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Chapter 7.
Linkage and Chromosome
Mapping
Outline of
Linkage, Recombination, and the
Mapping of Genes on Chromosomes
• Linkage and Meiotic Recombination
– Genes linked together on the same
chromosome usually assort together.
– Linked genes may become separated
through recombination.
• Mapping
– The frequency with which genes become
separated reflects the physical distance
between them.
• Mitotic recombination
– Rarely, recombination occurs during
mitosis.
– In eukaryotes mitotic recombination
produces genetic mosaics.
Recombination Results When CrossingOver during Meiosis Separates Linked
Genes
• In 1909, F. Janssens observed chiasmata, regions in
which nonsister chromatids of homologous
chromosomes cross over each other.
• T. H. Morgan suggested these were sites of
chromosome breakage and change resulting in genetic
recombination.
Chiasmata Mark the Sites of
Recombination
Reciprocal Exchanges between
Homologous Chromosomes are the
Physical Basis of Recombination
• In 1931, genetic recombination depends on the
reciprocal exchange of parts between maternal and
paternal chromosomes.
– Harriet Creighton and Barbara McClintock studied
corn.
– Curtis Stern studied fruit flies.
– Physical markers to keep track of specific
chromosome parts.
– Genetic markers were points of reference to
determine if particular progeny were result of
recombination.
Creighton and McClintock’s Experiment
in Maize
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Unlinked Genes Show a Recombination
Frequency of 50%
Genes on different chromosomes
Unlinked Genes Show a
Recombination Frequency of 50%
Genes far apart on the same chromosomes
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Linkage in an Autosomal Gene
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Single Crossover I
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Single Crossover II
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Consequences of a Single Exchange between
Two Nonsister Chromatids
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Consequences of a Double Exchange
between Two Nonsister Chromatids
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Summary of Linkage and Recombination
• Genes close together on the same chromosome are linked
and do not segregate independently.
• Linked genes lead to a larger number of parental class than
expected in double heterozygotes.
• Mechanism of recombination is crossing over.
• Chiasmata are the visible signs of crossing over.
• Farther away genes are the greater the opportunity for
chiasmata to form.
• Recombination frequencies reflect physical distance
between genes.
• Recombination frequencies between two genes vary from
0% to 50%.
According to Sturtevant,
In a conversation with Morgan… I suddenly realized that the
variations in strength of linkage, already attributed by
Morgan to differences in the spatial separation of the genes,
offered 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 undergrad homework) in
producing the first chromosomal map.
Recombination Frequencies for Pairs
of Genes Reflect Distance between
Them
• A. H. Sturtevant: Percentage of recombination, or
recombination frequency (RF) reflects the physical
distance separating two genes.
– 1 RF = 1 map unit (mu) or 1 centiMorgan (cM)
According to the paper published by
Sturtevant in 1931,
1) yellow-white
2) white-miniature
3) yellow-miniature
0.5%
34.5%
35.4%
According to the
paper published by
Sturtevant in 1931,
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1)
yellow-white:
0.5%
2)
white-miniature:
34.5%
3)
yellow-miniature:
35.4%
Map of the yellow body (y), white eye (w),
and miniature wing (m)
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Mapping: Locating Genes Along a
Chromosome
• Two-point crosses:
comparisons help establish
relative gene positions.
• Genes are arranged in a line
along a chromosome.
Mapping: Locating Genes Along a
Chromosome
Genes are arranged in a line along a chromosome.
Limitations of Two Point Crosses
• Difficult to determine gene order if two genes are
close together.
• Actual distances between genes do not always add up.
• Pairwise crosses are time and labor consuming.
Question
• A plant of genotype
A
B
a
b
a
b
a
b
is testcrossed to
• If the two loci are 10 m.u. apart, what proportion of progeny will
be AaBb?
Question
• The A locus and D locus are so tightly linked that no
recombination is ever observed between them.
• If AAdd is crossed to aaDD, and the F1 is intercrossed, what
phenotypes will be seen in the F2 and in what proportions?
Question
• The R locus and S loci are 40 m.u. apart.
• If a plant of genotype is selfed, what progeny phenotypes will be
seen and in what proportions?
R
S
r
s
Three Point Crosses: A Faster More
Accurate Method to Map Genes
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Determination of the Gene Sequence
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Mapping in Maize
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Mapping in Maize
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Parental Type
Double Crossover (DCO)
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Final Map by Three Point Mapping
1. Determine the two noncrossover classes.
2. Determine the sequence of genes using DCO.
3. Determine the distance between each pair of genes.
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Another Example of Three Point Crosse
Analyzing the Results of a Three Point
Cross
• Look at two genes at a time and compare to parental.
Analyzing the Results of a Three Point
Cross
Double Recombinants Indicate Order
of Three Genes
vg – b distance 252 + 241 + 131 + 118 X 100 = 17.7 m.u.
4197
252 + 241 + 13 + 9
vg – pr distance
X 100 = 12.3 m.u.
