genetics - clutch ch.4 genetic mapping and...

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GENETICS - CLUTCH

CH.4 GENETIC MAPPING AND LINKAGE

CONCEPT: OVERVIEW OF MAPPING

● Mapping is used to determine the position of gene loci on a chromosome □ Linked genes are genes that are inherited together

- They exist on the ___________________ chromosome (the chromosome is the unit of inheritance)

- Linked genes do not undergo independent assortment

- Complete linkage describes genes that are always inherited together

- F2 ratio from heterozygous cross is 1:2:1; test cross is 1:1

EXAMPLE: Linkage Group

□ Sometimes alleles on the same chromosomes _________________ inherited together

- Crossing over is a physical breaking and rejoining of homologous chromosomes

- Occurs during meiosis

- Produces genetic recombination which leads to a new combination of alleles

- Crossing over explains how genes on the same chromosome may not be inherited together

EXAMPLE: Example of crossing-over

P D r

Linkage Group

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□ Chromosomal mapping is performed by looking at the _______________________ of recombination

- The closer the genes are to each other, the less likely they are to cross over

- The farther the genes are to each other, the more likely they are to cross over

□ The recombination frequency can be used to determine the location of genes relative to each other

EXAMPLE:

P D r

Linkage Group

5 m.u. 8 m.u

13 m.u

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CONCEPT: CROSSING OVER AND RECOMBINANTS

Gamete Genotypes

● Mendel’s law of independent assortment states that the alleles of two genes assort independently EXAMPLE:

□ If the two genes are on the same chromosome, then they are physically linked and sorted into gametes

EXAMPLE:

AaBbGenotype

Phenotype Yellow Round

What are the genotypes of the gametes?

Genotype



A B

a bAB/ab

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□ Crossing over is the physical breaking and rejoining of sections of homologous chromosomes

EXAMPLE:

Discovery of Crossing Over ● Many studies have supported the idea of crossing over between homologous chromosomes □ McClintock and Creighton studied two traits of maize (corn)

- Their corn was heterozygous for color (Cc) and starchy (Wx) / waxy (wx)

- Chromosomes were:

□ One of the plants homologous chromosomes had two added markers

□ They mated their chromosome with a colorless, starchy plant

Genotype



A B

a bAB/ab

C

c

wx

Wx

C

c

wx

Wx

knob Foreign chromosome

C

c

wx

wx


X

c Wx

c Wx

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□ Many plants looked like the parental types, but some were recombinant

- Recombinant describes a mixing between the two parents (genotypes or phenotypes)

- The chromosomal markers identified the recombinant offspring

+

c wx c wx

c wx c Wx

colorless waxy colorless starchy

C

c

wx

wx


X

c Wx

c Wx

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□ The second major study was from Thomas Hunt Morgan and Alfred Sturtevant

- The studies fruit flies with two traits: Eye color, and wing length

- Color is red if p+ or purple if pp

- Wings are long if vg+ but short if vg vg (vestigial)

□ Morgan’s cross went like:

P: p+/p+ vg+/vg+ x p/p vg/vg

Gametes p+ vg+ p vg

F1 p+/p vg+/vg

F1 x tester p+/p vg+/vg x p/p vg/vg

Expected Offspring Ratios:

_______ p+ (red) ___________ Long wings _____ red/long wings

___________ vestigial _____ red/vestigial

________ p (purple) ___________ long wings _______ purple/long ___________vestigial _______purple/vestigial Observed Offspring Ratios:

- There was 10.7% recombinant ( (151+154)/2839 ) instead of the predicted 50%

Genotype Phenotype Offspring total (2839) Types p+ vg+ Red, Long wing 1339 Parental p vg Purple, vestigial 1195 Parental p+ vg Red, vestigial 151 Recombinant pr vg+ Purple, long wing 154 Recombinant

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□ Alfred Sturtevant figured out why it wasn’t equal

- It was because the two genes were on the same chromosome

- But what is the significance of the 10.7% recombination?

