1. CHAPTER 15 Gene Mapping In Eukaryotes
Peter J. Russell
A molecular Approach 2nd Edition
CHAPTER 15 Gene Mapping In Eukaryotes
edited by Yue-Wen Wang Ph. D. Dept. of Agronomy, NTU
台大農藝系 遺傳學

2. Discovery of Genetic Linkage
1. Genes on non-homologous chromosomes assort independently, but genes on the same chromosome (syntenic genes) may instead be inherited together (linked), and belong to a linkage group.
2. 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.
3. Current high-resolution maps include both gene markers from testcrosses, and DNA markers composed of genomic regions that differ detectably between individuals.
台大農藝系 遺傳學

3. Early Study of Genetic Linkage: Morgan’s Experiments with Drosophila
1. Both the white eye gene (w) and a gene for miniature wings (m) are on the Drosophila X chromosome. Morgan (1911) crossed a female white miniature (w m/w m) with a wild-type male (w + m+/ Y) (Figure 15.1).
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.
i. 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).
ii. Non-parental 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 non- parental flies result from recombination of linked genes.
台大農藝系 遺傳學

4. Fig Morgan’s experimental crosses of white-eye and miniature-wing variants of Drosophila melanogaster
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

5. c. Morgan proposed that:
i. During meiosis alleles of some genes assort together because they are near each other on the same chromosome.
ii. Recombination occurs when genes are exchanged between the X chromosomes of the F1 females.
d. A series of experiments supported Morgan’s hypothesis. In each case, parental phenotypes were the most frequent, while recombinant phenotypes occurred less frequently.
e. Some relevant terminology:
i. A chiasma (plural chiasmata) is the site on the homologous chromosomes where crossover occurs.
ii. Crossing-over is the reciprocal exchange of homologous chromatid segments, involving the breaking and rejoining of DNA.
iii. Crossing-over is also the event leading to genetic recombination between linked genes in both prokaryotes and eukaryotes.
f. 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 (Figure 13.2).
台大農藝系 遺傳學

6. Gene Recombination and the Role of chromosomal Exchange
1. Morgan’s results were only circumstantial evidence. Proof that physical exchange between chromosomes results in genetic recombination came in the 1930s.
Animation: Relationship Between Genetic Recombination and Chromosomal Exchange
台大農藝系 遺傳學

7. Corn Experiments
1. Creighton and McClintock (1931) worked with corn (Zea mays) plants in which the two chromosomes under study differed cytologically.
2. The study used a corn strain heterozygous for two genes on chromosome 9 (Figure 15.2):
a. One gene determines seed color (C for colored seeds, c for colorless).
b. The other gene is involved in starch synthesis. The wild-type allele (Wx) produces amylose, and the combination of amylose and amylopectin forms normal starch in a corn seed. The waxy mutant (wx) lacks amylose, and has waxy starch containing only amylopectin.
3. In this corn strain, the appearance of each chromosome 9 homolog correlated with its genotype:
a. One chromosome 9 had the genotype c Wx, and a normal appearance.
b. Its homolog had the genotype C wx, and cytological markers at each end of the chromosome. The end near the C locus had a darkly staining knob, and the other end, nearer the wx locus, had a translocated piece of chromosome 8.
4. When testcrossed, recombinant phenotypes were evident, and could be correlated with cytological features:
a. Whenever the genes had recombined, the cytological features had also recombined.
b. In the parental (non-recombinant.) type progeny, no exchange of cytological markers was evident.
5. This was direct evidence of physical exchange between homologs resulting in genetic recombination.
台大農藝系 遺傳學

8. Fig. 15.2 Evidence of the association of gene recombination with chromosomal exchange in corn
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

9. Drosophila Experiments
1. Identical observations were made by Stern a short time later in Drosophila melanogaster, using a similar approach involving crosses between strains with defined genetic and cytological markers on their X chromosomes (Figure 15.3).
2. The two linked gene loci were:
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 non-bar (round). Heterozygotes (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 the car locus.
3. In Stern’s crosses:
a. Male parents carried recessive alleles for both eye-color (car) and eye-shape (+) on a single X chromosome. Phenotype is carnation, non-bar eyes.
b. Female parent carried two abnormal and cytologically distinct X chromosomes, with a genotype of + + / B car, and a phenotype of wide-bar red eyes.
i. One X chromosome had a translocated fragment of Y chromosome. It carried the wild-type alleles (+ +, red and non-bar) 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 (Figure ). Its alleles were the two mutants, car and B.
c. 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 non-recombinant and two recombinant. A Punnett square shows the segregation of alleles:
台大農藝系 遺傳學

10. + (non-bar)
car
Normal X Y
+ (non-bar) + (red)
X with Y translocation
No recombination
+ + / + car
non-bar red
+ + / Y
B car
Short X
B car / + car
bar
carnation
B car / Y
+ (non-bar) car
Recombination
+ car / + car
non-bar
+ car / Y
non-bar carnation
B+(red)
Short X with
Y translocation
B+/+car
red
B+/Y
bar red
d. 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.
4. This confirmed that physical crossing-over between chromosomes results in genetic recombination (Box 15.1).
台大農藝系 遺傳學

