1. Copyright © 2010 Pearson Education Inc.
Chapter 14 Genetic Mapping in Eukaryotes
Copyright © 2010 Pearson Education Inc.

2. Some relevant terminology:
A chiasma (plural, chiasmata) is the site on the homologous chromosomes where crossover occurs.
Crossing-over is the reciprocal exchange of homologous chromatid segments, involving the breaking and rejoining of DNA.
Crossing-over is also the event leading to genetic recombination between linked genes in both prokaryotes and eukaryotes.

3. Mapping and Linkages
Genes on nonhomologous chromosomes assort independently, but genes on the same chromosome (syntenic genes) may instead be inherited together (linked) and belong to a linkage group.
Classical genetics analyzes the frequency of allele recombination in progeny of genetic crosses.
a. New associations of parental alleles are recombinants, produced by genetic recombination.
b. Testcrosses determine which genes are linked, and a linkage map (genetic map) is constructed for each chromosome.
c. Genetic maps are useful in recombinant DNA research and experiments dealing with genes and their flanking sequences

4. Mapping today
Current high-resolution maps include both gene markers from testcrosses and DNA markers composed of genomic regions that differ detectably between individuals.
Genomic sequencing allows determination of exact positions of genes. These physical maps use molecular tools, rather than data from cross-over studies.

5. Early Studies of Genetic Linkage
Morgan’s Experiments with Drosophila
Both the white eye gene (w) and a gene for miniature wings (m) are on the Drosophila X chromosome.
Crossed a female white miniature (w m/w m) with a wild-type male (w+m+/Y).
a. In the F1, all males were white-eyed with miniature wings (w m/Y), and all females were wild type for both eye color and wing size (w+m+/w m).
b. F1 interbreeding is the equivalent of a testcross for these X- linked genes, since the male is hemizygous recessive, passing on recessive alleles to daughters and no X-linked alleles at all to sons.

6. The outcome

7. The F2 nonparental phenotypes
In the F2, the most frequent phenotypes for both sexes were the phenotypes of the parents in the original cross (white eyes with miniature wings and red eyes with normal wings).
Nonparental phenotypes (white eyes with normal wings or red eyes with miniature wings) occurred in about 37% of the F2 flies.
Well below the 50% predicted for independent assortment, this indicates that nonparental flies result from recombination of linked genes.

8. Morgan proposed
During meiosis, alleles of some genes assort together because they are near each other on the same chromosome.
Recombination occurs when genes are exchanged between the X chromosomes of the F1 females.
Crossing-over occurs at the four-chromatid stage of prophase I in meiosis. Each crossover event involves two of the four chromatids. All chromatids may be involved in crossing-over, as chiasmata form along the aligned chromosomes.

9. Gene Recombination and the Role
Stern worked with two linked gene loci in Drosophila melanogaster. The linked loci were car and B:
a. The car (carnation) gene is recessive. Homozygotes have carnation-colored eyes, rather than wild-type red. The car locus is near the “left” end of the X chromosome.
b. The B (bar-eye) gene is incompletely dominant. Homozygotes (B/B) have a bar- shaped eye rather than wild-type nonbar (round). Heterozygotes (B/B+) have a wide-bar (kidney- shaped) eye. The B locus is farther from the “left” end of the X chromosome (thus nearer the centromere) than is the car locus.

10. What is with the X?
Male parents carried recessive alleles for both eye color (car) and eye shape (1) on a single X chromosome (car B+/Y). Phenotype is carnation, nonbar eyes (wild type).
Female parent carried two abnormal and cytologically distinct X chromosomes, with a genotype of car+ B+/car B, and a phenotype of kidney-shaped red eyes.
i. One X chromosome had a translocated fragment of Y chromosome. It carried the wild-type alleles (car+ B+, red, and nonbar) for both traits.
ii. The second X chromosome had lost a region by translocation to chromosome 4. This chromosome was visibly shorter than a normal X chromosome. Its alleles were the two mutants, car and B.

11. The conclusion
Gamete formation would produce two types in males, X with both recessive alleles, and Y with neither of the alleles.
Females produce four types, two nonrecombinant and two recombinant. A Punnett square shows the segregation of alleles.
Cytological examination of progeny showed:
i. Both males and females with nonbar carnation eyes had a normal X chromosome, along with a second normal X in females, or a Y in males.
ii. Female flies with wide-bar red eyes and males with bar red eyes had a short X chromosome with the Y translocation, along with a normal X or Y.
This confirmed that physical crossing-over between chromosomes results in genetic recombination.

