2.5 The Dihybrid Test Cross

While the cross of an F1 x F1 gives a ratio of 9:3:3:1, there is a better, easier cross to test for independent assortment: the dihybrid test cross. In a dihybrid test cross, independent assortment is seen as a ratio of 1:1:1:1, which is easier to score than the 9:3:3:1 ratio. This test cross will also be easier to use when testing for linkage.

Like in monohybrid crosses (Chapter 1), you can do test crosses with dihybrids to determine the genotype of an individual with dominant phenotypes, to see if they are heterozygous or homozygous dominant. This type of cross is set up in the same fashion; an individual with an unknown genotype in two loci is crossed to an individual that is homozygous recessive for both loci.

Two punnet squares showing different outcomes of a test cross depending on homozygous or heterozygous dihybrid organism
Figure 2.5.1 Punnett Square for a Test Cross. The tester in both cases is the male with the genotype r/r ; y/y. On the left, the unknown has a genotype of R/R; Y/Y. On the right, the unknown has the genotype R/R ; Y/y.

Take a look at the video, Two-Gene Test Cross Explained, by Nicole Lantz (2020) on YouTube, for some worked examples.

Punnett squares should be done ahead of the crosses, so you know what to expect for any of the possible outcomes. Using the example from the rest of this chapter, you cross a double homozygous recessive pea plant (r/r ; y/y. green and wrinkled) to an unknown individual that has two dominant phenotypes (R/_ ; Y/_. yellow and round). There are four possible genotypes the unknown individual could be: R/R ; Y/Y or R/R ; Y/y or R/r ; Y/Y or R/r; Y/y. The Punnett squares for the first two are shown in Figure 2.5.1. Notice on the left, you only get the dominant phenotype for both, so you know both genes in the unknown are homozygous dominant. On the right, you get only the dominant phenotype for round peas — but you get 50% yellow and 50% green peas, showing that the unknown is homozygous for round, but heterozygous for colour of the peas. Figure 2.5.2 is blank for you to fill in the other two gamete and genotype possibilities.

Blank punnet squares for learner to complete
Figure 2.5.2 Blank Punnett Squares to Fill in the Other Two Possibilities of the Test Cross.

Media Attributions


Canham, L. (2017). Figures: 7. Punnett square for a test cross; 8. Blank Punnett squares to fill in the other two possibilities of the test cross [digital images]. In Locke, J., Harrington, M., Canham, L. and Min Ku Kang (Eds.), Open Genetics Lectures, Fall 2017 (Chapter 17, p. 6-7). Dataverse/ BCcampus. http://solr.bccampus.ca:8001/bcc/file/7a7b00f9-fb56-4c49-81a9-cfa3ad80e6d8/1/OpenGeneticsLectures_Fall2017.pdf

Nicole Lantz. (2020, May 5). Two-gene test cross explained [Video file]. YouTube. https://youtu.be/GM0by2axiLM


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Open Genetics by Natasha Ramroop Singh, Thompson Rivers University is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, except where otherwise noted.

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