UDBB 2233 Title : Exp 1-Chi Square Analysis Exp 2- Mendelian Genetics of Drosophila and Maize Name : Mohd.

Zulfadhli B. Azizan Lau Wai Yip Siow Zi Kang Cheah Jun Zhe Ang Yee Von Chan Jo Yee Group Lecturer : : Practical 1 Mr. Kam Yew Chee 1004656 1004923 1002558 0905802 1007253 0908033

Experiment 1 Title: Chi-Square Analysis Introduction: The extent an observed data set fits or differs from the predicted or expected occurrences can be evaluated by testing the goodness of fit of the data. When the data do not fit exactly, we will want to find out how much deviation can be allowed before we reject a null hypothesis. One of the simplest statistical tests to assess the goodness of fit of the null hypothesis is chi-square analysis (x2), which tests the difference between observed (O) and expected (E) values. This x2 value is then used to estimate how frequently the observed deviation can be expected to occur strictly as a result of chance, allowing us to determine whether progeny phenotype ratios fit our assumptions about their genotypes. The formula for calculating the chi-square value is: x2= where O = the observed number of individuals in a particular phenotype, E = the expected number in that phenotype, and = the summation of all possible values of (O-E)2/E for the various phenotypic categories The degree of freedom (df), which is equal to n 1, where n is the number of categories (the number of phenotypes considered), will then be determined. Degrees of freedom must be taken into account because the greater the number of categories, the more deviation is expected by chance alone. The calculated _2 value can now be interpreted at a corresponding probability value (p), which can be obtained from _2 table (Appendix) for (n1) degree of freedom. The p value can be thought as a percentage. For example, a p value of 0.26 indicates that were the same experiment repeated many times, 26% of the trials would be expected to exhibit chance deviation as great as or greater than that seen in the initial trial. Conversely, 74% of the repeats would show less deviation than initially observed as a result of chance. The interpretation of the p value reveals that a hypothesis is never proved or disproved absolutely. Instead, a relative standard must be set to serve as the basis for either rejecting or failing to reject the hypothesis. This standard is often a probability value of 0.05. If the calculated _2 value is smaller than the critical value given in the table at (n1) df, then we accept the null hypothesis and conclude that the difference between observed and expected results is not significant, and vice versa.

Objectives: To 1.Calculate x2 to determine whether a data set approximates a theoretically expected ratio. 2.Interpret a calculated x2 value at an appropriate number of degrees of freedom.

Materials: Non-biological materials: container of equal quantities of colored and white beads, petri dishes, two equal-value coins and a calculator. Procedure : Activity 1: A beaker contained 150 colored and 150 white beads mixed were given. 1. One petri dish-full random sample of beads was removed from the beaker. 2. The beads of the different colors were segregated and counted. 3. The data was recorded in table 1.1. The expected numbers were calculated based on the size of the sample and the known ratio of colored to white beads in the entire population. The table 1.1 was completed and x2 was calculated. Activity 2: 1. Two equal-value coins were together tossed 30 times. 2. The data were recorded in table 1.2 and the expected numbers were calculated. The probability of two or more independent events occurring simultaneously is the product of their individual probabilities were noted. 3. The table 1.2 were completed and x2 were calculated. Activity 3: A beaker contained 100 beads of two different colors in the ratio of 3:1 . The beads ere thoroughly mixed. 1. One tablespoon-full random sample of beads were removed from the beaker. 2. The beads of different colors were segregated and counted. 3. The data in were recorded in table 1.3 and then the expected numbers based on the size of the sample were calculated and the known ratio of colored to white beads in the entire population. The table 1.3 were completed and x2 were calculated. Activity 4: The following are the approximate frequencies of the various ABO blood groups in a hypothetical population: 41% A, 9% B, 3% AB, and 47% O. The number of students in the class who have blood group A, B, AB and O were recorded in table 1.4 . The x2 were calculated to 2 decimal places and completed in table 1.4 .

