Why it's hard to produce diamond (lava charcoal) corns: a case of genetic linkage?

This exchange happened in Joe Pierce's 2015 season thread, but I thought I would post it here so it would have some more visibility.

Joe noticed that he was having no luck producing a diamond corn snake.

I bred a group of 1.2 het Lava Pewters for years and never produced a single Diamond. Everything else I could produce from the project, but not one of them.

I saved some ph's back like these two. Maybe, just maybe one Diamond this year.

From Steve Roylance:

I have had a similar experience. I have 2.4 Het Lava Pewters and have bred them 4 years in a row (some even double clutched) and still have not hit the target!! Diamond!

I even held back some Poss Hets and bred them and still didn't hit a Diamond lol!

My response, slightly edited for clarity:
"Sometimes when people get results like that, it makes me wonder if any of the corn snake mutations are "linked," a term describing two loci that are close together on the same chromosome. When this is the case, the alleles at those two loci are no longer inherited independently of each other, which has implications when you are trying to make a double homozygous animal.

For example, say you crossed a lava (vvCC) and a charcoal (VVcc) to make het lava het charcoals (VvCc). If the lava and charcoal loci are on the same chromosome, in the het offspring one member of the chromosome pair has the V and c alleles (inherited from the charcoal parent), and the other chromosome has the v and C alleles (inherited from the lava parent). Normally, you would expect these hets to be able to make Vc, VC, vC, and vc sperm/eggs, but linkage would prevent this, because the v allele is stuck on the chromosome with the C allele, and the V allele is stuck on the chromosome with the c allele, so when the members of the chromosome pair separate and go their separate ways into the gametes, v and C (or V and c) get dragged together into the same egg/sperm. So only two types of egg/sperm are produced, vC and Vc, making it impossible to produce diamonds, as no gamete carries both lava and charcoal mutations simultaneously. When two vC gametes unite, you get lavas not het charcoal. When two Vc gametes unite, you get charcoals not het lava. When a vC and Vc gamete unite, you get het lavas het charcoals, but again the mutant charcoal allele is not on the same member of the chromosome pair as the mutant lava allele.

How do we explain the existence of diamonds, if it should be impossible? The reason it's not impossible in reality is this: During egg/sperm formation, members of a chromosome pair line up and exchange segments of genetic material with each other, mixing up the allelic arrangements on those chromosomes in a process called crossing over. If an exchange happens between the lava and charcoal loci in the het lava charcoal animals described above, you would be able to produce VC and vc gametes (these gametes are called recombinant because their allelic arrangement is different from the original arrangement of Vc or vC in the parental chromosomes). However, the closer two loci are on the same chromosome, the less likely it is that a crossover event happens between them. If they are really far apart, a recombination happens at the maximum rate of 50%, generating results that are indistinguishable from independent assortment (the normal pattern of inheritance you get when genes aren't on the same chromosome). If they are really close together, recombination is very infrequent. Whether lava and charcoal are linked is just speculation but accurate and detailed clutch data could disprove it.

If the linkage were very tight you would expect this result:
If you breed a charcoal to a lava to produce double hets, and cross these double hets to each other, you would get

1/4 lavas NOT het charcoal (inherited a vC chromosome from both parents), 1/4 charcoals NOT het lava (inherited a Vc chromosome from both parents), 1/2 het diamonds (received a Vc from one parent and a vC from the other).

Some types of normals (VVCC, VvCC, VVCc) and diamonds (vvcc) would be rare, as would charcoals het lava (Vvcc) and lavas het charcoal (vvCc), because those would require the inheritance of one or two recombinant chromosomes (vc or VC). Is this anyone's experience?

