howdy and welcome back to chapter 7 of comprehensive genetics in this section we'll talk about what really nailed down the chromosomal theory of inheritance and how that led to the production of the first linear map of a chromosome in the early 1900s mendel's work was rediscovered and several scientists began doing experiments in earnest to look at how mendel's ratios worked with different genes and different organisms and this included reginald punnett william bateson and edith rebecca saunders and they began doing experiments with pea plants and one of their experiments involved purple flowers with long pollen and red flowers with round pollen so two different traits looking at basically a dihybrid cross in the f1 we should get dihybrid individuals and then in the f2 after self fertilization the expectation was that we would see a 9 to 3 to 3 to 1 ratio what they saw though was in fact not a 9 to 3 to 3 to 1 ratio and what they concluded from this was that if these genes were not independent from each other they were actually dependent on each other therefore instead of being unlinked they were likely linked so dependent and linked it's not one thing it's likely the other around the same time thomas hunt morgan began his work with drosophila and he also was setting up dihybrid test crosses which he expected to see one to one to one to one ratios which he did not observe and we'll look at more of that in just a few minutes but he also noted that there are only four chromosomes in drosophila and if all genes are unlinked then that means that we can only have four genes so he said it's likely that we would have more than four genes therefore some of those genes would have to be linked on the same chromosome and he eventually showed that there were multiple genes on the x chromosome and and had to be linked so we can look at this in terms of mendel's second law versus this chromosome theory that genes are existing on chromosomes versus independent assortment under mendel's second law if we have two individuals looking at two different genes and we end up with our dihybrid test cross here in the f1 generation we should get a one to one to one to one ratio and that is based on each of these genes independently assorting from one another so that we get four separate combinations in the hybrid parent according to the chromosome theory if genes are on the same chromosome then they should always travel together and we should get only the combinations that we observe originally so if we have the big r with the big y then we should have gametes that have the big r with the big y if we have little r with little y we should have gametes with little r and little y so we should see a one to one ratio of those two different combinations so these are the two options that people were thinking about mendel's second law with independent assortment and then the chromosome theory where things are linked on the same chromosome when morgan started setting up his crosses he saw something that really was quite different than either of these expectations so for example we start with our p1 cross that is wild-type a wild-type fly crossed with a black vestigial fly as shown in the picture here in the f1 we are going to get our hybrid individual and its dihybrid and then we're going to do our test cross and again the expectation is one to one to one to one if these genes are independent which is falling under mendel's theory but if these two things are linked on the same chromosome then chromosomal theory says we should see a one-to-one ratio well the data that morgan got was completely different from either of these theories and that was that he saw four different phenotypes not at a one to one to one to one ratio but he saw two that had large numbers and two that had small numbers and this data doesn't really fit either theory so something else is going on here beyond just independent assortment or being linked on the same chromosome what morgan decided was likely occurring was that we have what we call parental types that are occurring without any exchange of genetic information this is these are the ones that fall under the chromosome theory of inheritance but we also have two other types that are the result of genetic exchange which we can call a crossover and what that's going to look like is shown here on the right we have prior to gamete formation so this would be in prophase one of meiosis where our our two homologues have somewhat found each other and they begin to exchange information we're going to see a crossover between two non-sister chromatids where we're physically going to swap this plus and this b with each other and this is going to resolve itself by late prophase and going into metaphase so these chromosomes will now go through the remainder of meiosis the homologues will segregate and split and what we'll end up with is four completely different gamete types the two in the middle here will be what we are calling those recombinant types the two on the outside are what we'll call the parental types morgan didn't really have evidence for this he just said this is likely what is happening because these two genes are likely on the same chromosome and we're have because they're not obviously independent of sort independently assorting from one another so something else has to be going on here since they're dependent on each other but we're having that we're seeing these two types that are at a lower frequency we must be having some kind of genetic exchange that's occurring at some frequency evidence did come a little bit later uh by jansen and darlington both of which looked at what we call the chiasma type theory and we'll look a little bit more closely at that in just a few minutes but basically the idea is that they were observing these structures during meiotic division and when using salamander chromosomes which are really large it's a lot it's easier to see under a microscope but they were able to see these crossover structures and they said this must be where we're having a physical exchange between these chromosomes between the maternal and the paternal chromosomes but it it's it's going to look something like this where we have a crossover early in prophase one and the chiasma type this is what we're calling the chiasma this cross-shaped structure the x-shaped structure that we observe um if we look back here again those x-shaped structures and we're saying that we're getting a physical exchange where the a here and the plus are going to swap places with each other so that we get