LINKAGE, RECOMBINATION & GENE MAPPING

Linkage
When two or more genes reside on the same chromosome, they are said to
be linked. They may be linked together on one of the autosomes or connected
together on the sex chromosome. Genes on different chromosomes are distributed
into gametes independently of one another (Mendel's Law of Independent
Assortment). Genes on the same chromosome, however, tend to stay together
during the formation of gametes. Thus, the results of testcrossing dihybrid
individuals will yield different results, depending upon whether the genes are linked
or on different chromosomes.
EXAMPLE 6.1 Genes on different chromosomes assort independently during
meiosis, giving a 1 : 1 : 1 : 1 testcross ratio.

EXAMPLE 6.2 Linked genes do not assort independently, but tend to stay together
in the same combinations as they were in the parents. Genes to the left of the slash
line (/) are on one chromosome and those to the right are on the homologous
chromosome. Very closely linked genes may not recombine in the formation of
gametes.

Large deviations from a 1 : 1 : 1 : 1 ratio on the testcross progeny of a dihybrid

could be used as evidence for linkage. Linked genes do not always stay together,
however, because homologous nonsister chromatids may exchange segments of
varying length with one another during meiotic prophase. Recall from Chapter 1 that
homologous chromosomes pair with one another in a process called "synapsis" and
that the points of genetic exchange, called "chiasmata," produce recombinant
gametes through crossing over.

Crossing Over
In preparation for meiosis, the DNA of each chromosome replicates, producing two
genetically identical (barring mutation) sister chromatids. During prophase I,
homologous chromosomes form pairs called synapases [Fig. 6-1 (a)] with the aid of
proteins in the synaptonemal complex. Very large protein complexes,
called recombination modules [about 90 nanometers (nm) in diameter], occur at
intervals along the synaptonemal complex; each of these recombination modules is
thought to function as a multienzyme "recombination machine" that affects synapsis
and recombination. Anick is the removal of a phosphodiester bond between adjacent
nucleotides in a DNA strand. Endonucleases in the recombination modules nick a
single strand of each chromatid, allowing nonsister strands to be exchanged [Fig. 6-
1(b)], and thus affecting the recombination of linked genes. A DNA polymerase may
extend the exchanged strands, and an enzyme called DNA ligase repairs the nicks
[Fig. 6-1 c)]. If the top chromatid strand is rotated by 180°, a cross-shaped
structure called a chi (Χ) form can be seen under the microscope. This structure is
also referred to as the Holliday model [Fig. 6-1(d)]. An endonuclease nicks the two
previously uncut strands at tetranucleotide sequences 5'-(A/T)TT(G/C)-3'. Gaps and
nicks are then repaired, creating four recombinant chromatids [Fig. 6-1(e)] which
will segregate during the second meiotic division to be incorporated into different
gametes. Note that if only the A and B loci are being studied in the progeny of
dihybrid parents (AB/ab), two of the four possible gametes will retain the linkage
relationships of the dihybrid parents (AB and ab) and are thus referred to
as parental ornoncrossover types; the two other gametes will
be recombinant or crossover types (Ab andaB). Thus, each crossover or chiasma
event is expected to produce four gametes (AB, Ab, aB, ab) with equal frequencies.
However, if a crossover between the two genes under study does not occur in every
meiosis, then among all of thegametes (both those with and without crossovers in
this region) produced by a dihybrid individual, the frequency of noncrossover-type
gametes will exceed that of crossover-type gametes.
The alleles of double heterozygotes (dihybrids) at two linked loci may appear in
either of two positions relative to one another. If the two dominant (or wild-type)
alleles are on one chromosome and the two recessives (or mutants) on the other
(AB/ab), the linkage relationship is calledcoupling phase, or cis configuration.
When the dominant allele of one locus and the recessive allele of the other occupy
the same chromosome (Ab/aB), the relationship is termed repulsion phase,
or trans configuration. Parental and recombinant gametes will be of different
types, depending upon how these genes are linked in the parent.
Chiasma Frequency
A pair of synapsed chromosomes (bivalent) consists of four chromatids called a
tetrad. Every tetrad usually experiences at least one chiasma somewhere along its
length. Generally speaking, the longer the chromosome, the greater the number of
chiasmata. Each type of chromosome within a species has a characteristic (or
average) number of chiasmata. The frequency with which a chiasma occurs between
any two genetic loci also has a characteristic or average probability. The further
apart two genes are located on a chromosome, the greater the opportunity for a
chiasma to occur between them. The closer two genes are linked, the smaller the
chance for a chiasma occurring between them. These chiasmata probabilities are
useful in predicting the proportions of parental and recombinant gametes expected
to be formed from a given genotype. The percentage of crossover (recombinant)
gametes formed by a given genotype is a direct reflection of the frequency with
which a chiasma forms between the genes in question. Only when a crossover forms
between the gene loci under consideration will recombination be detected.

