In his 1865 paper, Experiments on Plant Hybridization, Gregor Mendel proposed two basic concepts concerning the nature of heredity, equal segregation and independent assortment. These concepts have become known as Mendel's First and Second Laws.
The first, the law of equal segregation, holds that two members of a gene pair (now called alleles) segregate from each other into the gametes such that one half of the gametes carry one member of the gene pair and the other half of the gametes carry the other member.
The second, the law of independent assortment, states that during gamete formation the segregation of alleles of one gene is independent of the segregation of alleles of a second gene.
At the time of their introduction, the importance of these concepts of heredity proposed by Mendel was unrecognized. In the early 1900s, Thomas Hunt Morgan began studying inheritance in a species of fruit fly, Drosophila melanogaster. His work and that of others on Drosophila led to the wider acceptance of the chromosome theory of inheritance.
In fact, because of experiments similar to those of Morgan, we now know that Mendel's Second Law applies only to genes which are not located on the same chromosome or, at the least, are very far apart on the same chromosome (the genes are not linked). This concept of the genetic linkage of traits will be explored in this experiment.
Today, we will set up experimental crosses of D. melanogaster (similar to those of Morgan) that will allow us to study the inheritance of some visible mutations. The mutations used in this experiment effect genes coding for eye color, wing shape and bristle morphology.
Over the next several weeks, we will determine which of these phenotypes
(wild type or mutant) are dominant and which are recessive, and whether
the traits are autosomal or sex-linked. If the three traits are found to
be linked (or located on the same chromosome), we will determine
how far apart these traits are located from one another on the chromosome.
Life Cycle of Drosophila
|D. melanogaster is a holometabolous insect, i.e. it undergoes
complete metamorphosis during its life cycle. The life cycle consists of
four distinct stages,egg, larva, pupa and adult.
Approximately 2 days after emerging from its pupal case, an adult female begins laying eggs (up to 400-500 eggs over 10 days); eggs are fertilized by a single sperm, although females can store enough sperm from a single insemination for the major portion of her reproduction. After hatching, the fertilized egg develeops into an instar or larva (a white segmented worm-shaped burrower). This larva undergoes two molts during which time it sheds its cuticle, and thus during this period of development larva are known as first instar (prior to first molt), second instar (between first and second molts) or third instar (post second molt). After the second molt, the instar feed until ready to pupate. The third instar crawl from the medium (and usually attach to the wall of the culture vial), the cuticle hardens and a pupal case (puparium) is formed. Metamorphosis occurs within the puparium. During the pupal stage, adult structures begin to develop from previously dormant tissues known as the imaginal discs (or anlagen). When metamorphosis is complete, the adult emerges (ecloses) from the anterior end of the puparium. Early after eclosion, the flies are light in color, the wings are not expanded and the abdomen is elogated. Within a few hours the wings expand, the abdomen becomes more rounded and the color darkens. After maturity, the flies are fertle and may live for several weeks.
The rate of development is dependent on the growth temperature; for example at 20 oC the entire life cycle takes approximately 2 weeks for completion while at 25 oC the life cycle is completed in 10 days. A female can store and use sperm from a single insemination for the major portion of her reproduction. Thus, it is necessary to select virgin females for genetic crosses. Older males will mate with newly emerged females; therefore, all adult flies are removed from a culture 8-12 hours prior to collection of female flies used to set up crosses.
The genetics of sex determination in D. melanogaster are similar
to that of many other organisms including humans, with one major exception.
In D. melanogaster, there are four pairs of chromosomes, three autosomal
pairs and a pair of sex chromosomes. Like humans, males are the heterogametic
sex with an X and Y chromosome; females are homogametic having a pair of
X chromosomes. However unlike humans in which the presence of a Y chromosome
determines maleness, in D. melanogaster, the dosage of X chromosomes
determines sex. For example, both XY and XO flies are phenotypically male.
In contrast, both XX and XXy flies are phenotypically female.
There are a number of visible criteria used to determine the sex of adult D. melanogaster. In general, females are larger than male flies; the abdomen of females is often noticeably swollen with maturing eggs and males are more darkly pigmented on the posterior portion of the dorsal side of the abdomen. However, these differences are subject to age and variation and are thus less reliable for sex determination.
As in most insects, an examination of the genitalia is a reliable method of determining the sex of adult flies. However, determining sex by this method can be time-consuming and subject to error by those who are not experienced in insect morphology.
The fastest and most reliable criterion for differentiating sex of adult flies is the presence of sex combs on the first tarsal segment of the front legs of males. The sex combs are absent in female flies.
It is imperative that you be able to reliably differentiate between male and female flies on the basis of sex combs!
In future phenotyping and crosses a mistake in sexing one fly can ruin
the experiment! Have your teaching assistant check your work before
setting up any genetic crosses!
The mutations that will be used in this experiment effect genes coding for eye color, wing shape and bristle morphology. Thus, we will be examining alleles at three different loci. Today, we will set up crosses between virgin triple mutant females and wild type males. We will then follow genetic crosses through the F2 generation.
You must be able to reliably identify ALL the mutant phenotypes involved in the crosses.
Although the mutant females used today carry all three mutations, this
will not necessarily be the case in subsequent generations. Thus,
today will be your opportunity to learn to identify all of the mutations
using individuals that are certain to have them.
|Eye color is the most easily identified mutation.
The top fly has the white eye ("w") phenotype and the bottom has the red eye (wild type or "+") phenotype.
|Wing morphology is also fairly easy to identify.
The top fly has wild-type ("+") wings and the bottom has miniature ("m") wings.
Note: flies that have just eclosed (emerged) from their pupae may have wings that have not yet fully expanded. In that situation, the folded wings will look like small black blobs on the backs of the flies. Since it will be impossible to identify their phenotype, they should be excluded from use in crosses or analyses of phenotype frequencies.
|Bristle morphology is the most difficult trait to
identify. Flies with the forked bristle phenotype ("f", left fly;
compare with wild type "+" on right) may have many normal looking bristles
in addition to forked bristles. Thus it is important to examine the
bristles on each fly thoroughly when deciding on the bristle phenotype.
The presence of a SINGLE forked bristle indicates that the fly has that
The distribution of forked bristles on the flies is not random.
They occur most commonly at the posterior end of the dorsal side of the
thorax (the position indicated by the arrow).
There are several other mutant phenotypes available for linkage mapping in Drosophila. Some of these mutant phenotypes are shown below: