Transcript Genetic mapping in eukaryotes

Chapters 14 - Genetic Mapping in Eukaryotes:

Discovery of genetic linkage

Gene recombination and chromosomal exchange

Holliday intermediate model of genetic recombination

Constructing genetic maps

Tetrad analysis

Gene conversion

Mitotic recombination
Genetic Mapping in Eukaryotes:

Genes on non-homologous chromosomes assort independently.

Genes on the same chromosome (syntenic) are physically linked
(linkage group) and may be inherited together.
Classical genetics:

Analyzes the frequency of of allele recombination in genetic crosses.

New combinations of parental alleles are produced by recombination
(crossing-over during meiosis I).

Test-crosses are used to determine which genes are linked and
create a linkage map showing the relative distances of markers on
each chromosome.
How does linkage affect Mendelian segregation patterns?
Discovery of genetic linkage in Drosophila (Thomas H. Morgan, ~1911)
•
Morgan determined that the gene for white-eyes (w) and a gene for
miniature wings (m) occur on the X-chromosome.
•
Cross female white-miniature (wm/wm) & wild type male (w+m+/Y):
wm x w+m+
wm
(Y)
•

w+m+ & wm
wm
(Y)
F1  wild type (w+m+/wm) females and white-eyed miniature wing
(wm/ Y) males.
Discovery of genetic linkage in Drosophila (Thomas H. Morgan, ~1911)
F1 x F1 
w+m+
wm
x
wm
(Y)
F2 “parental” genotypes and phenotypes (same allele states as F1):
w+m+
wm
wild-type female
(n = 439)
w+m+
(Y)
wild-type male
(n = 352)
wm
wm
white-eye/miniature female
(n = 359)
wm
(Y)
white-eye/miniature male
(n = 391)
Morgan also observed F2 non-parental genotypes and phenotypes:
w+m
wm
wild-type eye/miniature female
(n = 235)
w+m
(Y)
wild-type eye/miniature male
(n = 210)
wm +
wm
white-eye/wild-type wing female
(n = 218)
wm+
(Y)
white-eye/wild-type wing male
(n = 237)
F1 x F 1 
w+m
wm+
x
wm
(Y)

Non-parental combinations of linked genes are called recombinants.

50% recombinant phenotypes are expected if independent
assortment occurs.

Morgan observed 900/2,441 (36.9%) recombinant phenotypes and
concluded that the two genes must be linked.
Fig. 14.1, Morgan’s experimental cross of white-eyes and miniature wings.
Morgan also observed and hypothesized:
1.
Parental phenotypes always were more common in many other
types of crosses, and recombinant phenotypes occurred less
frequently.
2.
During meiosis, some alleles assort together because they lie
adjacent to each other on the same chromosome.
3.
The closer two genes are on the chromosome, the more likely they
are to remain together during meiosis.
4.
Recombinants are produced by crossing-over.
Mechanism of crossing-over gives rise to recombinant (nonparental) genotypes and phenotypes for linked genes.
Some basics terminology about crossing-over:
1.
Crossing over occurs in prophase I of meiosis I.
2.
Chiasma (pl. chiasmata) is the site where crossing-over occurs.
3.
Crossing-over is a reciprocal exchange of DNA, involving breaking
and rejoining of homologous chromatids.
4.
Crossing-over leads to recombination between linked genes and
produces novel genetic variation.
Gene recombination and chromosomal exchange:
Convincing experimental evidence for Morgan’s hypothesis was not
obtained until the 1930s.
Harriet B. Creighton & Barbara McClintock (1931) studied corn (Zea
mays).
1.
Creighton & McClintock crossed a strain of corn heterozygous for
two genes on chromosome 9.
2.
One gene determines color (C =colored, c = colorless) and the
other determines starch (Wx =standard, wx = mutant).
3.
A large piece of chromosome 8 also had broken off and reattached
to one of the two 9 chromosomes (translocation). At the opposite
end, the same chromosome 9 also possessed dark-staining knob
(2 visible cytogenetic markers).
4.
As a result, recombinant phenotypes could be correlated with
cytological features visible under the microscope.
5.
Whenever recombinant phenotypes appeared, cytological features
also showed recombination.
Fig. 15.2 2nd edition, Creighton’s and McClintock’s Corn Experiment
Several weeks later:
Curt Stern reported identical results in Drosophila
Stern’s Drosophila experiment included:
1.
Two linked gene loci for eye color (car = carnation) and shape (B
= bar-shaped)
2.
Female carried two cytologically distinct chromosomes:
1.
One X chromosome possessed a translocated Y fragment.
2.
The other X chromosome was visibly shorter.
Fig. 14.2, Stern’s
Drosophila
Experiment
*Recombinant phenotypes
and recombinant
cytological features show
perfect correlation.
Molecular mechanism for crossing-over (Robin Holliday, 1960s):
1.
Homologous chromosomes “recognize” and align.
2.
Single strands of each DNA (one on each chromosome) break and
anneal to the opposite chromosome forming Holliday intermediate.
3.
As chromosome ends pull apart, branch point migrations occur to
create a 4-arm intermediate structure.
4.
4-arm intermediate is cut by endonucleases in one of 2 planes.
5.
Model predicts that physical exchange between two gene loci at
the ends of the chromosomes should occur about 50% of the time.

