1. LINKAGE AND GENETIC MAPPING IN EUKARYOTES

2. genetic linkage map
Based on recombination data
What we will study now

3. LINKAGE AND CROSSING OVER
In eukaryotic species, each linear chromosome contains a long piece of DNA
A typical chromosome contains many hundred or even a few thousand different genes
The term linkage has two related meanings
1. Two or more genes can be located on the same chromosome
2. Genes that are close together tend to be transmitted as a unit
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4. Chromosomes are called linkage groups
They contain a group of genes that are linked together
The number of linkage groups is the number of types of chromosomes of the species
For example, in humans
22 autosomal linkage groups
An X chromosome linkage group
A Y chromosome linkage group
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5. What is crossing over?
When non-sister chromatids of homologous chromosomes exchange DNA segments

7. Crossing Over May Produce Recombinant Phenotypes
Occurs during prophase I of meiosis at the bivalent stage
In diploid eukaryotic species, linkage can be altered during meiosis as a result of crossing over
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8. (a) Without crossing over, linked alleles segregate together.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. B B b b B b a a A a A A
Diploid cell after chromosome replication
Diploid cell after chromosome replication
Meiosis
Meiosis B B B B A A A a b b b b a a a A
Possible haploid cells
Possible haploid cells
(a) Without crossing over, linked alleles segregate together.
(b) Crossing over can reassort linked
alleles.

9. These haploid cells contain a combination of alleles NOT found in the original chromosomes
This new combination of alleles is a result of genetic recombination
These are termed parental or non-recombinant cells
These are termed nonparental or recombinant cells
Figure 5.1
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10. Bateson and Punnett Discovered Two Traits That Did Not Assort Independently
In 1905, William Bateson and Reginald Punnett conducted a cross in sweet pea involving two different traits
Flower color and pollen shape
This is a dihybrid cross that is expected to yield a 9:3:3:1 phenotypic ratio in the F2 generation
However, Bateson and Punnett obtained surprising results
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11. Figure 5.2
A much greater proportion of the two types found in the parental generation
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12. Evidence for Linkage
The first direct evidence of linkage came from studies of Thomas Hunt Morgan
Morgan investigated several traits that followed an X-linked pattern of inheritance in?
You guessed it
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13. His experiment involved three traits: Body color, Eye color, Wing length
yy ww mm
y+y w+w m+m
F1 generation x
y w m Y
y+ w+ m+ Y
F1 generation contains wild-type females and yellow-bodied, white-eyed, miniature-winged males.

14. Morgan’s explanation: All three genes are located on the X chromosome
P Males
P Females
Morgan observed a much higher proportion of the combinations of traits found in the parental generation
Morgan’s explanation:
All three genes are located on the X chromosome
Therefore, they tend to be transmitted together as a unit
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15. Morgan Provided Evidence for the Linkage of Several X-linked Genes
1. Why did the F2 generation have a significant number of nonparental combinations?
2. Why was there a quantitative difference between the various nonparental combinations?
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16. White eyes, miniature wings 716
Let’s reorganize Morgan’s data by considering the pairs of genes separately
Gray body, red eyes
1,159
Yellow body, white eyes
1,017
Gray body, white eyes 17
Yellow body, red eyes 12
Total
2,205
But this nonparental combination was rare
Red eyes, normal wings
770
White eyes, miniature wings
716
Red eyes, miniature wings
401
White eyes, normal wings
318
Total
2,205
It was fairly common to get this nonparental combination
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17. Morgan made three important hypotheses to explain his results
1. The genes for body color, eye color and wing length are all located on the X-chromosome
They tend to be inherited together
2. Due to crossing over, the homologous X chromosomes (in the female) can exchange pieces of chromosomes
This created new combination of alleles
3. The likelihood of crossing over depends on the distance between the two genes
Crossing over is more likely to occur between two genes that are far apart from each other
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18. Figure 5.4
These parental phenotypes are the most common offspring
These recombinant offspring are not uncommon
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because the genes are far apart

