BIOL 205 Lecture 11 Handout (1)

7/27/2019 BIOL 205 Lecture 11 Handout (1)

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BIOL 205 Mendelian and Molecular

Genetics

Lecture : 11

Three point testcrossDeducing gene order

Interference

Using ratios as diagnosticsMapping with molecular makers


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Throw knives at this chromosome!

The larger the distance between two loci the morechance of recombination

Recombination frequencies less than 50% indicate

linkage. Remember unlinked genes on different

chromosomes show 50% recombinant (due toindependent assortment). So as we approach 50%

recombination, genes that are linked but far from each

other will appear to be unlinked.


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Figure 4-10

Longer regions have more crossovers and thus higher

recombinant frequen

cies


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Three Point test cross

v= vermillion eyes

cv= crossveingless

ct= cut wing edges

P v+/v+. cv/cv. ct/ct X v/v. cv+/cv+. ct+/ct+

Q. Are they linked and if so

what order on thechromosome

Gametes: v+. cv. ct v. cv+. ct+

F1 trihybrid: v+/v. cv/cv+. ct/ct+

(inputs)

(order or linkage not known)


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Trihybrid females are test crossed with triple recessive males:

v+/v. cv/cv+. ct/ct+ v/v. cv/cv. ct/ctX

F1 Trihybrid female Tester male*

How many gametes from trihybrid?

23= 2X2X2= 8

*Test cross is usually done with tester male because

Drosophila males do not show meiotic recombination.


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How many gametes from trihybrid? Use branch diagram

v cvcv+

ct

ct+

ct

ct+

v. cv. ct

v. cv. ct+

v. cv+. ct

v. cv+. ct+

v+ cvcv+

ct

ct+

ct

ct+

v

+

.

cv

.

ct

v+. cv. ct+

v+. cv+. ct

v+. cv+. ct+

Parental

Parental

Recombinants


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Trihybrid females are test crossed with triple

recessive males:

v+/v. cv/cv+. ct/ct+ v/v. cv/cv. ct/ctX

F1 Trihybrid female Tester male


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Gametes

v+. cv. ctv. cv+. ct+

v. cv. ct+

v+. cv+. ct

v. cv. ct

v+. cv+. ct+

v. cv+. ct

v+. cv. ct+

580

592

45

40

89

94

3

5

cvand ct

Recombinant for loci

R

R

1448

vand cv

R

R

R

R

268

(18.5%)

vand ct

R

R

RR

191

(13.2%)

RR

93

(6.4%)


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v ct cv

13.2 m.u. 6.4 m.u.

Q. Why doesnt the vand cvRF add up to 19.6%?


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Figure 4-11

When we calculated the RF value forvand cvwe did not

count the v ct cv+ and v+ct+cvgenotypes; after all, with

regard to vand cv, they are parental combinations (v cv+

and v+cv). This leads to an underestimation of the

recombinant frequency.

Normally this not a problem as the sum of the two shorter

distances gives us the best estimate of overall distance


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v ct cv

13.2 m.u. 6.4 m.u.

Recombination between v and cv :

45+40+89+94+3+3+5+5= 284/1448= 0.196


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Are the crossovers in adjacent chromosome

regions independent events or does a crossover in

one region affect the likelihood of there being a

crossover in an adjacent region?

Generally, crossovers inhibit each other somewhat

in an interaction called interference.

In some regions, there are never any observed

double recombinants (complete interference).

Interference values anywhere between between 0(no interference) and 1 (complete) and are found in

different regions of the chromosome and in

different organisms.


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A recombination-based map of one of the chromosomes ofDrosophila

cv ct 20 -13.7= 6.3m.u.

v ct 33 - 20= 13 m.u.


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Deducing gene order by inspectionGene order can usually be deduced by inspection,

without a recombinant frequency analysis. Typically, forthree linked genes, we have the eight genotypes at the

following frequencies:

two at high frequency

two at intermediate frequency

two at a different intermediate frequency

two rare

(Parental)(Recombinants)

(Recombinants from double crossovers)With three genes only three gene orders are possible, each

with a different gene in the middle position.

The gene in the middle is that it is the allele pair that has

flipped position in the double-recombinant classes.


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Different gene orders give different double recombinants

v ct cv

13.2 m.u. 6.4 m.u.


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Figure 4-13c

A map of the 12 tomato chromosomes


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Mapping with Molecular Markers

Changes in the DNA

Eg. Single nucleotide polymorphism (SNPs) GC to ATEg. DNA repeats eg. Microsatellite DNA repeats

In humans, there are thought to be about 3 million SNPs

distributed more or less randomly at a frequency of 1 in every

300 to 1000 bases, providing a useful set of markers for fine-

scale mapping.

Where are they?

They can affect a gene leading to a phenotype. Eg.

PKU or Albinism. You can be either homozygous orheterozygous for a SNP.

But SNPs do not have to be in a gene or affect a

gene.


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Why do we use molecular markers?

1)They have a position on the the chromosome2)We can detect them by sequencing, RFLP or

PCR

3)The phenotype is the actual behavior of theDNA on Gel electrophoresis4)Molecular markers can be used as a pseudo test

cross because we can determine the

homozygous, or heterozygous states of eachmorph.


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How do we detect SNPs?

If you know the sequence in the region of theSNPyou can just sequence.

RFLPs: restriction fragment length

polymorphisms (RFLPs), which areSNPs located at a restriction enzyme's

target site.

They do not have to be in a gene, butwe can use them to ask is our gene is

linked to a RFLP?


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Figure 4-15a

An RFLP linked to a mouse disease gene


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Figure 4-15b


Q. Is D linked to M1 or M2?


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Figure 4-15c



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Minisatellite and microsatellite markersmost genomes contain a great deal of repetitive DNA. called

simple sequence length polymorphisms (SSLPs). They arealso sometimes called variable number tandem repeats orVNTRs.

SSLPs commonly have multiple versions; sometimes 4

versions (2 from each parent) can be tracked in a pedigree.

Two types of SSLPs are useful in mapping and other genomeanalysis: minisatellite and microsatellite markers.minisatellite marker: is based on variation in the number of

tandem repeats of a repeating unit from 15 to 100 nucleotides

long. In humans, the total length of the unit is from 1 to 5 kb.

Detected by Southern Blot.microsatellite marker: is based on variable numbers oftandem repeats of an even simpler sequence, generally a

dinucleotide. Detected by PCR.


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The most common microsatellite type is a repeat

of CA and its complement GT, as in the following

example:

5 C-A-C-A-C-A-C-A-C-A-C-A-C-A-C-A 3

3 G-T-G-T-G-T-G-T-G-T-G-T-G-T-G-T 5


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Figure 4-19a

A microsatellite locus can show linkage to a disease gene

P


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P/p . M/M p/p. M/M


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Figure 4-20

Phenotypic and

molecular markers

mapped on human

chromosome 1


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BIOL 205 Lecture 11 Handout (1)