GENERAL GENETICS

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The practical outcome of

≅ 1.5 century of genetics

and ≅50 years of molecular biology

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Application of genetics -1 (lesson from the field)

A new species

cobs

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Application of genetics -2

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Application of genetics -3

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Application of genetics -4

rDNA stands for: derived from recombinant DNA technology

ALL STARTED WITH …

MENDELISM

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MENDELIAN METHOD

or

MENDELIAN GENETICS

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1842 attends the Olmutz Institute of Phylosophy.

Among the other topics (phylosophy, ethics) he selected

also Physics.

Johann Gregor Mendel (1822-1884)

1843 enters the monastery of Saint Thomas – Brünn - Royal Society for the improvement of agriculture

1851 enters the University of Wien, where he attended, in addition to 3 courses of Physics (as an assistant of Christian Doppler), also:

Chemistry, Math, Paleontology, Botanics and Phyisiology (multidisciplinaryapproach)

This will provide a solid background for his future work.

Scientific Method HypothesisExperimental workResultsAnalysis of the data (by means of mathematics)

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1853 back to Brünn, where he will face the scientific problem:

“What is inherited and how is inherited?”

Fundamental choice model organismPisum sativum (fam. Papilionatae)

1. It is easy to cultivate

2. Developing time relatively short (≅ 3 months)

3. The flower is suitable for manipulationsexual elements very well defined:

anther (male ) containing POLLEN

pistill (female)

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Stem length Flower position

Pod color Pod shapeSeed color

Seed shape

Flower color

Along stem

At tip

tall dwarf

Inflated Wrinckled

Choice of 7 characters that can be easily distinguished by visual inspection

(Was he lucky, or smart ?!)

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Hypothesis:

Inside each cell there are well defined elements (Elemente) (or factors) that control the characters (Merkmal).

These Elemente work together as a couple and the two Elemente are derived from bothparents. When the germ cell forms, the two factors are separated (segregation) and eitherone or the other is transferred to reproductive cells.

Experimental Approach :

Production of Plant Hybrids using 7 well-defined Merkmal (true breeds)

Instruments:

• Pincers, to remove stamen and pollen,

• Brush, to sprinkle stigma with pollen.

• Cotton hood, to prevent external fertilization.

Mendel used some terms derived from German language to define what we commonly know today as a gene:

Merkmal (characteristic, mark)

Anlage (plan, design or predisposition)

Elemente

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1854 starts teaching at the Realschule of Brünn (secondary school comparable to a Liceo Scientifico) Physics and Natural Science

1856 after two years of hard work on 34 varieties of seeds, he managed to obtain 22 true breeds and some preliminary results.

An individual that produces identical offspring when self-fertilizedor hybridized with an individual of like genotype

(in genetic terms: homozygous)Crossed Pollination

from 1856 until 1863 he carries out with true-breeding plants a set of experiments of seeding, cross fertilization and analysis of first generation (F1) and second generation (F2) plants with the aim of

“… observing how different merkmal are transmitted to the offspring, and deduce the rulesgoverning the appearance of these merkmal in the next generations of individuals”

In other words: “ how hybridization can produce new species?”

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5,474 smooth seeds and 1,850 rough seeds2,96 : 1

1) Crossing true breeds produced always the same trait;

2) F1 progeny appeares as one of the twoparental phenotypes (e.g. smooth seeds);

3) The character that was not present in F1 reappeared in the second generation (F2) withapprox. a 3:1 frequency (75% smooth seeds, 25% rough seeds);

4) The trait which is expressed in F1 progeny isdefined dominant, and the one which is NOT expressed is called recessive.

F1 plants have a genotype S/r, where S= dominant allele, determining the smooth character of the seed and r = recessive allele determining the rough character.

F1

F2

Results Conclusions

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Dominant allele Recessive allele

Homo-zygous (same zygotic constitution)?

Hetero-zygous (different zygotic constitution)?

Mendel used as nomenclature an uppercase letter (S big) for dominant allele and a lowercase letter (s small) for the recessiveallele

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Number of S alleles in the population?

Number of s alleles in the population?

