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Prior history To appreciate Mendels work, one must keep in mind the prevailing theories of inheritance Preformationism: the idea that gamete contains an intact organism, was first proposed in the late 1600s. A preformed human infant or homunculus contained within a sperm (Male centric view of the world) Blending inheritance: essences of both sperm and egg mixed to form offspring intermediate between the parents. Strains of plants and animals generated by blending Mendel ignored development, focused solely on transmission of traits 1 Darwin and Heredity Darwin: Among individuals of any species, there are differences (variations) Evolution cannot occur unless there are differences among individuals If individuals are identical and remain so generation after generation, there can be no evolution. Variation is important Variation must be transmitted from parent to offspring Mechanism of inheritance is important for understanding evolution (1860s) Mechanism of inheritance not understood Certain traits suddenly appear in an individual These traits are then transmitted to their progeny Porcupine man- protuberances on body Not due to the environment- only the man and his offspring have these traits 2 Pangenesis Pangenesis: (1860s) Whole organism reproduces itself Gemmules determine characteristics (traits) of organism Germ cells contain gemmules of all sorts and these are transmitted to the next generation. Fertilization- gemmules unite and produce new cells of the types from which they are produced. 3 Cytology Cytology: 1600s Organisms are composed of cells 1830s The most distinct structure in a cell is the nucleus. ALL cells have a nucleus. Role of nucleus was controversial because it disappears during cell division. 1840s Cells are formed by the division of preexisting cells (Mitosis) 1850s Sperm and Ovum are cells. 1873 Mitosis described in detail- nuclear and chromosome dynamics described 1883 Fertilization in sea urchin showed that sperm and ovum fuse. This links parents to offspring Problem of ploidy:. Nuclei of parents and progeny are diploids. Are nuclei of germ cells haploid? 1885 Meiosis described- Reductional division of chromosomes keeps number of chromosomes constant. No similar phenomenon seen in any other cellular organelle. 4 Cytology’s contribution to Mendel Heredity is a consequence of genetic continuity Germ cells are the vehicle of transmission of traits from one generation to the next. Germ cells contain half the number of chromosomes found in body cells. Fertilization involves union of sperm and eggs. Fertilization involves union of nuclei. Nucleus is crucial. During division it resolves into long chromosomes that split lengthwise. Chromosomes do not lose their individuality. They are inherited intact. Diploid embryo descends from maternal/paternal fusion of haploid gametes/chromosomes 5 The origin of genetics: The study of genetics begins when Gregor Mendel, in 1865, addressed the question : "How are characters passed on from one generation to the next?” Mendel was the first to make a serious attempt of experimentally answering the question of heredity and not only were his answers correct, they were a complete and compelling proof. Mendel published in 1866 but little attention was paid to his work until 1900, when it was simultaneously rediscovered by three scientists, one in Holland, one in Austria, and one in Germany. There are often impressions that Mendel was removed from the scientific community, or that his papers were not well circulated. This was not true. Over 200 copies of Mendels papers have been discovered in different libraries. All three of Mendel's rediscovers had read Mendel's work prior to publishing their own work. 6 Gregor Mendel was born on either 20th or 22nd July, 1822 in Heizendorf (today Hynice in the Czech Republic). From 1851 to 1853, Gregor Mendel studied zoology, botany, chemistry, and physics at the University of Vienna. He studied botany under Prof. Unger