4197
131 + 118 + 13 + 9
b – pr distance
X 100 = 6.4 m.u.
4197
Correction for Double Crossovers (DCO)
vg – b distance
131 + 118 + 13 + 9
X 100 = 6.4 m.u.
4197
Interference: The Number of Double
Crossovers May be Less Than
Expected
• Sometimes the number of observable double
crossovers is less than expected if the two exchanges
are independent.
– Occurrence of one crossover reduces likelihood that
another crossover will occur in adjacent parts of the
chromosome.
– Chromosomal interference: crossovers do not
occur independently.
– Interference is not uniform among chromosomes or
even within a chromosome.
Interference and the Coefficient of
Coincidence
• Coefficient of coincidence = ratio between actual
frequency of double crossover and expected frequency of
double crossover
• coefficient of coincidence (C):
Observed DCO/Expected DCO
• Interference (I) = 1 – coefficient of coincidence (C)
If interference = 0: observed and expected frequencies
are equal.
If interference = 1: no double crossovers can occur.
Mapping in Maize
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Measuring Interference
DCOexp = (0.223) x (0.434) = 0.097 = 9.7%
DCOobs = 7.8%
C = Observed DCO/Expected DCO = 0.078/0.097 = 0.804
I = 1 – C = 1 – 0.804 = 0.196
19.6% fewer double crossovers occurred than expected.
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Question
• The allele b gives Drosophila flies a black body and b+ gives
brown, the wild-type phenotype. The allele wx of a separate gene
gives waxy wings and wx+ gives non-waxy, the wild-type
phenotype. The allele cn of a third gene gives cinnabar eyes and
cn+ gives red, the wild-type phenotype. A female heterozygous
for these three genes is testcrossed, and 1000 progeny are
classified as follows:
5 wild type; 6 black waxy cinnabar; 69 waxy cinnabar; 67 black;
382 cinnabar; 379 black waxy; 48 waxy; 44 black cinnabar
• Determine the order of genes on the chromosome.
• Determine the genetic map.
• Calculate coefficient of coincidence and interference.
Question
• A three-point testcross was made in corn. The results and a
partial recombination analysis are shown (p = purple leaves, + =
green; v = virus-resistant, + = sensitive; b = brown, + = plain).
P
F1
++ ++ ++ x pp vv bb
+p +v +b x pp vv bb
• Determine which genes are linked.
• Draw a map that shows distances in map units.
• Calculate interference if appropriate.
Summary of Three-Point Cross Analysis
• Cross true breeding mutant with wild-type.
• Analyze F2 individuals (males if sex linked).
– Parental class: most frequent
– Double crossovers: least frequent
• Determine order of genes based on parentals and
recombinants.
• Determine genetic distance between each pair of
recombinants.
• Calculate coefficient of coincidence and interference.
Genes Chained
Together by
Linkage
Relation-ships
are Known as
Linkage Groups
Do Genetic and Physical Maps
Correspond?
• Order of genes in correctly predicted by physical
maps.
• Distance between genes is not always similar to
physical maps.
– Double, triple, and more crossovers
– Only 50% recombination frequency observable
in a cross
– Variation across chromosome in rate of
recombination
• Mapping functions compensate for inaccuracies,
but not often imprecise.
The Theoretical and Actual Percentage of
Recombinant Chromatids vs. Map Distance
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Undetected Double Crossover
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Somatic Cell Hybridization
• The technique of somatic cell hybridization is extensively
used in human genome mapping, but it can in principle be
used in many different animal systems.
• In fact, the membranes of the two cells may fuse together
and the two cells become one: a binucleate heterokaryon.
• The nuclei eventually fuse, creating synkaryon.
• Because the mouse and human chromosomes are
recognizably different in number and shape, the two sets in
the hybrid cells can be readily distinguished.
• However, in the course of subsequent cell divisions, for
unknown reasons the human chromosomes are gradually
eliminated from the hybrid at random.
Human Chromosome Mapping
• If a specific human gene product is synthesized in a synkaryon
containing one to three chromosomes, then the gene responsible
for that product must reside on one of the human chromosomes
remaining in the hybrid cell.
• If the human gene product is not synthesized in a synkaryon, the
gene responsible is not present on those human chromosomes
that remain in this hybrid cell.
Synteny Testing
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Tetrad Analysis in Fungi
• In diploid organisms, each individual represents only one of the
four potential gametes generated by each parent in a single
meiotic events.
• Thus, our analysis has depended on inferences derived from
examining the phenotypes of diploid progeny resulting from
random unions of random products of meiosis.
• Model organisms for understanding the mechanism of
recombination because all four haploid products of meiosis are
contained in ascus.
• Ascospores within ascus germinate into haploid individuals.
– Saccharaomyces cerevisiae : baker’s yeast
– Neurospora crassa : bread mold
Saccharaomyces cerevisiae Life Cycle
Neurospora crassa Life Cycle
Tetrads Can Be Characterized by the Number
of Parental and Recombinant Spores They
Contain
• If PD = NPD, the two genes must be unlinked, either because
they reside on different chromosomes or because they lie very far
apart on the same chromosome.