KEY WORDS

Genotype: the genetic make-up of an individual (e.g. RR, Rr or rr) used with reference to a particular characteristic

Phenotype: the physical expression of an organism’s genes (e.g. green or yellow seeds)

Homozygous: possessing a pair of identical genes (e.g. RR and rrplants are homozygous)

Heterozygous: possessing a pair of different genes (e.g. Rr)

Allele: alternative forms of a gene ( or a DNA sequence) found at a given locus on a chromosome

Female: the circle with the cross at the bottom represents the mirror of Venus, the goddess of love

Male: the circle with the arrow point sticking outwardrepresents the shield and spear of Mars, the god of war.

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1866 he published the results and interpretations of his experiments in a scientific article titled:

“Versuche über Pflanzen-Hybriden” in the Proceedings of the BrünnNatural History Society.

He also sent 40 reprints of this work to distinguished scientists (e.g. Charles Darwin): he got a reply from only one, Karl Wilhelm von Nägeli, professor of Botany at the University of Munchen.

p g F2 (numeri) F2 (rapporto) Carattere F1 Dominanti Recessivi Totale Domin./Recess.Semi: lisci /rugosi Tutti lisci 5.474 1.850 7.324 2.96 : 1 Semi: giallo/verdi Tutti gialli 6.022 2.001 8.023 3.01 : 1 Involucri del seme:grigi/bianchi Fiori: porpora/bianchi

Tutti grigi Tutti porpora

705 224 929 3.15 : 1

Fiori: assiali/terminali Tutti assiali 651 207 858 3.14 : 1 Baccelli: pieni/irregolari Tutti pieni 882 299 1.181 2.94 : 1 Baccelli: verdi/gialli Tutti verdi 428 152 580 2.82 : 1 Stelo: lungo/corto Tutti lunghi 787 277 1.064 2.84 : 1 Totale 14.949 5.010 19.959 2.98 : 1

(7)(1)

(6)

(3)(2)

(4)(5)

RESULTS OBTAINED WITH HYBRIDIZATION EXPERIMENTS (7 CHARACTERS)

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WHY 3:1 RATIO ?

The genotypes of the offspring are obtained bycombining the gamete from each column with the gamete from each row.

The use of Punnet squaresto predict the outcomes of genetic experiments whenthe parental genotypesare known.

R. Punnet – Prof. of Genetics at Cambridge

(co-discovered genetic linkage)

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ga

Number of A alleles ?

Number of a alleles ?

GENETIC BASIS OF A PHENOTYPE

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Key terms:

GENOTYPE = the geneticmakeup of an individual organism

PHENOTYPE = the observableoutward appearance of anorganism, which is controlled bythe genotype and its interaction with the environment

Notes concerning the experimental designof Mendel’s experiments

Selection of “true breeds” & initial use ofmonohybrids (i.e. crosses between plantsdiffering for a single character)

Prevention of self- and spurious pollinationby removal of anthers (no pollen) and by placing“hoods” on flowers

The stigmata are “brushed” with pollen derivedfrom the desired plant

Apply interdisciplinary approach to evaluateresults

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Parental generation → crosses→ First filial generation (F1) of monohybrids

→ all offsprings display the same (dominant) character

→ self-fertilize monohybrids

→ second filial generation (F2)

→ both phenotypes appear, always in the same ratio ≅ 3

Mendel’s interpretation based on “cell theory”

→ both characters are present in the zygote

→ segregation occurs in the gametsReciprocal cross experiments indicate that segregation of alleles is the same in both male and female parents

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Results: 9 (IR): 3 (Ir): 3 (iR) : 1 (ir)

An expansion of the 3 : 1 ratio

I = seed color (yellow or green)

R = seed shape (smooth or rough)

Punnett square illustratingthe genoptypes and phenotypes of the F2 generation from Mendel’s dihybrid experiment

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DI-HYBRID EXPERIMENTS

Each gene behaves exactly as it does in a monohybrid cross

The product law can be used to predict the frequency with which two independent events will occur simultaneously

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INDEPENDENT ASSORTMENT

Remember cell theory ?