19. 5-19 Figure 5.4 These recombinant offspring are fairly uncommon
because the genes are very close together
These recombinant offspring are very unlikely
1 out of 2,205
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20. Creighton and McClintock Experiment They demonstrated physical evidence of cross-overs.wx
Normal chromosome 9
Parental chromosomes c Wx
Abnormal chromosome 9
Crossing over c wx
Knob
Interchanged piece from
chromosome 8
Nonparental chromosomes
(a) Normal and abnormal chromosome 9 C Wx
C = Colored
c = colorless
Wx = Starchy endosperm
wx = waxy endosperm
(b) Crossing over between normal and
abnormal chromosome 9

21. GENETIC MAPPING IN PLANTS AND ANIMALS
Genetic mapping is also known as gene mapping or chromosome mapping
Its purpose is to determine the linear order of linked genes along the same chromosome
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22. A simplified genetic linkage map of Drosophila melanogaster
Each gene has its own unique locus at a particular site within a chromosome
Figure 5.8
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23. Genetic linkage map
Based on recombination data to determine the relative position of genes on the chromosome

24. Physical Maps Use nucleotide sequences to map genes
Will talk about this later

25. Experimentally, the percentage of recombinant offspring is correlated with the distance between the two genes
If the genes are far apart  many recombinant offspring
If the genes are close  very few recombinant offspring
Map distance =
Number of recombinant offspring
Total number of offspring
X 100
The units of distance are called map units (mu)
They are also referred to as centiMorgans (cM)
One map unit is equivalent to 1% recombination frequency
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26. Chromosomes are the product of a crossover during meiosis in the heterozygous parent
Recombinant offspring are fewer in number than nonrecombinant offspring
Figure 5.9
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27. Number of recombinant offspring
The data at the bottom of Figure 5.9 can be used to estimate the distance between the two genes
Map distance =
Number of recombinant offspring
Total number of offspring
X 100 =
X 100
= 12.3 map units
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28. The first genetic map was constructed in 1911 by Alfred Sturtevant
He was an undergraduate who worked in Morgan’s
lab.
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29. Sturtevant wrote: “In conversation with Morgan … I suddenly realized that the variations in the length of linkage, already attributed by Morgan to differences in the spatial orientation of the genes, offered the possibility of determining sequences [of different genes] in the linear dimension of the chromosome. I went home and spent most of the night (to the neglect of my undergraduate homework) in producing the first chromosome map, which included the sex-linked genes, y, w, v, m, and r, in the order and approximately the relative spacing that they still appear on the standard maps.”

31. Figure 5.10
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32. The Data 5-53 Alleles Concerned Number Recombinant/ Total Number
Percent Recombinant Offspring
y and w/w-e
214/21,736
1.0
y and v
1,464/4,551
32.2
y and r
115/324
35.5
y and m
260/693
37.5
w/w-e and v
471/1,584
29.7
w/w-e and r
2,062/6,116
33.7
w/w-e and m
406/898
45.2
v and r
17/573
3.0
v and m
109/405
26.9
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33. Interpreting the Data
In some dihybrid crosses, the percentage of nonparental (recombinant) offspring was rather low
For example, there’s only 1% recombinant offspring in the crosses involving the y and w or w-e alleles
This suggests that these two genes are very close together
Other dihybrid crosses showed a higher percentage of nonparental offspring
For example, crosses between the v and m alleles produced 26.9% recombinant offspring
This suggests that these two genes are farther apart
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34. Sturtevant assumed that the map distances would be more accurate among genes that are closely linked.
Therefore, his map is based on the following distances
y – w (1.0), w – v (29.7), v – r (3.0) and v – m (26.9)
Sturtevant also considered map distances amongst gene pairs to deduce the order of genes
Percentage of crossovers between w and r was 33.7
Percentage of crossovers between w and v was 29.7
Percentage of crossovers between v and r was 3.0
Therefore, the gene order is w – v – r
Where v is closer to r than it is to w
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35. Sturtevant began at the y gene and mapped the genes from left to right
Sturtevant collectively considered all these data and proposed the following genetic map
Sturtevant began at the y gene and mapped the genes from left to right
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36. As the percentage of recombinant offspring approaches a value of 50 %
A close look at Sturtevant’s data reveals two points that do not agree very well with his genetic map
The y and m dihybrid cross yielded 37.5% recombinants
But the map distance is 57.6
The w and m dihybrid cross yielded 45.2% recombinants
But the map distance is 56.6
So what’s up?
As the percentage of recombinant offspring approaches a value of 50 %
This value becomes a progressively more inaccurate measure of map distance
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37. When the distance between two genes is large
Figure 5.11
When the distance between two genes is large
The likelihood of multiple crossovers increases
This causes the observed number of recombinant offspring to underestimate the distance between the two genes
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38. Figure 5-12a Copyright © 2006 Pearson Prentice Hall, Inc.
Figure 5-12a Three types of double exchanges that may occur between two genes. Two of them, (b) and (c), involve more than two chromatids. In each case, the detectable recombinant chromatids are bracketed.
Figure 5-12a Copyright © 2006 Pearson Prentice Hall, Inc.