12. Constructing Genetic Maps
Genetic recombination experiments can be used in genetic (or linkage) mapping.

13. Detecting Linkage through Testcrosses
Linked genes are used for mapping. They are found by looking for deviation from the frequencies expected from independent assortment.
A testcross (one parent is homozygous recessive) works well for analyzing linkage:
a. If the alleles are not linked, and the second parent is heterozygous, all four possible combinations of traits will be present in equal numbers in the progeny.
b. A significant deviation in this ratio (more parental and fewer recombinant types) indicates linkage.

14. The Concept of a Genetic Map
In an individual heterozygous at two loci, there are two arrangements of alleles:
a. The cis (coupling) arrangement has both wild- type alleles on one homologous chromosome and both mutants on the other (e.g., w+m+ and w m).
b. The trans (repulsion) arrangement has one mutant and one wild type on each homolog (e.g., w+m and w m+).
c. A crossover between homologs in the cis arrangement results in a homologous pair with the trans arrangement. A crossover between homologs in the trans arrangement results in cis homologs.

15. Drosophila again
Drosophila crosses showed that crossover frequency for linked genes (measured by recombinants) is characteristic for each gene pair. The frequency stays the same, whether the genes are in coupling or in repulsion.
a. Morgan and Sturtevant (1913) used recombination frequencies to make a genetic map.
i. A 1% crossover rate is a genetic distance of 1 map unit (mu). A map unit is also called a centimorgan (cM).
Ii. Geneticists use recombination frequency as a way to estimate crossover frequency. It is not an exact measure, however.
Iii. The farther apart the two genes are on the chromosome, the more likely it is that crossover will occur between them, and therefore the greater their crossover frequency.

16. The first genetic map
Based on crosses in Drosophila involving the three sex-linked genes:
i.w gives white eyes.
ii.m gives miniature wings.
iii.y gives yellow body.
The crosses gave the following recombination frequencies:
i.w X m was 32.7.
Ii.w X y was 1.0.
Iii.m X y was 33.7.
Map is therefore:

17. Gene Mapping with Two-Point Testcrosses
With autosomal recessive alleles, when a double heterozygote is testcrossed, four phenotypic classes are expected. If the genes are linked, the two parental phenotypes will be about equally frequent and more abundant than the two recombinant phenotypes

18. When things are on X…
Mapping of genes with other mechanisms of inheritance is also done with two-point testcrosses:
a. For X-linked recessives, a female double heterozygote (a+b+/a b) is crossed with a male hemizygous for the recessive alleles (a b/Y).
b. For either X-linked case, it is possible to cross the females with males of any type. As long as only male progeny are analyzed, the father’s X will be irrelevant.
c. Phenotypes obtained in any of these crosses will depend on whether the alleles are arranged in coupling (cis) or repulsion (trans).
d. Recombination frequency is used directly as an estimate of map units. The same principles of mapping apply in using both gene markers and DNA markers.
i.The measure is more accurate when the alleles are close together.
Ii.Scoring large numbers of progeny increases the accuracy.
Mapping in all types of organisms shows genes arranged with a 1:1 correspondence between linkage groups and chromosomes.

19. Generating a Genetic Map
A genetic map is generated from estimating the crossover rate in a particular segment of the chromosome. It may not exactly match the physical map, because crossover is not equally probable at all sites on the chromosome.
Recombination frequency is also used to predict progeny in genetic crosses. For example, a 20% crossover rate between two pairs of alleles in a heterozygote (a+b+/a b) will give 10% gametes of each recombinant type (a+b and a b+).
A recombination frequency of 50% means that genes are unlinked. There are two ways in which genes may be unlinked:
a. They may be on separate chromosomes.
b. They may be far apart on the same chromosome.

20. What if….
If the genes are on the same chromosome, multiple crossovers can occur. The further apart two loci are, the more likely they are to have crossover events take place between them. The chromatid pairing is not always the same in crossover, so that 2, 3, or 4 chromatids may participate in multiple crossover.
To determine whether the genes are on the same chromosome, or on different ones, other genes in the linkage group may be mapped in relation to a and b and used to deduce their locations.