something new here and here after the resolution of the crossovers those will then segregate and split and again we get one two three four different gametes these are our recombinant types a parental and a parental but each of the four gametes will now be distinct when meiosis is complete further evidence did that really made this um a reality came late much later in the 1940s and 50s with barbara mcclintock and harriet creighton they showed cytological proof of crossover and what they did was barbara mcclintock as as we'll talk about her multiple times she she could have won multiple nobel prizes this is not what she won a nobel prize for however she probably could have um and this is based on her work with corn chromosomes she noted one of her chromosomes in corn had this little knob at the end of it which is basically just a repeat region that's kind of bound up on itself she also noted that sometimes that chromosome with the knob also had an extra piece a part of a different chromosome in this case chromosome 8 that was attached to it and she could observe these in the different structures that would be formed when we have translocations and we'll talk about those later in the semester but for now what you need to know is that there's two morphological markers on this chromosome the knob and an extra piece of chromosome eight and so she was going to look to see if we have a crossover and we have an exchange of these morphologies does that also include a change of an exchange of the genes and she was able to prove that yes it indeed it did because she also had on these chromosomes the genes for color that c gene that we've talked about before in the corn pigment pathway and another gene for waxy and starchy which just determines whether you're going to have a nice plump corn kernel or a kind of shrively corn kernel and so she's able to follow these phenotypes along with the morphology on the chromosomes so if we have a exchange between these two chromosomes we should exchange the genetic information as well as the morphological marker and you can see this c gene went with the knob whereas we we left the translocation behind with the the waxy recessive gene so you can see we have an exchange of the genes as well as an exchange of the morphological markers and she was able to follow this in the plants as well and see that indeed these two things were occurring at the same time so this is really good evidence cytological proof that crossover leads to an exchange of genetic information so what mendel or what morgan was was talking about is is actually real even though he didn't have the evidence of it at the time it didn't come till decades later but he was able to show that this is likely a physical exchange even though he did not have the proof and he said we can calculate this percent of recombination that occurs by taking the part that is the recombinant over the total so this is the part and this is the whole so this is very similar to saying if if i gave you a jar of m ms calculate the percent of blue m m's out of that jar you would count the blues divide by the whole number and you would get the frequency if you multiply by 100 you get an actual percentage and so the next question is if we see this recombination frequency between black and vestigial is this always going to be true for every pair of genes is it a constant number or does this number change depending on the genes that we're looking at so this leads into a really cool story about one of morgan's students named alfred sturdivant and he decided to take a set of data home one night and completely blow off his his homework because he was just a sophomore freshman or sophomore around this time and he just blew off his homework and he decided to sit down with this data and figure it out and so what they noted was that in in different experiments with different gene pairs we got different recombination frequencies well what do these recombination frequencies mean and so that's what alfred sturdivant figured out he figured out that we could relate a distance between the genes to how much recombination occurred and he his logic went like this if he could tell the distance from a to b and the distance from b to c then he should know he should be able to figure out the distance from a to c so this is this is how we're going to start we're going to start with our black and vestigial at 17 so if we relate that to a distance and say that is 17 map units apart then we can also come in here and say okay black and cinnabar are nine map units apart or nine percent and cinnabar and vestigial are eight well these two numbers if i added them together add up to 17 so let's put cinnabar right here in the middle and we'll say this is nine and this is eight okay next we have vestigial and lobed where we have five percent between vestigial and lobed and then black and lobed is 21 well that must mean that lobed is out here where this is 5 and this is 21. now 17 plus 5 equals 22 but this is pretty close so it's not always going to be exact but it's going to be close and so what sturdivant said was that the genes must be arrayed in a linear fashion recombination frequency can be used as a relative distance and the frequencies must therefore be additive okay so it's like i know the distance between here and navisoda and i also know the distance from navasota to houston so therefore i know the distance from college station to houston it's the same kind of idea and this was really foundational for what's going to come after this this is the first linear map that was put together of a chromosome and it took many years for people to kind of be okay with understanding this and it was very simple and very elegant how sturdivant laid it all out and he did this for several different uh chromosomes all four in fact he put together different maps and he went on to run his own fly lab this is turtleman in his own fly lab um later in his life uh but we're gonna look at what this means and what we can use it for as we are here now in the 21st century all right so are these map units real or relative well this is it's kind of like driving let's say driving to houston if i'm driving versus somebody else driving like my husband it may be the same distance but the relative time that we get there is very different so the same idea is is here these are not real distances they are relative to the recombination that's occurring between them and yes we're equating the map units or the centimorgans as sterling nicely named after his boss we're equating that to our