0:01

Welcome back to introduction to genetics and evolution.

Â In the previous videos we talked about the process of recombination and

Â specifically the sub-process of crossing over.

Â We talked about using the fraction of

Â recombinant offspring to figure out how far apart genes were.

Â In this video we'll talk about how to leverage that calculation to generate

Â a map using three or more genes.

Â And basically to figure out the order genes are along a chromosome

Â without having to get its full DNA sequence.

Â So what if you have three genes?

Â Let's say that you have three genes, A, B and C.

Â And let's say you know the phase of these genes where

Â all the capitals came from one pair, all the lower cases came from another pair.

Â You have the heterozygote, so it's big A little a, big B little b, Big C little c.

Â But in this phase as depicted here, and you're crossing it to the little a,

Â little b, little c.

Â Just like what we did in the previous video as a test cross.

Â 0:59

Okay, now you know the parental phase is ABC, capitals or all lowercase's.

Â But what is the linear order?

Â Now what you don't know is, is this the order?

Â Is B between A and C, or is it this order where A is between B and C,

Â or is it this order where C is between A and B?

Â 1:30

Now these top two individuals are definitely parental, right?

Â Because they have the full combination.

Â Capital A, capital B, capital C and lowercase a and lowercase b and

Â lowercase c.

Â These other ones all have some sort of recombination event that's

Â happened in there.

Â We don't know the details.

Â Let's put some numbers in there to see what happened.

Â Here are some numbers.

Â Maybe you scored either molecular markers or phenotypic markers and

Â you get these sorts of numbers here.

Â 1:58

Now, the top two, the parental types, are always gonna be the most abundant.

Â You will nearly always see that.

Â At worst they'll be equal to the others, but more typically they will be more

Â abundant if you're looking at genes along the same chromosome.

Â We have a bunch of much rarer types going down.

Â Now what I'd like to do is I'd like to dissect the problem.

Â Rather than trying to look at A, B, and C simultaneously, let's just ignore C for

Â a minute and just look at A and B, okay?

Â 2:38

The next one is actually parental.

Â This is important.

Â The next one is parental-like.

Â They're still big A, big B, and little a, little b, even though we know the C is out

Â of phase with the others with respect to the parents, but

Â again, we're breaking down the problem just to look at A and B.

Â Okay.

Â And the bottom one here as recombinant.

Â Big A, little b, and little a and big B are together.

Â So when we add these up, what fraction are recombinant.

Â Well, it's 15 plus 13 plus one plus one so it adds up to 30.

Â That's a total of 30.

Â The total here is 1,000 if you add up all those numbers together.

Â So, our recombination fraction, in this case, would be 3%, or 0.03.

Â So that is the recombination distance between A and B.

Â Okay? So we calculate that,

Â that's between A and B.

Â Three centimorgans apart, or .03 recombination fractions.

Â What about between A and C, between B and C?

Â 3:39

Well, we can repeat the same process, let's go ahead and

Â do that, between A and C which ones are recombinant.

Â This would be recombinant between A and C.

Â This would be recombinant between A and C.

Â 3:54

This would be recombinant, and this would be recombinant between A and C.

Â The bottom one would actually be parental for A and C.

Â Because you see the capital A and capital C are together.

Â Or lowercase a and lowercase c are together.

Â So, between A and C, we have 18 and 13 is 31.

Â 31 and 15.

Â It's 46.

Â 46 over 1,000.

Â Okay.

Â 4:17

What about between B and C?

Â Which ones are recombinant?

Â Which ones are parental?

Â I'll change the color of my diagram here.

Â Between B and C, this would be parental, this would be parental.

Â This would be recombinant.

Â 4:31

This would be recombinant, cuz again big B, little c, little b, big C.

Â This would be recombinant, this would be recombinant.

Â So in this case we have 20/1000 would be recombinant between B and C.

Â So let's put all these numbers together.

Â 4:56

So, we can, from this, figure out the layer or order genes, right?

Â What should happen is the distance from, from one at one end to the middle plus

Â from the middle to the other end, should be about the same as the total distance.

Â Imagine that you're going from, say New York to Florida.

Â So you have three points, New York, Washington D.C. and Florida.

Â If you add the distance from New York to Washington D.C., and

Â distance from Washington D.C.

Â to Florida, that should be about the same as the distance from New York to Florida.

Â So in this case, we can do that.

Â We can say, because of this A and C must be on the outside.

Â So you have to have A at one end, C at the other end, and

Â therefore necessarily, B will be in the middle.

Â So this would be the correct order.

