Genetics Notes
Chapter 7 -- Linkage, Recomination, and Eukaryotic Gene Mapping

These notes are provided to help direct your study from the textbook. They are not designed to explain all aspects of the material in great detail; that is what class time and the textbook is for. If you were to study only these notes, you would not learn enough genetics to do well in the course.

Linkage

Soon after Mendel's rules were rediscovered, it was found that some loci did not assort independently. (figures 7.3, 7.5, and 7.7)
While looking at figure 7.7, some points to consider are . . .
  1. The F2 generation does not fit the predicted ratio of 1:1:1:1 that we would expect from a dihybrid testcross. Thus, this cross violates the Rule of Independent Assortment
  2. Two phenotypes are in very high frequency have the same phenotypes as the original parents (P1). These are called non-recombinants or parentals.
  3. Two phenotypes are in low frequency and combine the phenotypes of the two original parents (P1). These are called recombinants or non-parentals.

The simplest explanation is that these two loci lie close to each other on the same chromosome. They are linked on the same chromosome. 
              # of recombinants
      100 x   -----------------  = % of recombinants
                # of offspring
1% recombination = 1 map unit, or 1 centimorgan, in honor of T.H. Morgan, one of the first persons to propose this linkage, and first to win a Nobel prize in genetics.

Mechanism for recombinant gametes: Recombinant gametes, which eventually form recombinant offspring, result from crossing-over in Prophase I (figure 7.6). In zygonema, bivalents are formed via the synaptonemal complex and the homologues are paired. By pachynema, we can see the tetrads, and by diplonema, we can see the chiasmata where crossing-over occurred.

trans or replusion configuration = In the dihybrid, the mutant alleles are across from each other on separate chromosomes (figure 6.8). (trans = repulsion)

cis or coupling configuration = In the dihybrid, the mutant alleles are on the same chromosome (figure 6.8). (cis = coupling)

When crossing-over, we make the following three assumptions.
  1. It leads to recombinations of linked genes in reproduction, which can be seen in the results of the crosses in figures 7.7 and 7.8.
  2. It takes place after chromosomes have replicated (in the four strand stage). Each cross-over event involves only two of the four chromatids.
  3. It is a process that involves an exchange of parts of homologous chromosomes

Timing of crossing-over -- assumption #2

The tetrad analysis of Neurospora provided strong evidence that crossing-over occurred after replication and during meiosis. 2 possibilities were originally proposed
  1. crossing-over occurred in G1 phase before replication of the sister chromatids
  2. crossing-over occurs after replication of the DNA, which occurs during S phase.
The two theories predict different types of tetrads (or patterns of spores) will be formed during meiosis in Neurospora.

Tetrad analysis

To address this question we can use an analysis of the pattern of spores in the asci of fungi in the group Ascomycetes (Figure 6.19 and Table 6.4).
  1. many fungi produce a tetrad of spores as products of a single meiotic cell
  2. may consist of ordered or unordered arrangement, where ordered spores retain a fixed position with the ascus.
Most of these fungi are haploid and are only diploid for a brief period in their life cycle.

A cross between an ab strain and an a+b+ strain will yield a parental ditype pattern of spores in the ascus, if no crossing-over occurred.

The same cross would yield only the nonparental ditype pattern, if crossing-over occurred before DNA synthesis (2-strand stage).

Finally, the same cross would yield either the tetratype pattern if crossing-over occurred after DNA synthesis (4-strand stage), or the nonparental ditype pattern if 2 cross-overs occurred in the 4-strand stage.

Thus the presence of the tetratype pattern, which can only occur with a cross-over in the 4-strand stage, indicates that crossing-over occurs after DNA replication.

Physical Exchange of Genetic Material. -- assumption #3

Two experiments were conducted, one by Creighton and McClintock on corn, and the other by Stern on Drosophila. The purpose was to relate the physical exchange between homologous chromosomes (as seen by microscopy) to the genetic recombination as seen in the phenotypes. Your book presents the work of Crieghton and McClintock (page 170), while a very similar experiment by Stern is summarized below.

Stern was able to isolate two strains of Drosophila, each having different X chromosomes that could be distinguished under the microscope. One strain had a piece of chromosome 4 attached to the X chromosome. The other strain had a piece of the Y chromosome attached to the X. He now had a system in which he could distinguish these chromosomes from each other and from the normal X chromosomes. In addition, one of the abnormal strains was homozygous recessive for carnation eye color and homozygous for the dominant trait bar-eyes.

First, he mated two strains to produce F1 females that were heterozygous for those chromosomes. The phenotypes of the F1s were wildtype at carnation-eyed locus and bar-eyed at bar-eyed locus (both dominant traits). Then he mated this heterozygous F1 to a male with carnation eyes (a testcross).
  1. He found that recombinant flies that were wild type at the carnation locus carried at least part of the chromosome with the Y chromosome attached.
  2. He also found that recombinant flies that were bar-eyed carried at least part of the chromosome with chromosome 4 attached.

