Genetics 201 & 202 - Using Punnett Squares for Multiple Genes

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Genetics 201: Multi-gene Autosomal

When tracking multiple genes, the biggest difference is that we have to track every combination of genes the parents could possibly donate. But unlike single-gene tracking that we've done so far, multiple mutations can travel separately or together, so we can put some of them into the same parent cell.

To differentiate between sets of autosomal chromosomes, we place a colon between the genes on the Punnett square. For now, I am just going to use color-coding to show how the genes are set up, and how they travel. Here, I have added the parents to the parent chromosome box, both wild type, with 2 pairs of chromosomes shown.

To populate the parent gene boxes, you need to represent both sides of the colon. Each side contains a set of genes from a pair of chromosomes. Since both parent chromosomes have the same gene, you only have to represent it once, but this won't always be the case. We'll get to that later.

For now, populate the parent gene boxes.

To fill in the offspring cells, we're still taking the genes from the cock row and combining them with the genes from the hen column, but the difference is that each side of the colon is combined separately with the matching side of the color for the other parent bird.

For the first offspring cell, we take the WT from the left side of the cock's colon and combine it with the WT from the left side of the hen's colon, to get WT/WT on the left side of the offspring's colon.

Then we add a colon because we're moving to the next pair of chromosomes, and we take the WT from the right side of the cock's colon and combine it with the WT from the right side of the cock's colon, to get WT/WT on the right side of the offspring's colon. This gives us a WT/WT:WT/WT genotype for the top left offspring cell. This means the offspring is wild type.

Then we do the same thing for all the rest of the cells!

It's a little more complicated than single-gene inheritance, but still looks fairly easy!

I was asked why you can't, for example, take a blue WT from the left side and a brown WT from the right side of the parent hen's genome, and the answer is- you technically can, because they're the same gene, and the color coding is merely there to make it easier to see how to put the genes into the table. It only matters when the genes are not the same, which we'll see in a moment.

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Unfortunately the tricky part of multi-gene autosomal Punnett squares comes when dealing with heterozygous birds.

For this example, we will pretend the cock bird is het for 2 different genes, "A" and "B."

Again, I've color coded the parent genes so you can see how to split the parent genes up, and to account for all combos. Because the genes on each side of the colon are NOT all the same within their chromosome pair, we have more possibilities- the cock bird must get 4 gene boxes instead of 2.

The cock may donate two wild type genes (1st box), just one A mutation gene (2nd box), just one B mutation gene (3rd box), or both an A and a B mutation gene (4th cell). The hen, as a wild type, will only donate a WT in all slots.

So, again, we just combine the genes from the cock column with the genes in the hen row, careful to match each side of the colon with its same side of the other parent.

• The actual order doesn't necessarily matter, as long as all the genes are accounted for and all genes are on the correct side of the colon. I prefer to place the WT designation first in any hets, since it matches saying 'wild type het/split mutation,' which is why some of these are not in the "same" order.

For the above, all the offspring will be blue, visually, but you have actual wild types (WTWT:WTWT), wild type het A (WT/A:WTWT), wild type het B (WTWT:WT/B), and wild type het for both A AND B (WT/A:WT/B). Visually, they all look identical.

In this case, each offspring genotype is still 25% of the total number of offspring, even though there are more than 4 boxes. The first column of offspring all share a genotype, meaning 25% of the total offspring will be full wild types. Same goes for the other columns. This is not always the case. Sometimes every box in an entire Punnett square is different from every other box. You can find the percentage by dividing 100 by the number of offspring boxes (in this case 8, so each box is 12.5%), and multiplying that number by the number of boxes that match that genotype (each genotype has 2 boxes, so 25%).

You can use the above method for any purely-autosomal multi-gene combination. If you have more than two genes to track, all you need to do is add another colon, and account for all gene combos in the parents. Then it's just a matter of filling in boxes. It's not necessarily harder, but it takes longer and there's more room for mistakes the bigger the Punnett square.

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Peafowl 202: Multi-gene Autosomal and Sex-linked

When combining autosomal and sex-linked genes, it's basically the same as above, but you're tracking males and females in the offspring.

Here is a parent box, color-coded with autosomal and sex chromosome slots. Keep in mind that you don't need to make every text color combination, just every gene combination. Because both birds are homozygous for all their genes, we only need 4 parent boxes to do this.

