an early dihybrid cross

Genetica per Scienze Naturalia.a. 04-05 prof S. Presciuttini

An early dihybrid cross In the early 1900s, William Bateson and R. C. Punnett were studying inheritance In the early 1900s, William Bateson and R. C. Punnett were studying inheritance

in the sweet pea. They studied two genes: one affecting flower color (P, purple, in the sweet pea. They studied two genes: one affecting flower color (P, purple, and p, red) and the other affecting the shape of pollen grains (L, long, and l, and p, red) and the other affecting the shape of pollen grains (L, long, and l, round). They crossed pure lines P/P · L/L (purple, long) × p/p · l/l (red, round), round). They crossed pure lines P/P · L/L (purple, long) × p/p · l/l (red, round), and selfed the Fand selfed the F11 P/p · L/l heterozygotes to obtain an F2. P/p · L/l heterozygotes to obtain an F2. The following table The following table

shows the proportions of each phenotype in the F2 plantsshows the proportions of each phenotype in the F2 plants::

Phenotype Genotype Observed Expectedpurple, long P/- L/- 4831 3910.5purple, round P/- l/l 390 1303.5red, long p/p L/- 393 1303.5red, round p/p l/l 1338 434.5Total 6952 6952

• The F2 phenotypes deviated strikingly from the expected 9:3:3:1 ratio. What is The F2 phenotypes deviated strikingly from the expected 9:3:3:1 ratio. What is going on? This does not appear to be explainable as a modified Mendelian ratiogoing on? This does not appear to be explainable as a modified Mendelian ratio caused by some kind of gene interaction.caused by some kind of gene interaction.


Testing the segregation ratios in the Bateson-Punnett cross

Analysis of phenotype Analysis of phenotype proportions in Fproportions in F22 shows that both shows that both traits independently follow a traits independently follow a typical 3:1 Mendelian ratio.typical 3:1 Mendelian ratio.As a possible explanation for this, As a possible explanation for this, Bateson and Punnett proposed Bateson and Punnett proposed that the Fthat the F11 had actually produced had actually produced more more P · LP · L and and p · lp · l gametes than gametes than would be produced by Mendelian would be produced by Mendelian independent assortment. Because independent assortment. Because these genotypes were the gametic these genotypes were the gametic types in the original pure lines, types in the original pure lines, the researchers thought that the researchers thought that physical physical couplingcoupling between the between the dominant alleles dominant alleles PP and and LL and and between the recessive alleles between the recessive alleles pp and and ll might have prevented their might have prevented their independent assortment in the Findependent assortment in the F11..

Null hypothesis: phenotypes in 9:3:3:1 ratioPhenotype Genotype Observed Expected Chi-sqpurple, long P/- L/- 4831 3910.5 216.678purple, round P/- l/l 390 1303.5 640.186red, long p/p L/- 393 1303.5 635.988red, round p/p l/l 1338 434.5 1878.739Total 6952 6952 3371.591

P= 0

Null hypothesis: phenotypes in 3:1 (purple/red) ratioPhenotype Genotype Observed Expected Chi-sqpurple P/- 5221 5214.0 0.009red p/p 1731 1738.0 0.028Total 6952 6952 0.038

P= 0.846Null hypothesis: phenotypes in 3:1 (long/round) ratioPhenotype Genotype Observed Expected Chi-sqlong L/- 5224 5214.0 0.019round l/l 1728 1738.0 0.058Total 6952 6952 0.077

P= 0.782


Comfirmation of coupling in Drosophila The confirmation of Bateson and Punnett's hypothesis had to await the The confirmation of Bateson and Punnett's hypothesis had to await the

development of development of DrosophilaDrosophila as a genetic tool. as a genetic tool. After the idea of coupling was first proposed, TAfter the idea of coupling was first proposed, T.. H H.. Morgan found a Morgan found a

similar deviation from Mendel's second law while studying two similar deviation from Mendel's second law while studying two autosomal genes in autosomal genes in Drosophila.Drosophila. One of these genes affects eye color (One of these genes affects eye color (prpr, purple, and , purple, and prpr++, red), and the other , red), and the other

affects wing length (affects wing length (vgvg, vestigial, and , vestigial, and vgvg++, normal). The wild-type alleles of , normal). The wild-type alleles of both genes are dominant. Morgan crossed both genes are dominant. Morgan crossed prpr//prpr · · vgvg//vgvg flies with flies with prpr++//prpr++ · · vgvg++//vgvg++ and then testcrossed the doubly heterozygous F and then testcrossed the doubly heterozygous F11 females: females: prpr++//prpr · · vgvg++//vgvg (female)(female) × × prpr//prpr · · vgvg//vgvg (males) (males)..

