12.4: Dihybrid Crosses

To determine whether traits are inherited together or separately, Gregor Mendel crossed pea plants that differed in two traits. These parental plants were homozygous for both traits but displayed different phenotypes. The first generation of offspring were all dihybrids, heterozygotes exhibiting the two dominant phenotypes. When self-fertilized, the dihybrids consistently produced progeny with a 9:3:3:1 ratio of four possible phenotype combinations. This ratio suggested that inheriting one trait did not affect the likelihood of inheriting the other, establishing Mendel’s law of independent assortment.

Mendel’s Dihybrid Crosses Demonstrate the Principle of Independent Assortment

Gregor Mendel’s monohybrid crosses, between pea plants that differed in a single trait, demonstrated that (1) organisms randomly inherit one of two copies of each gene from each parent (Mendel’s first law, segregation), and (2) the dominant allele can mask the recessive allele’s effects on phenotype (the principle of uniformity).

To determine whether two traits were inherited separately or together, Mendel also performed crosses with pea plants that differed in two traits, such as pea color and pea shape. For these dihybrid crosses, Mendel first mated plants that were true breeding (i.e., homozygous) for different traits of the same two characteristics. For example, he crossed plants that bred true for round, yellow peas (RRYY genotype) with those that bred true for wrinkled, green peas (rryy genotype). This parental (P₀) generation produced offspring (F₁ generation) that were all heterozygous with dominant phenotypes. These dihybrids had RrYy genotypes and round, yellow peas.

Mendel then induced self-pollination in the F₁ dihybrids. Of the sixteen possible parental allele combinations, nine produce offspring with both dominant traits, yellow and round peas. Six fertilization events confer one dominant trait, with three producing yellow (dominant), wrinkled peas, and three creating green, round (dominant) peas. The one remaining possibility results in green, wrinkled peas, the two recessive phenotypes.

The proportion of phenotypes that Mendel observed in F₂ plants was consistently similar to this 9:3:3:1 ratio, which is expected only if each fertilization event is equally probable. Thus, observing this phenotypic ratio suggests that inheriting one of these traits (e.g., yellow or green pea color) does not influence the likelihood of inheriting one of the others (e.g., round or wrinkled peas). This finding is the crux of Mendel’s second law, the principle (or law) of independent assortment.

Linkage and Recombination Influence Trait Co-inheritance

Genes on separate, non-homologous chromosomes are independently assorted into gametes during meiosis. However, genes close to one another on the same chromosome are more likely to be distributed into the same gametes; a phenomenon called linkage. Thus, inheriting one trait can be linked to the likelihood of inheriting another. Mendel never reported linkage, although not all of the traits he studied are determined by loci on different chromosomes.

The alleles determining pod color and pea shape are on chromosomes 5 and 7, respectively, and are thus unlinked. For most of the other traits, the lack of linkage can be accounted for by recombination, which can cause the inheritance patterns of genes on the same chromosome to mimic independent assortment. During prophase I of meiosis, chromosome pairs line up, cross over, and swap homologous genetic segments, a process known as recombination. The closer two loci are to each other on a chromosome, the more likely they are to be on the same recombined segment and thus inherited together. Likewise, loci that are far apart are more likely to be inherited separately due to more recombination events dividing them.

Returning to Mendel’s traits, pea and flower color are determined by two chromosome 1 loci that are far apart. Similarly, the locus for flower position is far from the other chromosome 4 loci, pod shape and plant height. Due to recombination, it is unsurprising that linkage never manifested in these crosses. Loci for pod shape and plant height, however, are close enough to each other on chromosome 4 that some linkage is likely. Mendel never published the results of this particular crossing, so it is possible that he simply never carried out these experiments, making him one cross short of discovering linkage.

In pea plants Gregor Mendel also performed dihybrid crosses, breeding experiments between organisms that vary by two traits, like seed shape and seed color.

For example, if one parent is homozygous dominant for round and yellow seeds and the other is homozygous recessive for wrinkled and green seeds, all plants of the F1 generation area dihybrids, meaning that they're heterozygous for two traits.

The F1 plants display the dominant phenotype for both traits and have round and yellow seeds.

When they self fertilize, their progeny, the F2 generation, includes plants with round, yellow seeds, round green seeds, wrinkled yellow seeds, and wrinkled green seeds, in a nine to three to three to one ratio.

Importantly, the relationship of round to wrinkled seeded plants and yellow to green seeded, is three to one for both, indicating that these traits segregated independently, which demonstrates the principle of independent assortment.