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LSM 1102 (MOLCULAR GENETICS) (PLANT GENETICS) - PRACTICAL 6 Mendelian Genetics

NOTES ON THE CHI-SQUARE TEST

The purpose of the chi-square (2) test is to determine whether experimentally obtained data constitute a good fit to, or a satisfactory approximation of, a theoretical expected ratio. Thus the 2 test enables one to determine whether it is reasonable to attribute deviations of a perfect fit to chance. Obviously, if deviations are small, they can be more reasonably attributed to chance than if they are large. The question we try to answer with the 2 test is How small the deviations be attributed to chance alone? The fomula for the 2 test is as follows: 2 =

(O E )2 E

Where O = the observed number of individuals in a particular phenotype E = the expected number of individuals in that phenotype = the summation of all possible values of (O-E)2/E for the various phenotypic categories EXAMPLE In a cross of tall tomato plants to dwarf ones, the F1 consisted entirely of tall plants. The F2 consisted of 102 tall and 44 dwarf plants. Do the F2 data fit a ratio of 3:1? To answer this question the 2 was calculated; the calculations are summarized in Table 1 below. Set the 2 hypotheses: (Null hypotthesis) H0 = the observed data fit the 3:1 ratio (Alternate Hypothesis) H1 = the observed data do not fit the 3:1 ratio Table 1. Summary of the calculation of 2 Phenotype Tall (T_) Dwarf (tt) Total Exp. ratio 1 No. of O 102 44 146 plants E 109.5 36.5 146.0 (O - E ) -7.5 7.5 (O - E)2 (O - E)2/E 56.25 0.5137 56.25 1.5411 2 2.0548 =

The calculated 2 value is 2.0548, but what does this 2 value mean? If the observed numbers (O) were exactly equal to the theoretcial expected numbers (E), the fit would be perfect and 2 would be zero. Thus a small value of 2 indicates that the observed and the expected ratios are in close agreement, whereas a large value indicates marked deviation from the expected ratio. Because chance deviations from the theoretical values are expected to occur, the question to be answered is Are the observed deviations within the limits expected by chance? Generally, statisticians have agreed on the arbitrary limits of odds of 1 chance in 20 (probability = 0.05) for drawing the line between acceptance and rejection of the hypothesis as a satisfactory explanation of the data tested. A 2 value of 3.841 for a two-class ratio corresponds to a probability of 1 in 20, or 0.05. One would expect a 2 value of 3.841 due to chance alone is only about 5% of similar trials if the hypothesis is true. When 2 exceeds 3.841 for a two-class ratio, the probability that the deviations can be attibuted to chance alone is less than 1 in 20. The hypothesis of fit between the observed and expected ratios (Ho) is thus rejected. In the practice example, 2 = 2.0548 which is considerably less than 3.841, therefore you can attribute the deviations to chance alone and accept the null hypothesis (Ho) that the data fit the 3:1 ratio. Where did the value of 3.831come from from? Statisticians have published extensive tables listing 2 values (see Table 2). Notice that the first row are probability (P) values. Down the left handside (first column) are degrees of freedom values (df = 1,2,,30). The number of degrees of freedom is generally one less than the number of phenotypic classes. In the example, with two classes in the ratio (3:1), you have one degree of freedom in interpreting 2. Thus on the one-degree-of freedom row and under the 0.05 column you find the 2 value of 3.841, which is the maximum value of 2 that we are willing to accept and still attribute the deviations observed to chance alone. How to conclude? If the calculated 2 value is smaller than the critical 2 value, we accept the null hypothesis and conclude that the observed data give support to the null hypothesis. If the the calculated 2 value is greater than the critical 2 value, we reject the null hypothesis and conclude that the observed data do not give support to the null hypothesis. In the example, 2 was observed to be 2.0548. Looking at the one-degree-of freedom row, you will see that this value of 2 falls between the 0.05 (2 = 3.841) and the 0.20 (2 = 1.642) probability columns.This means that the probability that the deviations may be attributed to chance alone is between 5% to 20%; that is, if you were to do this same experiment 100 times, you would expect to observe deviations as large as you have obtained or larger in between 5 and 20 of the 100 trials due to chance alone. Thus the null hypothesis is accepted. The observed data of this experiment fit the 3:1 F2ratio. With this background in the meaning, calculation and interpretation of 2 you can solve similar problems.