The use of the The use of the testcrosstestcross is extremely important. Because one parent (the tester) is extremely important. Because one parent (the tester) contributes gametes carrying only recessive alleles, the phenotypes of the contributes gametes carrying only recessive alleles, the phenotypes of the offspring reveal the gametic contribution of the other, doubly heterozygous offspring reveal the gametic contribution of the other, doubly heterozygous parent. Hence, the analyst can concentrate on meiosis in one parent and forget parent. Hence, the analyst can concentrate on meiosis in one parent and forget about the other. This contrasts with the analysis of progeny from an Fabout the other. This contrasts with the analysis of progeny from an F11 self, self, where there are two sets of meioses to consider: one in the male parent and one where there are two sets of meioses to consider: one in the male parent and one in the female.in the female.


Results of Morgan’s experiment Morgan's results follow; the alleles contributed by the FMorgan's results follow; the alleles contributed by the F11 female female (double (double

heterozygous) heterozygous) specify the Fspecify the F22 classes: classes:

Obviously, these numbers deviate drastically from the Mendelian prediction of a Obviously, these numbers deviate drastically from the Mendelian prediction of a 1:1:1:1 ratio, and they indicate a coupling of genes. The two largest classes are the 1:1:1:1 ratio, and they indicate a coupling of genes. The two largest classes are the combinations combinations prpr++ · vg · vg++ andand pr · vg pr · vg, originally introduced by the homozygous , originally introduced by the homozygous parental flies. You can see that the testcross clarifies the situation. It directly reveals parental flies. You can see that the testcross clarifies the situation. It directly reveals the allelic combinations in the gametes from one sex in the Fthe allelic combinations in the gametes from one sex in the F11, thus clearly showing , thus clearly showing

the coupling that could only be inferred from Bateson and Punnett's Fthe coupling that could only be inferred from Bateson and Punnett's F11 self. The self. The

testcross also reveals something new: there is approximately a 1:1 ratio not only testcross also reveals something new: there is approximately a 1:1 ratio not only between the two parental types, but also between the two nonparental types.between the two parental types, but also between the two nonparental types.


Repeating the cross Morgan then Morgan then repeatrepeateded the crossing experiments but chang the crossing experiments but changeded the combinations of the combinations of

alleles contributed as gametes by the homozygous parents in the first cross. In this alleles contributed as gametes by the homozygous parents in the first cross. In this cross, each parent was homozygous for one dominant allele and for one recessive cross, each parent was homozygous for one dominant allele and for one recessive allele:allele:

The following progeny were obtained:The following progeny were obtained:

Again, these results are not even close to a 1:1:1:1 Mendelian ratio. Now, however, Again, these results are not even close to a 1:1:1:1 Mendelian ratio. Now, however, the largest classes are those that have one dominant allele or the other rather than, as the largest classes are those that have one dominant allele or the other rather than, as before, two dominant alleles or two recessives. But notice that once again the allelic before, two dominant alleles or two recessives. But notice that once again the allelic combinations that were originally contributed to the Fcombinations that were originally contributed to the F

11 by the parental flies provide by the parental flies provide

the most frequent classes in the testcross progeny. the most frequent classes in the testcross progeny.


A hypothesis about coupling In the early work on coupling, Bateson and Punnett coined the term In the early work on coupling, Bateson and Punnett coined the term repulsionrepulsion to to

describe this situation, because it seemed to them that, in this case, the nonallelic describe this situation, because it seemed to them that, in this case, the nonallelic dominant alleles "repelled" each other the opposite of the situation in coupling, dominant alleles "repelled" each other the opposite of the situation in coupling, where the dominant alleles seemed to "stick together." What is the explanation of where the dominant alleles seemed to "stick together." What is the explanation of these two phenomena: coupling and repulsion?these two phenomena: coupling and repulsion?

Morgan suggested that the genes governing both phenotypes are located Morgan suggested that the genes governing both phenotypes are located on the on the same pair of homologous chromosomessame pair of homologous chromosomes.. Thus, when Thus, when prpr and and vgvg are introduced from are introduced from one parent, they are physically located on the same chromosome, whereas one parent, they are physically located on the same chromosome, whereas prpr++ and and vgvg++ are on the homologous chromosome from the other parent. are on the homologous chromosome from the other parent.