- It is because the area between the two genes is 10.7% of the length of the chromosome

- So we say these two genes are 10.7 map units apart

Genotype

Phenotype Red eyes and long wings


p+ vg+

p vgp+p/vg+vg F1

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Crossing Over Terminology

● Crossing over occurs between two ________________________ □ Crossing over occurs between two chromatids

- Sister chromatids are two copies of the same chromosome

- Non-sister chromatids are two copies, one from each homologous pair □ During Meiosis, homologous pairs line up and undergo crossing over

- Tetrads is the term describing the four paired chromatids

- Dyads is a pair of two chromatids

- Bivalent refers to the pair of homologous chromosomes

- Chiasmata is the structure that forms between dyads during crossing over

- Usually between non-sister chromatids, but can also occur between sister

EXAMPLE:

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□ Linked genes have certain terminology

- Cis conformations means that dominant alleles of two genes are on the same chromosome

- AB/ab or ++/ab

- Trans conformation means that two different alleles of two genes are on the same chromosome - Ab/aB or +b/+a □ Linked alleles are written differently

- Alleles on the same homolog have no punctuation between them (Ab instead of A/b)

- The “/” separates to homologs, instead of two genes

- If linkage is unknown you write like A/a ⋅ B/b

PRACTICE:

1. Which of the following gametes can be formed from the genotype AaBb if AB and ab are linked? a. AB, ab b. Ab, aB c. Aa, Bb

2. An experiment that was performed found the recombination frequency between two genes was 12.5%. What is the distance (in mapping units) between two genes?

a. 25 b. 6.25 c. 12.5 d. 10

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3. Which of the following terms describes two copies of the same chromosome?

a. Non-sister chromatids b. Sister-chromatids c. Bivalent d. Dyads

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CONCEPT: MAPPING GENES

● Measuring recombination is traditionally the best way to map gene loci on a chromosome

□ Recombination frequencies is the frequency of recombinant offspring produced in a cross

□ Morgan’s cross went like:

P: p+/p+ vg+/vg+ x p/p vg/vg

Gametes p+ vg+ p vg

F1 p+/p vg+/vg

F1 x tester p+/p vg+/vg x p/p vg/vg

Observed Offspring Ratios:

□ What is the significance of the 10.7% recombination?

- The area between the two genes is 10.7% of the length of the chromosome

- So we say these two genes are 10.7 map units (m.u.) apart

- Therefore, physical distance is directly correlated with recombination frequencies

- The closer two genes are, the less likely they are to cross over and recombine

Map Distance =

Number of recombinant offspring

Total number of off spring

X 100

Genotype Phenotype Offspring total (2839) Types

p+ vg+ Red, Long wing 1339 Parental

p vg Purple, vestigial 1195 Parental

p+ vg Red, vestigial 151 Recombinant

pr vg+ Purple, long wing 154 Recombinant

151+ 154

2839

X 100 10.7%=

p+ vg+

10.7 m.u

GENETICS - CLUTCH


□ Recombination frequencies can also determine __________________ a gene is linked

- Linkage is likely occurring if the recombination frequencies are less than 50%

- Linkage is likely not occurring if the recombination frequencies are close or equal to 50%

□ Recombination frequencies are never greater than 50% because:

- Independent assortment equally assorts alleles, but cannot cause more than 50% recombination

EXAMPLE:

Modern Mapping

● There are ____________________ types of gene loci maps

□ Recombination maps use recombination frequencies to determine gene loci

- Can be used to map 2+ gene loci

□ Physical maps use the action genomic sequence to determine gene loci

- Involves sequencing the entire chromosome, or genome of an organism

□ Mapping via certain genomic markers can also be used

- Single Nucleotide Polymorphisms are single nucleotide changes that can be markers

- Restriction fragment length polymorphisms (RFLPs) are sequences that restriction enzymes cut

- Restriction enzymes are proteins that cut DNA at one specific sequence

- Microsatellites are short repetitive sequences found throughout the genome

GENETICS - CLUTCH


PRACTICE

1. The genetic distances between three genes (ab, nm, kf) were determined using a two-point mapping cross. Determine the sequence of the three genes.