To see how I got that result, when genes are tightly linked you can kind of treat them as the same locus on a Punnett square. The gametes produced by the double het animals would be either mutant for lava and wt for charcoal, or wt for lava and mutant for charcoal. So either Vc or vC. Here is the square, treating the charcoal and lava loci as a unit:

____Vc__vC
Vc| VVcc VvCc
vC| VvCc vvCC

So only 3 genotypic/phenotypic classes.
VVcc=charcoal, not het lava
vvCC=lava, not het charcoal
VvCc=normal, het lava het charcoal.

This assumes perfect linkage, so even if the loci are linked, deviations from this result are normal. But this result is very different from what you would expect from independent assortment (the way we normally calculate the result of crosses involving more than one locus). Offspring deviating from the above square had to have inherited a recombinant chromosome, allowing us to calculate the "recombination frequency" between lava and charcoal. In a normal cross that involves 2 loci assumed to be on different chromosomes, the recombination frequency is 50% (you can prove this by comparing the results from a normal Punnett square to the one above), so frequencies between 0 (loci are really close together) and 50% (loci are really far apart or on different chromosomes) are suggestive of linkage.

There is a somewhat hilarious implication of this. If lava and charcoal were actually linked, if you did hit a diamond in the above cross, you might be tempted to mate it with its charcoal or lava littermates to produce more diamonds. But the Punnett square above should caution you against that, since the charcoals are probably not het lava, and the lavas are probably not het charcoal!

But you have to mate it to something, and there is still hope: all offspring of your diamond will automatically inherit a chromosome with the vc arrangement (since the diamond has two vc chromosomes, which is why it's vvcc). Thus, those offspring can easily produce vc gametes without recombination, allowing you to easily produce more diamonds when you mate a diamond to its offspring. If your pairing was diamond x homozygous wt, the offspring will be VvCc, but one of their chromosomes will be vc and the other will be VC. Thus, assuming tight linkage, roughly half their gametes will carry the vc chromosomes and half will cary VC. So your odds of getting more diamonds when you cross the diamond to its het diamond offspring should be closer to 1/2 than to the 1/4 you would normally predict! Note that in this case you DON'T get the same result if you cross the diamond to its het diamond SIBLINGS. In that case you would only get lavas and charcoals! However, a diamond produced from a diamond parent can be paired with its siblings to easily produce more diamonds. The arrangement of the alleles on the homologous chromosomes has to be considered when predicting the outcome of crosses involving linked genes, not just the genotype of the parents.

Sorry for the long post, sometimes a simple concept leads you down several roads."

From Steve:

Very Interesting. I was hoping you'd chime in on this one.

It does appear so far that my Lava and Charcoal hold backs from this projects are NOT Het for each other. I'm breeding some more this year from this project so we'll see what happens in a couple months when babies hatch.

Thanks for the info!

I think the observation that the charcoals/lavas Steve held back were not het for lava/charcoal supports my hypothesis of linkage, although more data is needed. He is testing possible hets from at least 5 more holdbacks this year.

This explanation might seem pretty technical to some of you, but my initial audience was Joe and Steve, who seem to have done their homework, and Mitchell Mulks, who has a formal background in the life sciences. So I will be happy to clarify anything that doesn't make sense.

Also, would love to have clutch data from people performing breedings that could potentially have produced diamonds, to see if the results conform to what we'd expect from genetic linkage. Corn snakes have 18 chromosome pairs, and there are more than 18 described mutations in corn snake, so it would not be surprising at all that at least two of them share a chromosome, and are close enough together to exhibit linkage.

Thanks for reading!

Please see post #6, for an expanded explanation!

This is very interesting and now makes think twice about my plans of a diamond project from scratch. But maybe I will just to see if I get the same results.

Originally Posted by Jereme

This is very interesting and now makes think twice about my plans of a diamond project from scratch. But maybe I will just to see if I get the same results.

Go for it! Someone definitely needs to finally produce one, so that more can be readily produced from my instructions. Even producing a lava that proves het charcoal or a charcoal that proves het for lava will suffice. But they will need to be crossed to another lava het charcoal or charcoal het lava, or to their own offspring, or to a dimaond, in order to produce more diamonds. This should increase the value of diamonds, I would think.