Table 2. Table of 2 values Df 1 2 3 4 5 6 7 8 9 10 15 20 25 Reference Gardner E. J., Mertens T. R. and Hammersmith R. L. (1985) Laboratory Investigations, Investigation 4 ; the Chi-square test. Pp 25-37. 8th Edition, MacMillan Publishing Company, New York. P=.99 .000157 .0201 .115 .297 .554 .872 1.239 1.646 2.088 2.558 5.229 8.260 11.524 .95 .00393 .103 .352 .711 1.145 1.635 2.167 2.733 3.325 3.940 7.261 10.851 14.611 .80 .0642 .446 1.005 1.649 2.343 3.070 3.822 4.594 5.380 6.179 10.307 14.578 18.940 .50 .4550 1.386 2.366 3.357 4.351 5.348 6.346 7.344 8.343 9.342 14.339 19.337 24.337 .20 1.642 3.219 4.642 5.989 7.289 8.558 9.803 11.030 12.242 13.442 19.311 25.038 30.675 .05 3.841 5.991 7.815 9.488 11.070 12.592 14.067 15.507 16.919 18.307 24.996 31.410 37.652 .01 6.639 9.210 11.345 13.277 15.086 16.812 18.475 20.090 21.666 23.209 30.578 37.566 44.314

SORGHUM GENETICS (one-tray per group) Some of the traits obey Mendelian principles Monhybrid cross, R locus (3 red leaves : 1 green leaves F2ratio) Each group of students are provided with a petri dish containing seedlings of F2 generation of the cross between a plant with red leaves and green leaves. R locus determines oresence of red pigment (anthocyanin) in leaves R (dominant) = red leaves, r (recessive) = green leaves P F1 F2 Red (RR) green (rr) Red (Rr) Red (R_) : green (rr)

You will count the number of red and green seedlings and record your data in the table below: Table 1. Record of F2 data and summary of the calculation of 2 from your group totals. Phenotype Red (R_) Green (rr) Total Exp. ratio 1 No. of O plants E Deviation (O - E ) Deviation2 (O - E)2 2 = (O E)2/E

(i) Set the Null (Ho) and Alternate Hypotheses (H1). (ii) Carry out the Chi-square test (set probability at 0.05) (iii) Draw your conclusions regarding the inheritance of red pigment in sorghum. Do your observed data fit the 3 : 1 expected ratio? Which hypothesis do you accept?

TOBACCO GENETICS
1. Monhybrid cross, A locus (3 green leaves : 1 albino leaves F2 ratio) Complete dominance Two petri-dishes per group of tobacco seedlings Parents : Green leaf (AA) vs albino leaf (aa) P F1 F2 Green (AA) Yellow (aa) Yellow-green (Gg) green (A_) : albino (aa)

You will count the number of green and albino seedlings and record your data in the table below: Table 2. Record of F2 data and summary of the calculation of 2 from your group totals. No. of plants Deviation Deviation2 Phenotype Exp. ratio O E (O - E ) (O - E)2 (O E)2/E Green (AA) Albino (aa) Total 1 2 = (i) Set the Null (Ho) and Alternate Hypotheses (H1). (ii) Carry out the Chi-square test (set probability at 0.05) (iii) Draw your conclusions regarding the inheritance of leaf color in tobacco. Do your observed data fit the 3 : 1 expected ratio? Which hypothesis do you accept? If the observed ratio did not fit the expected give some possible reasons for this deviation.

TOBACCO GENETICS
2. Monhybrid cross, G locus (1 : 2 : 1 ratio F2 ratio) Incomplete dominance Two petri-dishes per group Tobacco seedlings (F2 ratio green: yellow-green: yellow ratio) Parents : Green leaf (GG) vs yellow leaf (gg) GG = green leaves, Gg = yellow-green leaves, gg = yellow leaves P F1 F2 Green (GG) Yellow (gg) Yellow-green (Gg) green (GG) : yellow green (Gg) : yellow (gg)

You will count the number of green, yellow-green and yellow seedlings and record your data in the table below: Table 2. Record of F2 data and summary of the calculation of 2 from your group totals. No. of plants Deviation Deviation2 Phenotype Exp. ratio O E (O - E ) (O - E)2 (O E)2/E Green (GG) Yellow-green (GG) Green (rr) Total 1 2= (i) Set the Null (Ho) and Alternate Hypotheses (H1). (ii) Carry out the Chi-square test (set probability at 0.05) (iii) Draw your conclusions regarding the inheritance of leaf color in tobacco. Do your observed data fit the 1 : 2 : 1 expected ratio? Which hypothesis do you accept?

