This document discusses three-point test cross analysis for genetic mapping.
- A three-point test cross allows geneticists to simultaneously map the relative positions of three linked genes on a chromosome using a single set of crosses.
- Analyzing the progeny phenotypes from a three-point test cross involving a trihybrid individual allows determination of the gene order and distances between genes.
- The gene with the "odd one out" phenotype in double crossover progeny must be in the middle position of the three genes. Recombination frequencies are calculated to construct a genetic map.
It is the fundamental law of population genetics and provides the basis for studying Mendelian populations ( Mendelian population: A group of sexually inbreeding organisms living within a circumscribed area). It describes populations that are not evolving.
It is the fundamental law of population genetics and provides the basis for studying Mendelian populations ( Mendelian population: A group of sexually inbreeding organisms living within a circumscribed area). It describes populations that are not evolving.
Inability of a plant with functional pollen to set seed when self-pollinated.
Hindrance to self-fertilization.
Prevents inbreeding and promotes outcrossing.
Reported in about 70 families of angiosperms including crop species.
Introduction :
Mendel and subsequent workers assumed that a character was governed by a single gene.
But it was later discovered that many characters in almost all the organisms are governed by two or more genes. Such gene affect the development of concerned characters in various ways.
The phenomenon of two or more gene affecting the expression of each other in various ways in the development of a single character of on organism is known as gene interaction.
GENETICS
CYTOGENETICS
Definition of Linkage, Coupling and Repulsion hypothesis, Linkage group- Drosophila, maize and man, Types of linkage-complete linkage and incomplete linkage, Factors affecting linkage- distance between genes, age, temperature, radiation, sex, chemicals and nutrition, Significance of linkage.
The tendency of two or more genes to stay together (i.e., the co-existence of two or more genes) in the same chromosome during inheritance is known as LINKAGE. The linked genes are present on the same chromosome are said to be SYNTENIC. The linked genes do not show independent assortment.
LINKAGE v/s INDEPENDENT ASSORTMENT
The frequency of linkage or the strength recombination is influenced by several factors (agents).
According to Hardy (England,1908) and Weinberg (Germany,1909), gene and genotype frequency of a Mendelian population remain constant generation after generation unless there is selection,mutation,migration or random drift.
Inability of a plant with functional pollen to set seed when self-pollinated.
Hindrance to self-fertilization.
Prevents inbreeding and promotes outcrossing.
Reported in about 70 families of angiosperms including crop species.
Introduction :
Mendel and subsequent workers assumed that a character was governed by a single gene.
But it was later discovered that many characters in almost all the organisms are governed by two or more genes. Such gene affect the development of concerned characters in various ways.
The phenomenon of two or more gene affecting the expression of each other in various ways in the development of a single character of on organism is known as gene interaction.
GENETICS
CYTOGENETICS
Definition of Linkage, Coupling and Repulsion hypothesis, Linkage group- Drosophila, maize and man, Types of linkage-complete linkage and incomplete linkage, Factors affecting linkage- distance between genes, age, temperature, radiation, sex, chemicals and nutrition, Significance of linkage.
The tendency of two or more genes to stay together (i.e., the co-existence of two or more genes) in the same chromosome during inheritance is known as LINKAGE. The linked genes are present on the same chromosome are said to be SYNTENIC. The linked genes do not show independent assortment.
LINKAGE v/s INDEPENDENT ASSORTMENT
The frequency of linkage or the strength recombination is influenced by several factors (agents).
According to Hardy (England,1908) and Weinberg (Germany,1909), gene and genotype frequency of a Mendelian population remain constant generation after generation unless there is selection,mutation,migration or random drift.
Dihybrid Crosses, Gene Linkage and RecombinationStephen Taylor
For the IB DP Biology course AHL: Genetics unit. To get the editable pptx file, please make a donation to one of my preferred charities. More information at http://sciencevideos.wordpress.com/about/biology4good/
3. A smart undergrad…
and an excellent mentor
• T. H. Morgan and A. H. Sturtevant:
– Cross over frequency is proportional to
distance between loci
– Recombination frequency (RF) can be used
to determine distance between genes located
on the same chromosome and construct a
genetic map
• 1% RF = 1 map unit (m.u.)