Activity 5: A container contained several thousand beads of 4 different colors in the ratio 9:3:3:1 were given. The beads were thoroughly mixed. 1. One spatula-full random sample of beads were removed from the container. 2. The beads of different colors were segregated and counted. 3. The data were recorded in table 1.5 and then x2 were calculated. Experiment 2 Title: Mendelian Genetics of Drosophila and Maize (A) Drosophila Genetics Introduction: The vinegar or fruit fly, Drosophila melanogaster, has been widely used for demonstrating the classical Mendelian laws. This small fly undergoes a complete metamorphosis from egg, larva, pupa, through imago in 10 to 14 days at 25oC. The fertilized eggs normally hatch into white larvae in 2224 hours after they have been laid. The larvae burrow in the culture medium and feed on it for about four days. They then pupate on a dry spot, usually at the sides of the container. The pupation lasts for another four to seven days before the adults emerge from the pupa case. The newly emerged adults are pale in colour with unexpanded wings, but mature in about 810 hours with a characteristic darkened body. The females usually do not mate before 8 hours after emergence and do not lay eggs until they are two days old. In addition to a short life cycle, Drosophila is highly prolific, and has a lot of genetic variability with many morphological variants. Names and symbols are used to identify the mutant genes carried by the flies. Below are some commonly used notations in Drosophila genetics. a. Wild-type allele of any locus is indicated by the plus (+) symbol. b. Mutant alleles are shown with italicized letters. A capital letter designates a dominant gene, the lowercase letter represents recessive allele. E.g., B represents the dominant Bar gene; Bd represents the dominant Beaded gene; b designates the recessive black gene; bt designates the recessive bent gene. To represent a wild-type locus, a superscript + symbol is used after the letter. E.g., b+ shows that the wild-type gene is dominant over its recessive black gene, b; B+ indicates that the wild-type gene is recessive to its dominant Bar gene, B.

c. If more than two alleles are observed for a gene locus, mutant alleles can be distinguished by small letters in superscript after the alphabet for the genes. E.g., w+ represents a wild-type eye-colour gene; w represents a mutant white-eye gene; wa represents a mutant apricot-coloured eye gene d. The genotype of a gene locus for a diploid organism is represented by two symbols. E.g. homozygote (wild-type) b+/b+; homozygote (black mutant) b/b; heterozygote (wild-type) b+/b. e. To show two unlinked loci in a diploid, separate the two pairs of symbols. E.g. b/b, se/se (black and sepia) f. If two loci are linked, they are indicated by a slash. E.g., b vg/b vg (black and vestigial, coupling phase); b vg+/b+ vg (phenotype: wild-type, repulsion phase). g. Y chromosome is shown as Y, e.g., White-eye, miniature winged male can be written as w m/Y, where both w and m are located in X chromosome, or as Xw m. h. To show a phenotype, a line is written on top of the symbols of particular alleles. E.g., _ B (fly with Bar eye), _ __ e vg (ebony and vestigial fly) Description of phenotype does not indicate homozygosity, heterozygosity, and linkage group.