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a) Principle of SEGREGATIONIn the formation of pollen and ovules, the alleles of each pair“segregate” from each other into different gametes

b) Principle of PARENTAL EQUIVALENCEIn the formation of both male and female gametes, segregation of alleles is the same (exceptions: sex-linked genes, mitochondrial genes)

c) Principle of INDEPENDENT SEGREGATION or INDEPENDENT ASSORTMENTThe inheritance of alleles at one locus does not influence the inheritanceof alleles at another locus

Mendel’s Principles

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Segregation explained using cell theory

F1

n

2 n

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PRE-

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:M

ITO

SIS

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ES Haploid

Diploid

Meiosis ensures that when an individual is heterozygous at any locus, half of its gametes will carry one allele (A), and half will carry the other (a).

2 n

n

F1

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Meiosis not only preserves the genome size of sexuallyreproducing eukaryotes but also provides 3 mechanisms to diversify the genomes of the offspring.

Each chromatid contains a single molecule of DNA. So the problem of crossing over is really a problem of swapping portions of adjacent DNA molecules. It must be donewith great precision so that neither chromatid gains or loses any genes. In fact, crossing over has to be sufficiently precise that not a single nucleotide is lost or added at the crossover point if it occurs within a gene. Otherwise a frameshift would result and the resulting gene would produce a defective product or, more likely, no product at all.

In this photomicrograph (grasshopper chromosomes), a tetrad shows 5 chiasmata.

1. Crossing Over (Prophase I)Chiasmata represent points where earlier (and unseen) nonsister chromatids hadswapped sections. The process is called crossing over. It is reciprocal; the segmentsexchanged by each nonsister chromatid are identical (but may carry different alleles).

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When the chromosomes of the pair cross over, the chiasmata do not always occur at the same points along the lengths of the chromosomes; there are different sites at which chiasmata may occur: this gives endless different chromosome combinationsafter meiosis and NO two chromosomes will be identical in the gametes.

Diagram 3 illustrates this point: the same pair of chromosomes, one black and one white, have formed chiasmata at different sites, producing different chromosomes tothose in Diagram 2.

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2. Random AssortmentIn meiosis I, the orientation of paternal and maternal homologues at the metaphase plateis random. Therefore, although each cellproduced by meiosis contains only one of each homologue, the number of possiblecombinations of maternal and paternalhomologues is 2n, where n = the haploidnumber of chromosomes. In this diagram, the haploid number is 3, and 8 (23) different combinations are produced.

Random assortment of homologues in humans produces 223 (8,388,608)different combinations of chromosomes.Furthermore, because of crossing over, none of these chromosomes is "pure" maternal or paternal. The distribution of recombinant and non-recombinantsister chromatids into the daughter cells at anaphase II is also random.So it is safe to conclude that of all the billions of sperm produced by a man during his lifetime (and the ≅ 500 eggs that mature over the life of a woman), no two have exactly the same gene content.

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Ch.1

Ch.1Ch.2

Ch.2

Ch.3

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3. FertilizationBy reducing the number of chromosomes from 2n to n,the stage is set for the union of two genomes. If the parentsdiffer genetically, new combinations of genes can occur in their offspring.

Taking these three mechanisms together, it is safe toconclude that no two human beings have ever shared anidentical genome unless they had an identical sibling; that is a sibling produced from the same fertilized egg(monoovular twins).

The behavior of chromosomes during meiosis (2n → n) and fertilization (n + n → 2n) provides the structural basisfor Mendel's rules of inheritance.

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Can we PREDICT if a purple flowered plant isheterozygous or homozygous?

A TESTCROSS can beused to determinewhether an individualis heterozygous (Aa) or homozygous (AA)

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The tester organism ishomozygous recessive

(aa white-flowered plant)

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THE MOLECULAR BASIS OF DOMINANCE vs RECESSIVENESS

(This is related to Incomplete Dominance)

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a recessive allele

A dominant allele

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The effect of dominance

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Discovery of Mendel’s data

- Hugo De Vries (NL)

- W. Bateson (ENG)

- K.H. Correns (D)

- E. von T.-Seysenegg (A)

- A. Garrod (ENG)

- Giuseppe Cuboni (1903)

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“Inborn Errors of Metabolism” 1908

GENETICS OF CHARACTERS SHOWING ALTERNATIVE FORMS

[Discontinuous variation]

GENETICS OF QUANTITATIVE CHARACTERS (e.g. body size, human weight, height). The majority of measures fall in the middle of a bell-shaped frequency distribution with a range of observation tailing away on eitherside → continuous variation

[many gene loci and environmental factors]44

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Thomas Hunt Morgan

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Deviations from Mendel's Predicted Ratios

DISCOVERY OF SEX LINKAGE

Genes on the Same Chromosome, DO NOT

Assort Independently always

The term "Drosophila", means "dew-loving"

“melanogaster” means “black belly”

Drosophila melanogaster as MODEL ORGANISM:

- Very easy to isolate and cultivate

- Larvae feed not on the vegetable matter itself but on the yeasts and microorganisms present on the decayingbreeding substrate.