= 1 centimorgan (cM)
5. Example from last time…..
Final Step: Draw the Map
You can then draw a map showing the distance between
the two genes
19 cM
P
L
This is Two Point Linkage Analyses
6. Limitations of the two-point cross
approach
• Difficult to determine gene order if two
genes are very close together (small
differences could be result of sampling
error)
– Requires examination of large progeny
– Needs statistical analyses
• Actual distances do not always add up
7. 5.3 Three-Point Test-Cross Analysis Maps Genes
• A two-point test cross is not the most effective way
to build genetic maps
• By performing three-point test-cross analysis,
geneticists can efficiently map three linked genes
simultaneously
www.mun.ca
8. Determining gene order by threepoint crosses
• If genes w, y, and m
are in line, then one of
them must be in the
middle flanked by the
other two
• Greatest genetic
distance (y-m)
separates two genes
on the outside [third
gene (w) must be in
the middle]
9. Three-point crosses
• Allows positioning of
three genes in
relation to each other
using just one set of
crosses
• Allows correction for
double crossovers
(DCOs), which result
in double exchanges
of genetic material
10. A Three-Point Testcross Can Be Used to
Map Three Linked Genes
• Constructing a Genetic Map with the Three-Point Testcross
11. Finding the Relative Order of Genes by ThreePoint Mapping
• In a cross between a trihybrid individual
and one homozygous recessive for all
three genes, the gene configuration need
not be known in advance
• Incomplete linkage produces eight different
gamete genotypes, with unequal
frequencies if the genes are linked
• Parental types will be observed most
frequently, the recombinant types less
frequently
12. Three Point Mapping Criteria
• Wild-type organism producing crossover
gametes must be heterozygous at all three
loci
• Progeny phenotypes must reflect the
genotypes of the parental gametes (No
epistasis! No altered penetrance! Etc.)
• Number of offspring must be large enough to
facilitate recovery of all potential recombinants
(double crossovers!)
13. Example: a b c / a b c a b c / a b c
(Test Cross 1)
• Parental gametes are
produced when there is no
crossing over between the
genes: a b c and a b c
• A single crossover
between a and b produces:
a b c and a b c
• A single crossover
between b and b produces:
a b c and a b c
• A double crossover (in
both intervals) produces:
a b c and a b c
14. Example: a b c / a b c a b c / a b c
(Test Cross 2)
• Parental gametes are
produced when there is no
crossing over between the
genes: a b c and a b c
• A single crossover
between a and b produces:
a b c and a b c
• A single crossover
between b and c produces:
a b c and a b c
• A double crossover (in
both intervals) produces:
a b c and a b c
• NOTE! In trihybrid
cross, parental
phenotypes are not
necessarily all wild-type!
15. Frequencies of Gamete Classes
• When three genes are linked, each of the six recombinant gamete classes
are produced at frequencies significantly lower than that predicted by
chance
• Within each crossover class (e.g., single crossover between genes a and
b) both gamete types that result are equally frequent
• Double crossover classes are the least frequent because both crossover
events must occur to produce these
Least number →
Most number →
16. Constructing a Three-Point
Recombination Map
• Rollins Emerson (1935) mapped three
genes in maize: one for green (V-) or
yellow seedling (vv); one for rough
leaf (Gl-) or glossy leaf (gl gl); and
one for normal fertility (Va-) or
variable fertility (va va)
• He made trihybrids of genotype V Gl
Va/v gl va and crossed these to v gl
va/v gl va
• He analyzed the progeny to map the
genes
Great organism to study!
18. Analysis of Data – Five Questions
1. Are the data consistent with the proposal of
genetic linkage?
2. What are the alleles on the parental
chromosomes?
3. What is the gene order on the chromosome?
4. What are the recombination frequencies of the
gene pairs?
5. Is the frequency of the double crossovers
consistent with independence of the single
crossovers?
•
Each of these questions is answered in
analyzing the three-point test-cross data
19. 1. Are the Data Consistent with the Proposal of
Genetic Linkage?