This hypothesis also explains repulsion. In that case, one parental chromosome This hypothesis also explains repulsion. In that case, one parental chromosome carries carries prpr and and vgvg++ and the other carries and the other carries prpr++ and and vgvg. Repulsion, then, is just another . Repulsion, then, is just another case of coupling: in this case, the dominant allele of one gene is coupled with the case of coupling: in this case, the dominant allele of one gene is coupled with the recessive allele of the other gene.recessive allele of the other gene.

This hypothesis explains why allelic combinations from P remain together, but how This hypothesis explains why allelic combinations from P remain together, but how do we explain the appearance of nonparental combinations?do we explain the appearance of nonparental combinations?


Crossovers Morgan suggested that, when homologous chromosomes pair in meiosis, the Morgan suggested that, when homologous chromosomes pair in meiosis, the

chromosomes occasionally exchange parts in a process calledchromosomes occasionally exchange parts in a process called crossing-overcrossing-over. The . The figure below figure below illustrates this physical exchange of chromosome segments. The two illustrates this physical exchange of chromosome segments. The two new combinations are called new combinations are called crossover products.crossover products.

Data like those just presented, showing coupling and repulsion in testcrosses and in FData like those just presented, showing coupling and repulsion in testcrosses and in F11

selfs, are commonly encountered in genetics. Clearly, results of this kind are a departure selfs, are commonly encountered in genetics. Clearly, results of this kind are a departure from independent assortment. Such exceptions, in fact, constitute a major addition to from independent assortment. Such exceptions, in fact, constitute a major addition to Mendel's view of the genetic world.Mendel's view of the genetic world.

The residing of genes on the same chromosome pair is termedThe residing of genes on the same chromosome pair is termed linkagelinkage.. Two genes Two genes on the same chromosome pair are said to be on the same chromosome pair are said to be linkedlinked..

It is also proper to refer to the linkage of specific alleles: for example, in one It is also proper to refer to the linkage of specific alleles: for example, in one AA//aa · · BB//bb individual, individual, AA might be linked to might be linked to bb; ; aa would then of necessity be linked to would then of necessity be linked to BB..


Recombination In modern genetic analysis, the main test for determining whether two In modern genetic analysis, the main test for determining whether two

genes are linked is based on the concept of recombination.genes are linked is based on the concept of recombination. Meiotic recombinationMeiotic recombination is any meiotic process that generates a is any meiotic process that generates a

haploid product with a genotype that differs from both haploid haploid product with a genotype that differs from both haploid genotypes that constituted the meiotic diploid cell.genotypes that constituted the meiotic diploid cell.

The product of meiosis so generated is called aThe product of meiosis so generated is called a recombinantrecombinant.. Mendelian independent assortment Mendelian independent assortment may be reinterpreted in terms of may be reinterpreted in terms of

recombination.recombination. If we observe a recombinant frequency of 50 percent in a testcross, we can infer If we observe a recombinant frequency of 50 percent in a testcross, we can infer

that the two genes under study assort independently. The simplest interpretation that the two genes under study assort independently. The simplest interpretation of such a result is that the two genes are on separate chromosome pairs.of such a result is that the two genes are on separate chromosome pairs.

However, However, genes that are far apart on the genes that are far apart on the samesame chromosome pair chromosome pair can act virtually independentlycan act virtually independently and produce the same result. and produce the same result.


Recombination by crossing-over Recombinants arise from meioses in which Recombinants arise from meioses in which nonsister chromatidsnonsister chromatids cross over cross over

between the genes under study.between the genes under study. For genes close together on the same chromosome pair, the physical linkage of For genes close together on the same chromosome pair, the physical linkage of

parental allele combinations makes independent assortment impossible and hence parental allele combinations makes independent assortment impossible and hence produces recombinant frequencies significantly lower than 50 percent.produces recombinant frequencies significantly lower than 50 percent.

The recombinant frequency arising from linked genes ranges from 0 to 50 percent, The recombinant frequency arising from linked genes ranges from 0 to 50 percent, depending on their closeness. depending on their closeness. RRecombinant frequencies greater than 50 percentecombinant frequencies greater than 50 percent cannot be cannot be observed.observed.

Note in Note in the fthe figure that igure that crossing-over generates two crossing-over generates two reciprocal products, which reciprocal products, which explains why the explains why the reciprocal reciprocal recombinant classesrecombinant classes are are generally approximately generally approximately equal in frequency.equal in frequency.