This was very powerful support for the concept that crossing-over involved a physical exchange of material between non-sister chromatids.

Three-point cross

Now that we have seen the consequences of crossing-over, and have examined some of the physical properties of crossing-over, we can go on to chromosome mapping. Most of the crosses we have dealt with have been two-point crosses. Through a long and laborious breeding experiment, we could produce maps of chromosomes. However, we can do it much faster using a three-point cross. Also, the three-point cross allows us to evaluate the effects of double cross-overs.

Be sure to study very carefully the problems in the text, especially figures 7.13 and 7.14. To work these problems, it is very important that you approach them in a very consistent and systematic fashion. Failure to do so will have you hopelessly mired in confusion.

Determining gene order
an example of a worked problem is in figure 7.13
  1. The pair of phenotypes with the highest frequency is always the non-recombinant group.
  2. The pair of phenotypes with the lowest frequency is always the double cross-over group. The probability of a double cross-over is approximately the product of the probability of the single cross-over.
  3. compare the wild type class b+pr+c+ to the purple double cross-over class b+prc+ and we can see that the purple locus does not match indicating that the purple locus is in the middle.
Also note that as the parental phenotypes are composed of a gamete from the female that is either b pr c or b+ pr+ c+, the alleles are in the cis configuration.

Determining Map Distance
The next step is to set-up a table that is titled " number of recombinants between". The percent recombination is calculated as before. 
              # of recombinants
      100 x   -----------------  = % of recombinants
                # of offspring

thus for the distance from b to pr

                     887
      100 x   -----------------  = 5.9%  or 5.9 m.u.
                   15,000
The distance from b to c, the two outside loci, (25.4 m.u.) is the sum of the distance from b to pr (5.9 m.u.) and the distance from pr to c (19.5 m.u.).

Coefficient of coincidence
Are cross-overs occurring independently of each other, or does one cross-over affect the likelihood of a second cross-over in the same strand?

This is addressed by asking Is the number of observed double cross-overs equal to the expected number? The product rule yields the probability of a double cross-over. Then multiplying the probability of a double cross-over times the number of offspring for the cross yields the expected number of double cross-overs. The ratio of the observed number of double cross-overs divided by the expected number of double cross-overs is the coefficient of coincidence.

Human chromosomal maps

The analysis of pedigrees can be used to map a human chromosome, and to determine the percent recombination between two loci. This process involves examining the percent of recombination between the locus of interest and several markers that are scattered around the genome. The likelihood of recombination is evaluated via the calculation of a lod score, which is the logarithm of the probability of these offspring given linkage divided by the probability of these offspring given independent assortment. A lod score greater than 1 indicates linkage and a lod score greater than 3 is seen as statistically significant. Pedigree analysis is also used in genetic counseling. Review the calculation of a lod score as shown on pages 182 and 183.

Evaluation of risk in genetic counseling
  1. a female who is known to be a carrier of hemophilia (normal father, carrier mother) wants to know if it is possible to determine if the baby she is carrying has hemophilia
  2. determine that she is heterozygous at the GPD locus (GPD-A, GPD-a) H = normal, h = hemophilia HhAa
  3. from previous mapping studies, it is known that these two loci lie about 5 map units apart
  4. determine the genotype of her father at the GPD locus. The father was not hemophiliac and thus had the H allele. If he has the a allele at the GPD locus, then a allele at GPD is linked to the H allele at the hemophilia locus in the mother and the A allele at the GPD locus is linked to the h allele at the hemophilia locus (h is linked to A and H is linked to a).
If the loci were not linked, we would be able to tell the woman that her son would have a 50% chance of being hemophilic. When she becomes pregnant . . .
  1. aspirate some amniotic fluid - this can be done at 3 months past conception
  2. examine the fetal cells in the fluid to determine the sex (by karyotyping the cells)of the fetus - if the fetus is female then there is no problem, assuming the father is normal
  3. if the fetus is male, examine the fetal cells in the fluid to determine which form of GPD is present
  4. if GPD-A is present, then the fetus has a 95% chance of being hemophilic. (crossing-over does not allow us to make 100% prediction)
  5. if GPD-a is present, then the fetus has a 95% chance of being normal (this is much better than the 50/50 guess before this technique was perfected).
This is also used for other genetic traits such as Huntington's chorea, though this trait occurs on an autosome and the process is somewhat more complicated.

Increased knowledge of the DNA sequences of genes and the advent of polymerase chain reaction techniques have allowed genetic counselors to examine directly the gene of interest to see if abnormalities occur. As these techniques are developed the above method is supplanted by this new ability.

Last update on 13 February 2005
Provide comments to Dwight Moore at mooredwi@emporia.edu
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