TOMATO GENETICS
1. Tomato seedlings (F2 3 hairy : 1 hairless ratio) Parents: Normal plants (hairy on stems and leaves, HH) vs Mutant (hairless, hh) Note: Seedlings should be 21 days or older. The character is best seen in good light against a black background. The hairy gene shows complete dominance. P F1 F2 Hairy (HH) hairless (hh) Hairy (Hh) Hairy (H-) : hairless (hh)

You will count the number of hairy and hairless seedlings and record your data in the table below: Table 4. Record of F2 data and summary of the calculation of 2 from your group totals. No. of plants Phenotype Exp. ratio O E (O - E ) (O - E)2 (O E)2/E Hairy (H-) Hairless (hh) Total 1 2 = (i) Set the Null (Ho) and Alternate Hypotheses (H1). (ii) Carry out the Chi-square test (set probability at 0.05) (iii) Draw your conclusions regarding the inheritance of hairy/hairless stem in tomato. Do your observed data fit the 3 : 1 expected ratio? Which hypothesis do you accept? If it does not fit, offer some explanations as to the possible reasons for the deviation.

2. Tomato seedlings (F2 Green leaf : Green-yellow leaf: yellow leaf ratio) Parents : Green leaf (GG) vs yellow leaf (gg) GG = green leaves, Gg = yellow-green leaves, gg = yellow leaves This locus shows incomplete dominance. P F1 F2 Green (GG) Yellow (gg) Yellow-green (Gg) green (GG) : yellow green (Gg) : yellow (gg)

You will count the number of green, yellow-green and yellow seedlings and record your data in the table below: Table 5. Record of F2 data and summary of the calculation of 2 from your group totals. No. of plants Phenotype Exp. ratio O E (O - E ) (O - E)2 (O E)2/E Green (GG) Yellow-green (Gg) Green (gg) Total 1 2 = (i) Set the Null (Ho) and Alternate Hypotheses (H1). (ii) Carry out the Chi-square test (set probability at 0.05) (iii) Draw your conclusions regarding the inheritance of leaf color in tomato. Do your observed data fit the 1 : 2 : 1 expected ratio? Which hypothesis do you accept?

NOTES ON CORN GENETICS

The substantial genetic variability in corn (maize) obeys Mendelian principles; both seedling and aleurone characters may be used. The patterns of mendelian inheritance can be demonstrated by studying segregation ratios in F2 generation from standard monohybrid and dihybrid crosses. Monohybrid crosses involving one gene factor (one pair of contrasting characters) give rise to the 3:1 F2 segregating ratio. Dyhybrid crosses involvce two factors (two pairs of segregating characters) give rise to 9:3:3:1 F2 segregating ratio (no linkage or epistasis). Epistasis gives rise to various modifications of the 9:3:3:1 ratio. I. SEGREGATING CORN SEEDLINGS Because maize has a relatively long life cycle, taking 3 or 4 months to complete, you will not be able to conduct actual experimental matings with corn. Your Instructor has provided you with trays of of corn seedlings germinated from F2 kernels purchased from a number of biological supply companies. You will count the number of seedlings in the different phenotypes and then to interpret the data in terms of Mendelian principles. Dihybrid cross, T and G loci (9:3:3:1 F2 ratio) T = Tall, t = dwarf G = Green, g = albino This is a two-factor cross between a homozygous Tall/Green parent (genotype TTGG) with a dwarf/albino parent (genotype ttgg). The cross results in an F1 generation which is heterozygous (TtGg). The F1 plants are selfed, resulting in a F2 generation. P F1 F2 Tall/Green (TTGG) dwarf /albino (ttgg) Tall/Green (TtGg) 9/16 Tall/Green : 3/16Tall/albino : 3/16 dwarf/Green : 3/16 dwarf/albino (T_G_) (T_gg) (ttG_) (ttgg)

You will be given a tray of corn seedlings containing F2 tall/green, tall/albino, dwarf/green and dwarf/albino plants. You will count the number of the 4 types of seedlings and record your data in the table below: Table 4. Record of F2 data and summary of the calculation of 2 from your group totals. Phenotype Tall/Green Tall/albino Dwarf/Green Dwarf/albino Total Exp. ratio 9/16 3/16 3/16 1/16 1 No. of seedlings O (Obs) E (Exp) (O - E ) (O - E)2 (O - E)2/E

2 =

(i) Set the Null (Ho) and Alternate Hypotheses (H1). (ii) Carry out the Chi-square test. (iii) Draw your conclusions regarding the inheritance of height and colour of corn plants. Do your observed data fit the 9:3:3:1 expected ratio? Which hypothesis do you accept?