Transcript Document

Discovery of Genetic Linkage
• 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.
Discovery of Genetic Linkage
• Classical genetics analyzes the frequency of
allele recombination in progeny of genetic
crosses
– New associations of parental alleles are
recombinants, produced by genetic
recombination.
– Tests crosses determine which genes are linked,
and a linkage map (genetic map) is constructed
for each chromosome.
MORGAN’s EXPERIMENTS
• Both the white eye gene (w) and a gene for miniature
wings (m) are on the X chromosome.
• Morgan (1911) crossed a female white miniature (w
m/w m) with a wild-type male (w+ m+/ Y).
– In the F1, all males were white-eyed with miniature wings
(w m/Y), and all females were wild-type for eye color and
wing size (w+ m+/w m).
MORGAN’s EXPERIMENTS
– F1 interbreeding is the equivalent of a test cross 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.
– What is the expected ratio of phenotypes in F2, if white and
miniature are on different chromosomes?
• In 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).
• 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.
Fig. 13.1 Morgan’s experimental crosses of white-eye and miniaturewing variants of
Drosophila melanogaster
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
MORGAN’S PROPOSAL
• 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 X
chromosomes of the
F1 females
• Parental phenotypes occur
most frequently, while
recombinants less.
• Terminology
– Chiasma: site of crossover
– Crossing over: reciprocal
exchange of homologous
chromatid segments
– Crossing-over occurs at
prophase I in meiosis; each
event involves two of the
four chromatids. Any
chromatids may be involved
in crossing over.
Fig. 13.2 Mechanism of crossing-over
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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
– 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.
– A significant deviation in this ratio (more parental and
fewer recombinant types) indicates linkage.
Fig. 13.7 Testcross to show that two genes are linked
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.7 Testcross to show that two genes are linked
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Chi-square for analysis of linkage
• A null hypothesis (‘the genes independently
assort’) is used because it is not possible to predict
the phenotype frequencies produced by linked
genes.
– If two genes are not linked, a testcross should yield a
1:1 ratio of parentals: recombinants.
– Formula is X2 = sum (Obs-Exp)^2/Exp
– If P>0.05, deviation between Obs and Exp is not
significant
– If P<=0.05, deviation is statistically significant; such
that genes may be linked.
Concept of Genetic Map
• In an individual heterozygous at two loci, there are
two arrangements of alleles:
– 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).
– Trans (repulsion) arrangement: has one mutant and one
wild-type on each chromosome (e.g., w+ m and w m+)
– A crossover between homologs in cis arrangement
results in a homologous pair with the trans
arrangement. A crossover between homologs in the
trans arrangement results in cis homologs.
Drosophila Crosses
• They showed that cross over frequency
for linked genes (measured by
recombinants) is characteristics for
each gene pair. The frequency stays
the same, whether the genes are in
coupling or in repulsion.
– Morgan and Sturtevant (1913) used
recombination frequencies to make a
genetic map.
• A 1% crossover rate is a genetic distance of 1
map unit (mu). A map unit is also called a
centimorgan (cM). Geneticists use
recombination frequency as a way to estimate
crossover frequency. 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.
First Genetic Map
• Three X-linked genes
– White (w): white eyes
– Miniature (m): miniature wings
– Yellow (y): yellow body
• Crosses gave the following recombination
frequencies:
– White x miniature was 32.6
– White x yellow was 1.3
– Miniature x yellow was 33.9
MAP: m-----------------------------------w---y
Gene Mapping Using 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.
• For autosomal dominants, a double
heterozygotes (A B/A+B+) is testcrossed with a
homozygous wildtype (recessive) individual
(A+B+/A+B+)
• For X-linked recessives, a female double
heterozygote (a+ b+/a b) is crossed with a
hemizygous recessive male (a b/Y).
• For X-linked dominants, a female double
heterozygote (A B/A+ B+) is crossed with a
male hemizygous for the wild-type (A+ B+).
• Phenotypes obtained in these crosses will
depend on whether the alleles are in cis or trans
position.
GENETIC MAP
• Recombination frequency is used directly as
an estimate of map units.
– The measure is more accurate when alleles are
close together.
– Scoring large numbers of progeny increases
accuracy.
GENERATING A LINKAGE
MAP
• Genetic map is generated from estimating the
crossover rate in a particular segment of a 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+).
LINKED or NON-LINKED?
• A recombination frequency of 50% means
that genes are unlinked. There are two ways
in which genes maybe unlinked:
– They may be on separate chromosomes.
– They may be far apart on the same
chromosome.
MULTIPLE CROSSOVERS
• 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.
Fig. 13.8 Demonstration that the recombination frequency between
two genes located
far apart on the same chromosome cannot exceed 50 percent
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.8 Demonstration that the recombination frequency between
two genes located
far apart on the same chromosome cannot exceed 50 percent
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.8 Demonstration that the recombination frequency between
two genes located
far apart on the same chromosome cannot exceed 50 percent
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.9 Three-point mapping, showing the testcross used and the
resultant progeny
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Mapping using three-point
testcrosses
• 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
– In the progeny, each gene has two possible
phenotypes. For three genes there are (2)^3=8
expected phenotypic classes in the progeny.
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:
– Two classes are parental (p+ r+ j+/ p r j and p r j / p r j)
and will be the most abundant.
– 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.
