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Test Crosses

By: Ilona Miko, Ph.D. (Write Science Right) © 2008 Nature Education 
Citation: Miko, I. (2008) Test crosses. Nature Education 1(1):136
When you see a dominant trait, the underlying genetic make-up can still be ambiguous. See how researchers use test crosses to find out the genotype behind the phenotype.
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It is not always possible to determine what genes an organism is carrying by simply looking at its appearance. After all, gene expression is a complex process that is dependant on many environmental and hereditary factors. For example, Gregor Mendel's experiments with pea plants showed how dominant traits can mask recessive ones, thus causing him to muse how "rash it must be... to draw from the external resemblances of hybrids conclusions as to their internal nature" (Mendel, 1866).

Today, scientists use the word "phenotype" to refer to what Mendel termed "external resemblance" and the word "genotype" to refer to an organism's "internal nature." Thus, to restate Mendel's musing in modern terms, we cannot infer an organism's genotype by simply observing its phenotype. Indeed, Mendel showed that phenotypic traits can be hidden in one generation, yet reemerge in subsequent generations. This occurs because some alleles are dominant over others, which means that their phenotype will mask the phenotype associated with the recessive alleles.

Because of dominance, there is not a one-to-one correspondence between the alleles that an organism possesses (i.e., its genotype) and the organism's observed phenotype. Consider, for instance, the genes that code for eye and body color in the fruit fly Drosophila melanogaster. In these flies, the brown-eye allele (b) is recessive to the normal red-eye allele (B). Similarly, the ebony body color allele (e) is recessive to the normal (yellow-brown) body color allele (E). Because ebony has 100% penetrance, a fly that has dark black body color has the homozygous genotype ee. However, a fly that has a normal body color may have the homozygous genotype EE or the heterozygous genotype Ee.

Things get slightly more complex when considering two genes. For instance, a wild-type fly (with red eyes and a yellow body) has one of four possible genotypes: EEBB, EEBb, EeBB, and EeBb. There is no way to tell these genotypes apart visually, but there is a well-established experimental technique to determine the fly's genetic makeup. Specifically, to detect the underlying genotype of an organism with a dominant phenotype, one must do a type of breeding analysis called a test cross.

The test cross is another fundamental tool devised by Gregor Mendel. In its simplest form, a test cross is an experimental cross of an individual organism of dominant phenotype but unknown genotype and an organism with a homozygous recessive genotype (and phenotype). In order to understand how test crosses work, it helps to consider several examples, including those that involve just one gene of interest, as well as those that involve multiple genes.

Single-Gene Test Crosses

Recall that in the fruit fly Drosophila melanogaster, the ebony-body allele (e) is recessive to the normal yellow-body allele (E). Say you are given a male fly with a yellow body. How could you use a test cross to determine this fly's genotype?

In order to set up your test cross, you must first realize that the male fly has one of two possible genotypes: Ee or EE. Because the male exhibits the dominant body color phenotype, you must cross it with a female with the homozygous recessive phenotype and genotype. Thus, the male fly is crossed with an ebony-bodied female of genotype ee. Depending on the male fly's underlying genotype, this cross will yield one of two possible sets of outcomes, as depicted in Tables 1 and 2.

Table 1: Outcome if Male Fly Is Heterozygous (Ee)

    Female Gametes
    e e
Male Gametes E Ee Ee
e ee ee

Table 2: Outcome if Male Fly Is Homozygous (EE)

    Female Gametes
    e e
Male Gametes E Ee Ee
E Ee Ee

In the first outcome (Table 1), the male had the genotype Ee. By the principle of segregation, he made two types of gametes—one that contained E, and another that contained e—in equal frequencies. The female "tester" fly, on the other hand, had genotype ee, so she made only one type of gamete (which contained e). Thus, the progeny of this cross would be expected to be 50% ebony bodied (ee) and 50% yellow bodied (Ee), thus reflecting the type and frequency of their father's gametes.

