1. Mendel’s experiments

1. 1. Mendel’s experiments • Mendel’s experiments were devised to explain the mechanisms of inheritance: “The object of the experiment was to observe these variations in the case of each pair of differentiating characters, and to deduce the law according to which they appear in successive generations”. • Mendel's studies constitute an outstanding example of good scientific technique. He chose research material well suited to the study of the problem at hand, designed his experiments carefully, collected large amounts of data, and used mathematical analysis to show that the results were consistent with his explanatory hypothesis. The predictions of the hypothesis were then tested in a new round of experimentation. • The techniques of analysis used by Mendel remained unchanged for most part of the XX century; model organisms changed with time, but the basic methodology remained always the same. The existence of genes was originally inferred (and is still inferred today) by observing precise mathematical ratios in the descendants of two genetically different parental individuals. • Only with the advent of fast DNA sequencing in the 1980’s, genes could be analyzed directly without performing experimental crosses.

2. 2. The reasons of a choice • Mendel studied the garden pea (Pisum sativum) for two main reasons • Peas were available in a wide array of varieties that could be easily identified and analyzed. • Peas can either self (self-pollinate: the male parts - pollen, contained in anthers –can fertilizethe female parts – ovules, contained in pistils) or be cross-pollinated (the anthers from one plant are removed before they have opened to shed their pollen, and pollen from the other plant is transferred to the receptive stigma with a paintbrush). Thus, the experimenter can choose to self or to cross the pea plants. • Other practical reasons for Mendel's choice of peas were: • they are inexpensive and easy to obtain, • take up little space, • have a short generation time, • produce many offspring. • Such considerations enter into the choice of organism for any piece of genetic research.

3. 3. Differences among plant peas The various forms of pea plants available for crossing showed differences in: • length and color of the stem; • size and form of the leaves; • position, color, size of the flowers; • length of the flower stalk; • color, form, and size of the pods; • form and size of the seeds; • color of the seed-coats and of the albumen [cotyledons]. Some of these characters do not permit of a sharp and certain separation, since the difference is of a "more or less" nature, which is often difficult to define. Such characters could not be utilized for the separate experiments; these could only be applied to characters which stand out clearly and definitely in the plants. Lastly, the result must show whether they, in their entirety, observe a regular behavior in their hybrid unions, and whether from these facts any conclusion can be reached regarding those characters which possess a subordinate significance in the type.

4. 4. Characters selected for experiments • Form of the seeds The depressions, if any, occur on the surface, or they are irregularly angular and deeply wrinkled • Color of the seed albumen (endosperm) The albumen is either pale yellow, or it possesses an intense green tint. This difference of color is seen in the seeds as their coats are transparent • Color of the seed-coat/ color of the flower This is either white, with which character white flowers are constantly correlated; or it is gray, gray-brown, leather-brown, with or without violet spotting, in which case the color of the standards is violet, that of the wings purple, and the stem in the axils of the leaves is of a reddish tint • Form of the ripe pods These are either simply inflated, not contracted in places; or they are deeply constricted between the seeds and more or less wrinkled (P. saccharatum) • Color of the unripe pods They are either light to dark green, or vividly yellow, in which coloring the stalks, leaf-veins, and calyx participate • Position of the flowers They are either axial, that is, distributed along the main stem; or they are terminal, that is, bunched at the top of the stem and arranged almost in a false umbel; in this case the upper part of the stem is more or less widened in section (P. umbellatum) • Length of the stem The length of the stem is very various in some forms; it is, however, a constant character for each, in so far that healthy plants, grown in the same soil, are only subject to unimportant variations in this character. In experiments with this character, in order to be able to discriminate with certainty, the long axis of 6 to 7 ft. was always crossed with the short one of 3/4 ft. to 1 and 1/2 ft

5. 5. Plants differing in one character • Mendel chose seven different characters to study. The word character in this regard means a specific property of an organism; geneticists use this term as a synonym for characteristic or trait. • For each of the characters that he chose, Mendel obtained lines of plants, which he grew for two years to make sure that they were pure. A pure line is a population that shows no variation in the particular character being studied; that is, all offspring produced by selfing or crossing within the population are identical for this character. These two characters can be directly scored in the crossed plants

6. 6. Dichotomous phenotypes • Each pair of Mendel's plant lines can be said to show a character difference, i.e. a contrasting difference between two lines of organisms (or between two organisms) in one particular character. • Contrasting phenotypes for a particular character are the starting point for any genetic analysis. The differing lines (or individuals) represent different forms that the character may take: they can be called character forms, character variants, or phenotypes. • The term phenotype (derived from Greek) literally means "the form that is shown"; it is the term used by geneticists today. • The description of characters is somewhat arbitrary. For example, we can state the color-character difference in at least three ways:

7. 7. Reciprocal crosses • In one of his early experiments, Mendel pollinated a purple-flowered plant with pollen from a white-flowered plant. Individuals from which an experiment is started are called the parental generation (P). • All the plants resulting from this cross had purple flowers. This progeny generation is called the first filial generation (F1 ).The subsequent generations produced by selfing are symbolized F2 , F3 , and so forth. • Mendel made reciprocal crosses. In most plants, any cross can be made in two ways, depending on which phenotype is used as male or female. For example, the following two crossesare reciprocal crosses:

8. 8. Monohybrid crosses • Next, Mendel selfed the F1 plants (the pollen of each flower fertilized its own stigma). He obtained 929 pea seeds from this selfing (the F2 individuals) and planted them. • Some of the resulting plants were white flowered; the white phenotype had reappeared. • Mendel then did something that, more than anything else, marks the birth of modern genetics: he counted the numbers of plants with each phenotype. • Mendel counted 705 purple-flowered plants and 224 white-flowered plants. He noted that the ratio of 705:224 is almost exactly a 3:1 ratio (in fact, it is 3.1:1).

9. 9. Dominance defined • Mendel repeated the experiment for the six other character differences. He found the same 3:1 ratio in the F2 generation for each pair. In all cases, one parental phenotype disappeared in the F1 and reappeared in one-fourth of the F2 . • Mendel used the terms dominant and recessive to describe this phenomenon. The purple phenotype is dominant to the white phenotype and the white phenotype is recessive to purple. Thus the operational definition of dominance is provided by the phenotype of an F1 established by intercrossing two pure lines. The parental phenotype that is expressed in such F1 individuals is by definition the dominant phenotype.

10. Chi-square analysis of Mendel’s single-plant observations Mendel reported the following results for the F2 progeny of single plants:

11. 10. A diagram of Mendel’s monohybrid experiments x P F1 self 6022 yellow peas 2001 green peas F2 519 sampled and selfed self 166 peas with yellow progeny only 353 peas with 3:1 yellow/green progeny green peas with green progeny only F3 All the F2 greens were evidently pure breeding, like the green parental line; but, of the F2 yellows, two-thirds were like the F1 yellows (producing yellow and green seeds in a 3:1 ratio) and one-third were like the pure-breeding yellow parent. Thus the study of the individual selfings revealed that underlying the 3:1 phenotypic ratio in the F2 generation was a more fundamental 1:2:1 ratio

12. 11. Mendel’s theory Mendel's explanation is a classic example of a creative model or hypothesis derived from observation and suitable for testing by further experimentation. He deduced the following explanation: 1. The existence of genes. There are hereditary determinants of a particulate nature. 2. Genes are in pairs. Alternative phenotypes of a character are determined by different forms of a single type of gene. In adult pea plants, each type of gene is present twice in each cell, constituting a gene pair. In different plants, the gene pair can be of the same alleles or of different alleles of that gene. 3. The principle of segregation. The members of the gene pairs segregate (separate) equally into the gametes, or eggs and sperm. 4. Gametic content. Consequently, each gamete carries only one member of each gene pair. 5. Random fertilization. The union of one gamete from each parent to form the first cell (zygote) of a new progeny individual is random; that is, gametes combine without regard to which member of a gene pair is carried.

13. 12. The 1:2:1 ratio These points can be illustrated diagrammatically for a general case by using A to represent the allele that determines the dominant phenotype and a to represent the gene for the recessive phenotype (as Mendel did). The use of A and a is similar to the way in which a mathematician uses symbols to represent abstract entities of various kinds. In this figure, these symbols are used to illustrate how the preceding five points explain the 1:2:1 ratio. The members of a gene pair are separated by a slash (/). This slash is used to show us that they are indeed a pair; the slash also serves as a symbolic chromosome to remind us that the gene pair is found at one location on a chromosome pair

14. 13. The backcross • Mendel's next job was to test his model. He did so in the seed-color crosses by taking an F1 plant that grew from a yellow seed and crossing it with a plant grown from a green seed. • This type of cross, of an individual of uncertain genotype with a fully recessive homozygote, is now called a testcross or a backcross. Because one of the parents contributes only recessive alleles, the gametes of the unknown individual can be deduced from progeny phenotypes • A 1:1 ratio of yellow to green seeds could be predicted in the next generation. If Y stand for the allele that determines the dominant phenotype (yellow seeds) and y stand for the allele that determines the recessive phenotype (green seeds), we can diagram Mendel's predictions, as in the figure beside. • In this experiment, Mendel obtained 58 yellow (Y /y ) and 52 green (y /y ), a very close approximation to the predicted 1:1 ratio and confirmation of the equal segregation of Y and y in the F1 individual.