Unit content
Three-point test crosses for inferring gene order
A three-point test cross follows three linked loci at once. Its major advantage over separate two-locus crosses is that it can reveal the linear order of the loci on the chromosome.
Suppose a heterozygote carries three loci $A/a$, $B/b$ and $C/c$, and is test-crossed to
$$abc/abc.$$
Because the tester contributes only $abc$ gametes, each offspring class reveals the three-locus gamete produced by the heterozygote.
The most frequent classes are usually parental
Consider these offspring counts:
| Gamete class | Count |
|---|---|
| $ABC$ | 355 |
| $abc$ | 343 |
| $Abc$ | 76 |
| $aBC$ | 72 |
| $ABc$ | 68 |
| $abC$ | 64 |
| $AbC$ | 12 |
| $aBc$ | 10 |
The two largest classes, $ABC$ and $abc$, are inferred to be the parental classes.
The two smallest classes, $AbC$ and $aBc$, are typically produced by double crossovers and are especially informative about gene order.
Compare parental and double-crossover classes
Compare
$$ABC\quad\text{with}\quad AbC.$$
Only the allele at locus $B$ has changed.
Likewise, compare
$$abc\quad\text{with}\quad aBc.$$
Again, only $B$ has changed.
Therefore $B$ is the middle locus, and the gene order is
$$A-B-C.$$
The chromosome phase can then be written as
$$ABC/abc$$
in that order.
Why the middle gene flips in a double crossover
With order $A-B-C$, one crossover in interval $A-B$ and another in interval $B-C$ can exchange the middle portion twice relative to the chromosome ends. The endpoint alleles $A$ and $C$ return to the parental association while the middle allele $B$ is switched.
That is why the rare double-crossover classes differ from the parental classes at only the middle locus.
A robust three-point workflow
For a sufficiently large, well-behaved test-cross dataset:
- identify the two most frequent reciprocal classes as parental;
- identify the two least frequent reciprocal classes as double crossovers;
- compare parental and double-crossover genotypes;
- the locus that changes relative to the other two is the middle locus;
- rewrite all classes using the inferred chromosome order before calculating interval recombination frequencies.
Three-point mapping turns recombinant offspring patterns into a statement about chromosome geometry: which locus lies between the other two?