1. Verifying and Documenting a New Mutation A presentation for The Angelfish Society August 16, 2009 Meeting By Tamar Stephens

2. Introduction  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.  You wonder, is this an uncommon variation of one of the phenotypes you expected, or is it something altogether new?  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!  You want to find out for sure if this is something new – but what do you do?

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

4. Is it really a new mutation?

5. Make sure it really is a new trait!  Study the TAS phenotype library to see if you find a phenotype that it resembles.  Ask on the forums, post photos, and ask if anyone has seen anything like it before.  Ask your friends  Send photos to the Standards Committee

6. Rule Out Known Factors 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 in the parent fish, expression of a gene that is masked through epistasis 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 in the parents a trait that has partial penetrance and did not express in the parents or a result of an environmental condition. or a result of an environmental condition.  Let’s look at these factors

7. Is it a hidden recessive?  Any variety of angelfish can carry a single dose of albino (a), gold (g), pearlscale (p) and/or halfblack (h) without these traits expressing.  Some varieties, such as marbles and gold marbles, can carry a single dose of stripeless (S) without any obvious effect on the phenotype.  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 half- black.

8. Is it a trait that was masked by epistasis?  Epistasis results when a gene at one locus alters or masks the expression of a gene at another locus.  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.

9. Is it an uncommon expression of a known trait?  Silver or zebra can have broken or incomplete bars.  Gold marble shows a lot of variation in the expression within a spawn. Gold marble has random black markings. Some will have a lot of black, and some will have only a speck of black. 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 have partial stripes. Some will appear to mimic half-black. Some will appear to mimic half-black. The amount and location of black coloration is highly variable in gold marble.

10. Is it the result of an environmental condition?  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.  Continuous light can prevent expression of stripes in silver angels.  Some phenotypes are very sensitive to environmental conditions: orange in koi; expression of half-black; expression of pearlscale; comb- tail. The expression of orange in koi angelfish can be influenced by environmental conditions.

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

12. What do you do now?  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!  Document the appearance of this fish. Take photos of it at various stages as it grows.  Record the phenotypes and anything you know about the genotypes of the parents.  When it reaches maturity, cross it with multiple mates to create a larger gene pool of individuals with the allele.

13. Questions to answer about a suspected new mutation  How, specifically, does it alter the phenotype?  Is it on a previously unidentified locus, or is it an allele of a known gene?  Is it dominant, partially dominant, or recessive?  How does it interact with other alleles at same locus?  How does it interact with genes/alleles at other loci?

14. All crosses should be carried forward for two generations. 1. Do a P1 Cross (P = parental. This is a cross between two non-sibling angelfish.) Observe the offspring (F1’s) and record what you see. Observe the offspring (F1’s) and record what you see. If more than one phenotype is present, count the number of each phenotype. If more than one phenotype is present, count the number of each phenotype. 2. Do an F1 Cross (F = filial, a cross between two siblings from the first cross) Observe the offspring (F2’s) and record what you see. Observe the offspring (F2’s) and record what you see. If more than one phenotype is present, count the number of each phenotype. If more than one phenotype is present, count the number of each phenotype.

15. Ideally: Cross with a Wild Type  This is the best and most direct way to obtain some angelfish that exhibit only the new trait.  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.  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.  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.

16. Example – “Platinum” mutation  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.  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.)

17. Wild x Platinum 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 sperm) will either be + - + from the wild type or g - u from the platinum.  The resulting F1 offspring then will all have the genotype: +/g - +/u  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.

18. F1 Cross  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: + - + + - + + - u + - u g - + g - + g – u g – u  Let’s put these into a punnet square and see what we will get for offspring.

19. Punnet Square – F1 Cross + - + + - u g - + g - u + - + +/+-+/+silver+/+-+/usilver+/g-+/+silver+/g-+/usilver + - u +/+-+/usilver+/+-u/u*new*+/g-+/usilver+/g-u/u*new* g - + +/g-+/+silver+/g-+/usilverg/g-+/+goldg/g-+/ugold g - u +/g-+/usilver+/g-u/u*new”g/g-+/ugoldg/g-u/u*platinum* The Punnet square shows all the possible outcomes of this hypothetical cross.

20. F1 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.

21. Do 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

22. Calculate percent of each type  A ratio of 9:3:3:1, in percents, would yield 56%, 19%, 19%, 6%.  To determine the percent of each type, divide the number of that phenotype by the total number in the spawn, then multiply by 100.  There were 211 silver in a spawn of 369. 211/369 x 100 = 57%.  Now we do this calculation for all four phenotypes in the F2 generation.

23. Percent of each phenotype  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 silver2115756 “blue” silver 681819 gold722019 platinum1856 We can tell just by looking at the results that actual offspring counts are very close to the predicted outcome.

24. Why is it important to do offspring counts?  If you don’t get approximately the predicted ratio in each spawn, then maybe the genetics is more complicated, such as a gene linkage.  Maybe a double dose of the new gene impairs the immune system or makes the fry more vulnerable in some way.  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.

25. Further crosses  To fully document the genetics of the new mutation, crosses need to be made to determine: Whether it is an allele of a known gene Whether 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 confirm their genotypes.  Just remember to do a P1 cross and an F1 cross and to record what you observe in the offspring.  Next let’s look at just what information you should record.

26. What should you record?  Record all of your information in a computer file, in a notebook, or both.  P1 CROSS: All the information you have about the genotypes and phenotypes of the parents. Include photographs of the parents. 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. 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: Phenotypes of the angelfish used for the F1 cross. Photograph them. 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. Record the phenotypes you see in the F2 offspring – record the numbers of each phenotype. Take photographs of examples of each phenotype.

27. How do you get a new mutation recognized by TAS?  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.

28. What should you submit?  The following guidelines are on the TAS website under “Standards”: 1. Initial crosses should include test crosses to prove the background genetics of the original parents. 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. 2. Crosses should test for alleles with as many existing genes as 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 predictable. 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. 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. 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.

29. What if you don’t have all this information?  The Standards Committee will accept incomplete information, as long as it is sufficient to make a case for the new mutation.  The committee can evaluate the information you have and give you advice on what to do next to fill in data gaps.  Just remember: Do P1 and F1 crosses, Do P1 and F1 crosses, Take photos, do offspring counts, and keep good records of all crosses and the results. Take photos, do offspring counts, and keep good records of all crosses and the results.

30. Some mutations for which the genetics have not been fully documented.  Are you looking for a good project? The genetics of the following phenotypic traits has not been fully documented: Platinum Platinum Combtail Combtail Frank Grainer’s “sport” Frank Grainer’s “sport” Black “crossovers” Black “crossovers” Hump on the heads of males Hump on the heads of males  Suspected Genetic Defects for which genetics has not been fully documented Missing ventrals in gold and gold marble Missing ventrals in gold and gold marble Missing ray extensions in tails Missing ray extensions in tails

31. That’s all for now folks!