"Verifying the Genetic Probabilities of Corn and Drosophila Breeding"
Introduction
Mendelian Genetics is an accepted theory of genetic reproduction. In 1857, scientific researcher, Gregor Mendel, began breeding peas in a monastery garden where he lived (Campbell Reece, Mitchell 239). Peas were a choice specimen to breed because of their clear and obvious traits. Mendel chose to focus on genetic characters that did not contain a gradient between possibilities; this way the outcome of the pea breeding would be clear (Campbell Reece, Mitchell 240). Applying these simple rules to heritable exploration, Mendel set out to prove his theory.
A garden pea's flower is similar to most any other plant flower. Its stamen (male sex organ) produces the sperm which pollinates and lands on a plant's stigma (top of female sex organ). After landing on the stigma, the pollen releases two sperm which fertilize the plants ovary. The breeding of a plant can be controlled by regulating the pollen that lands on the stigma. If the stamen is removed and pollen from a different flower is set on top of the stigma, the source of the offspring produced will be unquestionable (Campbell Reece, Mitchell 240). The ability to accurately breed garden peas gave Mendel an explanation for heredity. Rather than proving a hypothesis that was well known, he could work in reverse using the data to find the hypothesis.
A plant chromosome count is expressed by the term 2n (or "Diploid"), meaning "two of every chromosome." Mendel discovered that two alleles (versions of a gene) segregate during plant meiosis, and both the male and the female provide the final offspring with half of their genetic makeup (one-half of male and one-half of female makes for one complete offspring). If a plant is made up of the same allele on both of its chromosomes when they segregate, then the selection between the two will not matter. Often times, the plant's genotype consists of differing alleles (only one is expressed), and the segregation can cause an allele not expressed in the parental phenotype to be expressed in the offspring. This concept was termed, the "law of segregation." A genotype can be hidden behind the appearance of a phenotype; it was best said by Psychologist Valerie Grant: "the core of dominance can be identified as acting overtly so as to change the views or actions of another" (Grant 98).
When a plant's chromosomes (2n) are split (1n) and combine with another plants split chromosomes (1n) there is a pairing that occurs which is made up of probability. A single allele bred with another allele (a monohybrid cross) will cause four chromosome-pairing outcomes. If more than one allele is bred with another (dihybrid cross), the outcome will not be affected by the fact there is more than one allele being combined into the offspring. As long as the alleles are on separate chromosomes, there is no effective difference other than more combinations of offspring due to more alleles. This is Mendel's second law, the "law of independent assortment."
Mendel's discoveries have revolutionized what we now know in the field of science and have brought about many other sub-categories of science such as Genetics. Although speaking about his great discoveries in 1865 and publishing an article in 1866, his discoveries were not fully understood until sixteen years after his death. When three European botanists spontaneously rediscovered his findings, they were able to spread his explanation of what was termed, Mendelism (Britannica 11: 899).
In practice, Mendelian Genetics accurately calculate the probabilities of offspring from breeding two parents with non-linked genes. Phenotypes can however be affected by alleles that are on the same chromosome, this is known as sex autosomal linkage. When breeding corn, if the parents are dominates, known as true-breeding, the offspring's alleles (ex. color or texture) are predictable by Mendelian Genetics. Drosophila, a type of fly, can have predicted offspring using Mendelian Genetics when the genes are not on the same chromosome; however, if they are on the same chromosome, which is the X-chromosome, then the expected Mendelian ratios will be greatly off.
Sex-linkage can not be predicted but can be analyzed (Biology Labs On-Line). Once numerous offspring for a sex-linked trait are produced, the order of the alleles can be identified as well as the map distance between each allele. Before breeding the specimen, the map distance is unknown, so the number of different possibilities that will occur due to crossover is also unknown. Analyzing probabilities of offspring with sex-linkage also has another obstacle: a male and a female do not have everything in common! The human female has two X-chromosomes, but the male has XY. The Y-chromosome almost never carries any traits. This makes a geneticist's job both easier and more difficult. We will see an example of this in the results section.
Corn cobs can be found which differ from the regular soft yellow cobs everyone has grown to love. Corn kernels can have an allele that gives color to the recessively yellow kernels and some have an allele that stops the conversion of excess sugar to starch, resulting in a wrinkled kernel. Similarly, alleles in Drosophila can cause a fly to have any one of many strange appearances. When crossing specimens with abnormalities, it is key to remember that a recessively heterozygous will be phenotypically expressed as the dominant.