EXAMPLE 6.5 Crossing over outside the A–B region fails to recombine these
markers.

When a chiasma forms between two gene loci, only half of the meiotic products will
be of crossover type. Therefore, chiasma frequency is twice the frequency of
crossover products.

Chiasma % = 2(crossover %) or Crossover % = 1/2(chiasma %)

EXAMPLE 6.6 If a chiasma forms between the loci of genes A and B in 30% of the
tetrads of an individual of genotype AB/ab, then 15% of the gametes will be
recombinant (Ab or aB) and 85% will be parental (AB or ab).
EXAMPLE 6.7 Suppose progeny from the testcross Ab/ab × ab/ab were found in the
proportions 40% Ab/ab, 40% Ab/ab, 10% Ab/ab, and 10% ab/ab. The
genotypes Ab/ab andab/ab were produced from crossover gametes. Thus, 20% of all
gametes formed by the dihybrid parent were crossover types. This means that a
chiasma occurs between these two loci in 40% of all tetrads.
Multiple Crossovers
When two-strand double crossovers occur between two genetic markers, the progeny
show only parental phenotypes [Fig. 6-2(a)]. However, a third gene locus c between
the outside markers allows detection of double crossovers [Fig. 6-2(b)].

If there is a certain probability that a crossover will form between the A and C loci
and another independent probability of a crossover forming between
the C and B loci, then the probability of a double crossover is the product of the two
independent probabilities.
EXAMPLE 6.8 If a crossover between the A and C loci occurs in 20% of the tetrads
and between C and B loci in 10% of the tetrads in an individual of genotype ACB/acb,
then 2% (0:2 × 0:1) of the gametes are expected to be of double-crossover
types AcB and aCb.
Odd numbers of two-strand crossovers (one, three, five, etc.) between two gene loci
produce detectable recombinations between the outer markers, but even numbers of
two-strand crossovers (two, four, six, etc.) do not.

Limits of Recombination
If two gene loci are so far apart in the chromosome that the probability of a chiasma
forming between them is 100%, then 50% of the gametes will be parental type
(noncrossover) and 50% recombinant (crossover) type. When such dihybrid
individuals are testcrossed, they are expected to produce progeny in a 1 : 1 : 1 : 1
ratio as would be expected for genes on different chromosomes. Recombination
between two linked genes cannot exceed 50% even when multiple crossovers occur
between them.

I All linkage map distances are calculated as the percentage recombination

between two loci. This is reflected in the percentage of recombinant offspring.

A. If two genes are completely linked, all of the offspring will be parental (have the
same linkage as the parents); there will be no recombinantoffspring. Thus the
linkage map distance between the two genes would be zero LMU (Linkage Map
Units).
B. If the genes are on separate chromosomes (or are far enough apart that crossing
over between them occurs in virtually all meioses), half of the offspring will be
recombinant, the other half parental. Thus the distance between them would
calculate as 50 LMU, and 50 LMU reflects independence.
C. Linkage Map Units are not additive, and there is no physical distance which is
equal to one LMU.
1. Locations of crossovers are not random; crossovers are repressed close to the
centromere, and the presence of one crossover will repress a second one close by.

