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Dihybrid Cross

Between 1856 and 1863, Gregor Johann Mendel, now known as the ‘Father of Modern Genetics’, experimented on garden pea plants and observed their inheritance pattern from one generation to the other. Accordingly, he proposed three laws: 1) Law of Dominance, 2) Law of Segregation, and 3) Law of Independent Assortment, collectively known as Laws of Inheritance.

Among all, the law of independent assortment was discovered while performing dihybrid crosses between the pea plants.

What is a Dihybrid Cross

A dihybrid cross is a breeding experiment involving two organisms that are identical hybrids for two traits or characters. A hybrid organism is heterozygous, which means it carries two alleles of a particular gene. Traits are characters determined by segments of DNA called genes. An allele is an alternative gene form inherited from each parent during sexual reproduction. A dihybrid cross determines the genotypic and phenotypic combination of offspring for two particular genes that are unlinked.

Here, the individuals are homozygous for a particular trait. One parent contains homozygous dominant alleles, while the other contains homozygous recessive alleles. The offspring, or F1 generation, formed after crossing the two parents is heterozygous for the specific traits being studied. Thus, all of the F1 individuals possess a hybrid genotype, expressing dominant phenotypes for each trait. 

How to Set Up Dihybrid Cross

  1. Signify each allele using characters. Capital letters are used for the dominant allele, lower case letters for the recessive allele.
  2. Write the genotype and phenotype of the parents (P generation). Always pair alleles for the same gene and write capital letters first and then small letters (like AaBb).
  3. Write all potential gamete combinations for both parents.
  4. Use a Punnett square to work out potential genotypes of offspring. Include the different gamete combinations for each parent (like AaBB has two combinations, AB and aB).
  5. Write the phenotype ratios of potential offspring.

Examples of Dihybrid Cross

Mendel’s Experiment

He chose to cross a pea plant pair with two pairs of contrasting traits. These traits are seed color and seed shape. One of the plants is homozygous for the dominant traits of round seed shape (RR) and yellow seed color (YY). Thus, together the genotype is expressed as (RRYY). The other plant is homozygous for the recessive traits of wrinkled seed shape (rr) and green seed color (yy). Together the genotype is expressed as (rryy).

F1 Generation

Dihybrid Cross

A true-breeding pea plant with round seed shape and yellow seed color (RRYY) is cross-pollinated with another true-breeding plant with wrinkled seed shape and green seed color (rryy). The resulting F1 generation is all found to be heterozygous for round seed shape and yellow seed color (RrYy). All F1 offspring were found to be phenotypically identical, producing yellow seed color and round seed shape.

F2 Generation

Mendel continued with his experiment with the self-pollination of F1 progeny plants. To his surprise, the plants exhibited a 9:3:3:1 phenotypic ratio of seed shape and seed color. Nine out of the sixteen plants were found to exhibit round, yellow seeds. Three of them exhibited round green seeds. Another three gave wrinkled, yellow seeds, and the remaining one plant gave wrinkled green seeds. Thus, F2 generation exhibited four different phenotypes and nine different genotypes.  

Genotypic and Phenotypic Ratios

Genotypes determine the phenotype in an organism. Thus, a plant exhibits a specific phenotype based on whether its alleles are dominant or recessive. One dominant allele leads to a dominant phenotype being expressed. The other way for a recessive phenotype to appear is for a genotype to possess two recessive alleles. Both homozygous and heterozygous dominant genotypes are expressed as dominant.

In the experiment performed by Mendel, yellow (Y) and round (R) are dominant alleles, and green (y) and wrinkled (r) are recessive. The possible phenotypes and their genotypes are given below:

PhenotypesNumber of PlantsGenotypes
1) Yellow and Round 9YYRR, YYRr, YyRR, YyRr
2) Yellow and Wrinkled3YYrr, Yyrr
3) Green and Round3yyRR, yyRr
4) Green and Wrinkled1yyrr

How to Make a Dihybrid Cross Punnett Square

The above result is represented using a 4 x 4 Punnett square. All the four possible combinations of gametes for yellow seed color and round seed shape pea plant are placed from top to bottom of the first column. For green seed color and wrinkled seed shape, pea plant in the top row from left to right. The Punnett square is given below:


Purpose of Mendel’s Experiment

His experiment with dihybrid crosses shows all the possible offspring that can come from two given parents. Crosses involving multiple traits also help us understand the type of inheritance pattern that governs each trait. It also works to determine a specific phenotypic ratio and how many offspring have a specific trait. Dihybrid crosses gave rise to independent assortment law, which states that pairs of alleles are inherited independently if their loci are on separate chromosomes. In other words, the genes are unlinked.

What is a Dihybrid Test Cross

It is a cross that helps to explore the genotype of an organism based on the offspring ratio. When an organism’s genotype expressing a dominant trait is not known to be homozygous or heterozygous, we need to perform a test cross. A dihybrid test cross is done involving two pairs of contrasting characters.

In a test cross, an individual with an unknown genotype is crossed with a homozygous recessive individual. The unknown genotype can be obtained by analyzing the phenotypes in the offspring. The result of a dihybrid test cross-ratio is represented using a Punnett square. If the unknown genotype is heterozygous, a test cross with a homozygous recessive individual will result in a 1:1:1:1 ratio of the offspring’s phenotypes.


Here, one of the heterozygous parents for seed color and seed shape (RrYy) is crossed with a homozygous plant for both the traits (rryy). The test cross produces four possible genetic combinations RrYy, Rryy, rrYy, and rryy in a ratio of 1:1:1:1.

Thus, a pea plant’s genotype producing dominant round and yellow seeds can be determined when test crossed with a wrinkled green plant having recessive alleles for both the traits. 

Monohybrid Cross vs. Dihybrid Cross

The significant differences between a monohybrid and a dihybrid cross are listed below:

BasisMonohybrid CrossDihybrid Cross
1. Number of Traits StudiedA single pair of contrasting trait/character is studiedTwo pairs of contrasting traits/character are studied
2. Phenotypic ratio in F2 generation3:19:3:3:1
3. Genotypic ratio in F2 generation1:2:11:2:1:2:4:2:1:2:1  
4. ExampleCross between tall and dwarf stem pea plantCross between pea plants having yellow and round seed and green and wrinkled seed

Article was last reviewed on Friday, February 17, 2023

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