• Under independent assortment,
eight genetically distinct gametes
would be produced with equal
frequency: 1/8 726 90.75 for
each class
• The chi-square value from the data
in Table 5.3 is over 800, with df 7
and a p value of 0.005
• This is consistent with genetic
linkage; parental gametes are
more frequent and recombinant
gametes less frequent than
predicted
20. 2. What Are the Alleles on Parental
Chromosomes?
• The genotypes of original parents are
known in this case, so the alleles on
the parental chromosomes are V Gl Va
and v gl va
• The frequencies of the F2 progeny also
show this, as these are the most
abundant F2 phenotypes. So if we
didn’t know this already, we can
determine it from the data!
V Gl Va: 235!
v gl va: 270!
21. 3. What Is the Gene Order on the Chromosome?
• The double recombinants, or double crossover
progeny, can be used to determine the gene order
• To determine the order, genes can be listed in each
of three possible orders and the resulting double
crossover progeny determined (trial and error)
• Alternatively when parental alleles and the
double crossover genotypes are compared, only
one allele will differ; this is the gene in the
middle of the three (the ‘odd one out’)
22. Determining the Gene Order on the
Chromosome by Trial and Error
Double cross-overs:
yellow, rough, variable (v Gl va/v gl va) &
green, glossy, normal (V gl VA / v gl va)
•
normal, yellow, rough
variable, green, glossy
• Result: double-crossover gametes from this order
are not those predicted from the data
•
green, variable, rough
yellow, normal, glossy
• Result: double-crossover gametes from this order
are not those predicted from the data
23. The Correct Gene Order on the Chromosome
•
green, glossy, normal
yellow, rough, variable
Double cross-overs:
yellow, rough variable (v Gl va/v gl va) &
green, glossy, normal (V gl VA / v gl va)
• Result: double-crossover gametes from this order are those
predicted from the data
• Conclusion: this predicted gene order is the correct one!
• NOTE: glossy/rough is the odd one out! This is the
middle!
25. 4. What Are the Recombination Frequencies of
the Gene Pairs? (Start with the two small ones!)
• Count the number of crossovers that occurred between the two members
of each gene pair, including the double crossover classes
• For V-Gl the frequency, r, is 60 62 4 7/726 0.183 or 18.3 cM
•
So NOT yellow, glossy OR green, rough; count yellow, rough & green, glossy
• For Gl-Va the frequency, r, is 48 40 4 7/726 0.136 or 13.6 cM
26. The Recombination Frequencies of the Largest
of the Three Distances
• Count the number of crossovers that occurred between the two most distant
genes, including the double crossover classes
• So, V-Va (from order we determined!) has frequency r 60 62 48 m 40 4
7 4 7/726 0.320 or 32.0 cM
•
NOT green, normal OR yellow, variable, but everything else!
• For the larger distance, the double crossover progeny are added twice because
each represents two crossovers between V and Va
X
X
26
28. 5. Is the Frequency of Double Crossovers
Consistent with Independence of the Single
Crossovers?
• In most experiments the number of observed double
crossovers is less than expected
• This is caused by an effect called interference (I)
• In Emerson’s data, the expected double crossovers would be
the product of the two single-crossover frequencies
(0.183)(0.136) = 0.025 (2.5%) 726 18.2
• But we only have 11!
• Observed double crossovers/Expected double crossovers
the coefficient of coincidence, c.
29. Interference
c = Observed double crossovers/Expected double crossovers
In Emerson’s experiment, c 11/18.2 0.60
• I 1 c, or 0.40
• Interference identifies the double crossovers
expected but not produced (ie. How many are
missing?)
• Crossover in one chromosomal region inhibits
second crossover nearby
• In cases where I 0, negative interference has produced
more double crossovers than predicted
30. The relationship between
recombination frequency
and physical distance
between genes
-Recombination frequency
measured in organisms
underestimates the distance
between genes
31. Interference
Interference (I): the presence of a crossover interferes
with the formation of another crossover in the area
I=1-
Observed double crossover frequency
0.015
Expected double crossover frequency
A-B-Caabbcc
A-B-cc
aabbCA-bbCaaB-cc
aaB-CA-bbcc
379
354
94
80
42
36
8
7
25.2 cM
9.3 cM
18.9 cM
B
A
C
b
a
c
f (D.C.O) = (8 + 7) / 1000 = 0.015
32. Interference
Interference (I): the presence of a crossover interferes
with the formation of another crossover in the area
0.015
I=1-
0.166
Expected double crossover frequency
0.018
25.2 cM
9.3 cM
B
18.9 cM
A
9.3% = .093
C
18.9% = .189
0.093 x 0.189 = 0.018
Interference
IS happening!