a. Ab – nm – kf b. Ab – kf – nm c. Kf – ab – nm

2. True or False: Recombination frequencies are never greater than 50% a. True b. False

Gene Distance

ab - nm 45

ab – kf 3

nm – kf 42

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3. Using the following data collected from a test cross, calculate the recombination frequency.

a. 15.9% b. 10.3% c. 40.5% d. 32.7%

Phenotype Offspring

Parental 1400

Parental 1200

Recombinant 150

Recombinant 150

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CONCEPT: TRI-HYBRID CROSS FOR MAPPING

● A tri-hybrid cross, is a mating to look at the inheritance of _________ traits in the offspring

□ For unlinked genes, this should follow normal Mendelian inheritance

□ For linked genes, recombination frequencies in the offspring can be used to map all three gene loci

EXAMPLE:

In a cross of fruit flies, there are three traits of interest.

1. Eye color: v+ = red, v = vermillion (like orange/purpleish),

2. Wing veins: cv+= presence of wing crossvein, cv = absence of crossvein

3. Wing shape: ct+ = normal wing, ct = cut wing edges

P: v+/v+ cv/cv ct/ct x v/v cv+/cv+ ct+/ct+

Parental phenotypes: Red, crovsveinless,cut wings x vermillion, crossvein, normal wing

Gametes: v+ cv ct v cv+ ct+

F1 trihybrid v+/v cv/cv+ ct/ct+

F1 test cross v+/v cv/cv+ ct/ct+ x v/v cv/cv ct/ct

F1 phenotypes: Red, crossvein, normal wing vermillion, crossvienless, cut wings

Parental gametes:

Gametes Phenotypes Offspring # Recomb. v and cv Recomb v and ct Recomb cv and ct

v cv+ ct+ Parental 580

v+ cv ct Parental 592

v cv ct+ Recombinant 45

v+ cv+ ct Recombinant 40

v cv ct Recombinant 89

v+ cv+ ct+ Recombinant 94

v cv+ ct Recombinant 3

v+ cv ct+ Recombinant 5

Total 1448 268 191 93

v+ cv ct

v cv+ ct+

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Now, you determine the recombination frequencies:

1. For v and cv: RF = 268/1448 = 18.5

2. For v and ct: RF = 191/1448 = 13.2

3. For cv and ct: RF = 93/1448 = 6.4

What can we determine from these ________________________ frequencies?

1. All three genes are linked, because their RFs are less than 50%

2. The orientation and distances of the gene loci

□ Why does 13.2 + 6.4 = 19.6 and not the 18.5 (RF for v and cv)?

- We should have counted the double cross overs twice, instead of once!

Gametes Phenotypes Offspring # Recomb. v and cv

v ct+ cv+ Parental 580

v+ ct cv Parental 592

v ct+ cv Recombinant 45 R

v+ ct cv+ Recombinant 40 R

v ct cv Recombinant 89 R

v+ ct+ cv+ Recombinant 94 R

v ct cv+ Recombinant 3 R x2

v+ ct+ cv Recombinant 5 R x2

Total 1448 284

RF = 284/1448 = 19.6%

v+ cvct

v cv+ct+

GENETICS - CLUTCH


PRACTICE

1. The following table shows data from a cross (ABC x abc) examining three genes (a, b, and c). Calculate the recombination frequency for A and B

a. 20% b. 32% c. 37% d. 9.8%

2. The following table shows data from a cross examining three genes (a, b, and c). Calculate the recombination frequency for A and C

a. 32% b. 37% c. 20% d. 9.8%

Genotype Offspring A C A B B C

A B C 320

a b c 276

a B C 145

A b c 152

A b C 43

a B c 34

A B c 9

a b C 12


A B C 320

a b c 276

a B C 145

A b c 152

A b C 43

a B c 34

A B c 9

a b C 12

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3. The following table shows data from a cross examining three genes (a, b, and c). Calculate the recombination frequency for B and C

a. 20% b. 32% c. 37% d. 9.8%

4. The following table shows data from a cross examining three genes (a, b, and c). Determine the order of genes a. A B C b. B C A c. C A B