percent recombination and we're looking at just the relative distance between so a greater distance between two genes is going to mean we can have more crossover events between them so in this case we have five out of six versus v and w which are much closer together we only have space for two crossovers that are occurring here so this would be two out of six so the greater the distance the more likely we are to have crossovers occur between two genes there are some limitations however when we get to a point that is beyond um 50 that's not realistic so for example if we look from b to w if we were to add these two numbers together we would say 61 recombination well if we go back to that original kind of diagram that we had where we have bcw here that all the uppercase letters and then the lower case letters if we have just a single recombination here between b and c that's only going to occur in half of the game half of the chromatids so the most that we could have is 50 percent because we're always going to have at least one of each parental so we're always going to have at least 50 percent parentals at a minimum so we cannot have recombination that is beyond that 50 percent so if genes are greater than 50 map units apart we treat them as if they're independent so for b and w we would likely say that we would have almost independent assortment between them even though they're on the same chromosome you're going to have 25 of the the two parentals and 25 of the two recombinants so that's that's kind of how we're going to deal with it those limitations but we can't we can't have 61 recombination it's not possible when half of them have to be parentals so when we're looking at two-point mapping two-point mapping allows us to look at the distances between genes so this is what we mean by two points where we have the a and the c but we we might miss some things if we just have two-point mapping for example if i have a double cross over here between a and c then i'm going to get back my parentals in both cases so i'll have a parental here this with the double crossover will also be a parental because it's also ac and then we have plus plus which is a parental and then we have plus plus which is the other parental so all four of the gamete types will be parentals but when we throw in a third point now we can see that there's two crossovers between the a and the c because we can get a new combination now that is a plus with c and plus b with plus and those will be our recombinant types we'll still have our two parentals a b c and plus plus plus but now we have these two new types we're able to see the double crossover with that third point of reference this is also going to allow us to order three genes so we can instead of just saying these two genes are this map distance apart if we have that third reference point we can put things in a specific order these long when we have long map distances we're also going to have the problem where we underestimate when we have some of those double crossovers especially when we have only two points of reference like we talked about on the last slide so here if we have just a single crossover we'll get the big a with the little b and the little a with the big b so those are both recombinant types and we'll be able to see that but again if we have a large distance we might miss that we have two crossovers occurring here because we're going to get back the big a with the big b and the little a with the little b so those are still parental types and this is going to occur more often when we have larger map distances so larger map distances leave more room for double crossovers to occur and therefore we miss some of those crossovers because we're not we just can't see them based on the types so when we have map distances larger than 10 map units we are going to underestimate those drastically and it expounds upon itself the further apart these two genes are so it's going to increase as as we approach 50 map units and this is going to produce what we call large coefficients of coincidences which we'll talk about later but the idea here is that large map distances underestimate the number of crossovers that are occurring there so what is the purpose of all this talking about maps of chromosomes and what we're going to talk about in the next coming sections where we're talking about linkage mapping what is the purpose of all this this happened in the 1900s why do we care well the the coolest thing at the time was that we were able to localize genes to specific chromosomes and place them in an order and then understand how those traits were inherited but the really profound thing that happened because of this these maps that were created in the early 1900s is that it laid the foundation for the human genome project drosophila was actually one of the first genomes that was sequenced in the in the lead up to that human genome project to to do the proof of principle and that's because historically it was the first that had the genetic map so it was kind of a cool come full circle kind of thing where we have the genetic map that sturtevant did we have a cytogenetic map which is basically the staining the g banding um of the chromosome we have a physical map that really puts things in in more detail and then we have the dna sequence that we can lay on top of that so without the work that sturdivant and morgan did we might not have had the human genome project in the 1980s and 1990s which is is really cool um that it took it only it really only took less than a century to get us from point a to point b and now we have a full sequence of human genomes of multiple human genomes and it's just it's pretty incredible so understanding where that technology comes from and how these maps were generated is is going to um help us understand what's going on with the human genome and how we put how we can lay that dna sequence on top so it really is while it does seem like it's something that happened way long time ago and why do we even care it is important in understanding the technology that we have today and all the techno technological advancements that have resulted um over the past 20 or 30 years it's pretty incredible so we'll get into in the next section how we deal with this linkage and how we can use it for mapping um and we'll see how we can put together maps with two genes and maps with three genes and begin to understand how the genome how these genes get inherited from one generation to the next and so we'll we'll look at that in the next section [Music] you