-+/u

silversilver

+/g-+/++/g-+/+

silversilver

+/g-+/u+/g-+/u

silversilver

+ - u+ - u +/+-+/u+/+-+/u

silversilver

+/+-u/u+/+-u/u

*new**new*

+/g-+/u+/g-+/u

silversilver

+/g-u/u+/g-u/u

*new**new*

g - +g - + +/g-+/++/g-+/+

silversilver

+/g-+/u+/g-+/u

silversilver

g/g-+/+g/g-+/+

goldgold

g/g-+/ug/g-+/u

goldgold

g - ug - u +/g-+/u+/g-+/u

silversilver

+/g-u/u+/g-u/u

*new”*new”

g/g-+/ug/g-+/u

goldgold

g/g-u/ug/g-u/u

*platinum**platinum*

The Punnet square shows all the possible outcomes of this hypothetical cross.

F1 Cross – Hypothetical ResultsF1 Cross – Hypothetical Results 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.

From the Punnet square, we can see that we will hypothetically get four phenotypes in the F2 offspring in these ratios:

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 countsDo offspring counts Let’s suppose that we do see four phenotypes in the fry.

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.

Suppose there are a total of 369 fry in the spawn, and the numbers of each phenotype are as follows:

211 silver 68 “blue” silver 72 gold 18 platinum

Calculate percent of each typeCalculate percent of each type

A ratio of 9:3:3:1, in percents, would yield 56%, 19%, A ratio of 9:3:3:1, in percents, would yield 56%, 19%, 19%, 6%.19%, 6%.

To determine the percent of each type, divide the To determine the percent of each type, divide the number of that phenotype by the total number in the number of that phenotype by the total number in the spawn, then multiply by 100. spawn, then multiply by 100.

There were 211 silver in a spawn of 369. 211/369 x 100 There were 211 silver in a spawn of 369. 211/369 x 100 = 57%. = 57%.

Now we do this calculation for all four phenotypes in the Now we do this calculation for all four phenotypes in the F2 generation.F2 generation.

Percent of each phenotypePercent of each phenotype When we calculate the percent of each phenotype and compare When we calculate the percent of each phenotype and compare

with the predicted percent, we can see that we did get with the predicted percent, we can see that we did get approximately the predicted percent of each phenotype.approximately the predicted percent of each phenotype.

Phenotype:Phenotype: No. in spawnNo. in spawn Actual Actual percentpercent

Predicted Predicted percentpercent

silversilver 211211 5757 5656

““blue” silverblue” silver 6868 1818 1919

goldgold 7272 2020 1919

platinumplatinum 1818 55 66

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 Why is it important to do offspring counts?counts?

If you don’t get approximately the predicted ratio in each If you don’t get approximately the predicted ratio in each spawn, then maybe the genetics is more complicated, spawn, then maybe the genetics is more complicated, such as a gene linkage.such as a gene linkage.

Maybe a double dose of the new gene impairs the Maybe a double dose of the new gene impairs the immune system or makes the fry more vulnerable in immune system or makes the fry more vulnerable in some way.some way.

It is beyond the scope of this presentation to go into all It is beyond the scope of this presentation to go into all the possibilities, but the bottom line is that without the possibilities, but the bottom line is that without offspring counts, you can’t confirm that you have the offspring counts, you can’t confirm that you have the genetics figured out correctly. genetics figured out correctly.

Further crossesFurther crosses

To fully document the genetics of the new mutation, To fully document the genetics of the new mutation, crosses need to be made to determine:crosses need to be made to determine:

Whether it is an allele of a known geneWhether it is an allele of a known gene How it affects the phenotypic expression of alleles of other genes.How it affects the phenotypic expression of alleles of other genes.

You may need to do test crosses with the P1 parents to You may need to do test crosses with the P1 parents to confirm their genotypes.confirm their genotypes.

Just remember to do a P1 cross and an F1 cross and to Just remember to do a P1 cross and an F1 cross and to record what you observe in the offspring. record what you observe in the offspring.

Next let’s look at just what information you should record.Next let’s look at just what information you should record.

What should you record?What should you record? Record all of your information in a computer file, in a Record all of your information in a computer file, in a

notebook, or both.notebook, or both.