39. If you need help, watch the Boseman video below.

40. Chi Square Analysis
This method is frequently used to determine if the outcome of a dihybrid cross is consistent with linkage or independent assortment
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41. Trihybrid Crosses
Data from trihybrid crosses can also yield information about map distance and gene order
The following experiment outlines a common strategy for using trihybrid crosses to map genes
In this example, we will consider fruit flies that differ in body color, eye color and wing shape
b = black body color
b+ = gray body color
pr = purple eye color
pr+ = red eye color
vg = vestigial wings
vg+ = normal wings
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42. Trihybrid Crosses
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43. Step 1: Cross two true-breeding strains that differ with regard to three alleles.
Order of genes not important here.
Male is homozygous wildtype for all three traits
Female is mutant for all three traits
The goal in this step is to obtain aF1 individuals that are heterozygous for all three genes
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44. Step 2: Perform a testcross by mating F1 female heterozygotes to male flies that are homozygous recessive for all three alleles
During gametogenesis in the heterozygous female F1 flies, crossovers may produce new combinations of the 3 alleles
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45. Number of Observed Offspring
Step 3: Collect data for the F2 generation
Phenotype
Number of Observed Offspring
Gray body, red eyes, normal wings
+ + +
parental
Gray body, red eyes, vestigial wings
+ + vg
pr/vg
Gray body, purple eyes, normal wings
+ pr +
2 b/pr and pr/vg
Gray body, purple eyes, vestigial wings
+ pr vg
b/pr
Black body, red eyes, normal wings
b + +
b/pr
Black body, red eyes, vestigial wings
B + vg
1 b/pr and pr/vg
Black body, purple eyes, normal wings
B pr +
pr/vg
Black body, purple eyes, vestigial wings
B pr vg
parental
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46. The three genes exist as two alleles each
Therefore, there are 23 = 8 possible combinations of F2 offspring
If the genes assorted independently, all eight combinations would occur in equal proportions
It is obvious that they are far from equal
In the offspring of crosses involving linked genes,
Parental phenotypes occur most frequently
Double crossover phenotypes occur least frequently
Single crossover phenotypes occur with “intermediate” frequency
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47. The combination of traits in the double crossover tells us which gene is in the middle
A double crossover separates the gene in the middle from the other two genes at either end
Notice pr is no longer with the b and vg
And pr+ is not with the b+ and vg+.
In the double crossover categories, the recessive purple eye color is separated from the other two recessive alleles
Thus, the gene for eye color lies between the genes for body color and wing shape
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48. Which are the double cross-overs?
The ones with the least amount.