Working on a post that will show you how to follow the chromosomal arrangements a little better, and has a rudimentary illustration of what is going on during recombination to produce the vc gametes that are necessary to produce diamonds.

I am a fan of genetics, so I love reading your post.

And really want to see your post on basic genetics. I can't seem to not overwhelm my wife when I try to teach her, so I am hoping you do a better job.

I'll try my best!

Now I'm going to expand on my post to hopefully clarify some things.

The notation I am going to use for the genotypes is something like this: Vc/vC. That is a het lava het charcoal, but I've used the slash to indicate how the alleles are arranged on each member of the chromosome pair. So one chromosome is Vc and the other is vC, and the only gametes that animal can produce, without an apparently rare recombination event, are Vc and vC. So that animal would not be able to produce a diamond in the first generation, no matter what you mate it to, unless a rare recombination event occurred to produce a vc gamete.

Here are some crosses to illustrate how to use the concept:

1) Lava x Charcoal= Lava (vC/vC) x Charcoal (Vc/Vc)

The lava can only make vC gametes, and the charcoal can only make Vc gametes, so all offspring will be vC/Vc

2) Het lava het charcoal offspring from 1 crossed to each other = vC/Vc x vC/Vc
The only gametes that can be produced without recombination are vC and Vc. So offspring are

¼ vC/vC (lava not het charcoal)
¼ Vc/Vc (charcoal not het lava)
½ vC/Vc (het lava het charcoal)

Note that the allelic arrangement is the same as both parents, ie you never see a vc or VC chromosome. So we haven't made any headway. If you cross lavas not het charcoal to charcoals not het lava, you will not get any diamonds. There is a possibility of getting diamonds from cross 2, though. I will return to this later.

3) Crossing the lavas not het charcoal to the charcoals not het lava from above is the same as doing cross number 1. Crossing the het lava het charcoals is the same as doing cross number 2.

From cross number two, there is a small possibility of recombination. Normally those snakes can only produce vC and Vc gametes. But when a vC and Vc chromosome line up during meiosis before separating, something can happen. Basically what I've done below is draw a horizontal line representing the chromosome, and with the charcoal and lava loci (pronounced LOW-sigh, by the way) labeled. They are one below the other, and an X indicates crossing over (recombination). What is happening is that the chromosomes break where the X is drawn, and reconnect to their partner. So in the top chromosome, follow along until the X, then move down to the lower chromosome. That gives you one of the outcomes of a crossover. Follow the bottom chromosome until the X, and move up to the top chromosome, to get the other outcome. I color-coded parts of the chromosomes, so you easily understand what is happening. The result is two new chromosomes that have the same LOCI on them, but the alleles that occupy them are a different combination than what we saw before. When a crossover happens between a Vc and vC chromosome in the region between the lava and charcoal loci, the outcome is a VC chromosome and a vc chromosome.



So when these recombinant chromosomes separate during meiosis, a small amount of VC and vc gametes will be produced. A vc gamete is what you need to produce a diamond, because a diamond results from the union of a vc sperm and vc egg. You can see now why a recombination event is rare between loci that are close together on the same chromosome. Since recombination is a random event, the further apart two loci are, the more likely it is that the region a crossover occurs in will be between the two loci, instead of outside them. You might be wondering if the lava and charcoal loci were FAR apart on the same chromosome, would recombination always happen between them, resulting in gametes that never have the same allelic arrangement as the parents? This does not happen, because if loci are far apart, more than one recombination event can happen between them. An odd number of crossovers results in recombinant chromosomes relative to the two loci in question, and an even number of crossover events results in nonrecombinant chromosomes, relative to the two loci in question.

So in this cross, almost all gametes will be Vc and vC, but a very small portion will be vc or VC. So the odds of a diamond are very small, because it requires the union of TWO recombinant gametes.