To fill in the boxes, it's the exact same process as earlier.

In this instance, the top row is cocks, and the bottom row is hens.

The most important difference between a single-gene Punnett square and a multi-gene Punnett square is that birds are not automatically wild type just because they have a WT in the box. If any chromosome section is missing a WT notation, then the bird is that mutation. This only works because peafowl mutations are mainly recessive mutations; in dominant mutations, even 1 copy of the gene would change the phenotype.

Here is an example, where "A" is a mutation. Both parents are heterozygous for the autosomal mutation. The sex-chromosomes normally wouldn't need to be tracked, but we're tracking them to show you how they track in a multi-gene situation with sex-linked genes involved.

I've color-coded the offspring boxes this time. In this instance, 25% of male and 25% of female offspring will be full wild type, and the same for full "A" mutation. 50% of both sexes will be heterozygous for the "A" mutation, which means they will appear blue but carry the mutation still.

If you have more than two genes, you just add more colons, and follow the same method as before, to account for all gene combinations.

As a last note, once you get into tracking multiple traits, it REALLY helps to keep the same genes in the same spot relative to the other genes in the parent boxes. So, if "A" comes first for the cock, it should come first for the hen. However, as long as you account for all three spots in the offspring that are present in the parents, and you are translating from the same chromosome "spot," then the order doesn't technically matter. For example, when I am doing my own Punnett squares, I prefer to put the sex chromosomes at the end, so it's easier for me to see it, but it could go first if you prefer.

Here is an example of a three gene combo so you can see what I mean.

I color coded males vs females in the offspring. You can see, A is first in mom and dad, b is second, and the sex chromosomes are last. But as long as you can match the right pairs to each other in the offspring, the order doesn't really matter. It's just a LOT easier to make sure they're just in the same place for both parents and then for the offspring.

And that's it! More genes just means more colons and bigger strings of letters, but it's all the same method. Write the parent genome, split the chromosome pairs into individual genes in the gene boxes making sure to account for all combos, and recombine them into the offspring boxes.

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Advanced Complex Combinations

I will provide one example of a complicated, multi-gene Punnet square as an example of what they can do. Don't worry too much about understanding the below right away- it's here as an example of how complex they can be, and how many genes they can account for. Once you've done enough simple ones, you'll be able to come back here to see a nice big one!

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We'll use a taupe (oo:Z(pl)Z(pl)) blackshoulder (bsbs) pied (Wp) cock (bsbs:Wp:oo:Z(pl)Z(pl)) and a cameo (Z(c)w) split blackshoulder (WT/bs) pied (Wp) hen (WT/bs:Wp:Z(c)w).

Since the hen is het for an autosomal mutation, and has 2 different sex chromosomes (Z and w), we just have to be careful to include all the combinations. Since the cock has a gene the hen doesn't, we'll add a "blank" wild type spot to the hen's genes for tracking while we learn. To make it easier to fill in offspring boxes, we will put this "blank" wild type in the spot where the cock's opal genes are, to make her genes WT/bs:Wp:WTWT:Z(c)w instead. This will allow us to line up all four gene slots easier.

I've filled in the parent boxes and offspring boxes.

As you can see, even though there's a lot of genes in play, and a good number of results, the table isn't that big. That's because we technically only have one het (white/pied) in the male genes.

Below, I've added a column to the same pairing, to show which offspring are cocks, and which are hens.

A sex-linked gene can double the number of results in a multi-gene pairing, because it travels differently between cocks and hens, so it can look like there are a LOT more possibilities than there really are

Below is the same table as above, using the same parents, just without the rainbow colors. Instead, I have color-coded the offspring boxes, and written a "key" to the right with what the genotype's phenotype would be (what the bird looks like) where blue boxes are cocks and pink boxes are hens.

As you can see, there can be a LOT more going on under the surface than meets the eye!

And that's it! You now know how to use a Punnet square to track single genes, sex-linked genes, multiple autosomal genes, and multiple mixed genes (autosomal and sex-linked). From here, the next step is to learn what genes peafowl have and what sort of genes they are. Once you know that, you can use your shiny new Punnett square making skills to find the outcome of ANY peafowl pairing your heart desires.

This is also useful for reverse-engineering what genes your parent birds have, or reverse-engineering what genes you need a parent bird to have to produce the offspring you want!