Fig. 13.10 Consequences of a double crossover in a triple
heterozygote for three linked
genes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.11 Rearrangement of the three genes in Figure 13.9 to p j r
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.12 Rewritten form of the testcross and testcross progeny in Figure 13.9, based
on the actual gene order p j r
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Calculating the recombination
frequencies
• Cross data is organized to reflect the gene
order, and this example the region between
genes p and j is called region I, and that
between j and r is region II.
Calculating recombination
frequencies
• 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.
• 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.
Fig. 13.13 Genetic map of the p-j-r region of the chromosome computed from the
recombination data in Figure 13.12
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Interference and Coincidence
• 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 crossover occuring in the region.
Coefficient of coincidence
express the extent of interference
• Interference = 1-coefficient of coincidence. The
values are inversely related.
• 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.
• A value of 0 means that none of the expected
crossovers occurred, and interference is total.
Calculating accurate map
distances
• Recombination frequency generally
underestimates the true map distance:
– 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 take place.
– 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.
– Map distances for genes that are less than 7 mu apart
are very accurate. As distance increases, accuracy
declines because more crosses go uncounted.
Fig. 13.14 Progeny of single and double crossovers
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Mapping functions
• 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.
Genetic markers
• The number of physically observable genes in
humans is very small. Consequently, human
genetic maps based on these were not useful.
• The development of genetic loci that could be
observed at the level of DNA was essential to
modern human genetics.
• Two alleles (ie, D vs d) needed to be
detected, at the DNA level, to define a DNA
“marker” locus.
• There are several technical solutions to the
observation of DNA differences.
Genetic markers
• In a DNA marker, somewhere in the 100-1000
bp amplified region there must be a DNA
sequence difference (polymorphism)
between individuals.
• The most common DNA marker systems
examine the number of repeated units in a
simple sequence repeat motif, such as
CACACACACACACAC.
• Individuals can vary considerably in the
number of CA blocks, making these types of
DNA sequences very useful population
markers.
Genetic markers
• Single basepair differences, however, are
much more common in the genome and so
have great potential.
• Single basepair differences are often called
SNPs (Single Nucleotide Polymorphisms).
• However, the frequency of individuals being
different at a single base is much less than
CACACACA repeat motifs.
• Genetic markers are simply “signposts” along
the chromosomes that are readily detected
and comparable between laboratory
experiments.
Genetic markers
• Ideally, genetic markers should be readily
available at a high density across the genome
(>100,000).
• The markers should be easily communicated
between lab groups and easily quality
controlled.
• And, the work to obtain the marker information
should be low error and inexpensive.
SNP genetic marker data
Mitotic recombination
• Crossing over during mitosis was first
observed by Stern (1936) in Drosophila.
– The alleles involved are sex-linked and
recessive to the wild type:
• Y produces yellow body color instead of wild type
grey.
• Sn produces short, twisty bristles (“signed”) rather
than the wild-type long, curved ones. Bristles
follow body color (y+/- are black, and y/y are
yellow.
Mitotic recombination
• Female progeny from the cross
• y+ sn / y+ sn x y sn+ /Y
• Generally have wild type phenotype of grey
bodies and normal bristles, corresponding to their
genetoype (y+ sn / y sn+). But exceptions:
– Some flies had patches of yellow and/or signed bristles.
This could be explained by nondisjunction or
chromosomal loss.
– Other flies had twin spots, adjacent regions of bristles,
one yellow and the other signed, a mosaic phenotype.
The spots are reciprocal products of the same genetic
event, a mitotic crossing over.
– Mitotic crossover occurred either between the
centromere and the sn locus or between the sn and the y
locus.
Fig. 13.22 Body surface phenotype segregation in a Drosophila strain
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.23 Production of the twin spot and single yellow spot shown
in Figure 13.22 by
mitotic crossing-over
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Mechanism of Mitotic Crossing
over
• A rare event occurring only in diploid cells, mitotic
crossover can result when replicated chromatids come
together to form a structure similar to the four-strand
stage in meiosis.
• If the starting genotype is d+ e / d e+, the two possible
orientations of the resulting chromatids are:
– One cell with d+ e+ / d+ e+, and one with the d e/ d e. These
are the ones that are useful for mapping, because the recessive
phenotype can be observed in progeny of the d e / d e cells.
– Reversal of the alleles, d e+ / d+ e. Phenotypically
indistinguishable from non-recombinant cells, there are not
useful for mapping, but are nonetheless derived from a
crossover event.
Retinoblastoma
• Most common childhood eye cancer.
– Non hereditary (sporadic) form occurs in an individual
with no family history of the disease, and affects only
one eye (unilateral).
– Heteditary form affects both eyes (bilateral) and usually
occurs at an earlier age than sporadic.
– A single gene (Rb) on chromosome 13q14 involved.
• In hereditary retinoblastoma, tumor cells have mutations in
both copies of this gene, while other cells in the same
individual are heterozygous. The disease is caused by a second
mutation that affects the normal RB allele.
• The second mutation is often identical to the one on the other
chromosome, strong circumstantial evidence that the wild-type
copy of the gene is somehow replaced by the inherited mutated
allele. One possible explanation is mitotic recombination.
Fig. 13.24 Normal mitotic segregation of genes in a theoretical diploid cell with one
homologous pair of chromosomes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 13.25 Result of a mitosis of the same cell type as the cell in Figure 13.24 but in
which a rare mitotic crossing-over occurs
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.