One pattern (intermediate cut in one plane) yields the parental
arrangement.

The other (cut in the other plane) is recombinant.
Box 15.1 2nd edition,
Holliday model of
chromosome
recombination
Constructing genetic maps with recombination frequencies:
•
Number (%) of genetic recombinants produced reflects gene linkage
relationships.
•
Recombination experiments can be used to generate genetic maps.
•
Perform a test cross to determine that genes are linked.
•
Select a test cross including a homozygous recessive (or
homozygous dominant) individual for all genes involved.
•
If loci are not linked and the second parent is a heterozygote, all
4 phenotypes will occur in equal numbers in the F1 (2 loci) and
the ratio of parentals to recombinants will be 1:1.
•
Compare observed and expected using a goodness of fit test and
significant P-value < 0.05.
•
Significant deviations from these ratios indicate linkage.
•
Two arrangements of alleles exist for an individuals heterozygous at
two loci.
cis
w+m+
w m
trans
or
w+m
w m+
•
Cross-over of cis results in trans and vice versa.
•
Frequency of recombinants (%) is a characteristic of each gene pair,
regardless of cis or trans arrangements.
•
Sturtevant (1913) recognized that recombination frequencies could
be used to create a map.
•
1% cross-over rate = 1 map unit (mu) or centiMorgan (cM).
•
Map units (mu) and centiMorgans (cM) are relative measures.
•
Cross-over is more likely between distant genes than close genes.
First genetic map was for Drosophila:
•
•
3 sex-linked genes
•
w = white-eyes
•
m =miniature wings
•
y = yellow body
Recombination frequencies:
•
wxm
= 32.6
•
wxy
= 1.3
•
mxy
= 33.9
y
w
1.3 mu
m
32.6 mu
Use different types of test-crosses to map genes with different types of
linkage and dominance relationships:
Example 2-point test crosses (cross with homozygote):
Autosomal recessive:
Aa/Bb x
aa/bb
Autosomal dominant:
Aa/Bb x
AA/BB
X-linked recessive:
Aa/Bb x
ab/Y
X-linked dominant:
Aa/Bb x
AB/Y
•
Expected frequencies of parental and recombinant genotypes (in
absence of linkage) are 1:1.
•
Number (%) recombinants is used directly as the map unit.
•
Measurement is most accurate when genes are close together.
•
Scoring large numbers of progeny increases accuracy.
•
Direct correspondence between linkage groups and the linear
sequence of genes on a chromosome.
More about using test-crosses to determine linkage:
•
3-point test-crosses (Aa/Bb/Cc x aa/bb/cc) also are a common
technique, allow simultaneous analysis of several traits.
•
The recombination rates (i.e., linkage relationships) can be used to
predict progeny phenotypes (of interest to breeders).
•
For any test-cross, recombination rate cannot exceed 50%.
•
Observed recombination rate of 50% = no linkage.
•
No linkage indicates that genes are:
•
•
(1) on separate chromosomes
•
(2) very far apart on the same chromosome
•
(1) and (2) can be distinguished by mapping multiple genes in
the same linkage groups.
Recombination frequency generally underestimates true map
distance (corrected with mapping functions).
Fig. 14.10 - Mapping function are used for corrections because the
recombination frequency can underestimate the true map distance.
Fig. 14.7, How to
calculate recombination
frequencies:
Calculate (%):
p x j (%) =
(52+ 46 +4 + 2)/500
= 20.8%
j x r (%) =
(22 + 22 + 4 + 2)/500
= 10.0%
p x r (%) =
(p x j) + (j x r)
= 30.8%
r+
Fig. 14.8, How to
calculate recombination
frequencies:
Calculate (%):
p x j (%) =
(52+46+4+2)/500
= 20.8%
j x r (%) =
(22+22+4+2)/500
= 10.0%
p x r (%) =
(52+46+22+22+2(4+2))
= 30.8%
When does crossing over occur?