where he learned genetic crosses He studied physics under Prof. Doppler where he learnt statistics Mendel returned to Brno and began his experiments with the hybrid cultivation of pea plants in 1856. After spending eight years carrying out experimental work in the monastery garden, he reported on the results of his observations at the meetings of the Association for Natural Research in Brno on the evenings of February 8th and March 8th, 1865 Why Peas? 7 Model organisms A model organism is a species that has been widely studied, usually because it is easy to maintain and breed in a lab and/or has particular experimental advantages and are used to obtain information about species that are more difficult to study directly. Remember: processes are conserved!!!!! Genetic model organisms Amenable to genetic analysis Breed in large numbers Short generation time (large-scale crosses over several generations.) Many different mutants available Detailed genetic maps Baker's yeast (Saccharomyces cerevisiae), the fruit fly (Drosophila melanogaster) and the nematode (Caenorhabditis elegans) Experimental model organisms Produce robust embryos that can be studied and manipulated with ease. Used in developmental biology and biochemistry Chicken, Zebrafish and Xenopus laevis Genomic model organisms Occupy a pivotal position in the evolutionary tree Special quality of their genome. An example is the puffer fish (Fugu rubripes) which has a similar gene repertoire to humans but a much smaller genome (400 million base pairs instead of 3000 million Modern genetics: Location in evolutionary tree Genome size Ease of cloning a gene Ability to insert DNA back into genome Ability to monitor development Xenopus, dog, tetrahymena 8 The Pea Mendel chose the common garden pea to study patterns of inheritance. This was a excellent choice as a model system for the following reasons: 1 Many varieties 2 It can self pollinate and cross pollinate 3 Cheap 4 Short generation time He identified over 20 traits and studied 7 (why 7?) Seed shape: round versus wrinkled Seed color: yellow versus green Flower color: red versus white Pod shape: inflated versus pinched Pod color: yellow versus green Flower position: axial versus terminal Stem length: long versus short 9 True breeding The first two years of Mendel's work were devoted to selecting lines that breed true (pure lines) for a particular character or trait. He identified over 20 traits that bred true and studied 7 Breeding True: smooth seed | Self cross | smooth seed ------------------wrinkled seed | Self cross | wrinkled seed He identified plants that produced only smooth seeds and plants that produced only wrinkled seeds. He identified plants that produced only purple flowers and plants that produced only white flowers. 10 Mendel’s first cross P purple X male anther white ----> All purple female stigma F1 purple purple X -----> purple:white 705 purple:224 white ------------Reciprocal cross P purple X male white ----> purple F1 purple x purple -------> Purple:white female Blending may not be correct Reciprocal cross also gave the same result Therefore preformationism also not correct 11 Mendel’s first cross P yellow X green ----> All yellow Mendel crossed pure breeding yellow pea plants to pure breeding green pea plants. All of the progeny were yellow pea plants. Next he selfed these yellow plants allowing the pollen to fall on its own stigma. F1 yellow X yellow=6022 yellow ----> yellow:green green=2001 12 Keys to success He did these experiments with all seven traits!!!! The ratios obtained were between 2.82:1 and 3.15:1 This cross involving only one character, seed color, is called a monohybrid cross. Keys to success: 1 Generated a pure breeding line before starting the cross 2 Analyzed individual character 3 Counted progeny 13 Conclusions Although others were doing crosses at the time, Mendel work was unique Results and Conclusions: 1 In F1 all progeny showed only one of the two possible traits for all crosses 2 Males and females not important 3 Character missing in F1, reappeared in F2 Traits did not blend in the offspring but were transmitted in a discrete fashion and remained unchanged. Reciprocal crosses produced the same results, this indicated that each parent makes an equal contribution to genetic makeup of the offspring. The sperm does not contain a homunculus. 