In contrast, in the second outcome (Table 2), the male fly had the genotype EE, so he made only one type of gamete (E). Meanwhile, the female "tester" had genotype ee, so she also made only one variety of gamete (e). The progeny of this cross would thereby be expected to be 100% yellow heterozygotes (Ee), again reflecting the type and frequency of their father's gametes.

Two-Gene Test Crosses

Test crosses operate under the same principle no matter whether you are considering one gene or multiple genes; in all cases, you are crossing an individual of a dominant phenotype but unknown genotype to an individual that is homozygous recessive for all relevant genes. Because the "tester" individual makes one known type of gamete, the ratios of phenotypes among the progeny of the cross indicate the type and frequencies of gametes made by the individual with the unknown genotype. Once you know the gametes that this individual produces, you can "reconstruct" the individual's genotype.

Consider again the fruit fly Drosophila melanogaster, and recall that the ebony-body allele (e) is recessive to the normal yellow-body allele (E), while the brown-eye allele (b) is recessive to the normal red-eye allele (B). If you are given a male with a yellow body and red eyes, how can you determine its genotype?

In this example, there are now four possible genotypes that are associated with the dominant phenotype of yellow body/red eyes. These four genotypes can produce one, two, two, and four different gametes, respectively (Table 3). Moreover, in combination with the single gamete from the "tester" parent, these gametes will produce one, two, two, or four progeny phenotypes.

Table 3: Possible Male Gametes and Their Frequency

Case # Possible Genotype Frequency of EB Allele Frequency of Eb Allele Frequency of eB Allele Frequency of eb Allele
1 EEBB 1 0 0 0
2 EEBb 0.5 0.5 0 0
3 EeBB 0.5 0 0.5 0
4 EeBb 0.25 0.25 0.25 0.25

Now, say you carry out the test cross and obtain 400 progeny. You sort these progeny by phenotype and discover that you have 200 flies with a yellow body and red eyes, as well as 200 progeny with a yellow body and brown eyes. These progeny must have the genotypes described in Table 4.

Table 4: Offspring Phenotype and Genotype and Corresponding Parental Gametes

Phenotype Frequency Genotype Gamete from Tester Parent Gamete from Parent with Unknown Genotype
Yellow body,
red eyes
0.5 EeBb eb (1) EB (0.5)
Yellow body, brown eyes 0.5 Eebb eb (1) Eb (0.5)

You know that the homozygous recessive tester parent produces only one type of gamete (eb). Thus, the yellow-bodied, red-eyed progeny must be heterozygous at both loci (EeBb) due to the receipt of an EB allele from the unknown parent. Meanwhile, the yellow-bodied, brown-eyed progeny must be heterozygous at the body color locus but homozygous recessive at the eye color locus (Eebb). This could only happen if the progeny received an Eb gamete from the individual with the unknown genotype. Thus, you can deduce that the fly with the unknown genotype produced two types of gametes, EB and Eb, in equal frequencies. This means that you can reconstruct the fly's genotype as EEBb (case 2 in Table 3).

In sum, a test cross is a device that can be used to infer the Mendelian alleles present in parental gametes based on the observation of offspring phenotypes. Specifically, the ratio of phenotypes in a set of offspring reveals missing information about one of the parent's genotypes. Test crosses may also be used to determine whether two genes are linked, as well as to determine the underlying genotype if an allele's penetrance is less than 100%.

References and Recommended Reading

Mendel, G. Versuche über Plflanzen-hybriden. Verhandlungen des naturforschenden Ver-eines in Brünn, Bd. IV für das Jahr 1865, Abhand-lungen, 3-47 (1866) (Bateson translation)

Pierce, B. Genetics: A Conceptual Approach, 2nd ed. (New York, W. H. Freeman, 2006)

Sadava, D., et al. Life: The Science of Biology, 8th ed. (New York, W. H. Freeman/Sinaeur Associates, 2008)


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