However, some potentially useful offspring can be produced that require only one recombinant gamete instead of two. If a recombinant vc gamete unites with nonrecombinant Vc or vC gametes, you will get vc/Vc or vc/vC offspring. These are lavas het charcoal and charcoals het lava. These can be used to produce diamonds. However, most of the lava and charcoal offspring from cross #2 would be vC/vC or Vc/Vc, ie homozygous for one trait but NOT het for the other. You’d have to prove out the ph hets from the lavas and charcoals to find the rare ones that are recombinant and actually het for lava or charcoal. But from a het lava het charcoal x same (vC/Vc x vC/Vc) you are more likely to produce lavas het charcoal or charcoals het lava than you are a diamond, because one type requires the union of a recombinant and nonrecombinant gamete, whereas the other requires two recombinant gametes. But these lavas het charcoal and charcoals het lava can be used to easily produce diamonds.

Say you held back all of the lava ph charcoal and charcoal ph lava offspring, and managed to find one that was indeed homozygous for one trait and het for the other. Maybe you didn’t get lucky enough to get two that proved out their hets. If you did, though, you could mate them together. Assume you had a lava het charcoal and a charcoal het lava. The arrangement of their alleles on the chromosomes would be vC/vc for the lava het charcoal, and Vc/vc for the charcoal het lava. Crossing the two would give

4) Lava het charcoal (vC/vc) x charcoal het lava (vc/Vc)

¼ vC/Vc (normal het charcoal het lava)
¼ vC/vc (lava het charcoal)
¼ vc/Vc (charcoal het lava)
¼ vc/vc (diamond)

So a ¼ probability of producing diamonds!

Notice that the normals het charcoal het lava do not have an allelic arrangement that would be useful for rapidly producing more diamonds. They’d have to have another rare recombination event to produce any vc gametes.

You also produce more lavas het charcoal and charcoals het lava that have the vc arrangement on one of their chromosome pairs, so these could be mated to each other or back to the parents to create more diamonds.

Note that in this cross, recombination can’t come back to bite you. Since the parents were vC/vc and Vc/vc, recombination can’t change the allelic arrangements to undo all your hard work. Recombination between a vC and vc chromosome produces vc and vC chromosomes, the same two types that you had before.

In all likelihood, you won’t be able to prove out the ph for two separate charcoals/lavas, unless you have a huge collection. You’ll probably just get one or the other. Say you got a lava ph charcoal that proved out lava het charcoal. You can still create diamonds easily by mating it to anything, and crossing the offspring back to the lava het charcoal. Just say you mated it to a het charcoal het lava Vc/vC.

5) lava het charcoal (vC/vc) x het charcoal het lava (Vc/vC) gives

¼ vC/Vc (het lava het charcoal, same arrangement as parent)
¼ vC/vC (lavas not het charcoal)
¼ vc/Vc (charcoals het lava)
¼ vc/vC (lavas het charcoal)

This kind of stinks because you get ½ lavas, but only half of those have a vc chromosome, which you need to make more diamonds. But the charcoals should all have one vc chromosome. So mating them to each other or to the lava het charcoal parent would produce more diamonds.

You could get diamonds in this cross, if a recombination event occurred in the het charcoal het lava parent. You could also get normals (vc/VC), and those normals would be good for diamond-making.

Try this instead

6) Cross lava het charcoal (vC/vc) to a charcoal (Vc/Vc) and get

½ vC/Vc (het lava het charcoal, not a favorable arrangement for diamond making)
½ vc/Vc (charcoal het lava, favorable arrangement for diamond making)

In this cross, half of the offspring were suitable for making more diamonds, and they are all charcoal, which is an immediate visual indicator of which offspring to hold back. Note that any recombination event during gamete formation in the parents does not change anything. Recombination between a vC and vc chromosome generates a vc and vC chromosome, the same as what you started with. Same for the chromosomes of the charcoal parent, obviously, since both of their chromosomes are identical with regards to lava and charcoal. This is good, because you KNOW that all the charcoal offspring are suitable for diamond-making.

