Test Cross (Single, Two, Triple Gene)- Definition, Examples, Uses

Phenotypic characters can be easily separated just by looking at the individual. It’s not that easy to determine the genotypic character of that same individual. This is because, genetically a dominant character can be either homozygous or heterozygous, and it is impossible to separate them just by looking over phenotypic characters.  To determine the genotype (homozygous or heterozygous), Mendel developed a genetic cross method, known as “test-cross”.

A test cross is a very important genetic tool devised by Gregor Johan Mendel.  It is applicable in Mendelian genetic characters only, but not in non-mendelian genetics. 

Test Cross Definition

A test cross is a genetic method for determining the unknown genotype of a dominant individual. It is a breeding method between a (known genotype) homozygous recessive individual with an individual of the opposite mating type with an unknown dominant genotype.

Generally, dominant offspring is crossed with a known recessive parent or another recessive individual to determine the genotype of the offspring. Hence, it is a type of backcross. Alternately, it can also be used to determine the genotype of dominant parents also.

The resulted offspring’s phenotypic characters are studied and the genotype of the tested individual can be determined accordingly. If all the offspring after the test cross are dominant, then we can say that the genotype of the tested unknown individual is homozygous dominant. Whereas, if 50% of offspring show dominant and the rest 50% show recessive characters, then we can say that the genotype of the tested unknown individual is heterozygous dominant.   

Based on the number of genes or characters studied during the test cross, we can categorize the test cross into monohybrid test cross, dihybrid test cross, trihybrid test cross, and polyhybrid test cross.

A. Monohybrid Test Cross (Single Gene Test Cross)

Also called ‘single gene test cross’, is a type of testcross where only one type of gene or phenotypic character is studied.  Among different characters of test individuals, only one of the dominant characters is considered. In a monohybrid test cross, a 1:1 phenotypic ratio is obtained if the test individual is heterozygous.

Let’s consider an example where a red flower (dominant) bearing plant is crossed with a yellow flower (recessive) plant. Here, a red flower may be either homozygous (RR) or heterozygous (Rr), but the yellow one being recessive is always homozygous (rr).

To know the genotype of the red flower test cross is done and the following results are obtained:

1. If the red flower is homozygous dominant (RR)

Phenotypic ratio : Red : Yellow = 1 : 0

Here all the offspring are phenotypically red. Hence, the test concludes that the test flower was homozygous red (RR).

2. If the red flower is heterozygous dominant (Rr)

Phenotypic ratio : Red : Yellow = 1 : 1

Here half of the offspring are phenotypically red and half are phenotypically yellow. Hence, the test concludes that the test flower was heterozygous red (Rr).

B. Dihybrid Test Cross (Two Gene Test Cross)

Also called ‘two-gene test cross’, is a type of testcross where two types of genes or phenotypic characters are studied.  Among different characters of test individuals, only two of the dominant characters are considered. A test individual with two selected dominant phenotypic characters is crossed with double recessive parents and the phenotypic characters of F1 generations are studied.  In a dihybrid test cross, a 1:1:1:1 phenotypic ratio is obtained if the test individual is heterozygous.

Let’s consider an example where a red flower tall plant (dominant) bearing plant is crossed with a yellow flower dwarf plant (recessive) plant. Here, a red flower tall plant may be either homozygous (RRTT) or heterozygous (RrTt), but the yellow flower dwarf plant is always homozygous (rrtt).

To know the genotype of the individual with dominant characters test cross is done and the following results are obtained:

1. If the test plant is heterozygous dominant (RrTt)

Phenotypic ratio : Red/Tall : Red/Dwarf : Yellow/Tall : Yellow/Dwarf = 1 : 1 : 1 : 1

Here 25% of offspring are red and tall, 25% of offspring are red and dwarf, 25% of offspring are yellow and tall, and the rest 25% of offspring are yellow and dwarf.  Hence, the test concludes that the test individual was a heterozygous red flower tall plant (RrTt).

2. If the test plant is homozygous dominant (RRTT)

Phenotypic ratio : Red/Tall : Red/Dwarf : Yellow/Tall : Yellow/Dwarf = 1 : 0 : 0 : 0

Here all the offspring are red flower tall plants. Hence, the test concludes that the test individual was a homozygous red flower tall plant (RRTT).

C. Trihybrid Test Cross (Triple Gene Test Cross)

Also called ‘triple gene test cross’, is a type of testcross where three types of genes or phenotypic characters are studied.  Among different characters of test individuals, only three of the dominant characters are considered. A test individual with three selected dominant phenotypic characters is crossed with a triple recessive parent and the phenotypic characters of F1 generations are studied. In a trihybrid test cross, a 1:1:1:1:1:1:1:1 phenotypic ratio is obtained if the test individual is heterozygous.

Let’s consider an example where a red flower tall plant with normal leaves (dominant) bearing plant is crossed with a yellow flower dwarf plant with wrinkled leaves (recessive) plant. Here, a red flower tall plant with normal leaves may be either homozygous (RRTTNN) or heterozygous (RrTtNn), but a yellow flower dwarf plant with wrinkled leaves is always homozygous (rrttnn).

To know the genotype of the individual with dominant characters test cross is done and the following results are obtained:

1. If the plant is heterozygous dominant (RrTtNn)

Phenotypic ratio: red/tall/normal : red/tall/wrinkled : red/dwarf/normal : red/dwarf/wrinkled : yellow/tall/normal : yellow/tall/wrinkled : yellow/dwarf/normal : yellow/dwarf/wrinkled =  1 : 1 : 1: 1 : 1 : 1 : 1 : 1

Here, 12.5% are red flower tall plant with normal leaves, 12.5% are red flower tall plant wrinkled leaves, 12.5% are red flower dwarf with normal leaves, 12.5% are red flower dwarf with wrinkled leaves, 12.5% are yellow flower tall with normal leaves, 12.5% are yellow flower tall with wrinkled leaves, 12.5% are yellow flower dwarf with wrinkled leave, and rest 12.5% are yellow flower dwarf with wrinkled leaves. The result concludes that the test individual was heterozygous dominant (RrTtNn).

2. If the plant is homozygous dominant (RRTTNN)

Phenotypic ratio: red/tall/normal : red/tall/wrinkled : red/dwarf/normal : red/dwarf/wrinkled : yellow/tall/normal : yellow/tall/wrinkled : yellow/dwarf/normal : yellow/dwarf/wrinkled =  1 : 0 : 0: 0 : 0 : 0 : 0 : 0

Here, all the offspring are red flower tall plants with normal leaves. The result concludes that the test individual was homozygous dominant (RRTTNN).

Applications of Test Cross

• To determine the genotype of a dominant individual (F1 generation as well as a dominant parent)
• To know the types and numbers of gametes formed
• To separate pure and hybrid dominant breed

Limitations of Test Cross

• Requires a large number of offspring for confirmation
• Applicable only in case of Mendelian genetic inheritance
• Difficulty to study a large number of characters and genes at a single cross
• It is not suitable in the case of species with a very long generation time

References

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