During the following lab exercises, corn alleles and Drosophila alleles will be tested to verify if Mendel's genetics are acceptable in a Chi-Squared test with our lab results (when using non-linked genes). Offspring ratio's of Drosophila with sex-linked genes will be examined for analysis of the cross itself.
Materials and Methods
Using five different cobs of corn- whose parents were true breeding- Mendel's Punnett square test should predict the results that would appear in lab testing. By counting the number of kernels that contained color, lacked starch, both, or neither, we assume the genetic makeup for every kernel the cob of corn will produce will be approximately the same ratio as every other kernel produced by that same cob. After the data is collected, it should be combined with the data from every other group to increase the sample size.
In a similar experiment, a Virtual Flylab simulated the breeding of different types of Drosophila. By true-breeding a dominant and a recessive parent, the results will always be the same: four half recessive, half dominants. If the F1 offspring of this breeding are bred with a homozygous recessive parent or another F1 offspring then the offspring will be predictable assuming Mendelism. This lab should accurately match the predicted data (using a Punnet-square) in a Chi-Squared test.
During both of these labs, a null hypothesis will be tested. This hypothesis states that there will be no difference between the observed and expected results. If observed results are what are expected, we will accept this null hypothesis; if results are unexpected, we will reject it and attempt to determine why.
Results of Corn testing
The first cob examined was a cross between a true-breeding pigmented parent (blue) and a true breeding non-pigmented parent. A total of 3,245 kernels were analyzed; 2,504 (3,245 expected) of them were pigmented, and 741 (811 expected) of them were non-pigmented.
The second cob examined was a cross between an F1 generation of the previous cross with a heterozygous recessive parent (non-pigmented) parent. A total of 2,489 kernels were analyzed; 1,212 (1,245 expected) of them were pigmented, and 1,277 (1,245 expected) of them were non-pigmented.
The third cob examined was an F2 dihybrid cross of starch and pigmented traits. A total of 2,665 kernels were analyzed; 1,488 (1,493 expected) were pigmented and starchy, 517 (498 expected) were pigmented only, 501 (498 expected) were starchy only, and 149 (166 expected) were neither.
The fourth cob examined is a F1 dihybrid test cross of the same traits as the previous. A total of 1,665 kernels were analyzed; In a dihybrid test cross, all of the offspring should be one-fourth of the total (416). The results were 376, 407, 457, and 425.
The fifth cob examined was a cross between a pigmented and a pigment-causing, epistatic, trait.
A total of 3,953 kernels were analyzed; 805 (741 expected) were pigmented and 3,148 (3,212 expected) were un-pigmented.
Results of Drosophila testing
In a monohybrid test-cross of vestigial wings, all offspring produced had a normal phenotype. When the F1 generation were bred, 7,431 were wildtype and 2,502 had vestigial wings.
In a dihybrid test-cross of brown eyes and crossviens, all offspring produced had a normal phenotype. When the F1 generation was bred, 5,708 were wildtype, 1,837 were brown-eyed, 1889 had crossviens, and 691 were both brown-eyed and crossviened.
In a cross between a wildtype female and a white-eyed male, 4,947 of the offspring were female and 4,943 were male. When the F1 male and F1 female were bred, 5,028 were wildtype females, 2,500 were wildtype males, and 2,525 were white eyed males.
In a cross between a wildtype female and a male with vestigial wings and a black body, all offspring were wildtype. When the F1 female was bred with the parental male, 4,286 were wildtype, 790 were vestigial, 776 were black, and 4,223 were black and vestigial.
In a cross between a wildtype female and a male with a black body, brown eyes, and vestigial wings, all of the offspring were wildtype. When the F1 female was crossed with the parental male, 3,051 were wildtype, 554 were black, 202 were vestigial, 1,143 were black and vestigial, 1179 were brown-eyed, 192 were brown-eyed and black, 621 were brown-eyed and vestigial, and 3084 were brown-eyed, black, and vestigial. These alleles do not follow the Law of Independent Assortment and therefore they are linked
Discussion
The results from the second cob were predicted to be a 1:1 ratio of blue to yellow kernels. These results were near expected. The Chi-Squared P-value was 0.19262 and any test greater than 0.05 can accept its null hypothesis. The results of the first cob cross, the F2 generation of the first cob, should have been a 3:1 phenotype ratio, 3 being the homozygous dominant and both heterozygous recessives, and the 1 should result from the homozygous recessive possibility. The results from this cross were not very close to what was predicted; a Chi-Squared P-value of 0.004. The null hypothesis was rejected.