Linkage is Demonstrated by PDs
Outnumbering NPDs
How
Crossovers
between
Linked Genes
Generate
Different
Tetrads
• Thus, if two genes are linked, the only way to generate an
NPD tetrad is through a four-strand double exchange.
• Meioses with crossovers generating such a specific kind of
double recombination must be a lot rarer than no crossingover or single crossovers, which produce PD and T tetrads,
respectively.
• Linkage exists only when PD >> NPD, that is, when the
number of PD tetrads (carrying only parental type spores)
substantially exceeds the number of NPD tetrads (carrying
only recombinants).
Calculating Recombination Frequency
• All of the spores in an NPD tetrad are recombinant and half of
the four spores in a tetratype are recombinant.
RF = NPD + ½ T ´ 100
Total tetrads
RF = 3 + ½ 70 ´ 100 = 19 m.u.
200
Confirmation that Recombination
Occurs at the Four-Strand Stage
A Mistaken Model
Recombination before four-strand stage is not consistent with tetrads
containing recombinant spores would be NPDs instead of Ts.
Tetrad Analysis Demonstrates that
Recombination is Reciprocal
• In a cross between strains with different alleles at two
genes, every T tetrad carries one Ab and one aB spore,
while every NPD tetrad contains two of each type of
recombinant.
• We can thus concluded that meiotic recombination is
almost always reciprocal, generating two homologous
chromosomes that are inverted images of each other.
Ordered Tetrads Allow Mapping a
Gene in Relation to the Centromere
• When each of the four products of meiosis divides once by
mitosis, the two genetically identical cells that result lie
adjacent to each other.
• Because of this feature, starting from either end of the
ascus, you can count the octad of ascopores as four cell
pairs and analyze it as a tetrad.
• From the precise positioning of the four ascopore pairs
within the ascus, you can infer the arrangement of the four
chromatids of each homologous chromosome pair during
the two meiotic divisions.
Segregation Patterns in Ordered Asci
No Crossover
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Segregation Patterns in Ordered
Tetrads
Single Crossover I
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Single Crossover II
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Calculating Distance to Centromere
• Since second-division segregation patterns result from
meioses in which there has been a crossover between a
gene and its centromere, the relative number of asci with
this pattern can be used to determine the gene and
centromere distance.
% SDS =
½ SDS ´ 100
Total tetrads
Example of Genetic Mapping by
Ordered Tetrad Analysis
Question
• A Neurospora cross was made between one strain that
carried the mating-type A and mutant allele arg-1 and
another strain that carries the mating-type allele a and the
wild-type allele for arg-1 (+). Four hundred linear tetrads
were isolated, and these fell into the following seven
classes.
1) Deduce the linkage arrangement of the mating-type locus
and the arg-1 locus.
2) Include the centromere or centromeres on the map.
3) Label all intervals in map units.
Question
A cross is made between a haploid strain of Neurospora, of
genotype nic+ ad and another haploid strain of genotype nic ad+.
From this cross, a total of 1000 linear asci are isolated and
categorized as follows: (총 8점)
1) Map the ad and nic loci in relation to centromeres? (4점)
2) Are the ad and nic loci linked? (2점)
3) Map the ad and nic loci in relation to each other (2점)
1
nic+ ad
nic+ ad
nic+ ad
nic+ ad
nic ad+
nic ad+
nic ad+
nic ad+
808
2
nic+ ad+
nic+ ad+
nic+ ad+
nic+ ad+
nic ad
nic ad
nic ad
nic ad
1
3
nic+ ad+
nic+ ad+
nic+ ad
nic+ ad
nic ad+
nic ad+
nic ad
nic ad
90
4
nic+ ad
nic+ ad
nic ad
nic ad
nic+ ad+
nic+ ad+
nic ad+
nic ad+
5
5
nic+ ad
nic+ ad
nic ad+
nic ad+
nic+ ad
nic+ ad
nic ad+
nic ad+
90
6
nic+ ad+
nic+ ad+
nic ad
nic ad
nic+ ad+
nic+ ad+
nic ad
nic ad
1
7
nic+ ad+
nic+ ad+
nic ad
nic ad
nic+ ad
nic+ ad
nic ad+
nic ad+
5
Mitotic Recombination Can Produce
Genetic Mosaics
• Mitotic recombination is rare.
• Initiated by
– Mistakes in chromosome replication.
– Chance exposure to radiation.
• Curt Stern observed “twin spots” in
Drosophila: a form of genetic mosaicism.
– Animals contained tissues with
different genotypes.
Twin Spots in Drosophila
In a y sn+/y+ sn Drosophila female, most of the body is wild type,
but aberrant patches showing either yellow color or singed bristles
sometimes occur. In some cases, yellow and singed patches are
adjacent to each other, a configuration known as twin spots.
Mitotic Crossingover between sn and
Centromere in Drosophila
Crossingover between sn and y
Did Mendel Encounter Linkage?
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