BIOL 205 Mendelian and Molecular Genetics

Lecture : 11 Three point testcross Deducing gene order Interference Using ratios as diagnostics Mapping with molecular makers

Throw knives at this chromosome!

The larger the distance between two loci the more chance of recombination Recombination frequencies less than 50% indicate linkage. Remember unlinked genes on different chromosomes show 50% recombinant (due to independent assortment). So as we approach 50% recombination, genes that are linked but far from each other will appear to be unlinked.

Longer regions have more crossovers and thus higher recombinant frequencies

Figure 4-10

Three Point test cross

v= vermillion eyes cv= crossveingless ct= cut wing edges P v+/v+ . cv/cv . ct/ct
(order or linkage not known)

Q. Are they linked and if so what order on the chromosome X v/v . cv+/cv+ . ct+/ct+

Gametes: v+ . cv . ct (inputs)

v . cv+ . ct+

F1 trihybrid:

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

Trihybrid females are test crossed with triple recessive males:

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

F1 Trihybrid female

X v/v . cv/cv . ct/ct

Tester male*

How many gametes from trihybrid? 3 2 =

2X2X2= 8

*Test cross is usually done with tester male because Drosophila males do not show meiotic recombination.

How many gametes from trihybrid? Use branch diagram

v+

cv
cv+

ct ct+ ct ct+ ct

v+ .

cv ct
.

Parental

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

. .

Recombinants

cv
cv+

ct+ ct ct+

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

Parental

Trihybrid females are test crossed with triple recessive males:

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

F1 Trihybrid female

X v/v . cv/cv . ct/ct

Tester male

Recombinant for loci Gametes v and cv 580 592 45 40 89 94 3 5 1448 268 (18.5%) v and ct cv and ct

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

R R R R R R R R
191 (13.2%)

R R

R R
93 (6.4%)

v
13.2 m.u.

ct
6.4 m.u.

cv

Q. Why doesnt the v and cv RF add up to 19.6%?

Figure 4-11

When we calculated the RF value for v and cv we did not count the v ct cv+ and v+ ct+ cv genotypes; after all, with regard to v and 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

v
13.2 m.u.

ct
6.4 m.u.

cv

Recombination between v and cv : 45+40+89+94+3+3+5+5= 284/1448= 0.196

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.

A recombination-based map of one of the chromosomes of Drosophila

cv ct 20 -13.7= 6.3m.u. v ct 33 - 20= 13 m.u.

Gene order can usually be deduced by inspection, without a recombinant frequency analysis. Typically, for three linked genes, we have the eight genotypes at the following frequencies: two at high frequency (Parental)
two at intermediate frequency (Recombinants)
two at a different intermediate frequency two rare (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.

Deducing gene order by inspection

Different gene orders give different double recombinants

v
13.2 m.u.

ct
6.4 m.u.

cv

A map of the 12 tomato chromosomes

Figure 4-13c

Phenotypic ratios in progeny reveal the type of cross

Figure 4-14

Mapping with Molecular Markers

Changes in the DNA Eg. Single nucleotide polymorphism (SNPs) GC to AT Eg. 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 finescale mapping. Where are they? They can affect a gene leading to a phenotype. Eg. PKU or Albinism. You can be either homozygous or heterozygous for a SNP. But SNPs do not have to be in a gene or affect a gene.

Why do we use molecular markers?

1)They have a position on the the chromosome 2)We can detect them by sequencing, RFLP or PCR 3)The phenotype is the actual behavior of the DNA on Gel electrophoresis 4)Molecular markers can be used as a pseudo test cross because we can determine the homozygous, or heterozygous states of each morph.

How do we detect SNPs?

If you know the sequence in the region of the SNPyou can just sequence. RFLPs: restriction fragment length polymorphisms (RFLPs), which are SNPs located at a restriction enzyme's target site. They do not have to be in a gene, but we can use them to ask is our gene is linked to a RFLP?

An RFLP linked to a mouse disease gene

Figure 4-15a

An RFLP linked to a mouse disease gene

Q. Is D linked to M1 or M2?

Figure 4-15b

An RFLP linked to a mouse disease gene

Figure 4-15c

Minisatellite and microsatellite markers

most genomes contain a great deal of repetitive DNA. called simple sequence length polymorphisms (SSLPs). They are also sometimes called variable number tandem repeats or VNTRs. 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 genome analysis: 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 of tandem repeats of an even simpler sequence, generally a dinucleotide. Detected by PCR.

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

A microsatellite locus can show linkage to a disease gene

Figure 4-19a

P/p . M/M

p/p . M/M

Figure 4-20

Phenotypic and molecular markers mapped on human chromosome 1

The National Center for Biotechnology Information