33. Interference
Observed double crossover frequency
I=1Expected double crossover frequency
What does it mean?
If I > 0
Observed D.C. < Expected; 1 crossover decreases the
chance of a second crossover
If I = 0
Observed D.C. = Expected; No interference
If I < 0
Observed D.C. > Expected; 1 crossover increases the
chance of a second crossover
= rare
35. Determining Gamete Frequencies from Genetic
Maps
• The relationship between recombination frequency
and map distance can be used to predict
frequencies of recombinant gametes based on map
distances
We can predict
what the next
generation will
look like!
http://fromdahliastodoxies.blogspot.com
36. Gamete Frequencies from Genetic Maps
• Both recombinant
gametes (A b and a B)
should be observed
equally frequently, ½
(0.10) 0.05, or 5% each
• The parental gametes will
be observed when there
is no crossover between
the genes, or 100%
10% 90% of the time
• The two gametes, A B
and a b, will be seen in
equal proportion, in this
case ½ (0.90) 0.45, or
45% each
37. Whew, that can be a little confusing when done on slides….
LET’S DO A PROBLEM
TOGETHER!
38. Scales: Green (Y) or Yellow (y)
Eyes: Tan (B) or blue (b)
Scales: Smooth (R) or rough (r)
Phenotype
Offspring Number
Green, Tan, Smooth
1
Green, Blue, Rough
61
Yellow, Tan, Rough
88
Green, Blue, Smooth
96
Yellow, Blue, Smooth
367
Yellow, Blue, Rough
3
Yellow, Tan, Smooth
46
Green, Tan, Rough
338
39. Analysis of Data – Five Questions
1. Are the data consistent with the proposal of
genetic linkage?
2. What are the alleles on the parental
chromosomes?
3. What is the gene order on the chromosome?
4. What are the recombination frequencies of the
gene pairs?
5. Is the frequency of the double crossovers
consistent with independence of the single
crossovers?
•
Each of these questions is answered in
analyzing the three-point test-cross data
40. Practice Problems!
• Chapter 5: 7 & 13
• Chapter 5: 26 (answer in back of book!)
• There are LOTs of appropriate practice
problems for chapter 5!
41. Biological Factors Affecting Accuracy of Genetic
Maps
• Age, environment, and sex may
affect recombination frequency
• In female fruit flies, increased
age decreases recombination
frequency
• Fruit flies grown at temperatures
above or below the optimal 22°C
experience changes in
recombination frequency
• Levels of dietary calcium and
magnesium also affect rates of
crossover in flies
42. Influence of Sex on Rates of Recombination
• Recombination rates differ
between males and females for
most animals
• The heterogametic sex generally
has a lower recombination rate
than the homogametic sex
• The differences in recombination
rate are genome-wide, i.e., not
confined to the sex chromosomes
• In male fruit flies, there is no
crossing over at all
43.
44. How do we map human genes?
• Humans do not do controlled
matings.
• Humans produce small number of
offspring.
• Mid-1980s: Advances in
polymorphic DNA markers and in
gene-mapping software make
human mapping possible.
• Polymorphinc DNA markers:
restriction fragment length variants
(RFLP) and single nucleotide
polymorphisms (SNP)
45. Linkage Group
• Assigning a disease-causing
gene to a chromosomal
location is a first step toward
the cloning and sequencing of
the gene
• Linkage groups: clusters of
syntenic genes that are linked
to one another
• Linkage groups can be
assigned chromosomal
locations.
• Allelic phase: the arrangement
of alleles of linked genes on
the homologous parent
chromosomes.
When a disease-causing allele
is seen to segregate along with
a known genetic marker, allelic
phase can be determined
46. Allelic Phase Analysis
D travels with P1!
recombinant
If we don’t know all the markers, we
don’t know how it traveled!
NOTE: there are two THEs in this header and in the text book page 157, one is capitalized and one is not. I have left both in lower case here to match other headings in this section.