(B) Maize Genetics Introduction: The inheritance of variability in both maize seedlings and aleurone (endosperm) obeys the Mendelian principles. An expected 3:1 ratio has been observed in a recessive mutant allele for albinism (absence of chlorophyll) in the maize seedlings and a recessive allele for colourless aleurone segregating according the Mendels first law. Mendel also postulated that independent assortment of genes occurs when two traits are considered simultaneously, yielding an expected ratio of 9:3:3:1 among the dihybrid F2 progenies and dihybrid testcross ratio 1:1:1:1. Indeed, the dihybrid F2 kernels of corns yield a ratio of 9:3:3:1 for the phenotypes starchy (smooth) endosperm, sugary (wrinkled) endosperm, colourless starchy endosperm, and colourless sugary endosperm, respectively. Objectives: 1. To determines the sexes, indentify and differentiate the morphological traits of the 4 known mutant fly stocks . 2. To recognize and interpret maize F2 data and illustrate Mendels law of segregation and law of independent assortment. Materials: Mutant stocks of Drosophila that may be provided are Ebony body colour, Black body colour, Vestigial wing, Apterous (wingless), Curly wings, Sepia eye colour, Vermilion eye colour, White eye colour, Apricot (yellow) eye colour, Bar eyes, and Singed bristles, commercially prepared monohybrid, dihybrid and testcross F2 kernels of corns. Procedure: Activity 1 : The morphological traits of wild and mutant flies were compared. The table 2.1 were completed. The difference seen between the mutant and wild-type flies with appropriate symbol, sketches, or description. The wild-types flies that are considered normal was examined for all traits listed below. A plus sign was placed in the appropriate space to signify the normal condition. The flies from the given mutant stocks were examined carefully and compared with the wild types. The mutant flies are wild type for given traits were placed a plus sign in the appropriate space. The different traits were diagramed. Activity 2 : 2 possible illustration of endosperm were given in table 2.2 . The number of kernels were counted without removing from the corn ears in each phenotype. The data were recorded in table 2.3 and then hypothesis were formulated to explain the data collected.

Activity 3 : The kernels were groups into 4 different types. The number of kernels were counted and recorded without removing from the corn ears in each of different types of phenotypes in table 2.4 Result : Experiment 1 Table 1.1 Calculation of x2 for a sample removed from a large population consisting of equal numbers of coloured and white beads.  Classes Observed Expected Deviations (O-E)2 (Phenotypes) (O) (E) (O-E) Colored White Total: 46 42 88 44 44 88 2 -2 0 = 0.18 :1 : 3.84 4 4

X2 Degree of freedom p value : 0.05 Critical value I accept the hypothesis that the data approximate the expected ratio.

Table 1.2 Calculation of x2 on data from tossing coins. Classes Observed Expected Deviations (Phenotypes) (O) (E) (O-E) Heads on 4 7.5 -3.5 both coins Head on one, 14 15 -1 tail on the other coin Tails on both 12 7.5 4.5 coins Total : 30 30 0

(O-E)2 12.25 1 20.25

1.6333 0.0666 2.7

X2 = 4.3999 Degree of freedom : 2 P value : 0.05 Critical value : 5.99 I accept the hypothesis that the data approximate the expected ratio. Table 1.3 Calculation of x2 for a sample removed from a large population consisting of beads of two different colors. Classes Observed Expected Deviations   (O-E)2 (Phenotypes) (O) (E) (O-E) Colored 12 11.25 0.75 0.5625 0.05 White 3 3.75 -0.75 0.5625 0.15 Total : 15 15 0 X2 = 0.2 Degree of freedom : 1 P value : 0.05 Critical value : 3.84 I accept the hypothesis that the data approximate the expected ratio. Table 1.4 Calculation of x2 on blood group data from a hypothetical population. Observed Expected Deviations Phenotypes (O-E)2 (O) (E) (O-E) A 4 10.66 -6.66 44.36 B 10 2.34 7.66 58.68 AB 2 0.78 1.22 1.49 O 10 12.22 -2.22 4.93 Total : 26 26 0

4.16 25.08 1.91 0.40

P value : 0.05 I reject the hypothesis that the data approximate the expected ratio.

X2 = 23.23 Degree of freedom : 3 Critical value : 7.82

Table 1.5 Calculation of x2 for a sample removed from a large population consisting of beads of two different colors. Observed Expected Deviations  Phenotypes (O-E)2 (O) (E) (O-E) White 50 46.31 3.69 13.61 0.29 Yellow 127 138.94 -11.94 142.56 1.03 Red 432 416.81 15.19 230.74 0.55 Blue 132 138.94 -6.94 48.16 0.35 Totals : 741 741 23.88