Recombination frequency in Morgan’s crossesFirst

experimentSecond

experiment

Ppr +/pr + · vg +/vg +

xpr/pr · vg/vg

pr +/pr + · vg /vgx

pr/pr · vg + /vg +

F1 pr+/pr · vg+/vg pr+/pr · vg+/vg

F1 testcrossfemales x males

pr +/pr · vg +/vgx

pr/pr · vg/vg

F1 testcrossfemales x males

pr +/pr · vg +/vgx

pr/pr · vg/vgTotal

Parental (pr + · vg + ) 1339 Parental (pr+ · vg ) 1067Parental (pr · vg ) 1195 Parental (pr · vg+ ) 965Total parental 2534 2032 4566Recombinant (pr+ · vg ) 151 Recombinant (pr + · vg + ) 157Recombinant (pr · vg+ ) 154 Recombinant (pr · vg ) 146Total recombinant 305 303 608Total F2 progeny 2839 2335 5174

Recombinationfrequency 0.107 0.130 0.118


Interpreting linkage data As Morgan studied more linked genes, he saw that the proportion of As Morgan studied more linked genes, he saw that the proportion of

recombinant progeny varied considerably, depending on which linked recombinant progeny varied considerably, depending on which linked genes were being studied, and he thought that these variations in genes were being studied, and he thought that these variations in crossover frequency might somehow indicate the actual distances crossover frequency might somehow indicate the actual distances separating genes on the chromosomes.separating genes on the chromosomes.

Morgan assigned the study of this problem to a student, Alfred Morgan assigned the study of this problem to a student, Alfred Sturtevant. Morgan asked Sturtevant to make some sense of the data Sturtevant. Morgan asked Sturtevant to make some sense of the data on crossing-over between different linked genes.on crossing-over between different linked genes.

In one night, Sturtevant developed a method for describing relations In one night, Sturtevant developed a method for describing relations between genes that is still used today.between genes that is still used today. In Sturtevant's own words, "In the latter part of 1911, in conversation with In Sturtevant's own words, "In the latter part of 1911, in conversation with

Morgan, I suddenly realized that the variations in strength of linkage, already Morgan, I suddenly realized that the variations in strength of linkage, already attributed by Morgan to differences in the spatial separation of genes, offered attributed by Morgan to differences in the spatial separation of genes, offered the possibility of determining sequences in the linear dimension of a the possibility of determining sequences in the linear dimension of a chromosome. I went home and spent most of the night (to the neglect of my chromosome. I went home and spent most of the night (to the neglect of my undergraduate homework) in producing the first chromosome map."undergraduate homework) in producing the first chromosome map."


Sturtevant approach Imagine two specific genes positioned a certain fixed distance apartImagine two specific genes positioned a certain fixed distance apart, and, and imagine imagine

random crossing-over along the paired homologs.random crossing-over along the paired homologs. Sturtevant postulated a rough proportionality: the greater the distance between the Sturtevant postulated a rough proportionality: the greater the distance between the

linked genes, the greater the chance that nonsister chromatids would cross over in linked genes, the greater the chance that nonsister chromatids would cross over in the region between the genes and, hence, the greater the proportion of recombinants the region between the genes and, hence, the greater the proportion of recombinants that would be produced.that would be produced.

Thus, by determining the frequency of recombinants, we can obtain a measure of Thus, by determining the frequency of recombinants, we can obtain a measure of the map distance between the genes. the map distance between the genes.

Proportionality between chromosome distance and recombinant frequency. In every meiosis, chromatids cross over at random along the chromosome. The two genes T and U are farther apart on a chromosome than V and W. Chromatids cross over between T and U in a larger proportion of meioses than between V and W, so the recombinant frequency for T and U is higher than that for V and W.


The genetic map unit In fact, we can define one genetic map unit (m.u.) as that distance between genes In fact, we can define one genetic map unit (m.u.) as that distance between genes

for which one product of meiosis in 100 is recombinant. Put another way, a for which one product of meiosis in 100 is recombinant. Put another way, a recombinant frequency (RF)recombinant frequency (RF) of 0.01 (1 percent) is defined as 1 m.u of 0.01 (1 percent) is defined as 1 m.u

A map unit is A map unit is today today referred to as areferred to as a centimorgan centimorgan ( (cMcM) in honor of Morgan) in honor of Morgan A direct consequence of the way in which map distance is measured is that, if 5 map A direct consequence of the way in which map distance is measured is that, if 5 map

units (5 units (5 cMcM) separate genes ) separate genes AA and and BB whereas 3 m.u. separate genes whereas 3 m.u. separate genes AA and and CC, then , then BB and and CC should be either 8 or 2 should be either 8 or 2 cMcM apart. apart.