A B C 320

a b c 276

a B C 145

A b c 152

A b C 43

a B c 34

A B c 9

a b C 12


A B C 320

a b c 276

a B C 145

A b c 152

A b C 43

a B c 34

A B c 9

a b C 12

GENETICS - CLUTCH


CONCEPT: MULTIPLE CROSS OVERS AND INTERFERENCE

● Mapping the loci of 3+ genes can be more difficult, because of multiple cross overs

□ Multiple crossovers occur when more than one crossover causes 2+ changes in the gamete genotype

EXAMPLE:

□ Calculate information on double cross overs by:

1. Double counting the double crossovers when calculating the RF for the genes of farthest distance

- This will help you accurately map the genes (Trihybrid cross)

● Interference is when crossovers in one region of the chromosome affects the chance of crossover in an adjacent region

□ Independent crossovers (meaning, no interference) can be calculated using the double recombination frequency

EXAMPLE: Double cross over calculation with no interference

1. If the frequency of crossing over is 20% for genes A and B, and 30% for genes B and C, what is the frequency

of a double crossover between A and C?

- (0.2 x 0.3) x 100 = 6%

v+ cvct

v cv+ct+ v+ cv

ctv cv+

ct+

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□ If the calculated value does not equal the observed results, then that means interference is impacting the data

- Uses the coefficient of coincidence

EXAMPLE: Calculating the interference

Observed frequency: 4

Expected frequency: 6

1-(4/6) = 0.33 x 100 = 33%

□ Therefore, there were 33% fewer double crossovers occurred than expected

- Crossing over at one location, partially decreased the chance of crossover at an adjacent location

I= 1-

Observed frequency

or # of double recombinants

Expected frequency

or # of double recombinants

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PRACTICE:

1. A female with the following genotype can produce a number of different gametes. Choose the gamete produced if no crossovers have occurred. Genotype = a b +

a. a + c + + c b. + b c c. a b + d. + b +

2. A female with the following genotype can produce a number of different gametes. Choose the gamete produced if a single crossover has occurred. Genotype = a b +

a. + + c + + c b. + b c c. a b + d. a b c

GENETICS - CLUTCH


CONCEPT: CHI-SQUARE TEST FOR LINKAGE

● A chi-square test can be used to identify the likelihood of gene linkage

□ Chi-square test is used to evaluate if your experimental values are different from the predicted values

EXAMPLE:

I want to know if genes A and B are linked, so I do an experiment where I cross two heterozygous organisms. I get 50

offspring, 31 parental types, and 19 recombinant types. Is it likely that A and B are linked?

1. What are the expected numbers?

- If two genes are not linked then the recombination frequency is 50%. Therefore, there would be 50% parental

types and 50% recombinant types. So out of 50 offspring, 50/2 = 25. There would be 25 parental and 25

recombinants.

2. Calculate the chi-square value

3. Determine the P value.

a. Calculate degrees of freedom. DF=1

b. Find chi-square value on row 1. It is between 2.71 and 3.84

c. The appropriate p value is between 0.10 and 0.05 (10% to 5%)

Phenotype Observed Expected (o-e)2/e

Parental 31 25 1.44

Recombinant 19 25 1.44

Total 2.88

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4. Do we accept or reject the null hypothesis?

a. The null hypothesis states that the expected and observed values are not different. (This would mean that

the genes would NOT be linked)

b. Because the p values are greater than 0.05 (5%), so we accept the null hypothesis.

5. Therefore, we can say with 95% confidence the genes are NOT linked.

a. Remember: This does not confirm linkage, it just states the likelihood

PRACTICE Black(B) rabbit coat colors are dominant to white(b) coat colors. Long hair (H) is dominant to short hair (h). A breeder crosses a rabbit homozygous for white, short hair with a homozygous black rabbit with long hair. The F1 is backcrossed to the rabbit with white, short hair and the following progeny are produced. Use the chi-square test to answer the following questions.