- Fast development time = 10 - 12 days

- Sexual dimorphism

- Only 4 pairs of chromosomes

- Larvae salivary glands provide polytenic chromosomes.

1. narcotize (ether) 3. observe with stereomicroscope2. collect46

SEX LINKAGE

First experimental evidence of characters linked to sex was produced by T.H. Morgan in 1910 who found a white eyed male Drosophila and made the following crosses:

Parental wild female x white male

F1 all wild offspring – red eyes (mated together)

F2 all females were wild : 1/2 wild males and 1/2 white males

White eye character is recessive, but is found ONLY in males

“the fly room”

> 13.000.000 fliesexamined

47female male

white recessive inonly one type of cross !

3470 R783 W

all ♂

Based on these results, Morgan postulated that the gene for white eyes was on the X chromosome. Morgan deduced that the X-chromosome

carried a number of discrete hereditary units, or factors.

Other characters (variations) were found to be X-linked.

50% RED

50% WHITE

X = “straightchromosome”

Y = “bentchromosome”

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Morgan adopted the term gene, which was introduced by the Danish botanist Wilhelm Johannsen in 1909.This was the first experimental proof that: i) genes are located on chromosomes; ii) genes are possibly arranged in a linear fashion on chromosomes.

Alfred SturtevantCalvin Bridges

The abstract ideas of gene become tangible;

genes are discrete physical units

This figure depicts an abbreviated GENETIC MAP (or LINKAGE MAP) of a chromosome in Drosophila

Using crossover data, A. H. Sturtevant and his coworkersmapped other Drosophila genes in linear arrays at particulargenetic loci.

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Morgan found that some alleles do not separate (segregate); instead they tend to “travel together”

B = wild type body (grey)

b = mutant body (black)

Vg= wild type (normal wings)

vg= mutant (vestigial wings)

Experiment:

Test-cross with F1 individuals

Expected ratio: 1 : 1 : 1 : 1

2300 Drosophila examined

Expected results

Obtained results

tester

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Expected results

Obtained results

nonparental combinations recombinant

An exchange event between homologous chromosomes, crossingover, results in the recombination of genes in the homologous chromosomes.

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The tester organism ishomozygous recessive

(bb vgvg)

Frequency of recombination = frequency of nonparental combinations(recombinant phenotypes) of the traits over the total number of phenotypes

The FR values allow to determine:

1. The linear order of loci along the chromosome

2. The relative distance between them

1 + 2 = LINKAGE MAP53

B = wild type body (grey)

b = mutant body (black)

Vg= wild type (normal wings)

vg= mutant (vestigial wings)

2300 Drosophila examined

= 17 cM

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- Maps of relative positions, (the orders ) of linked genes on a chromosome can beconstructed by noting the frequencies of crossing-over between genes. The closertwo genes are together, the less likely they will show crossing-over.

- Conversely, the greater the distance between two genes on a chromosome, the greater the chance that a cross-over will happen between them.

-The probability of crossing over between any two genes can be expressed as a distance or value (the % of crossing-overs that occurs between 2 points on the chromosome).

-One map unit, (m.u.) is the distance between linked genes in the space where1% of crossing-over occurs, or is the distance between genes for which one resultof meiosis out of 100 is recombinant.

1 map unit = 1% recombinant = 1cM (centi-Morgan)

In humans 1 cM ≅ 1.000.000 bp

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The genetic mapping of linked genes is an important research tool in geneticsbecause it enables a new gene to be assigned to a chromosome and often to a precise position relative to other genes within the same chromosome. Geneticmapping in the pre-genomic era was the first step in the identification and isolation of a new gene and the determination of its DNA sequence.

The position on the map (LINKAGE MAP) where a gene is located is called the gene locus. On Drosophila chromosome 1, for instance, the locus of the cross-veinless wings (cv) is 13.7. The locus of cut wings (ct) is 20.0, so the distance is 6.3 m.u. The relationshipcould be shown like this:

cv ct__|___________________________|__

6.3

If the recombination frequency between cv and ct is 6.3, and ct and vermillioneyes (v) is 13, the order on the chromosome could either be cv-ct-v, or ct-cv-v. We can determine which of these is correct by measuring the recombinationfrequency between cv and v. If cv and v are found to recombine with a frequencyof 19.3 %, then we deduce that ct is located between them.

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57[1 cM = the distance between two loci determined by 1 % frequency of recombination]

MATHS & GENETICS

A theoretical framework to studypopulation genetics (1908)

Godfrey Harold Hardy

Wilhelm Weinberg

1. How can “O” be the most common of the blood types if it is a recessive trait?

2. If Huntington's disease is a dominant trait, shouldn't three-fourths of the population have Huntington's while one-fourth have the normal phenotype?

3. Shouldn't recessive traits be gradually “swamped out' so they disappear from the population?

p + q = 1

(p + q)2 = 1

(p + q)2 = p2 + 2pq + q2

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W.E. Castle (1903) and S. Chetverikov

F1

4/4 = 100% purple

F2

3/4 purple

1/4white

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VocabularyEvolution – changes in population allele frequencies over time. The population isthe smallest unit which can evolve.

Population – any group of organisms coexisting at the same time and place thatare capable of interbreeding with one another.

Allele frequency – proportion of a particular allele among allother alleles in a population. They are represented by the lettersp and q (range 0 – 1).

Gene Pool – all of the alleles at all loci in the population.Natural Selection – differential survival and reproduction of individuals in a population due to trait differences.Genetic Drift – changes in the gene pool of a small population due to chance. Random changes due to sampling errors in propagation of alleles.Bottleneck Effect – population undergoes a drastic reduction in size as a result ofchance events. A cause of genetic drift.Founder Effect – a small group of individuals becomes separated from the largerpopulation. A cause of genetic drift.Gene flow – movement of genes between populations. Gain or loss of allelesfrom a population due to migration of fertile individuals, or from the transfer ofgametes.Allele fixation – when a gene has only one allele. When one allele of a gene becomes the only allele, while the alternatives are eliminated from the population. 60

The Hardy-Weinberg Theorem states that:

the allele frequencies of a gene in a population will remain constant, aslong as evolutionary forces are not acting. H-W therefore provides a baseline (a null expectation) for a population that is not evolving.

For a population to be in H-W equilibrium, the following conditions or assumptions must be met:1. The population is very large; there is no genetic drift2. Matings are random3. There is no mutation4. There is no migration5. There is no selection

If one of these conditions is broken, an evolutionary force is acting tochange allele frequencies, and the population may not be in H-W equilibrium.

Natural populations probably seldom meet all of these conditions; H-W provides a nice model to study EVOLUTION via deviations from H-W equilibrium.

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Frequency of Allele O

Hardy-Weinberg Equation

Basic Relations: A = dominant allele a = recessive allele

p + q = 1 applies to all populations with only two alleles at one locus

where p = frequency of A allele and q = frequency of a allelep2 + 2pq + q2 = 1 this equation (binomial square) provides the

genotype frequencies.

Where p2 = frequency of homozygous AA genotype2pq = frequency of heterozygous Aa genotypeq2 = frequency of homozygous aa genotype

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q2pqq

pqp2p

qpIf p equals the frequency of allele A and q is the frequency of allele a, union of gametes would occur as follows:

In the above table the genotypic frequency for AA is p2, the genotypic frequency for Aa is 2pq and the genotypic frequency for aa will be q2

These are the values that are predicted by the law.

The prediction is that the frequencies of the two alleles will remain the same from generation to generation. The following is a mathematical proof of the prediction. To determine the allelic frequency, they can be derived from the genotypic frequencies as shown above.

p = f(AA) + ½f(Aa) (substitute from the above table)p = p2 + ½(2pq)p = p (p + q) (p + q =1; therefore q =1 - p)p = p [p + (1 - p)] (subtract and multiply) p = p

Therefore, gene frequencies do not change in one generation ! There would be less type O blood in that next generation, but not less O alleles !

