The 3-point test cross

•  Mapping three (linked) loci

•  Inferring gene order
•  Interference
–  Is the frequency of double-crossover less than
expected?
•  Map functions
–  Translating c = freq(recombination gametes) into
the expected number of crossovers m.
 Linkage 2
The three-point test cross
 Double crossovers
•  When considering three loci, we can
observe double crossovers that would
have been missed if only two loci are
followed
•  Hence, the frequency of “recombinant”
gametes underestimates the actual
frequency of crossingover.
 The three-point testcross

The mapping (and analysis) of three linked loci

Testcross as only
st e ss gametes from
This parent
These are the two parental
gametes (being the most
Common)

For three-point analysis,

key is to consider EACH
PAIR of loci separately!
 st - e locus pair
Parentals: st+ e+, st e

st+ e --> recombinant

st e+ --> recombinant

st+ e+ --> parental

st e --> parental

st+ e --> recombinant

st e+ --> recombinant
Freq(recom) = (50+52+43+41)/755
= 0.246
 e - ss locus pair
Parentals: e+ ss+, e ss

e ss --> parental

e+ ss+ --> parental

e+ ss --> recombinant

e ss+ --> recombinant

e ss+ --> recombinant

e+ ss --> recombinant
Freq(recom) = (5+3+43+41)/755
= 0.122
 st - ss locus pair
Parentals: st+ ss+, st ss

st+ ss --> recombinant

st ss+ --> recombinant

st+ ss --> recombinant

st ss+ --> recombinant

st+ ss+ --> parental

st ss --> parental
Freq(recom) = (50+52+5+3)/755
= 0.146
 Summary (so far)
•  Recombination frequencies between pairs
–  st - e, c = 0.246
–  e - ss, c = 0.122
–  st - ss, c = 0.146
•  Ordering: Which locus is in the middle?
–  Since st - e has the largest distances, suggests
these are on the ends, hence ss in the middle
–  Suggested order: st - ss - e
•  Formally show this by looking at the double-
crossovers
–  Rarest gametes correspond to doubles
 Formally showing gene order
•  Find the pair of gametes corresponding to
the double crossovers
–  These will be the rarest (indeed, may be so rare
that they are not seen in the sample)
•  Given the genotypes of the parents, try
different gene orders, and find which gene
order generates the observed class of double
crossovers.
Effect of different gene orders on the classes
corresponding to double crossovers
Most common pair =
parentals

Rarest pair = double

crossovers

Does placing ss in the middle

give this class as the
double crossovers?
These are the observed
doubles, hence (3) is the
Gene order
 Interference
•  Presence of one crossover may influence the
probability of nearby crossovers
–  This is called interference, and is the rule (rather
than the exception)
–  Under complete interference, NO doubles are
seen, only singles.
•  What is the expected recombination fraction
if no interference?
Trow’s formula
 If Crossovers occurs
independently
•  Trow’s formula holds:
–  c = c1 + c2 - 2c1c2
–  Note that recombination frequencies are not
additive, as c < c1 + c2 .
–  Why? Double crossovers are scored as parental
•  If n gametes scored, if no interference,
expected number of doubles is just
–  n c1 c2
 Coefficient of Coincidence
(COC)
•  What fraction of the expected number
of doubles are actually seen?

If no interference, Pr(Double) = c1c2

COC = 1 - inference
 Computing COC for our example
•  For our example, 8 doubles (out of 755) are seen
•  Map distances are
–  st - 0.146 - ss - 0.122 - e
•  Expected number of doubles under no interference
is
–  755*(0.146*0.122) = 13.44
•  COC is observed/expected, 8/13.44 = 0.59
–  We only observe 59% of the expected number of doubles
•  Interference = 1-COC = 0.41.
–  41% of the expected crossover do not occur
–  Complete interference (COC = 0) no doubles seen
–  No interference, Number doubles = n c1 c2
 Different types of doubles
•  Crossing over occurs after each homologous
chromosome has double to consist of two
sister strands
•  Hence, at the time of crossovering, a double
crossover event may involve two, three, or
four DNA steands
•  We can distinguish between these when all
products of a single meiosis can be
recovered as a group (fungal genetics)
 Mapping functions
•  When considering a pair of loci, we estimate c,
the fraction of gametes that we score as non-
parental
•  This UNDERCOUNTS the actual amount of
crossingover
–  All even number of crossovers = parental
–  All odd = recombinant
–  Using c to estimate the expected number of
crossovers ignores all events of 2 or more crossovers
–  Scores all parental = zero crossovers (could be 2, 4,
6, ..), all recombinants = 1 crossover (3, 5, ..)
 Mapping functions
•  Observe c = fraction of recombinant gametes
•  Want to estimate m = expected number of
crossovers
•  Mapping functions rate c and m
•  Genetic maps are given in terms of map units
(m)
•  Distances are additive in the m scale, not so
on the c scale
 Haldane’s mapping function
•  Makes the critical assumption of NO
INTERFERENCE
–  This implies that the expected number of
crossovers follows a Possion distribution
•  c = prob(recombinant gamete) = prob(odd
number of crossovers)
•  Haldane’s map function
–  c = [1-exp(-2m)]/2
–  m = -ln(1-2c)/2
–  For m small, m is essentially c (little chance of
more than one crossover).
–  However, for c large, m > c
C

100*m
 Example
•  Consider the st - e recombination fraction c
= 0.246
•  This translates into a haldane map distance of
–  m = -ln(1-2*0.246)/2 = 0.339
–  Denoted as a map distance of 33.9 centiMorgans
(cM)
–  Hence, expect an average of 0.339 crossovers per
gamete
–  Prob(no crossovers) = exp(-0.339) = (Obtained
from zero term of the Poisson) = 0.712
–  Hence, pro(1 or more crossovers) = 1-0.712 =
0.288
Derivation of Haldane’s formula