Inheritance of Two Genes (Dihybrid Cross)

• Mendel performed a cross involving two characters simultaneously, as seen in the monohybrid cross, where a single trait was considered while crossing.

1. A cross between two pure (homozygous) parents in which the inheritance pattern of two pairs of contrasting characters is considered simultaneously is called a dihybrid cross.
2. In dihybrid cross, two characters (colour and shape) are considered at a time.
3. Mendel considered the seed shape (round and wrinkled) and cotyledon colour (yellow & green) as the two characters.
4. In seed shape, round (R) is dominant over wrinkled (r); in cotyledon colour, yellow (Y) is dominant over green (y). Hence, the pure breeding round yellow parent is represented by the genotype RRYY and the pure breeding green wrinkled parent is represented by the genotype rryy.
5. During gamete formation, the paired genes of a character assort out independently of the other pair. During the F₁ × F₁ fertilisation, each zygote with an equal probability receives one of the four combinations from each parent.
6. The resultant gametes thus will be genetically different, and they are of the following four types:
   

   i)	Yellow round (YR)	9/16
   ii)	Yellow wrinkled (Yr)	3/16
   iii)	Green round (yR)	3/16
   iv)	Green wrinkled (yr)	1/16

    These four types of gametes of F₁ dihybrids unite randomly in the process of fertilisation and produce sixteen types of individuals in F₂ in the ratio of 9: 3: 3: 1 as shown in the figure.
7. Thus, in dihybrid cross, F₁ generation has:
   Genotype: Yy Rr
   Phenotype: Yellow-coloured and round seed.
8. When F₁ progeny was self-hybridized to give F₂ progeny, 
   a) 3/4^th of F₂ plants had yellow seeds and 1/4^th had green seeds.
   b) 3/4^th of F₂ plants had round-shaped seeds and 1/4^th had wrinkled seeds.
   Thus, The yellow and green colours are segregated in a 3: 1 ratio. Round and wrinkled seed shapes are also segregated in a 3: 1 ratio, like in a monohybrid cross.
9. Dihybrid ratio: The phenotypic ratio of different types of offspring (with different combinations) obtained in the F₂ generation of dihybrid cross is called the dihybrid ratio. Mendel’s 9: 3: 3: 1 dihybrid ratio is an ideal ratio based on probability, including segregation, independent assortment and random fertilisation.

In sexually reproducing organisms/plants, from garden peas to human beings, Mendel’s findings laid the foundation for understanding inheritance and revolutionised the field of biology. The dihybrid cross and its result led Mendel to propose a second set of generalisations that we called Mendel's Law of independent assortment.

The protein called starch branching enzyme (SBEI) is encoded by the wild-type allele of the gene (RR), which is dominant. When the seed matures, this enzyme SBEI catalyzes the formation of highly branched starch molecules. Normal gene (R) has become interrupted by inserting an extra piece of DNA (0.8 kb) into the gene, resulting in r allele. In the homozygous mutant form of the gene (rr) which is recessive, the activity of the enzyme SBEI is lost resulting in wrinkled peas. The wrinkled seed accumulates more sucrose and has high water content. Hence, the osmotic pressure inside the seed rises. As a result, the seed absorbs more water, and when it matures, it loses water as it dries. So it becomes wrinkled at maturation. When the seed has at least one copy of the normal dominant gene heterozygous, the dominant allele helps to synthesize starch, amylopectin an insoluble carbohydrate, with the osmotic balance which minimises the loss of water resulting in the smooth structured round seed.