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Allele frequency = p (0.5); q (0.5)

Genotype frequency = 25% Homozygous dominant; 50% Heterozygous; 25% Homozygous recessive

Phenotype frequency = 75% purple25% white

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F2

Probability of being homozygous AA: the probability of receiving A from the motherparent is p and the probability of receiving A from the father parent is p.

Combined probability is p2.

Probability of being homozygous aa: the probability of receiving “a” from the motherparent is q and the probability of receiving “a” from the father parent is q.

Combined probability is q2.

Probability of being heterozygous Aa: Combined probability is 2pq.65

aa(q2)

Aa(pq)a (q)

Aa(pq)

AA (p2)A (p)

Males

a (q)A (p)

Females

Table 1: Punnett square for H–W equilibrium

Start from q: 9% =0.09 = rr = q2

(f)r = q = Square root of 0.09 = 0.3 p + q = 1 → p = 1 - 0.3 = 0.7

2pq = 2 (.7 x .3) = .42 = 42% of the population are heterozygotes Rr(carriers)

If 9% of a population is born with a severe form of “disease r” (rr), what percentage of the population will be heterozygous (Rr) ?

What percentage of the population will be homozygous (RR) ?

In a certain population of 1000 fruit flies, 640 have red eyes while the remainder have sepia eyes. The sepia eye trait is recessive to red eyes. How many individuals would you expect to be homozygous for red eyecolor?

Calculations:Start from q: q2 for this population is 360/1000 = 0.36

q = √0.36 = 0.6p = 1 - q = 1 - 0.6 = 0.4

The homozygous dominant frequency = p2 = (0.4)(0.4) = 0.16Therefore, you can expect 16% of 1000, or 160 individuals, to be homozygousdominant for red eye color.

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Gene pool frequencies are inherently stable.

That is to say, they do not change by themselves.

Despite the fact that evolution is a common occurrence in natural populations, allele frequencies will remain unalteredindefinitely unless evolutionary mechanisms such as mutation and natural selection cause them to change.

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Example: f(A/A) f(A/a) f(a/a)

I 0.3 0.0 0.7

II 0.2 0.2 0.6

III 0.1 0.4 0.5

Frequency p of allele A in the three populations is:

I p = f(A/A) + ½ f(A/a) = 0.3 + ½ (0) = 0.3

II p = 0.2 + ½(0.2) = 0.3

III p = 0.1 + ½(0.4) = 0.3

Conclusion: these three populations have different genotypiccomposition, but share the same allelic frequencies.

Before Hardy and Weinberg, it was thought that dominant alleles must, over time, inevitably swamp recessive alleles out of existence. This incorrect theory was called "genophagy" (literally "gene eating"). According to this wrong idea, dominant alleles always increase in frequency from generation to generation.

Hardy and Weinberg were able to demonstrate with their equation that dominant alleles can just as easily decrease in frequency.

… as G. H. Hardy stated in 1908, 'There is not the slightest foundation for the idea that a dominant trait should show a tendency to spread over a whole population, or that a recessive trait should die out.'

Gene frequencies can be high or low no matter how the allele is expressed, and can change, depending on the conditions that exist.

It is the changes in gene frequencies over time that result in evolution.

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Amish community

Chi-square is used to test whether or not some observed distributionaloutcome fits an expected pattern. Since it is unlikely that the observed genotypefrequencies will be exactly as predicted by the Hardy-Weinberg equation, it isimportant to look at the nature of the differences between the observed and expected values and to make a judgment as to the "goodness of fit" betweenthem. In the chi-square test, the expected value is subtracted from the observedvalue in each category, and this value is then squared. Each squared value isthen weighted by dividing it by the expected value for that category. The sum ofthese squared and weighted values, called chi square (denoted as χ2), isrepresented by the following equation:

χ2 = Σ (observed - expected)2

expected

In the chi-square test, two hypotheses are tested. The null hypothesis (Ho) states that there is no difference between the two observed and expectedvalues; they are statistically the same and any difference that may be detectedis due to chance. The alternative hypothesis (Ha) states that the two sets ofdata, the observed and expected values, are different; the difference isstatistically significant and must be due to some reason other than chance.

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