14 Terms: Dominant and recessive: All F1 seeds were yellow but when selfed the F2 produced some green seeds. Mendel termed the trait that is expressed in the F1 as dominant The trait that is hidden but re-expressed in the F2 as recessive. The F1 plants must contain factors for green and yellow since both reappear in the F2. ------------------------------------------With these assumptions the simplest model is that the F1 contains two hereditary factors One for green and another for yellow We will use the Uppercase Y to represent the dominant yellow factor and the lower case y to represent the recessive green. 15 Genotype The parental yellow pea plants are a pure line- they only produce yellow pea plants when selfed. However the F1 yellow pea plants produce some green pea plants when selfed. The Yellow of the P generation is different from the yellow from the F1 generation If there are Two factors regulating this phenotype- we can represent these phenotypes as:P YY x yy (yellow) (green) F1 Yy (yellow) Yy (yellow) X Yy (yellow) F2 1YY: 1 Yellow: 2Yy: 2Yellow: 1yy 1Green It is necessary to make the distinction between the appearance of an organism and its genetic make-up. Phenotype refers to the appearance of an organism Genotype refers its genetic makeup 16 The parental yellow pea plants and F1 yellow pea plants have the same phenotype but a different genotype. The Principle of Segregation: If there are two factors for each trait in an individual, then without a mechanism to halve the number of factors in each generation, the factors would multiple with each generation and become unmanageable. Mendel therefore reasoned that during gamete formation the paired factors separate and each gamete receives only one of the two factors. Parent YY Gametes Y F1 yy Y y y Y y Sperm and egg then randomly combine to produce F2 progeny reconstituting two copies of each factor in the progeny Notice that while the parents have two factors, they produce gametes containing only a single factor and the progeny again have two factors He developed this idea without knowing about meiosis! 17 yy Green YY Yellow Grows into A plant Grows into A plant Generates Gametes Y Or Y Generates Gametes Gametes combine at Random y Or y Yy Yellow Yy Yellow Grows into A plant Generates Gametes Y Or y 18 Mendel's assumption of two factors and segregation makes a strong prediction concerning the genetic make-up of the of F2 yellow pea plants F1 Yy Y X y Y y Yy Y y Y y YY Yy F2 yy yY Mendel's prediction: Phenotype ratio: 3:1 (yellow:green) Genotype ratio: 1:2:1 1/4YY Yellow 1/2Yy yellow 3/4 1/4yy green 1/4 genotype Of the yellow F2 plants, 1/3 should be YY and 2/3 Yy 19 How would you test this prediction? What about the green plants? All should be yy Selfing Mendel selfed each of the green F2 plants Green yy F3 X X Green yy yy green 120 green F2 were selfed: all 120 plants gave rise ONLY to green progeny 20 Selfing Mendel selfed each of the yellow F2 plants There are two types of yellow YY OR Yy Yellow Yellow Yellow Yellow YY X YY Yy X Yy Gametes Y Y Gametes Y y Y y F3 YY yellow YY:Yy:yy 3 yellow:1green 1/3 of F2 yellow plants were YY 2/3 of F2 yellow plants were Yy Of the yellow F2 plants, 1/3 should be YY and 2/3 Yy That is what Mendel observed! 519 yellow F2 were selfed: 166 gave all yellow and 363 gave yellow/green F2 Phenotype ratio: 3:1 yellow:green F2 Genotype ratio: 1:2:1 1/4YY Yellow ¾ 1/2Yy yellow 1/4yy green ¼ genotype Is there another way to test this prediction? 