Sturtevant found this to be the case. In other words, his analysis strongly suggested Sturtevant found this to be the case. In other words, his analysis strongly suggested that that genes are arranged in some linear ordergenes are arranged in some linear order..

Because map distances are Because map distances are roughly roughly additive, calculation of additive, calculation of the the AA--BB and and AA--CC distances leaves distances leaves us with the two possibilities us with the two possibilities shown for the shown for the BB--CC distance. distance. Of Of course, these two possibilities course, these two possibilities can be discriminated by can be discriminated by examining the actual examining the actual recombination frequency recombination frequency between B and C.between B and C.


Linkage groups TThe map he map obtained by recombination frequency analysisobtained by recombination frequency analysis does not place the gene loci does not place the gene loci

at specific places on the chromosome; at specific places on the chromosome; it simply allows us toit simply allows us to determine the positions determine the positions of genes relative to one anotherof genes relative to one another (linkage groups) (linkage groups). The small cluster of three genes . The small cluster of three genes could in theory be anywhere on the actual chromosome. However, as more and could in theory be anywhere on the actual chromosome. However, as more and more recombination analyses are done with many more genes, the entire more recombination analyses are done with many more genes, the entire chromosome becomes "fleshed out." chromosome becomes "fleshed out." AAfter the genes close to each chromosome end fter the genes close to each chromosome end are placed in the map, and gene positions are determined by are placed in the map, and gene positions are determined by other techniquesother techniques, can , can absolute chromosome position be assigned.absolute chromosome position be assigned.

GGenetic analysis works in two directions. In the case of linkage analysis, we have enetic analysis works in two directions. In the case of linkage analysis, we have seen that by using RF measurements we can draw a genetic map; however, working seen that by using RF measurements we can draw a genetic map; however, working in the other direction, if we have the map we can predict frequencies of progeny in in the other direction, if we have the map we can predict frequencies of progeny in different genotypic classes.different genotypic classes.

For example, if we know that the For example, if we know that the DrosophilaDrosophila autosomal genes autosomal genes PPr/prr/pr and and VVg/vgg/vg are linked are linked on the same chromosome 11 cM apart, and if we testcross a trans dihybrid on the same chromosome 11 cM apart, and if we testcross a trans dihybrid PPrr v vgg/p/prr V Vgg to to pprr v vgg/p/prr v vgg, we can predict that there will be 11 percent recombinants. Furthermore, , we can predict that there will be 11 percent recombinants. Furthermore, there should be 5.5 percent each of there should be 5.5 percent each of PPrr V Vgg/p/prr v vgg and and pprr v vgg/p/prr v vgg because the two because the two recombinant types are recombinant types are reciprocalreciprocal products of the same type of crossover event. The products of the same type of crossover event. The remaining 89 percent will be nonrecombinant, 44.5 percent each of remaining 89 percent will be nonrecombinant, 44.5 percent each of PPrr v vgg/p/prr v vgg and and pprr VVgg/p/prr v vgg..


An example of linkage map AA genetic map of the genetic map of the DrosophilaDrosophila genome, showing how each linkage group genome, showing how each linkage group

corresponds to one chromosome pair. Values are given in map units corresponds to one chromosome pair. Values are given in map units (cM) (cM) measured measured from the gene closest to one end.from the gene closest to one end.

Larger values are Larger values are calculated as sums of calculated as sums of shorter intervals because shorter intervals because the recombinant frequency the recombinant frequency for any two loci cannot for any two loci cannot exceed 50 percent.exceed 50 percent.


The vestigial phenotypeOn the left is a picture of a fruit fly with very small wings. This trait On the left is a picture of a fruit fly with very small wings. This trait arose through a mutation in the arose through a mutation in the vestigialvestigial gene, which codes for wing gene, which codes for wing type. The fly on the left carries two copies of the mutant form of this type. The fly on the left carries two copies of the mutant form of this gene. The fly shown on the right is the wild type. It carries two normal gene. The fly shown on the right is the wild type. It carries two normal copies of this gene. copies of this gene.

an early dihybrid cross

Documents

pr pr vg vg

combinations pr vg

coupling of genes

dominant alleles p

recessive alleles p

heterozygous f1 females

punnetts f1 self

f1 pp ll heterozygotes