Some of Mendel’s crosses examined three pairs of contrasting characteristics. Such a cross is called a trihybrid cross. A trihybrid cross is a combination of three individual monohybrid crosses. For example, plant height (tall vs. short), seed shape (round vs. wrinkled), and seed color (yellow vs. green).

The F1 generation plants of a trihybrid cross are heterozygous for all three traits and produce eight gametes. Upon self-fertilization, these gametes have an equal chance to give rise to 64 different combinations of genotypes in the F2 generation. In cases like this, when there are more than two pairs of contrasting characteristics to be studied, a Punnett square is unwieldy and impractical. The forked line method can be used instead of a Punnett square to simplify predicting genotype and phenotype ratios.

While it is impossible to predict the actual number of individuals per genotype in the F2 generation, this method can predict the phenotypic ratio, 27:9:9:9:3:3:3:1. In a cross involving tall plants with round, yellow seed and dwarf plants with wrinkled, green seeds, one can expect to find 27 tall plants with round and yellow seeds, 9 short plants with yellow, round seeds, 9 tall plants with yellow, wrinkled seeds, 9 tall plants with green, round seeds, 3 short plants with yellow, wrinkled seeds, 3 short plants with green, round seeds, 3 tall plants with green, wrinkled seeds, and 1 short plant with green, wrinkled seeds.

There are rules to identify the gametes and genotypes of the offspring of the F1 and F2 generations, respectively. These rules apply to all the multi-hybrid crosses that obey the law of independent assortment and follow the dominant-recessive pattern. The number of gametes formed in F1 generation can be identified by using the 2^{n} formula, where n is the number of heterozygous gene pairs. For example, breeding between XxYy and XxYy heterozygotes has n of 2. Thus, the number of gametes formed by the F1 heterozygotes will be 2^{2}, which is four.

Similarly, breeding between XXYy and XXyY heterozygotes has n of 1 because X is not heterozygous. Hence, the number of gametes formed by the F1 heterozygotes will be 2^{1}, which is 2. Similarly, the genotype of the F2 generation can be identified using the 3^{n} formula.

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