49. Step 4: Calculate the map distance between pairs of genes
Number of recombs between pr and vg: = 124
Number of recombs between b and pr: = 61
Number of recombs between b and vg, all but double cross-overs: = 178
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50. Map Distance pr/vg = 124/1005 x 100 = 12.3 b/pr = 61/1005 x 100 = 6
b and vg = 179/1005 x 100 = 17.8
_____6____________12.3____________
b pr vg
The distance between b and vg was found to be The actual distance is 18.3 mu.

51. Interference
The slightly smaller lower value was a small underestimate because we did not consider the double crossovers in the calculation between b and vg.
The lower than expected value is due to a common genetic phenomenon, termed positive interference. The first crossover decreases the probability that a second crossover will occur nearby.

52. Need help with trihybrid linkage?
You do not need to compute interference.

53. GENETIC MAPPING IN HAPLOID EUKARYOTES
Much of our earliest understanding of genetic recombination came from the genetic analyses of fungi
Fungi may be unicellular or multicellular organisms
They are typically haploid (1n)
They reproduce asexually and, in many cases, sexually
The sac fungi (ascomycetes) have been particularly useful to geneticists because of their unique style of sexual reproduction
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55. 5-79 Figure 5.12 Meiosis produces four haploid cells, termed spores
These are enclosed in a sac termed an ascus
Figure 5.12
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56. The cells of a tetrad or octad are contained within a sac
In other words, the products of a single meiotic division are contained within one sac
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57. Types of Tetrads or Octads
The arrangement of spores within an ascus varies from species to species
Unordered tetrads or octads
Ascus provides enough space for the spores to randomly mix together
Ordered tetrads or octads
Ascus is very tight, thereby preventing spores from randomly moving around
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58. 5-82 Tight ascus prevents mixing of spores
Ascus provides space for spores to randomly mix together
Mold
Yeast
Unicellular alga
Figure 5.13
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59. Ordered Tetrad Analysis
Ordered tetrads or octads have the following key feature
The position and order of spores within the ascus is determined by the divisions of meiosis and mitosis
In crosses of tan and black Neurospora cultures, the spores appear tan or black in a certain order.
All black spores or all tan spores indicate no hybridization.
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60. Pairs of daughter cells are located next to each other
All eight cells are arranged in a linear, ordered fashion
Pairs of daughter cells are located next to each other
Figure 5.13
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61. 020
No hybridization
020. A linear, cylindrical, eight-spored ascus of Sordaria fimicola. Note ascosporogenesis (sexual reproduction in the Ascomycotina) involves the formation of ascospores, typically eight, by "free cell formation" within an ascus.

62. Non-crossovers

63. Cross-overs

64. Non cross-overs
Non cross-overs
Cross-overs

65. (1/2) (Number of cross-over asci) Map distance = X 100
To calculate this distance, the experimenter must count the number of cross-over asci, as well as the total number of asci
In cross-over asci, only half of the spores are actually the product of a crossover
Therefore
(1/2) (Number of cross-over asci)
Map distance =
X 100
Total number of asci
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66. For the last questions on the worksheet about the fungi.
11. The distance between the gene and centromere is ½(number of recombs)/total asci counted.
½ ( )/993 = 4.4 mu
12. (a) Six types: pro-1 - I just listed as “1”
(b) Since you have 9.8 map units, remember that number was derived by dividing the percent recombs by ½, so there are actually 19.6% recombs or a total of 196 asci that are recombs. Divide that by four types and you get an expected number of 49 of each type of recomb and 402 of each parent types.
13. Ascomycetes produce spores in a sac in an ordered tetrad so you can see the products of meiosis and subsequently mitosis of those meiotic spores.

67. Linkage on human chromosomes
Hampered by the inability to perform desired crosses and small number of progeny in most human families
Geneticists mostly use pedigrees which are often incomplete.

68. Nail-Patella Syndrome
One of the first documented demonstrations of linkage in humans
Linkage between this syndrome and ABO blood types

69. Pedigree of Nail-Patella Syndrome

70. More on physical maps later……