21 Test cross If instead of selfing the F2 plants, they are crossed to pure breeding green plants, what are the expected outcomes: F2 YY x yy Yy x yy yy x yy ------>all yellow (Yy) ------>1:1 yellow (Yy):green (yy) ------>all green (yy) 22 Genotype Probability F1 Yy X y Y Y F 2 Yy y Y y Y Y Y Y y y y Y y y Lets cross a Yy with a Yy pea plant What is the probability of obtaining a homozygous YY plant Chance of a Y sperm uniting with a Y egg Chance of sperm with Y allele 1/2 Chance of egg with Y allele Chance of Y and Y uniting -------------- 1/2 ½x½=¼ What is the probability of obtaining a heterozygous Yy plant Chance of sperm with Y allele and egg with y allele =(1/2x1/2)=1/4 OR Chance of sperm with y allele and egg with Y allele= (1/2x1/2)=1/4 Chance of Yy = 1/4+1/4= 1/2 23 More terms: Mendel's factors are now known as genes Alternative forms of a gene that determine A traits are known as---_alleles__ Individuals with two identical alleles are said to be -----_homozygous_ Individuals with two different forms of alleles are said to be— _heterozygous__ 24 The dihybrid cross and the principle of independent assortment: In the second set of experiments Mendel investigated the pattern of inheritance for two sets of characters simultaneously. A cross involving two sets of characters is called a dihybrid cross. Pea shape: smooth, wrinkled (Smooth is dominant to wrinkled) Cotyledons color: yellow, green (Yellow is dominant to green) P F1 Smooth yellow x wrinkled green smooth yellow selfed F2 315 smooth yellow 101 wrinkled yellow 108 smooth green 32 wrinkled green 9 3 3 1 25 9:3:3:1 ----> (3:1) (3:1) F2 315 smooth yellow 101 wrinkled yellow 108 smooth green 32 wrinkled green If we examine seed shape only (smooth, wrinkled) and ignore cotyledon color (yellow, green), in the F2, we expect to find: 3/4 smooth and 1/4 wrinkled: Take all the smooth peas from the four classes and add them up # Smooth = 315 + 108 = 423 # wrinkled = 101 + 32 = 133 423:133 is close to the 3:1 expected Now, if we only examine cotyledon color and ignore seed shape we expect 3/4 Yellow to 1/4 green. # Yellow = 315+101 # green = 108+32 416:140 is also close to the 3:1 expected ratio The wrinkled and green phenotypes INDIVIDUALLY behave as a standard recessives in a monohybrid cross. 26 The 9:3:3:1 ratio. The 9:3:3:1 ratio appears a lot more complex than the 3:1 ratios of the monohybrid cross. Correns insight was to realize the 9:3:3:1 ratio is nothing more than two 3:1 ratios combined at random. (3:1) x (3:1) That is if one examined the traits individually they formed a 3:1 ratio. To determine the mode of inheritance of the two genes in this dihybrid cross Mendel examined each of the traits separately: 27 Each trait behaves as a standard recessive found in a monohybrid cross. They do not affect one another Genes segregate independently!!!! GeneS in a gamete does not affect the segregation of geneY in that gamete Parent Gamete ss yy SS YY sy SY Ss Yy F1 Self cross Ss Yy x Ss Yy 28 In a heterozygous individual (Self cross) F1 self cross SsYy x SsYy The two genes combine in two ways during gamete formation This occurs at random giving rise to four classes of gametes SY Sy S Y s y or S y s Y sy Gamete sY SY sy Sy sY 25% 25% 25% 25% 29 Independent assortment If independent assortment is occurring, four different kinds of gametes will be produced in equal frequencies. The only rule is that S and s segregate to separate gametes and Y and y segregate to separate gametes (that is one does not get an Ss gamete or a Yy gamete) In a group of individuals SsYy males and SsYy females can produce four types of gametes in equal frequencies: SY, Sy, sY, sy These male and female gametes randomly fertilize each other to restore diploidy 30 Independent assortment of gene pairs YYSS x yyss YySs x YySs 9Y-S-:3Y-ss:3yyS-:1yyss Different gene pairs assort independently during gamete formation. The presence of a Y in a gamete does not influence the probability of a S or s being in that gamete. 