2. Some crossover events are always missed because it is possible that multiple
crossovers have occurred between the two points you are mapping; thus you will
underestimate the total amount of recombination. The distance calculated between
 two genes will always be somewhat dependent upon how many interior points
you are monitoring. (ie, you will calculate a greater distance between two genes
if you are doing a three point cross including a gene between the two in question
than if you are doing a simply two point cross.

II. Straightforward calculation of the percent recombination between two genes is

called a two point cross.

A. To perform a mating cross, mate a parent who is heterozygous for both genes to
one who is homozygous recessive for both genes. Thus the recombination in the
offspring will reflect crossing over of only one parent, reducing the complexity of
the problem.

B. X linked genes can be mapped by mating a female heterozygous for both genes in
question to any male, then scoring only the male offspring, which will directly
reflect what happens to the female’s X chromosomes.

C. Establish linkage by determining that the results of the mating do not indicate
independence.

D. Calculate the map distance between the two genes by calculating the percentage
of the offspring which have recombined the two genes.

III.
Most useful linkage mapping is done with three point crosses, which involve
mapping three genes relative to each other. The results of many three point
crosses can be combined together to create an overall map of many genes.
A.
Again, a useful mating for mapping would be between a parent heterozygous for
all three genes and a homozygous recessive partner, or for X linked genes,
between a triple heterozygous female and any male. Unless you have complete
linkage, you will get eight phenotypic classes of offspring.
B.
Confirm linkage by determining that the results of the cross do not meet
expectations for independence.
C.
Identify parental offspring and double crossover offspring. The most numerous
offspring classes will be the parental classes. There should be two, and they
should be reciprocals. The least numerous classes should be the double
crossover classes (those that resulted from crossing over between both pairs of
genes). Again, there should be two of these classes, and they should be
reciprocals.
D.
 Determine gene order by determining which of the three genes is in the middle.
This is done by comparing the parental and double crossover classes. Since the
double crossovers crossed over between the first and second genes, then
crossed back between the second and third genes, the only gene which is actually
recombined is the middle one.
1.
Select one parental class, then choose the double crossover class which is most
like it. They should differ only in one gene. This is the middle gene.
2.
Examine the other parental and the other double crossover; they should give you
the same result. See example.
E.
Rewrite all of your offspring phenotypic classes, putting the genes in the correct
order. It doesn’t matter which end gene you decide is the “first” gene, but you
must get the correct one in the middle.
F.
You have four single crossover classes. Two of them resulted from crossing over
between the first two genes; the other two from crossing over between the second
and third genes. Figure out which of these are which, again by comparing them to
the parental classes which are most like them. If a single crossover class differs
from a parental class only in the first gene, then it represents a crossover between
the first and second genes. If it differs only in the last gene, then it represents a
crossover between the second and third genes. Again, you should have to pairs of
reciprocal phenotypes. See example.
G.
Calculate the distance between the first two genes by calculating the percentage of
your offspring that recombined those two genes. Remember that the double
crossovers count as recombinants here.
H.
Calculate the distance between the second and third genes by calculating the
percentage of your offspring that recombined those two genes. Again, the double
crossovers count here.
I.
The distance between the end genes can be calculated simply by adding the two
intervening distances.

IV.
Note that the distance you calculated between your end genes is different from the
distance you would have calculated if you were simply doing a two point cross.
You would have scored only the single crossovers as recombining the end genes;
the double crossovers—each of which actually represents two crossovers between
those end genes—would have been scored as parentals, since the end genes are
still in the parental association.

Three-point Cross
This time we are looking at the A, B, and C genes (alleles A and a, B and b, C and c).
Again, complete dominance for all. Here's our mating:

AaBbCc x aabbcc

If the genes are independent, we should get eight classes of offspring, all pretty much
equal in number. However, here's what we get:

ABC: 95 aBC: 50
ABc: 5 aBc: 700
AbC: 700 abC: 5
Abc: 50 abc: 95

Obviously, not all phenotypic classes are equal, so we definitely have linkage.

