Todd Alderman
Dr. Elder
Genetics
October 18, 2005
Recombination Lab 6
Introduction:
The purpose of this lab is to better understand the concepts behind determining linkage between genes due to the fact that they are on the same chromosome. This is accomplished through a cross-over of the maternal and paternal genes in the organisms producing the sex cells. In Drosophilia melanogaster only the females experience the cross-over thus a form of testcross can be performed to detect the presence of linkage by studying the female offspring only. This is in direct conflict with Mendel’s law of independent assortment which states that one gene has no influence on a second. In the case of gene linkage, if two genes are located on the same chromosome they can quite possibly influence one another. Two genes far from each other on a chromosome are very likely to cross-over and thus influence each other. On the other hand if they are close together the chances of a cross-over goes way down. These frequencies in cross-over can be formulated to determine the distance between genes thus allowing us to see a map of the genes and sequence. All of this will be seen in the following experiments.
Procedure:
Directions were obtained from the assignments page of www.biologylabsonline.com experiment 6.
Results:
Flylab Lab Notes for Todd Alderman
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Results of Cross #1
Parents
(Female: EY) x (Male: SV)
Offspring
Phenotype Number Proportion Ratio
Female: + 5094 0.5069 1.028
Male: + 4955 0.4931 1.000
Total 10049
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This was simply the initial cross made by two true breeders in order to get a dihybrid organism thus providing a single genotype.
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Results of Cross #2
Parents
(Female: +) x (Male: SV;EY)
Offspring
Phenotype Number Proportion Ratio
Female: + 25 0.0025 1.190
Male: + 27 0.0027 1.286
Female: SV 2413 0.2409 114.905
Male: SV 2535 0.2531 120.714
Female: EY 2418 0.2414 115.143
Male: EY 2552 0.2548 121.524
Female: SV;EY 21 0.0021 1.000
Male: SV;EY 25 0.0025 1.190
Total 10016
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These results show a peculiar outcome if one were trying to apply Mendel’s second law because it does not hold true. Since this was a test cross and we were instructed not to look at the males, we see that the 1:1:1:1 does not hold true. Instead we see that some interaction is occurring which means that cross-over has occurred. This also means that we can analyze this data and extract a map of these two genes and the distance between them. Although we can not tell the order of the genes from the centromere a depiction of them would be seen as in the diagram below or in the reverse order of the diagram.
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Chromosome IV
Chi Square Hypothesis Using Cross #2
Phenotype Observed Hypothesis Expected Chi-Square Term
Female: + 25 0.5000 1252.0000 1202.4992
Male: + 27 0.5000 1252.0000 1198.5823
Female: SV 2413 0.5000 1252.0000 1076.6142
Male: SV 2535 0.5000 1252.0000 1314.7676
Female: EY 2418 0.5000 1252.0000 1085.9073
Male: EY 2552 0.5000 1252.0000 1349.8403
Female: SV;EY 21 0.5000 1252.0000 1210.3522
Male: SV;EY 25 0.5000 1252.0000 1202.4992
Total 10016 4.0000 10016.0000 9641.0623
Chi-Sqaured Test Statistic = 9641.0623
Degrees of Freedom = 7
Level of Significance = 0.0000
Recomendation: Reject your hypothesis
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Based on a normal testcross of a dihybrid, the ratio of offspring should have been equal between each phenotype. However, if we are to test this hypothesis using the Chi-squared test we see that it is horribly rejected. This leads us to begin considering that the genes are probably linked because they defy Mendel’s Second Law.
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Results of Cross #3
Parents
(Female: PR;VG;BL) x (Male: +)
Offspring
Phenotype Number Proportion Ratio
Female: + 4958 0.5007 1.003
Male: + 4944 0.4993 1.000
Total 9902
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This was simply the initial cross made by two true breeders in order to get a trihybrid organism thus providing a single genotype.