22. Gene Mapping with Three-Point Testcrosses
Typically, geneticists design experiments to gather data on several traits in one testcross. An example of a three-point testcross would be p+r+j+/p r j X p r j/p r j
In the progeny, each gene has two possible phenotypes. For three genes there are (2)3 = 8 expected phenotypic classes in the progeny.

23. Establishing the Order of Genes
The order of genes on the chromosome can be deduced from results of the cross. Of the eight expected progeny phenotypes:
a. Two classes are parental (p+r+j+/p r j and p r j/p r j) and will be the most abundant.
b. Of the six remaining phenotypic classes, two will be present at the lowest frequency, resulting from apparent double crossover (p+r+j/p r j and p r j+/p r j). This establishes the gene order as p j r .

24. Calculating the Recombination Frequencies for Genes
Cross data is organized to reflect the gene order, and in this example the region between genes p and j is called region I, and that between j and r is region II.
Recombination frequencies are now calculated for two genes at a time. It includes single crossovers in the region under study, and double crossovers, since they occur in both regions.

25. The distances are…
Recombination frequencies are used to position genes on the genetic map (each 1% recombination frequency = 1 map unit) for the chromosomal region.
Recombination frequencies are not identical to crossover frequencies and typically underestimate the true map distance.

26. Interference and Coincidence
Double crossovers do not occur as often as expected from the observed rate of single crossovers. Crossover appears to reduce formation of other chiasmata nearby, producing interference. Interference = 1 is total interference, with no other crossovers occurring in the region.
The coefficient of coincidence expresses the extent of interference.
a. Interference = 1 minus the coefficient of coincidence. The values are inversely related.
b. A value of 1 means the number of double crossovers that occurs is what would be predicted on the basis of two independent events, and there is no interference.
c. A value of 0 means that none of the expected crossovers occurred, and interference is total.

27. Calculating Accurate Map Distances
Recombination frequency generally underestimates the true map distance:
a. Double crossovers between two loci will restore the parental genotype, as will any even number of crossovers. These will not be counted as recombinants, even though crossovers have taken place.
b. A single crossover will produce recombinant chromosomes, as will any odd number of crossovers. Progeny analysis assumes that every recombinant was produced by a single crossover.
c. Map distances for genes that are less than 7 mu apart are very accurate. As distance increases, accuracy declines because more crosses go uncounted.

28. Mapping functions are mathematical formulas used to define the relationship between map distance and recombination frequency. They are based on assumptions about the frequency of crossovers compared with distance between genes.

29. Genetic Maps and Physical Maps Compared
Gene or DNA mapping based on crossover assumes that crossover sites are random. This is not entirely true, because hot spots and cold spots for crossover occur on chromosomes. Nonrandom distribution of crossing-over limits accuracy of mapping by this method.
DNA sequence is the best physical map.

30. Constructing Genetic Linkage Maps of the Human Genome
The lod Score Method for Analyzing Linkage of Human Genes
The lack of suitable human pedigrees showing segregation of defined linked traits makes mapping autosomes especially difficult, and so usually the lod (logarithm of odds) score method is used for statistical analysis of pedigree data.
a. A lod score compares the expected distributions of traits if they are linked, and if they are not linked.
b. The lod score is the log10 of the ratio of the two probabilities. The higher the lod score, the closer the two genes.
The map distance for linked markers is computed from the recombination frequency given by the highest lod score, by solving lod scores for a range of proposed map units.

31. Human Genetic Maps The Human Genome Project has used two approaches:
a. The mapping approach, based on landmarks used to construct physical maps that are correlated with sequencing data. This approach was important at the beginning of the genome project.
b. The shotgun approach, sequencing random fragments and then using computers to assemble the genome based on overlaps between fragments. This is the technique now routinely in use.

32. Human genetic mapping was revolutionized by:
a. Discovery of many polymorphic DNA markers.
b. Development of molecular tools to type the markers (determining molecular phenotypes). PCR is an important example.
c. Short tandem repeats (STRs), also called microsatellites, are 2–6-bp repeats that form polymorphic loci that can be detected by PCR.
Hundreds of markers may be typed in a given cross, and computer algorithms are used to determine linkage relationships.
A high-density human genetic map was completed in
a. A consortium of laboratories worked on the same set of DNA samples (mapping panel), so their data could be combined.
b. Localized 5,840 loci with resolution of about 0.7 mu.