Determining Gene Order

To determine gene order, you must first identify the parental classes and the double
crossover classes. Don't fall into the trap of assuming that the parentals must always
beABC and abc. Parental classes are always the largest ones, since the connection
between the genes will tend to preserve parental linkages above the expected level of
independence. In our example, the AbC and aBc classes are the parental classes. Notice
that these are reciprocals of each other--they have "opposite" phenotypes. This should
make sense.

The double crossover classes will always be the smallest classes, since they require a
crossover between A and B and a crossover between B and C. So our double crossover
classes are ABc and abC. Again, note that they are reciprocals.

Now we compare the parentals with the double crossovers. The only gene which has
recombined in a double crossover will be the middle one, because the chromatids will
have crossed over between the first two genes, then crossed back between the second and
third genes. Compare each double crossover with the parental which is most like it. For
double crossover ABc, the most similar parental is aBc. (It's the same in two of the
genes; the other parental is the same in only one gene.) Comparing these, we see that the
only gene that is different is the A/a gene. If we compare the other double crossover to
the other parental, we get the same result--only the A/a gene is difrerent. So A must be
the gene in the middle. Our actual gene order is B-A-C.

To keep from getting confused, it's an excellent idea to rewrite the offspring chart above
with the genes in the correct order, like this:

BAC: 95 BaC: 50
BAc: 5 Bac: 700
bAC: 700 baC: 5
bAc: 50 bac: 95

Map Distance

To figure out the distances, we must identify our single crossover classes. We've got four
of them: BAC, bAc, BaC and bac. Note that this is two pairs of reciprocals
(BACand bac are reciprocals and bAc and BaC are also reciprocals). Again, we want to
compare these to the parentals which are most like them to determine just where the
crossover occurred to produce them. Comparing single crossover BAC to parental bAC,
we see that it has recombined between the B and the A loci. Same for its reciprocal.
Comparing the BaC single crossover to the Bac parental, we see that it has recombined
between the A and the C genes, as has its reciprocal. Note also that the numbers of
offspring give you hints as to which crossover classes go together.

Now we are ready to calculate distances. We have four offspring classes which have
recombined between the B and the A genes. These are the single crossovers BAC (95)
and bac (95) as well as the double crossovers Bac (5) and bAC (5). This gives us a total
of 200 offspring who have crossed over between these two genes. There are a total of
1700 offspring, so we'd calculate this distance as (200/1700) x 100, which equals 11.8. So
we've calculated a distance of 11.8 LMU between genes B and A.

There are also four offspring classes which have recombined between
the A and C genes. bAc (50), BaC (50) and again, our double crossover classes BAc (5)
and baC (5), for a total of 110. Calculating: (110/1700) x 100 = 6.5 LMU between
genes A and C.

To calculate the distance between B and C, just add 11.8 and 6.5, to get 18.3 LMU.
1) determine genotype of parents
- examine initial cross that produced F1
- determine which genes are dominant, and which are recessive
- sex-linked or autosomal?
- male +++//Y
- male abc//Y
- is backcrosser homozygous recessive?
if not,
- focus only on progeny with recessive phenotypes
- can both parents undergo recombination?
(keyword "self-fertilize" or absence of keyword "crossed back to")
if so, calculate Rf for the gametes, not the offspring
- is there codominance?
treat as groups of 3 (AA, Aa, aa), not groups of 2 (A_, aa)

2) determine which genes are linked or unlinked

- 2n = # of phenotypes (n = # of genes)
- groups of 2: all genes linked
- groups of 4: 1 gene unlinked
- groups of 8: 2 genes unlinked

3) determine linkage phase

- look at parents of F1, if they're homozygous, then F1's linkage phase is just
mother//father
- look at most abundant progeny - usually NR, similar linkage phase as parents
* these only work when backcrosser is homozygous recessive.

4) gene order (only applies if 3+ linked genes)

- compare DCOs (least abundant progeny) to NR (most abundant progeny)
gene that's different between the two groups of progeny is in the middle

5) calculate map units of recombinants

Rf1 = (SCO1 + DCO)/(total progeny)
6) calculate interference if necessary
coefficient of coincidence = (observerd DCOs)/(expected DCOs)
Interference = 1 - coefficient of coincidence