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Results of Cross #4
Parents
(Female: +) x (Male: PR;VG;BL)
Offspring
Phenotype Number Proportion Ratio
Female: + 2092 0.2095 139.467
Male: + 2084 0.2087 138.933
Female: PR 19 0.0019 1.267
Male: PR 16 0.0016 1.067
Female: VG 270 0.0270 18.000
Male: VG 257 0.0257 17.133
Female: PR;VG 127 0.0127 8.467
Male: PR;VG 127 0.0127 8.467
Female: BL 146 0.0146 9.733
Male: BL 138 0.0138 9.200
Female: PR;BL 270 0.0270 18.000
Male: PR;BL 276 0.0276 18.400
Female: VG;BL 18 0.0018 1.200
Male: VG;BL 15 0.0015 1.000
Female: PR;VG;BL 2109 0.2112 140.600
Male: PR;VG;BL 2022 0.2025 134.800
Total 9986
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Again we look at the female offspring only to find that the 1:1:1:1 ration of Mendel’s Law of Independent segregation does not hold true. Now we look for how often cross-over occurred between different genes to find their frequency which is directly related to the distance between the three. In the case of three genes one must determine the oder of the genes by closely studying the data to see exactly what is going on. First we look at the largest two groups of offspring and recognize those to be the parental types meaning no cross-over took place. Second we look at the smallest group of offspring and recognize it to be the double recombinants meaning that the genes crossed over twice and the two that stayed constant are the two genes on the outside while the one that crossed over is the one in the middle. Last we look at the single cross-overs and analyze those to extract there frequency of cross-over and eventually there distance from each other. Compiling all this together will give a gene map that resembles the one below. Keep in my we still can not be sure of the distance of each gene from the centromere thus the order might be completely reverse. Regardless of this we are sure of the order they appear.
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Chromosome II
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Chi Square Hypothesis Using Cross #4
Phenotype Observed Hypothesis Expected Chi-Square Term
Female: + 2092 0.5000 624.1250 3452.2844
Male: + 2084 0.5000 624.1250 3414.7567
Female: PR 19 0.5000 624.1250 586.7034
Male: PR 16 0.5000 624.1250 592.5352
Female: VG 270 0.5000 624.1250 200.9285
Male: VG 257 0.5000 624.1250 215.9516
Female: PR;VG 127 0.5000 624.1250 395.9676
Male: PR;VG 127 0.5000 624.1250 395.9676
Female: BL 146 0.5000 624.1250 366.2784
Male: BL 138 0.5000 624.1250 378.6381
Female: PR;BL 270 0.5000 624.1250 200.9285
Male: PR;BL 276 0.5000 624.1250 194.1775
Female: VG;BL 18 0.5000 624.1250 588.6441
Male: VG;BL 15 0.5000 624.1250 594.4855
Female: PR;VG;BL 2109 0.5000 624.1250 3532.7118
Male: PR;VG;BL 2022 0.5000 624.1250 3130.8704
Total 9986 8.0000 9986.0000 18241.8294
Chi-Sqaured Test Statistic = 18241.8294
Degrees of Freedom = 15
Level of Significance = 0.0000
Recomendation: Reject your hypothesis
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Again, if we apply Mendel’s Law and expect an even ratio of each phenotype our hypothesis is strongly rejected proving that these three genes are linked and can then proceed with determining distances from each other.
Conclusion:
Recombinants are a strong tool for looking at genes. It can tell us if genes are linked together, how far apart they are from each other and ultimately has helped us map the human genome. If we apply the chi-squared test to the test cross of a di- or trihybrid and do not come up with an equal ration of offspring then the hypothesis for of Independent Segregation of the genes being analyzed will be rejected. Based the idea that Mendel’s Second Law (of independent segregation) doesn’t always hold true we can begin to look at why and are able to determine that based on frequency of genes crossing over with other genes, we can determine a distance that those two or even three genes are apart from each other. The largest group of offspring is considered the parental types meaning no cross-over takes place. The smallest group of offspring is considered to be the double recombinants meaning that the genes crossed over twice and the two that stayed constant are the two genes on the outside while the one that crossed over is the one between the other two. Last are the single cross-overs that are analyzed to extract there frequency of cross-over and eventually there distance from each other. With this information valuable knowledge can be gained about an organism’s entire genome and help us better understand how it works with heredity of traits and diseases.