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Understanding The Punnett Square

Written by: Michelle Ricketts

The Punnett square is a diagram that is used to predict an outcome of a particular cross or breeding experiment. Many biologists and breeders use this method to determine the probability of the progeny (offspring) having a particular genotype.

It gives a tabular summary of every possible combination of one maternal allele with one paternal allele for each gene being studied in the cross. These tables give the correct probabilities for the genotype outcomes of independent crosses where the probability of inheriting copies of each parental allele is independent.

Using the Punnett square is simply an adding tool to add 1 allele from one parent to an allele from the other parent that are located on the same locus to determine the resulting combinations for that particular locus.

Each parent can have a set of (2) alleles per locus. In most cases there will be more than one locus (loci-plural for more than one locus). Once you have determined the combinations for each locus, we then add those together separating each locus with a hyphen (-) to determine the genotypes of the progeny.

Monohybrid Cross is a mating between individuals who have different alleles at one genetic locus of interest. A genetic cross made to examine the distribution of one specific set of alleles in the resulting offspring.

(a) A monohybrid cross looks like this: D/+ x +/g

In the example below we show a Monohybrid cross. One parent is a Black Lace (D/+), the other parent is a wild type Silver with the recessive gene "Gold" (+/g). This sort of cross is good to do when one is studying the interaction of the alleles on a particular locus.


The genotype results would be:

  • 25% Black Lace (D/+)
    25% wild-type Silver (+/+)
    25% Hybrid Black with a recessive Gold gene (D/g)
    25% wild-type Silver with a recessive Gold gene (+/g).

(Please note: A Monohybrid cross is also used to "separate" alleles. This is done by crossing to wild-type. Such as D/g x +/+. This will result in 50% Black Lace (D/+) and 50% wild-type silver (+/g).)

However, how an individual looks and what their genetic code is... sometimes do not match up. This is the difference between "genotype" and "phenotype." The genotype is the actual genetic make up of an individual. The phenotype is what that individual looks like.

Even though the above cross shows 4 different genotypes, the actual phenotype results would be 25% Black Lace, 25% Hybrid Black (a darker fish than the Black lace), and 50% Silver with half of each phenotype carrying the recessive gene for Gold.

Dihybrid Cross - Hybridization using two loci with two alleles each.
Example: Hybrid Black with standard fins x Hybrid Black with long fins or D/g-+/+ x D/+-v/v.

(a) A dihybrid cross looks like this: D/g-+/+ x D/+-v/v.

(b) A trihybrid cross looks like this: D/g-+/+-St/St x D/+-v/v-+/+ (Please note: One parent may have alleles on a locus that the other parent does not. In that case, we use wild-type symbols to show that there isn't any alleles on that locus for that parent. Once you get the hang of it, it is often left out showing only the loci that do have alleles. Ex: D/g-+/+-St/St would simply be D/g-St/St.

higher-order hybrid crosses
(d) A tetrahybrid cross looks like this: Aa-Bb-Cc-Dd x Aa-Bb-Cc-Dd, etc.

- A more complicated cross showing alleles at 4 loci: *Please note: The grid below is a forked line method that I use to figure out my crosses rather than placing the female gametes down the left side. In this cross we show 4 loci, meaning there are 4 genetic locations of alleles combined between both parents.

Paternal D + Z + St + + +
Maternal + g S S + + V V
Results 25% D/+, 25% D/g,
25% +/+, 25% +/g
50% Z/S
50% +/S
50% St/+
50% +/+
100% +/V
Genotype Results:
  • D/+ - Z/S - St/+ - +/V  =  ( Streaked Clown Lace Veil )
    D/+ - Z/S - +/+ - +/V  =  ( Clown Lace Veil)
    D/+ - +/S - St/+ - +/V  =  ( Streaked Black Ghost Veil )
    D/+ - +/S - +/+ - +/V  =  ( Black Ghost Veil )
  • D/g - Z/S - St/+ - +/V  =  ( Streaked Hybrid Black Clown Veil )
    D/g - Z/S - +/+ - +/V  =  ( Hybrid Black Clown Veil )
    D/g - +/S - St/+ - +/V  =  ( Streaked Hybrid Black Ghost Veil )
    D/g - +/S - +/+ - +/V  =  ( Hybrid Black Ghost Veil )
  • +/+ - Z/S - St/+ - +/V  =  ( Streaked Clown Veil )
    +/+ - Z/S - +/+ - +/V  =  ( Clown )
    +/+ - +/S - St/+ - +/V  =  ( Streaked Ghost Veil )
    +/+ - +/S - +/+ - +/V  =  ( Ghost Veil )
  • +/g - Z/S - St/+ - +/V  =  ( Streaked Clown Veil )
    +/g - Z/S - +/+ - +/V  =  ( Clown Veil )
    +/g - +/S - St/+ - +/V  =  ( Streaked Ghost Veil )
    +/g - +/S - +/+ - +/V  =  ( Ghost Veil )

As you can see, this cross resulted in 16 different genotypes from one cross. Because each parent had completely different alleles on locus 1, it resulted in 4 different combination groups. Loci 2 & 3 both resulted in 2 genetic combinations because the genes added up to 50% each. Locus 4 resulted in 100% single dose for veil (V) therefore resulting in all progeny receiving one dose of the gene for veil (+/V).

Additional notes: It was thought for many years that the streaked gene could only be seen on dark fish. In a similar cross I made, I found that the Streaked gene modified the pattern on "Smokey" and "Stripeless" resulting in a fish that looked like a very light bronze marble.

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