P1 CROSS:P1 CROSS: All the information you have about the genotypes and phenotypes All the information you have about the genotypes and phenotypes

of the parents. Include photographs of the parents.of the parents. Include photographs of the parents. Record the phenotypes you see in the F1 offspring – record the Record the phenotypes you see in the F1 offspring – record the

numbers if you see more than one phenotype. Take photographs numbers if you see more than one phenotype. Take photographs of examples of each phenotype.of examples of each phenotype.

F1 CROSS:F1 CROSS: Phenotypes of the angelfish used for the F1 cross. Photograph Phenotypes of the angelfish used for the F1 cross. Photograph

them. them. Record the phenotypes you see in the F2 offspring – record the Record the phenotypes you see in the F2 offspring – record the

numbers of each phenotype. Take photographs of examples of numbers of each phenotype. Take photographs of examples of each phenotype. each phenotype.

How do you get a new mutation How do you get a new mutation recognized by TAS?recognized by TAS?

You need to submit your information to the Standards You need to submit your information to the Standards Committee. The TAS bylaws say the committee must Committee. The TAS bylaws say the committee must use the following guidelines to evaluate a new mutation:use the following guidelines to evaluate a new mutation:

a. A color mutation must be determined to be a major or significant a. A color mutation must be determined to be a major or significant mutation to earn a new gene name. In general, a slight mutation to earn a new gene name. In general, a slight modification of an existing color variety would not qualify. The modification of an existing color variety would not qualify. The mutation should produce statistically predictable phenotypes mutation should produce statistically predictable phenotypes and must be uniquely identifiable.and must be uniquely identifiable.

b.b. A major or significant mutation will be given the name used by A major or significant mutation will be given the name used by the person doing the documented gene research for that the person doing the documented gene research for that mutation. This research must be reproduced and documented mutation. This research must be reproduced and documented by at least one other person and the research for this gene by at least one other person and the research for this gene must be accepted by at least 4 Committee members. must be accepted by at least 4 Committee members.

What should you submit?What should you submit?

The following guidelines are on the TAS website under The following guidelines are on the TAS website under “Standards”: “Standards”:

1. Initial crosses should include test crosses to prove the 1. Initial crosses should include test crosses to prove the background genetics of the original parents.background genetics of the original parents.

2. Crosses should test for alleles with as many existing genes as 2. Crosses should test for alleles with as many existing genes as possible.possible.

3. Dominant or recessive characteristics should be clearly shown.3. Dominant or recessive characteristics should be clearly shown. 4. Photographs labeling phenotypes should accompany the study.4. Photographs labeling phenotypes should accompany the study. 5. Phenotypes should be uniquely identifiable and statistically 5. Phenotypes should be uniquely identifiable and statistically

predictable.predictable. 6. Actual progeny counts should be included for all crosses, with 6. Actual progeny counts should be included for all crosses, with

the statistical reasoning behind the conclusion given.the statistical reasoning behind the conclusion given. 7. Included should be the manner in which a person can obtain 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 some of the same stock used in the study, in order to verify the results. results.

What if you don’t have all this What if you don’t have all this information?information?

The Standards Committee will accept incomplete The Standards Committee will accept incomplete information, as long as it is sufficient to make a case for information, as long as it is sufficient to make a case for the new mutation.the new mutation.

The committee can evaluate the information you have The committee can evaluate the information you have and give you advice on what to do next to fill in data and give you advice on what to do next to fill in data gaps.gaps.

Just remember: Just remember: Do P1 and F1 crosses, Do P1 and F1 crosses, Take photos, do offspring counts, and keep good records of all Take photos, do offspring counts, and keep good records of all

crosses and the results. crosses and the results.

Some mutations for which the genetics Some mutations for which the genetics have not been fully documented.have not been fully documented.

Are you looking for a good project? The genetics of the Are you looking for a good project? The genetics of the following phenotypic traits has not been fully following phenotypic traits has not been fully documented:documented:

Platinum Platinum CombtailCombtail Frank Grainer’s “sport”Frank Grainer’s “sport” Black “crossovers”Black “crossovers” Hump on the heads of malesHump on the heads of males

Suspected Genetic Defects for which genetics has not Suspected Genetic Defects for which genetics has not been fully documentedbeen fully documented

Missing ventrals in gold and gold marbleMissing ventrals in gold and gold marble Missing ray extensions in tailsMissing ray extensions in tails

That’s all for now folks!That’s all for now folks!