Â Right? Because here we have,

Â distance from A to B is about three, distance from B to C is about two.

Â The total of the two is close to five, it would round up to five.

Â 5:52

So now you may be wondering why don't AB + BC,

Â why don't those two distances add up exactly right?

Â Why doesn't it add up exactly to 4.6?

Â Well the reason is something you actually noticed when

Â we were calculating these numbers.

Â Is that we have two things that we're calling parental, but

Â they're actually recombinant.

Â 6:13

Here in the bottom two, this individual and this individual,

Â we are counting as parental for A and C, but in fact they are double crossovers.

Â So not only should they be counted, but they should be counted twice, but

Â we didn't do that.

Â So this is why it doesn't quite add up exactly.

Â We'll come back to this in just a second, but

Â first let me emphasize a different point.

Â When you're trying to figure out the linear order of genes

Â there's a few tricks you can use.

Â 6:36

First, if all the loci are linked at some level and

Â I don't mean completely linked, but I mean they're on, for example,

Â the same chromosome and reasonable close together.

Â 6:44

The largest two, the combinations which are the most abundant.

Â So that's just what we saw last time with capital A,

Â capital B, capital C lowercase a, lowercase b, lowercase c.

Â Those will always be the original non-recombinant parentals.

Â So that will help you determine the phase.

Â The smallest two, basically the numbers that are the least frequent,

Â so this is what we saw in the previous slide of capital A and little b capital C.

Â Or lowercase a big B little c.

Â Those would be the double crossover.

Â 7:35

Well again, double crossovers, there were basically two things happening together.

Â So in this case we have the little a, big B, little c as a double crossover gamete.

Â So imagine that you have a 1% chance of recombination between A and B and

Â a 1% chance of recombination between B and C.

Â Then what would happen is you could actually multiply these two probabilities

Â so it'd be a 0.01% chance of a double crossover.

Â Essentially, you're taking a rare thing and

Â multiplying by the probability of another rare thing to get this double

Â crossover class, it'll always be the rarest class from any particular cross.

Â 8:11

And again, you can identify which marker's in the middle

Â because it'll be that rarest class.

Â The alleles at two markers will be parental relative to each other,

Â while the allele at the third will be recombinant.

Â So, we could have immediately looked at the previous slide and said oh,

Â I see that little a, big B, little c is the least frequent Category,

Â therefore this must be the double recombinant.

Â If you notice, the double recombinant,

Â you immediately know that this one must be the one that's in the middle.

Â 8:56

The parental group would be the most abundant group, so clearly in this case,

Â the most abundant group is capital A, capital B, capital C, and lowercase a,

Â lowercase b, lowercase c.

Â That establishes your phase already, so we know that the person who we're measuring

Â recombination, and would look something like this.

Â 9:22

So what we said is that this would necessarily be

Â the double recombinant class.

Â Given that, when we look at it, A and

Â B are in the same phase relative to the parents, C is in a different phase.

Â A and B are in the same phase relative to the parent, C is in a different phase.

Â So this case we can infer, the order is actually A, C, B,

Â and then we can calculate the distance between these pairs of genes.

Â 10:08

What would that mean?

Â Well there's a couple of possibilities here.

Â Now it could mean that A and B are reasonably close together,

Â and C is really far away on the same chromosome, right?

Â Just because you have recombination fraction of 50% doesn't

Â necessarily mean they're on different chromosomes.

Â Could also mean they are on different chromosomes.

Â 10:26

Right? So you could add this or

Â this is on one chromosome, this is another chromosome.

Â You can have this, or you could even have this other order.

Â Basically, one point I just wanna toss out there for you is that above about 40%

Â map distances are very inaccurate and maybe basically, 50%.

Â So, I wouldn't ever trust, when you're determining an order of genes,

Â any map distance you see that's more than 40%.

Â Because, at that point, you really just have no information.

Â So if you have something like this,

Â where A and B is 11%, B and C is 49, or even 43%, or something like that.

Â A and C is 50%, or even 45%.

Â Basically, you don't know how far apart these are.

Â You just know that A and B are linked.

Â So all the information you have in this case, is that A and B are nearby,

Â and you have no idea where C is.

Â C could be really far out this way on the chromosome, it could be really far out

Â that way on the chromosome, or it could be on a different chromosome.

Â I just want to emphasize that because some people use these numbers as though

Â they have this absolute precision to them but we're actually measuring rare events.

Â So when you get something on the order of 49%, or

Â really anything above about 40%, don't have too much faith in that number.

Â