X2 = 2.22 Degree of freedom : 3 P value : 0.05 Critical value : 7.82 I accept the hypothesis that the data approximate the expected ratio. Experiment 2 : Table 2.1 Comparison of wild-type and mutant Drosophila. Trait Wild-type Mutant 1 Mutant 2 Body Color Yellow Black Red Square shape with a blunt edge Normal Large Black line Yellow Black White Black line Yellow Black Red Square shape with a blunt edge Normal Small

Mutant 3 Yellow Black Red Square shape with a blunt edge Normal Very small

Mutant 4 Black line Yellow Black Red Square shape with a blunt edge Normal small

Eye Color Eye Shape

Round shape Normal Small

Wing shape Bristle and Shape

Table 2.2 Record of F2 data for maize (species 6500) endosperm trait. Phenotypes Number of Number of Genotypes of Deviations (O-E)2 kernels kernels the kernels (O-E) observed expected Purple 446 443 3 9 P_ Yellow Totals : 145 591 148 591 pp -3 9

 0.02 0.06 0.08

x2 = 0.08 Degree of freedom : 1 P value : 0.05 Critical value : 3.84 I accept the hypothesis that the data approximate the expected ratio.

The diagram of monohybrid cross and test cross of the species of 6500 maize Parent Gametes F1 Gametes F2 : : : : : PP,Pp,Pp,pp PP P Pp P,p x pp p x Pp P,p

Hence, we can determine that the parent genes of PP and pp will produce the F2 offspring with a ratio of 3:1 Table 2.3 Record of F2 data for maize (species 6502) endosperm trait. Phenotypes Number of Number of Genotypes of Deviations kernels kernels the kernels (O-E) observed expected Purple 291 291 0 P_ Yellow 291 291 0 pp Totals : 582 582

(O-E)2 0 0

0 0 0 2 x =0 Degree of freedom= 1 Critical value= 3.84

P value= 0.05 I accept the hypothesis that the data approximate the expected ratio.

The diagram of monohybrid cross and test cross of the species of 6502 maize Parent Gametes F1 Gametes F2 : : : : : Pp,pp Pp P,p Pp P,p x pp p x pp p

Hence, we can determine that the parent genes of Pp and pp will produce the F2 offspring with a ratio of 1:1

Activity 3: Let the gene P represents the purple traits while gene p represents the yellow traits and the gene P is dominant over the gene p. Let the gene S represents the smooth traits while gene s represents the wrinkled traits and the gene S is dominant over the gene s. Table 2.5 Data of independent assortment in maize and calculation of x2 for the maize 6600 Classes Genotypes Observed Expected Deviations   (O-E)2 (O-E) (phenotype) (O) (E) Purple 339 316.125 22.875 523.266 1.655 P_S_ smooth kernels Yellow 96 105.375 -9.375 87.89 0.834 ppS_ smooth kernels Purple 85 105.375 -20.375 415.14 3.940 P_ss wrinkled kernels Yellow 42 35.125 6.875 47.266 1.346 wrinkled ppss kernels Total: 562 562 0 x2= 7.775 Degree of freedom= 3 P value= 0.05 Critical value= 7.82

I accept the hypothesis that the data approximate the expected ratio.

The diagram of monohybrid cross and test cross of the species of 6600 maize Parent Gametes F1 Gametes F2 : : : : : PPSS PS PpSs PS,Ps,pS,ps x x ppss ps PpSs PS,Ps,pS,ps

PPSS,PPSs,PpSS,PpSs,PPSs,PPss,PpSs,Ppss, PpSS,PpSs,ppSS,ppSs,PpSs,Ppss,ppSs,ppss

Hence, we can determine that the parent genes of PPSS and ppss will produce the F2 offspring with a ratio of 9:3:3:1 Table 2.6 Data of independent assortment in maize and calculation of x2 for the maize 6602 Classes Expected Deviations O E 2 Genotypes Observed (O) (O-E)2 (O-E) (phenotype) (E) Purple smooth P_S_ 151 133.5 17.5 306.25 2.294 kernels Yellow ppS_ 128 133.5 -5.5 30.25 0.227 smooth kernels Purple 123 133.5 -10.5 110.25 0.826 wrinkled P_ss kernels Yellow ppss 132 133.5 -1.5 2.25 0.017 wrinkled kernels Total: 534 534 0 X2= 3.364 Degree of freedom= 3 P value= 0.05 I accept the hypothesis that the data approximate the expected ratio. Critical value= 7.82