Data from the dihybrid cross of starch and pigment traits (cob 4) resulted in the denial of the null hypothesis when the Chi-Squared P-value was calculated at only 0.04073, barely failing to amount higher than 0.05. When cob 4 produced F2 offspring, the Chi-Squared P-value again failed to meet the 0.05 requirement with only 0.04073. The data collected could have been greatly miscalculated or the cobs may not have had the parents expected. Other factors such as water requirements or even mutation could have played a role in the observed data.
The observed results from the fifth cob showed very little resemblance to the expected results. With a Chi-Squared P-value of only 0.009313, the data differentiated from what was calculated for a cross of such traits. This cross had an epistatic reaction that was taken in consideration; however, more kernels were pigmented than were expected to be. The epistatic allele, or an unknown external reaction, may have caused this result to be effected in ways not known.
In a monohybrid cross of Drosophila, the resulting ratio was almost exactly what was predicted. With a Chi-Squared P-value of 0.99999, the results could not have been closer. In a similar dihybrid cross of Drosophila, predicted ratios met the offspring ratio almost as well as the monohybrid cross, with a calculated Chi-Squared P-value of 0.9888886. These accurate results are proof that the methods used were performed correctly.
Results from a Sex-Linkage test matched the expected ratio substantially. An F1 cross had an expected ratio of 1:1 and an observed ratio of 1.001:1. A 2:1:1 F2 ratio was expected and a 2.011:1:1.01 ratio was observed; thus producing a 0.99992 Chi-Squared P-value.
In an Autosomal Linkage two point cross, the expected results differed greatly from the observed results. A 1:1:1:1 ratio was expected and a 5.523:1.048:1:5.442 ratio was achieved. This is understandable considering the alleles are located on the same chromosome; in fact, this difference in ratio is data necessary for determining the linkage on the chromosome and the expected ratio would have been more surprising when working with linked autosomes. The order is unimportant when dealing with only two alleles. Locus distance can be determined in linked autosomes by dividing the cross group sum with the total; in our example the distance is 0.1573 map units.
In a similar Autosomal Linkage three point cross, a ratio close to 16:3:1:6:6:1:3:16 ratio was observed. Linked genes were seen obvious in the matching 16's, 6's, 3's, and 1's, in the ratio. The 16's were identified as parental offspring, and the 1's were identified as double crosses. The 6's and 3's were thus the result from two different single crosses. The order of the alleles can be found most easily by viewing the phenotype of the two matching ratios; these phenotypes consist of two opposite alleles (i.e. vg,bl), and one allele (i.e. br) that is between them, resulting in a combination of bw-vg-bl. By determining the combination of alleles on the chromosome, adding the values such as 6.141 and 5.953 and adding the combined double cross ratio, we are able to divide by the total ratio and determine the map distance of one allele to another. The recombination frequency of area 1 (bw to vg) is a distance frequency of area 2 (vg to bl) is 0.255711 map units.
The overall outcome of both the corn experiment and the Virtual Flylab was mixed. The null-hypothesis was accepted in corn cob lab for cob 2, but rejected for cobs 1, 3, 4, and 5. Cob 3 and 4 were just under the limit of what can be accepted, but cob 5 was far beyond anything that could be expected. Such improvements as better parental breeding and identifying any mutation in the cobs might make it possible to produce better results in the future.
The Virtual Flylab produced remarkable, near perfectly predicted results for a monohybrid cross, dihybrid cross, and sex-linkage cross. Autosomal-linkage crosses- two-point cross and three-point cross- did not produce results that were expected, but only because the alleles were on the same chromosome, thus being linked genes (which initially have unpredictable ratios). Once the chromosomes were mapped, the original results were clarified.
Literature Cited
Grant, Valerie J. Maternal Personality, Evolution, and the Sex Ratio: Do Mothers Control the Sex of the Infant? London: Routledge, 1998.
Campbell, Reece, and Mitchell. Biology. Menlo Park: Benjamin/Cummings, 1999.
The New Encyclopedia Britannica. Macropûdia Vol. 11. Chicago: William and Helen Hemingway Benton, 1984.
California State University and Benjamin Cummings. Biology Labs On-Line. 2001. 10 October 2001. http://www.biologylab.awlonline.com
