Name: Date: Title: Gene Interactions in Corn. Introduction. The phenotype of an organism is determined, at least in part, by its genotype. Thus, given the genotype of an organism, and an understanding of how the various genes and alleles interact, we can predict the phenotype of the progeny of a particular cross. For example, given that flower colour in pea plants is controlled by a single gene, and that the allele that gives rise to purple flowers is dominant to that giving white flowers, we can predict that a cross between homozygous purple flowered plants and homozygous white flowered plants will give a purple flowered F1 generation, which, when self-pollinated, will give a three to one ratio of purple to white flowered plants in the F2 generation. In many cases the problem in genetics is reversed. We are able to observe the phenotypes that result from certain crosses, and from that information must deduce how many genes and alleles are involved, and how those genes and alleles interact. In this laboratory session we will investigate the genetics of certain aspects of kernel morphology in corn. The colour and appearance of corn kernels are affected by several gene loci. We will investigate interactions between these different loci using a number of dihybrid crosses. In these dihybrid crosses, individuals that are pure breeding for one phenotypic trait are crossed with individuals that are pure breeding for a different phenotypic trait. A/A;b/b x a/a;b/b The doubly heterozygous F1 progeny generally show a single phenotype, determined by the dominant alleles of the two genes. A/a;B/b If the genes are unlinked, and there are no interactions between the two loci, the F2 progeny from a self-cross of the F1 will show a nine to three to three to one ratio of phenotypes. Genotypes A/?;B/? A/?;b/b a/a;b/? a/a;b/b Phenotypes 9 3 3 1 However, this ratio is often altered as a result of interactions between the two loci involved. A number of different types of interaction are possible. One possibility is epistasis, where the genotype at one locus prevents or modifies expression of the other locus. In recessive epistasis, the homozygous recessive state at one locus affects expression of the other locus, resulting in a nine to three to four ratio. Genotypes A/?;B/? A/?;b/b a/a;b/? + a/a;b/b Phenotypes 9 3 4 Genetics Laboratory 06.1
In dominant epistasis, the dominant allele at one locus affects expression of the other locus, resulting in a twelve to three to one ratio. Genotypes A/?;B/? + A/?;b/b a/a;b/? a/a;b/b Phenotypes 12 3 1 Further complications occur when the two gene loci can produce identical phenotypes. If the dominant alleles of both loci produce identical phenotypes, and both loci show dominant epistasis, a fifteen to one ratio will be produced. Genotypes A/?;B/? + A/?;b/b + a/a;b/? a/a;b/b Phenotypes 15 1 This situation occurs when the two gene loci encode proteins that can substitute for each other. If the homozygous recessive genotypes of both loci produce identical phenotypes, and both loci show recessive epistasis, a nine to seven ratio will be produced. Genotypes A/?;B/? A/?;b/b + a/a;b/? + a/a;b/b Phenotypes 9 7 This situation occurs when the proteins encoded by the two gene loci are both required for a particular phenotype. If the dominant allele of one locus produces the same phenotype as the homozygous recessive genotype at the other locus, and the first locus shows dominant epistasis, a thirteen to three ratio will be produced. Genotypes A/?;B/? + A/?;b/b + a/a; b/b a/a;b/? Phenotypes 13 3 This situation occurs when the protein encoded by one gene suppresses the phenotype associated with the protein encoded by the other gene. You will be investigating the interactions between five loci that affect the morphology of corn kernels. Four of these loci affect the colour of the kernels by determining the pigmentation of the endosperm or the aleurone layer that surrounds it. The relative positions of the endosperm and aleurone in a corn kernel are shown in figure 1. Genetics Laboratory 06.2
Aleurone Endosperm Figure 1: Cross section of corn kernel, showing relative positions of aleurone and endosperm. The five loci and their alleles are listed below. Alleles are listed in order of dominance. C locus: affects pigmentation of aleurone C i - aleurone unpigmented, endosperm visible C - aleurone can be pigmented, endosperm can be obscured c - aleurone unpigmented, endosperm visible Pr locus: affects colour of aleurone Pr - aleurone purple pr - aleurone red R locus: affects pigmentation of aleurone. R - aleurone can be pigmented, endosperm can be obscured r - aleurone unpigmented, endosperm visible Su locus: affects morphology of kernel Su - smooth kernels su - wrinkled kernels Y locus: affects colour of endosperm Y - endosperm yellow y - endosperm white Genetics Laboratory 06.3
Methods. F2 corn ears from five different crosses will be available. Each cross involves two gene loci, each with two alleles. None of the loci are linked. The ancestry of each type of ear is shown below. In all cases, the original parents are pure breeding. (a) P purple smooth x yellow wrinkled F1 purple smooth F2 purple smooth purple wrinkled yellow smooth yellow wrinkled (b) P purple x white F1 purple F2 purple red white (c) P purple x yellow F1 purple F2 purple yellow white (d) P white x white F1 red F2 red white (e) P yellow x yellow F1 yellow F2 yellow purple (1) Select an F2 corn cob from one of the five crosses. Count the number of kernels in each of the phenotypic classes. If a kernel cannot be unambiguously assigned to a phenotypic class, record it separately. (2) Repeat this process with F2 corn cobs from each of the other four crosses. Genetics Laboratory 06.4
Report. Your report should be typed or neatly written. Marks may be deducted for illegibility and/or poor grammar and/or poor spelling. Your report should be stapled once in the top left corner. Binders or paper clips are not acceptable. Your report should be in the following format: Name and date. Title of experiment. Introduction to experiment. Description of methods and materials used. The above information is in this manual. You can attach these manual pages to your report in place of this information. Results obtained. For each cross record the number of kernels observed in each phenotypic class. You may combine your data with that of another student (provided the other student counted different corn ears) to increase your sample size. Analysis of results obtained and conclusions. For each cross, use the χ 2 test to determine which of the interactions listed in the introduction can be rejected. State clearly your conclusion regarding the type of interaction involved. Table 1: χ 2 values. d.o.f Probability 0.90 0.50 0.10 0.05 0.01 1 0.016 0.455 2.706 3.841 6.635 2 0.211 1.386 4.605 5.991 9.210 3 0.584 2.366 6.251 7.815 11.345 4 1.064 3.357 7.779 9.488 13.277 Discussion. Indicate the genotype at all five loci of the pure breeding parents, the F1 generation, and each of the F2 progeny phenotype classes. Use the abbreviations and format given in the introduction. You will need to take into consideration the information about the loci and the alleles given in the introduction, the information about the parental, F1, and F2 generations given in the methods, and the information about the parental genotypes given below. Genetics Laboratory 06.5
Cross A: The parents differ only at the R and Su loci. Both are C/C. Cross B: The parents differ only at the Pr and R loci. Both are C/C. Cross C: The parents differ only at the R and Y loci. Both are C/C. Cross D: The parents differ only at the C and R loci. Cross E: The parents differ only at the C and R loci. In the event that your data do not fit any of the ratios mentioned in the introduction, discuss possible explanations for the results obtained. Comment on any other discrepancies between the results observed and those expected. The report is due one week from today. Late reports will be penalized. You should each write your own report, although you may discuss the results with each other. If you require any help with your report, please come and see me. There is no penalty attached to this help. Genetics Laboratory 06.6