Possibilities:
(1) Prophase I of meiosis I: 4-chromatid stage after
chromosomes are duplicated
(2) Interphase prior to meiosis: 2-chromatid stage before
chromosomes are duplicated

Ordered tetrad experiments with Neurospora crassa (orange
bread mold) and the arrangement of ascospores in the ascus
indicate the pattern of crossing over.
Tetrad analysis of haploid eukaryotes:
•
Tetrad refers to the four haploid gametes produced by meiosis.
•
Tetrad may be ordered (e.g., Neurospora) or unordered (e.g., yeast).
•
In haploid organisms, phenotype corresponds directly with genotype
of each member of the tetrad (no dominance or recessiveness).
•
Haploidy simplifies interpretation of results and linkage mapping.
•
Three of the most common organisms used for tetrad analysis (each
have asexual and sexual mating types):
•
Yeast (Saccharomyces cerevisiae)
•
Green algae (Chlamydomonas reinhardtii)
•
Orange bread mold (Neurospora crassa)
Ordered Tetrad
Experiment with
Neurospora
Crossing-over
occurs at the 4chromatid stage,
and not the 2chromatid stage,
in prophase I of
meiosis after
chromosomes are
duplicated.
Gene conversion:
•
Process by which DNA sequence information is transferred from one
DNA helix (which remains unchanged) to another DNA helix, whose
sequence is altered.
•
Gene conversion is a type of Non-Mendelian Inheritance.
•
Evidence for conversion occurs when tetrad gamete genotype ratios
are 3:1 or 1:3 instead of 4:4, 2:4:2, or 2:2:2:2.
•
Example: m+/m+ x m/m
•
Can be caused by mismatch during a recombination event, mismatch
is excised (using exonuclease) and replaced (w/DNA polymerase),
overwriting the previous base pairs.

m+/m+/m+/m
Box 16.1 2nd
edition,
Gene conversion
Gene conversion vs crossing over:
Why does conversion occur?

We look to the gene duplication literature for some answers.
•
Paralogous genes with new, but potentially similar functions
(divisions of labor) originate by gene duplication.
Example - Fetal
hemoglobins (Hbs)
exhibit higher affinity for
O2 than adult Hbs, and
expression of isoforms is
developmentally
regulated.
Example - Species of birds that fly at extremely high
elevations (11,300 meters) like the Ruppell’s Griffon
possess a mixture of different hemoglobin isoforms.
HbA, HbA’, HbD, HbD‘ each have different O2 affinity.
Like fetal hemoglobins, these different Hb isoforms have
arisen from gene duplications.
http://en.wikipedia.org/wiki/Gene_duplication
Gene duplication relaxes of selective constraints on gene function.
Old gene copy can serve original function.
New gene copies can serve similar but novel function
(subfunctionalization & neofunctionalization).
Genes can also be lost.
Gene conversion between paralogous genes can maintain similarity of
structure and function over evolutionary time.
Balance between mutation/drift & gene conversion evolves.
Mitotic recombination:
•
Crossing-over sometimes occurs in mitosis (i.e., somatic cells)!
•
First observed by Curt Stern (1936) in heterozygous Drosophila
carrying sex-linked mutations for yellow body color (y+/y) and
singed bristles (sn/sn+).
•
Mitotic cross-over occurred in heterozygotes and appeared as a
mosaic of two different phenotypes in the same individual.
•
Possibly explained by non-disjunction, but mosaic regions were
always adjacent, and therefore likely to be products of the same
genetic event.
Fig. 16.6,
2nd edition
Fig. 16.7,
2nd edition
Mitotic cross-over
in Drosophila