The diagram of monohybrid cross and test cross of the species of 6600 maize Parent Gametes F1 Gametes F2 : : : : : PpSs PS,Ps,pS,ps PpSs PS,Ps,pS,ps PpSs,Ppss,ppSs,ppss x x ppss ps ppss ps
Written only the offspring that will produce wanted F2

Hence, we can determine that the parent genes of PpSs and ppss will produce the F2 offspring with a ratio of 1:1:1:1 Discussion :

In experiment 1, all x2 analysis are accepted except the activity 4. The rejection of the hypothesis is due to the small sample of 26 students cannot represents the whole population on the Earth. A sample must reach a minimum numbers to represents a populations, in this case, we reject the hypothesis due to this error. In contrary, others x2 analysis can be accepted and obeys the mendels law. In experiment 2 activity 1, the fly characteristics are determine. We seen a great deviation of characteristics between the fly species. This is due to the variations of genes in fly. Hence, we can know that different genes can produce different types of traits in fly. In this experiment, the comparison of the morphological trait in fly show the variations. In experiment 2 activity 2, it is the study of the Mendels Law of segregation. The law states that allele pairs segregate during gamete formation, and randomly unite at fertilization. There are 4 main concepts in this law : 1)A gene can exists in more than one form 2)Organisms inherit two alleles for each traits 3)When gametes are formed through meiosis, allele pairs separate leaving each cell with a single allele for each traits 4)When the two alleles of a pair are different, one is dominant and the other is recessive. As an example, for the maize 6500 species, parents cells consist of homozygous dominant gene (PP) and homozygous recessive gene (pp). The F1 generation will inherit the both of the dominant gene and recessive gene which resulting a heterozygous genes. When F1 mate with

each other, they will produce a F2 generations with a phenotypes of ratio 3:1 (refer to result in 6500 maize species). But in the maize species of 6502, when a heterozygous dominant gene parent mates with a homozygous recessive genes, this mating will produce an offspring of heterozygous gene that is dominant and homozygous gene which is recessive. Hence, the F2 generations will show a phenotype with a ratio 1:1 . (refer to results in 6502 maize species) In experiment 2 activity 3, the experiments are carried out to test the Mendels Second Law which is the law of independent assortment which states that the segregation of alleles of one allelic pair is independent of the segregation of the alleles of another allelic pair during gamete formation which involves dihybrid crosses. As examples is the maize species 6600 and 6602. For species 6600, it is similar to maize 6500 but only introducing one more genes which is known as dihybrid. When a dominant dihybrid homozygous (PPSS) mates with recessive dihybrid homozygous (ppss), it will produce a dominant dihybrid heterozygous. When they mates with each other, they will produce f2 generations that obey the Mendels Law Second Rule with a ratio of 9:3:3:1. For the maize species 6602, parents of dominant heterozygous dihybrid genes mates with homozygous dihybrid recessive gene. The F1 generations will mates with each other that have different genes (PpSs and ppss)only will produce the F2 generations with the ratio of 1:1:1:1 . Conclusion :

1. Chi square analysis used to determined whether the data approximating the expected ratio. 2. A Chi square analysis were carried out using a appropriate degree of freedom to compare with the critical value. 3. The fly were identified by observing their morphological traits. 4. F2 generations of maize can be used to illustrate the Mendels First and Second Law. References :

Bowler, P. J. (1989). The Mendelian Revolution : The EMergence of Hereditarian. Mendel, G. J. (2009). The Lost and Found Genius of Gregor Mendel, the Father of Modern Genetics. The Monk in the Garden .