31 Punnet diagram of a dihybrid cross male Female 1/4 SY 1/4 Sy 1/4 sY 1/4 sy 1/4 SY 1/4 Sy 1/4 sY 1/4 sy 1/16 SY/SY Smooth yellow 1/16 SY/Sy Smooth yellow 1/16 SY/sY Smooth yellow 1/16 SY/sy Smooth yellow 1/16 Sy/sY Smooth yellow 1/16 Sy/sy Smooth green 1/16 Sy/SY Smooth yellow 1/16 sY/SY Smooth yellow 1/16 sy/SY Smooth yellow 1/16 Sy/Sy Smooth green 1/16 sY/Sy smooth yellow 1/16 sy/Sy Smooth green 1/16 sY/sY wrinkled yellow 1/16 sy/sY wrinkled yellow 1/16 sy/sY wrinkled yellow 1/16 sy/sy wrinkled green 9 smooth yellow: 3 smooth green: 3 wrinkled yellow: 1 wrinkled green Therefore the 9:3:3:1 ratio is a natural outcome of applying Mendel's two laws 32 Punnet diagram of a dihybrid cross 1/4SY 1/4 SY 1/4 Sy 1/4 sY 1/4 sy 1/16 SY/SY Smooth yellow 1/16 Sy/SY Smooth yellow 1/16 sY/SY Smooth yellow 1/16 sy/SY Smooth yellow 1/4Sy 1/16 SY/Sy Smooth yellow 1/16 Sy/Sy Smooth green 1/16 sY/Sy smooth yellow 1/16 sy/Sy Smooth green 1/4sY 1/16 SY/sY Smooth yellow 1/16 Sy/sY Smooth yellow 1/16 sY/sY wrinkled yellow 1/16 sy/sY wrinkled yellow 1/4sy 1/16 SY/sy Smooth yellow 1/16 Sy/sy Smooth green 1/16 sy/sY wrinkled yellow 1/16 sy/sy wrinkled green 33 Significance of Ratios What is the biological significance of the 9:3:3:1 ratio? This ratio is only produced if TWO DIFFERENT GENE PAIRS assort independently of each other during gamete formation. The presence of one gene in a gamete does not influence the probability of another gene being found in that gamete Principle of segregation: for one gene, each individual has two copies. These two copies segregate from one another during gamete formation. Independent assortment: Segregation of one gene pair is independent of the segregation of any other pair of gene. 34 xxxxxx 35 Mendels laws Mendel inferred these gamete ratios by selfing SsYy individuals and looking at phenotypes of progeny. He could also have inferred these gamete ratios by crossing SsYy individuals to ssyy individuals. Crossing to the homozygous recessive individuals is known as a test cross 36 A test cross is easier that a self cross for the F2 SsYy x Gamete: 1/4 SY 1/4 Sy 1/4 sY 1/4 sy Test cross ssyy 1 sy What are the expected genotypic and phenotypic ratios of the progeny produced from this cross? 1 sy 1/4 SY 1/4 sy 1/4 SY/sy Smooth Yellow 1/4 sy/sy Wrinkled Green 1/4 sY 1/4 sY/sy Wrinkled Yellow 1/4 Sy 1/4 Sy/sy Smooth green What are the expected genotypic and phenotypic ratios of the progeny produced from this cross? To answer this, first describe the gamete classes and their frequencies produced from the SsYy and ssyy individuals. Use these to construct a Punnett square or branched diagram 37 38 Rules of Probability Independent events: The probability of two events occurring together What is the probability that both X and Y will occur together? Answer: First determine the probability of each event Then multiply them together. Mutually exclusive events: The probability of one or another event occurring. What is the probability of X or Y occurring? Answer: First determine the probability of each event Then add them together. Probability and Mendel’s Results Cross Yy xYy pea plants. What is the chance of Y sperm uniting with a Y egg ½ chance of sperm with Y allele ½ chance of egg with Y allele Therefore chance of Y and Y uniting = ½ x ½ = 1/4 ----------------------------------------What is the chance of Yy offspring ½ chance of sperm with y allele and egg with Y allele or ½ chance of sperm with Y allele and egg with y allele Therefore chance of Yy = (½ x ½) + (½ x ½) = 2/4, or 1/2 Yellow is dominant to green Smooth is dominant to wrinkled Calculating Phenotype probability A heterozygous smooth yellow pea is crossed to a heterozygous smooth yellow pea. (SsYy x SsYy) What is the expected ratio for a smooth green pea? 1/4SY 1/4 SY 1/4 Sy 1/4 sY 1/4 sy 1/16 SY/SY Smooth yellow 1/16 Sy/SY Smooth yellow 1/16 sY/SY Smooth yellow 1/16 sy/SY Smooth yellow 3/4 Yellow 1/4Sy 1/16 SY/Sy Smooth yellow 1/16 Sy/Sy Smooth green 1/16 sY/Sy smooth yellow 1/16 sy/Sy Smooth green 1/4sY 1/16 SY/sY Smooth yellow 1/16 Sy/sY Smooth yellow 1/16 sY/sY wrinkled yellow 1/16 sy/sY wrinkled yellow 1/4sy 1/16 SY/sy Smooth yellow 1/16 Sy/sy Smooth green 1/16 sy/sY wrinkled yellow 1/16 sy/sy wrinkled green 3/4x3/4= 9/16 3/4 Smooth 1/4 Green 3/4 Yellow 1/4 Wrinkled 3/4x1/4= 3/16 1/4x3/4= 3/16 1/4 Green 1/4x1/4= 1/16 41 Probability Yellow is dominant to green Smooth is dominant to wrinkled Heterozygous yellow Heterozygous smooth X Heterozygous yellow Homozygous wrinkled Probability of yellow wrinkled? 