1. What are the expected offspring numbers if the two genes are not linked, and therefore assort independently? a. 73 parental, 73 recombinant b. 146 parental, 0 recombinant c. 60 parental, 86 recombinant d. 102 parental, 44 recombinant

Phenotype Offspring

Black, Long 40

Black, Short 20

White, Long 24

White, Short 62

Total 146

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2. Calculate the chi-square value for the above problem.

a. 23.00 b. 5.89 c. 0.02 d. 0.467

3. In this example, how many degrees of freedom should be used? a. 1 b. 2 c. 3 d. 4

Phenotype Offspring

Black, Long 40

Black, Short 20

White, Long 24

White, Short 62

Total 146

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4. Using the appropriate chi-square value and degrees of freedom, do the coat color and hair length genes assort independently?

a. Yes, both genes assort independently b. No, both genes do not assort independently (meaning they are linked)

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CONCEPT: MAPPING WITH MARKERS

● Mapping with ____________________ attempts to link chromosomal regions without the need to link alleles □ A molecular marker is a small DNA segment that has unique identifiable properties

- Can be polymorphic, meaning that they can differ between individuals in a population

- Very useful in identifying location of unknown genes, with unknown alleles

□ Restriction fragment length polymorphisms (RFLPs) occurs when DNA from individuals are cut differently

- Different lengths are due to slight differences in the DNA that are recognized by restriction enzymes

- Due to: mutations, deletions, duplications, etc…

- RFLPs are obtains by taking an individuals DNA, cutting it, and then comparing lengths on a gel or blot

□ RFLPs can be mapped through crosses and analysis of ___________________________ phenotypes

- Exactly the same as linkage mapping, but instead of looking at phenotypes, scientists look at DNA

- Generates an RFLP map, which is a linkage map of RFLP markers in the genome of an organism

EXAMPLE:

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□ Microsatellites are short tandem repetitive sequences that can also be used as markers to _______ the genome

- A CA repeat of 5-50 is found repeated about every 10,000 bases

- PCR is used to identify the length and position of the microsatellites in the organisms or genomic region

- Like RFLPs, microsatellites can be followed through crosses and offspring production to map

PRACTICE:

1. What is a molecular marker? a. A fluorescent probe attached to regions of a chromosome b. A gene of interest c. A small DNA segment with unique properties d. A fluorescent protein that marks regions of the cell

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2. Which of the following markers is not useful for mapping genes? a. Restriction fragment length polymorphisms b. Microsatellites c. Single Nucleotide Polymorphisms d. Enhancers

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CONCEPT: POSITIONAL CLONING

● Positional cloning is an approach that identifies which candidate genes may be the one that causes the ____________ 1. Scientists use mapping techniques to identify a specific chromosomal region associated with a disease

- This technique establishes linkage between DNA markers and disease phenotype

2. Additional markers are used within this locus to more precisely locate the gene of interest

3. Chromosomal walking uses overlapping DNA fragments to identify the gene of interest

- Begin with clone A, which is closest to the marker

- Identify an overlapping clone (called clone B) that walks closer to the gene

- Repeat with as many overlapping clones as you need to walk to the gene, one clone at a time

4. Identify a small number of candidate genes within this region that could cause the disease phenotype

5. Examine each gene individually to determine which one actually causes the phenotype

EXAMPLE:

Marker

Chromosome Genes

Gene of Interest

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PRACTICE:

1. The purpose of positional cloning is to what? a. Identify a large section of chromosome that may be responsible for a phenotype b. Identify one or a few candidate genes that may be responsible for a phenotype c. Identify a RNA sequence that may be responsible for a phenotype d. Identify a protein that may be responsible for a phenotype

2. Which of the following is a crucial step of positional cloning? a. Sequencing the genome b. Chromosomal walking c. Identifying microsatellites d. Generating RFLPs

GENETICS - CLUTCH


genetics - clutch ch.4 genetic mapping and...

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