Obtaining Parental Lines - Autosomal Traits

Mutant Allele is Completely Dominant

The Scenario:

A new phenotype occurs in a population.  This phenotype is due to a new mutation. We will call this new allele A₁. Previous work has demonstrated that the A₁ allele is an autosomal dominant gene.  As a Geneticist -  How do you obtain a Pure - breeding Parental Line?

Fixing a new dominant allele:

We have evidence from previous work that the A₁ allele is inherited as an autosomal dominant allele in relation to the wildtype allele.  We can also assume that we have a wild-type population that is homozygous for the recessive allele (genotype of A₂A₂).

 It is possible to obtain a homozygous line for the dominant phenotype through breeding analyses.  The exact breeding procedure(s) will depend on the fecundity and the mating systems of your organism.  In general, the more progeny the organism produces, the better.  Females who can produce several sequential broods or litters by different males are more desirable than females who produce only a single brood or who mate only once and then store sperm.

Defined Phenotypes- the heterozygote.

Getting the appropriate homozygous line for a dominant trait requires that the experimenter be very flexible. In general, individuals with the dominant phenotype will be heterozygous .  Perhaps, you have a large number of organisms who are known to be heterozygous since they came from a cross of a mutant organism to a wildtype organism.  You decide against inbreeding because you really want the gene in a heterogeneous background. 

First you intercross the known heterozygotes (A₁A₂ x A₁A₂). This should give you 1/4 of your progeny who are A₁A₁, 1/2 of the progeny who are A₁A₂ and 1/4 of the progeny who are A₂A₂.  You discard the A₂A₂ progeny (wildtype phenotype) since they do not have the allele that you want to make homozygous.  You are left with a pool of individuals with the dominant phenotype.  One-third of these individuals are A₁A₁ and two-thirds of these individuals are A₁A₂.

 How can you tell A₁A₁ from A₁A₂ individuals?

Test cross (inactive link): If the organism produces fairly large litters (8 or more progeny) and the males and females can remate (without storing sperm) you can test cross individuals to the homozygous recessive line (wildtype lines).  If any of the resultant progeny show the recessive phenotype, you can discard the dominant individual as a founder for your homozygous line.  If all of the progeny have the dominant phenotype, you have a pretty good idea that the tested individual was homozygous.  If the organism is long-lived, you might do several test crosses with the same individual (easier for males than for females).  Test crosses work well with zebra fish and mice. Once you have a group of individuals that are homozygous A₁A₁, you can let them interbreed to produce your parental line.

Individual crosses:

Drosophila females generally mate once and they store sperm.  Therefore it is impractical to do a test cross.  You can overcome this obstacle with pairwise crosses.  Individuals with the dominant phenotype from a heterozygote by heterozygote mating (that is, individuals have either the A₁A₁ or A₁A₂ genotype) are mated in pairs.  These matings will be of three types (as seen below).  Each cross will have a probability of occurring that depends upon the expected frequencies of the A₁A₁ (1/3) or A₁A₂ (2/3) genotypes.  On a practical note, you might set up 50 to 100 such crosses. This procedure works best for organisms such as Drosophila where each pair of flies might have 100 to 200 progeny.

For every nine matings that you set up, you will discard 4/9 of the families since the recessive phenotype showed up among the progeny (Mating 3).  Of the remaining 5/9 matings, only one of them, on average, is what you want.  However, since all of the progeny have the dominant phenotypes, you cannot tell them apart.

Progeny from all of the remaining matings are allowed to interbreed (10 or more pairs per mating) and their progeny are examined for the presence of the recessive phenotype.  In any mating where there are only dominant phenotypes, you have probably reached homozygosity.  Any line in which a wildtype phenotype occurs is discarded. Once your line is established, you might check a random sample of the progeny by a testcross (inactive link).

You may never get a homozygous line: If an organism has low fecundity (one progeny or two at a time) it may be impossible to ever know that it is homozygous.

Selfing:

Some organisms can donate both eggs and sperm (selfing).  The nematode C. elegans, for example, can reproduce by selfing.  In addition, a few males are produced each generation.  If you have a C. elegans with a dominant phenotype it has either an A₁A₁ or A₁A₂ genotype.  A single hermaphroditic nematode can be placed in a Petri dish and allowed to self.  Nematodes can produce many progeny.  If a wildtype recessive nematode shows up among the progeny, then the single hermaphrodite was A₁A₂ and you can discard that Petri dish with all the progeny.  If all of the progeny are mutant, then the single hermaphrodite was A₁A₁.  Progeny from these plates can be allowed to self or allowed to mate with the rare A₁A₁males.

Mendelian Genetics Index