GENETICS CHAPTER 5Part 2

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)

Genes follow linear sequence

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

Example from last time…..

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

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

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Determining gene order by three-point 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]

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

A Three-Point Testcross Can Be Used to Map Three Linked Genes

• Constructing a Genetic Map with the Three-Point Testcross

Finding the Relative Order of Genes by Three-Point 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

• 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!)

Three Point Mapping Criteria

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

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!

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 →

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!

Double cross-overs: yellow, rough variable (v Gl va/v gl va) & green, glossy, normal (V gl VA / v gl va)

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

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

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!

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’)

Determining the Gene Order on the Chromosome by Trial and Error

•

• Result: double-crossover gametes from this order are not those predicted from the data

•

• Result: double-crossover gametes from this order are not those predicted from the data

Double cross-overs: yellow, rough, variable (v Gl va/v gl va) & green, glossy, normal (V gl VA / v gl va)

normal, yellow, roughvariable, green, glossy

green, variable, roughyellow, normal, glossy

The Correct Gene Order on the Chromosome

•

• 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!

Double cross-overs: yellow, rough variable (v Gl va/v gl va) & green, glossy, normal (V gl VA / v gl va)

green, glossy, normalyellow, rough, variable

Map in progress……..

V Gl va

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

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

26

X

X

OUR MAP!

V Gl va

18.3 cm 13.6 cm

32.0 cm

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.

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

The relationship between recombination frequency and physical distance between genes

-Recombination frequency measured in organisms underestimates the distance between genes

Interference

Interference (I): the presence of a crossover interferes with the formation of another crossover in the area

I = 1 - Observed double crossover frequency

Expected double crossover frequency

A-B-C-

aabbcc

A-B-cc

aabbC-

354

94

379

80

A-bbC-

aaB-cc

aaB-C-

A-bbcc

36

8

42

7

B C

25.2 cM

A

9.3 cM 18.9 cM

b ca

f (D.C.O) = (8 + 7) / 1000 = 0.015

0.015

Interference

Interference (I): the presence of a crossover interferes with the formation of another crossover in the area

I = 1 - 0.015

Expected double crossover frequency

B C

25.2 cM

A

9.3 cM 18.9 cM

9.3% = .093 18.9% = .189

0.093 x 0.189 = 0.018

0.0180.166

Interference IS happening!

Interference

If I > 0

I = 1 - Observed double crossover frequency

Expected double crossover frequency

If I = 0

If I < 0

Observed D.C. = Expected; No interference

Observed D.C. > Expected; 1 crossover increases the chance of a second crossover

Observed D.C. < Expected; 1 crossover decreases the chance of a second crossover

What does it mean?

= rare

V Gl va

18.3 cm 13.6 cm

32.0 cm

We come across a map, what can we do with it?

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

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

LET’S DO A PROBLEM TOGETHER!

Whew, that can be a little confusing when done on slides….

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

Scales: Green (Y) or Yellow (y)Eyes: Tan (B) or blue (b)Scales: Smooth (R) or rough (r)

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

Practice Problems!

• Chapter 5: 7 & 13• Chapter 5: 26 (answer in back of book!)• There are LOTs of appropriate practice

problems for chapter 5!

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

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

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)

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

D travels with P1!

recombinant

Allelic Phase Analysis

If we don’t know all the markers, we don’t know how it traveled!

Questions?