12.5: Crossing Techniques Revealing Unknown Genotypes
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Classical Genetics
Not only did Mendel solve the mystery of inheritance as units (genes), he also invented several testing and analysis techniques still used today. Classical genetics is the science of solving biological questions using controlled matings of model organisms. It began with Mendel in 1865 but did not take off until Thomas Morgan began working with fruit flies in 1908. Later, starting with Watson and Crick’s structure of DNA in 1953, classical genetics was joined by molecular genetics , the science of solving biological questions using DNA, RNA, and proteins isolated from organisms. The genetics of DNA cloning began in 1970 with the discovery of restriction enzymes.
True Breeding Lines
Geneticists make use of true breeding lines just as Mendel did (Figure \(\PageIndex{6}\)a). These are in-bred populations of plants or animals in which all parents and their offspring (over many generations) have the same phenotypes with respect to a particular trait. True breeding lines are useful, because they are typically assumed to be homozygous for the alleles that affect the trait of interest. When two individuals that are homozygous for the same alleles are crossed, all of their offspring will all also be homozygous. The continuation of such crosses constitutes a true breeding line or strain. A large variety of different strains, each with a different, true breeding character, can be collected and maintained for genetic research.
Monohybrid Crosses
A monohybrid cross is one in which both parents are heterozygous (or a hybrid) for a single (mono) trait. The trait might be petal colour in pea plants (Figure \(\PageIndex{6}\)b). Recall from chapter 1 that the generations in a cross are named P (parental), F 1 (first filial), F 2 (second filial), and so on.
Punnett Squares
Given the genotypes of any two parents, we can predict all of the possible genotypes of the offspring. Furthermore, if we also know the dominance relationships for all of the alleles, we can predict the phenotypes of the offspring. A convenient method for calculating the expected genotypic and phenotypic ratios from a cross was invented by Reginald Punnett. A Punnett square is a matrix in which all of the possible gametes produced by one parent are listed along one axis, and the gametes from the other parent are listed along the other axis. Each possible combination of gametes is listed at the intersection of each row and column. The F 1 cross from Figure \(\PageIndex{6}\)b would be drawn as in Figure \(\PageIndex{7}\). Punnett squares can also be used to calculate the frequency of offspring. The frequency of each offspring is the frequency of the male gametes multiplied by the frequency of the female gamete.
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Figure \(\PageIndex{7}\): A Punnett square showing a monohybrid cro ss. (Original-Deholos (Fireworks)-CC:AN)
Test Crosses
Knowing the genotypes of an individual is usually an important part of a genetic experiment. However, genotypes cannot be observed directly; they must be inferred based on phenotypes. Because of dominance, it is often not possible to distinguish between a heterozygote and a homozgyote based on phenotype alone (e.g. see the purple-flowered F 2 plants in Figure \(\PageIndex{6}\)b). To determine the genotype of a specific individual, a test cross can be performed, in which the individual with an uncertain genotype is crossed with an individual that is homozygous recessive for all of the loci being tested.
For example, if you were given a pea plant with purple flowers it might be a homozygote ( AA ) or a heterozygote ( Aa ). You could cross this purple-flowered plant to a white-flowered plant as a tester , since you know the genotype of the tester is aa . Depending on the genotype of the purple-flowered parent (Figure \(\PageIndex{8}\)), you will observe different phenotypic ratios in the F 1 generation. If the purple-flowered parent was a homozgyote, all of the F 1 progeny will be purple. If the purple-flowered parent was a heterozygote, the F 1 progeny should segregate purple-flowered and white-flowered plants in a 1:1 ratio.
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Figure \(\PageIndex{8}\): Punnett Squares showing the two possible outcomes of a test cros s. (Original-Deholos (Fireworks)-CC:AN)