1/2 smooth 3/4 yellow 1/2 wrinkled 1/4 green Heterozygous yellow Homozygous smooth X Heterozygous yellow Homozygous wrinkled Probability of yellow wrinkled? 1 smooth 3/4 yellow 0 wrinkled 42 1/4 green Calculation Red is dominant to white eye Flat wing is dominant to delta wing A heterozygous Red eyed flat winged fly is crossed to a heterozygous Red eyed flat winged fly. What is the expected ratio for a Red eyed flat winged fly? 43 xxxxxx 44 More Phenotype probabilities Yellow is dominant to green Smooth is dominant to wrinkled Tall is dominant to short Heterozygous yellow Heterozygous smooth Heterozygous tall X Heterozygous yellow Homozygous wrinkled Heterozygous tall Probability of green wrinkled short? 3/4 tall 1/2 smooth 1/4 green 1/2 wrinkled 1/4 short 3/4 tall 1/4 short 3/4 tall 1/2 smooth 3/4 yellow 1/4 short 3/4 tall 1/2 wrinkled 45 1/4 short Genotype probability P YYRRTTSS yyrrttss F1 YyRrTtSs YyRrTtSs What is the probability of obtaining the genotype YyRrTtss in the F2? -Four loci Each loci is heterozygous i.e. 2 types of gametes - 24 = 16 possible gamete combinations for each parent Thus, a 16 16 Punnet Square giving rise to 256 genotypes F1 gametes YyRrTtSs X YyRrTtSs RYTS RYTs RYtS RYts RYTS RYTs RYtS RYts RyTS RyTs RytS Ryts RyTS RyTs RytS Ryts rYTS rYTs rYts rYTS rYTS rYTs rYts rYTS ryTs rYtS ryTs rYtS rYts ryts rYts What is the ratio of different genotypes in the F2? ryts Branched diagram for genotypes F1 Alternatively – Loci Assort Independently Look at each gene locus separately. X Yy y Y Y F 2 P Yy y Y y Y Y Y Y y y y Y y y YYRRTTSS yyrrttss F1 YyRrTtSs YyRrTtSs What is the probability of obtaining the genotype YyRrTtss in the F2? Yy X Yy Rr Rr 1YY:2Yy:1yy 1RR:2Rr:1rr 2/4 Yy 2/4 Rr Tt Tt Ss Ss 1TT:2Tt:1tt 1SS:2Ss:1ss 2/4 Tt 1/4 ss Probability of obtaining individual with Rr and Yy and Tt and ss. 2/4 2/4 2/4 1/4 = 8/256 (or 1/32) Genotype probability P RRYYTTSS rryyttss F1 RrYyTtSs RrYyTtSs What is the probability of obtaining a completely homozygous Dominant or completely homozygous recessive genotype in F2? Genotype could be RRYYTTSS or rryyttss Rr Rr Yy Yy Tt Tt Ss Ss 1RR:2Rr:1rr 1YY:2Yy:1yy 1TT:2Tt:1tt 1SS:2Ss:1ss 1/4 RR 1/4 YY 1/4 TT 1/4 SS 1/4 rr 1/4 yy 1/4 tt 1/4 ss (RR x YY x SS x TT) + (rr x yy x ss x tt) (1/4 1/4 1/4 1/4) + (1/4 1/4 1/4 1/4) = 2/256 Specific breeds of dogs are also associated with specific diseases Dobermans: narcolepsy Scotties: Haemophilia Terriers: copper metabolism (menke disease) Labrador: hip dysplasia Beagle: seizure risk The ratio from a doberman cross suggests that narcolepsy is most likely mediated by a single gene! (3:1 ratio) Why are Mutts healthier than true breeds? 49 Hybrid Vigor Hybrid vigor: The first cross between two purebred lines is often healthier than either parent Breed1 a-B-C-d-E a-B-C-d-E F1 Breed2 A-b-c-d-E A-b-c-d-E a-B-C-d-E A-b-c-d-E If GeneA causes narcolepsy and geneC causes haemophilia F1 will be normal. 50 Corn Commercial corn is a F1 hybrid because of hybrid vigor Two inbred lines are mated to generate a F1 that is sold AAbb F1 x aaBB AaBb hybrid vigor 1930’s increase in corn output because of these hybrid varieties Line1 -disease resistant- rust and mold resistance but low yield Line2- large crop but susceptible to disease Hybrid- disease resistant and large yield----Sold to farmer The F1 hybrids do not breed true! The farmer cannot plant the seeds he gets from the F1 hybrid in the next season. They will not be disease resistant with high yields! Gametes AB Ab aB ab The seeds will generate plants with A-Bhigh yield, disease resistant A-bb high yield, susceptible to disease aaBlow yield, disease resistant aabb low yield, susceptible to disease 51