11. Fig Stern’s experiment to demonstrate the relationship between genetic recombination and chromosomal exchange in Drosophila melanogaster
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

12. Box Fig. 15.1 Holliday model for reciprocal genetic recombination
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

13. Constructing Genetic Maps
1. Genetic recombination experiments can be used in genetic (or linkage) mapping.
台大農藝系 遺傳學

14. Detecting Linkage through Testcrosses
1. Linked genes are used for mapping. They are found by looking for deviation from the frequencies expected from independent assortment.
2. 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.
3. Chi-square analysis is used to analyze testcross data and determine whether a deviation is “significant.” A null hypothesis (“the genes are not linked”) is used because it is not possible to predict phenotype frequencies produced by linked genes.
a. If two genes are not linked, a testcross should yield a 1:1 ratio of parentals : recombinants.
b. The formula is: χ2 = Σ d2 / e
i. Σ is “sum of.”
ii. d = deviation value = (o - e).
iii. o = the observed number.
iv. e = the expected number.
c. The χ2 value and the degrees of freedom (df) for the data set are used with a table of chi-square probabilities to determine the probability (P) that the deviation of observed from expected values is due to chance.
i. If P ＞ 0.05 (probability of more than 5 in 100 that deviation was by chance alone) the deviation is not considered statistically significant.
ii. If P ＜ 0.05, the deviation is considered statistically significant, and not due to chance. The null hypothesis is likely to be invalid.
iii. If P ≦ 0.01, deviation is highly statistically significant, and the data are not consistent with the hypothesis, which must be rejected. If the hypothesis “the genes are not linked” is rejected, the only remaining option is that the genes are linked.
台大農藝系 遺傳學

15. The Concept of a Genetic Map
1. 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.
2. 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 (Figure 13.6).
b. The first genetic map was 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.
c. The crosses gave the following recombination frequencies:
i. w x m was 32.6.
ii. w x y was 1.3.
iii. m x y was 33.9.
d. Map is therefore: m————————————————w—y.
台大農藝系 遺傳學

16. Gene Mapping Using Two-Point Testcrosses
1. 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(Figure 15.4).
2. 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+/ab) is crossed with a male hemizygous for the recessive alleles (ab/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.
i. The measure is more accurate when the alleles are close together.
ii. Scoring large numbers of progeny increases the accuracy.
e. Mapping in all types of organisms shows genes arranged with a 1;1 correspondence between linkage groups and chromosomes.
台大農藝系 遺傳學

17. Fig. 15.4 Testcross to show that two genes are linked
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

18. Generating a Genetic Map
1. 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.
2. 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+ ).
3. 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.
4. 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 (Figure 15.5).
5. To determine whether the genes are on the same chromosome, or different ones, other genes in the linkage group may be mapped in relation to a and b, and used to deduce their locations.
台大農藝系 遺傳學

19. Fig Demonstration that the recombination frequency between two genes located far apart on the same chromosome cannot exceed 50 percent
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

20. Gene Mapping Using Three-Point Testcrosses
1. Typically, geneticists design experiments to gather data on several traits in 1 testcross. An example of a three-point testcross would be p+ r+ j+/p r j X p r j/p r j (Figure 15.6).
In the progeny, each gene has two possible phenotypes. For three genes there are (2)3 = 8 expected phenotypic classes in the progeny.
Animation: Three-Point Mapping
台大農藝系 遺傳學

21. Fig. 15.6 Three-point mapping, showing the testcross used and the resultant progeny
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

22. Establishing the Order of Genes
1. 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 (Figures 15.7).
台大農藝系 遺傳學

23. Fig. 15.7 Consequences of a double crossover in a triple heterozygote for three linked genes
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

24. Calculating the Recombination Frequencies for Genes
1. 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 (Figure 15.8).
2. 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.
3. Recombination frequencies are used to position genes on the genetic map (each 1% recombination frequency = 1 map unit) for the chromosomal region (Figure 15.9).
4. Recombination frequencies are not identical to crossover frequencies, and typically underestimate the true map distance.
台大農藝系 遺傳學

25. Fig Rewritten form of the testcross and testcross progeny in Figure 13.9, based on the actual gene order p j r
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

26. iActivity: Crossovers and Tomato Chromosomes
Fig Genetic map of the p-j-r region of the chromosome computed from the recombination data in Figure 13.12
iActivity: Crossovers and Tomato Chromosomes
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

27. Interference and Coincidence
1. Characteristically, 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.
2. The coefficient of coincidence expresses the extent of interference.
a. Interference = 1 - 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.
台大農藝系 遺傳學

28. Fig. 13.14 Progeny of single and double crossovers
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

29. Calculating Accurate Map Distances
1. Recombination frequency generally underestimates the true map distance (Figure 15.10):
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.
2. 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 (Figure 15.11).
台大農藝系 遺傳學

30. Fig. 15.11 A mapping function for relating map distance and recombination frequency
台大農藝系 遺傳學
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.