When you do get a diamond, here are a few crosses and what the expected results are.

7) Diamond (vc/vc) x het lava het charcoal (vC/Vc)

½ vc/vC (lavas het charcoal)
½ vc/Vc (charcoals het lava)

Both types are good for making more diamonds. However, since once parent was vC/Vc, recombination in that parent can produce vc and VC gametes. So a small fraction of your offspring would be

vc/vc (diamond)
vc/VC (het lava het charcoal).

Notice that any het lava het charcoal offspring from cross 7 are DIFFERENT from their het lava het charcoal parents! They can be used to easily create more diamonds, unlike the parent.

8) Diamond (vc/vc) x wt

All offspring are vc/VC, and can make more diamonds when crossed to each other, a diamond, a lava het charcoal, or a charcoal het lava.

9) Diamond (vc/vc) x lava het charcoal (vC/vc)

½ vc/vC (lavas het charcoal)
½ vc/vc (diamonds)

10) Diamond (vc/vc) x het lava het charcoal offspring from cross 8 (vc/VC)

1/2 normals het charcoal lava (vc/VC)
1/2 diamonds (vc/vc).

Recombination in the het lava het charcoal could produce vC and cV gametes, so a small proportion of the offspring would be lavas het charcoal and charcoals het lava.

Notice that with unlinked genes, for example amel and cinder, if you mated amel cinder x het amel het cinder, you would only get 1/4 offspring that are homozygous for both mutations. For closely linked genes, it is 1/2, if the allelic arrangement is favorable in the het parent, or close to zero, if it is not.

I hope this helps! Let me know if you have any questions, so that I can improve this post.

Did I make everyone's head explode :/

If you happen upon this post, and manage to slog through it all, I'd really appreciate it if you chimed in just to say if it makes sense to you or not!

It does make sense....although it also causes dizziness :-) ........ But one thing is clear. To make this into a project requires a lot of room for a lot of animals. In the end that could increase the value of a diamond but because it requires a budget and room it could steer people away from this project and ultimately value would drop. From a genetics point of view it is a great project. I luv that part

I think the value should increase, because it is hard to produce a diamond, but once you have one, or a lava het charcoal, or a charcoal het lava, you can easily produce more diamonds, and diamond combines. Someone with a large collection that manages to produce one of those genotypes can control the market (inasmuch as there is one) for diamonds and diamond makers, at least for a few years.

Another point I forgot to make. This is a bit of an oversimplification, but bear with me. Imagine that there is only a 1% chance that a het lava het charcoal (vC/Vc) produces a vc gamete. The cross is vC/Vc x same. The odds of making a lava het charcoal or a charcoal het lava are only about 1/50. This is because a vc gamete will make a lava het charcoal or a charcoal het lava when it unites with either of the nonrecombimant gametes. So the odds of getting those is 1/50 (it's not 1/100, because either parent can make a vc gamete, doubling your odds). But the odds of getting a diamond are the odds of two vc gametes uniting, so (1/100)^2 or 1 in 10,000!!! So clearly it is a good idea to test the possible hets of lava and charcoal offspring, rather than hope for a miracle in the form of a diamond!

Pairing charcoal ph lava or lava ph charcoal to a het lava het charcoal works, as does pairing the charcoals to the lavas, since if only one of them is actually a het, it still proves out, and you'll be able to tell who it was, and are testing two animals with one cross.

I don't think the recombination frequency is that low, though. Unless the two genes are REALLY close together.

Initialy value would increase yes and the breeder would control "the market". He has invested quite some time and money so its fair he would get a return. But for people buying that animal and trying to do the same it would take that same time and money (probably little less) so that could steer them away from a diamond project and value could drop.

Should never be about the money of course but I can imagine a breeder has to look at it from that perspective at some time.

I love both the lava and the charcoal but a diamond could be too expensive for a hobbyist.