No Linkage of Yellow Body and Sepia Eye Mutations in Drosophila: Yellow Body X-linked and Sepia Eye Autosomal

Written by Brandon

Abstract:

The theory of independent assortment was first discovered by Gregor Mendel; however when the idea of genetic inheritance was revisited by Thomas Morgan, it was determined that it was not universally true of all alleles and for all organisms. Morgan’s experiments with Drosophila showed a difference in trait inheritance. Chromosomal Theory of Inheritance suggested that multiple genes are inherited in a linked fashion that is predictable and observable. This study focused to determine linkage between the recessive mutant alleles of yellow body and sepia eyes in Drosophila. Through performing a di-hybrid parental cross using true breeding strains, the yellow body allele was determined to be sex-linked and the sepia eyes allele was autosomal, both originating from separate chromosomes. Following with an F₁-cross and a Chi-square test, it was determined that there was no linkage between the two mutant alleles.

Introduction:

Modern genetics originated from the theories of Gregor Mendel and his extensive and historically notable experimentation with pea plants (Klug et al, 2009). By crossing true breeding strains of pea plants, Mendel discovered that traits are inherited through both parents in sexually reproducing species (Klug et al, 2009). Mendel determined that traits are inherited by unit factors, or alleles, in pairs that are derived from each parent and that each of these paired alleles determine inherited physical characteristics of the offspring (Klug et al, 2009). Each parent passes on only one unit factor to their offspring through segregation during the formation of gametes (Klug et al, 2009).  Mendel also discovered that alleles exhibit dominant and recessive relationships when paired (Klug et al, 2009). Mendel’s work during the 1800’s was not well noted in the scientific community until the 1900’s, his work has deemed him a father of the modern study of genetics (Klug et al, 2009).

When Mendel’s work was rediscovered in the 1900’s by Thomas Morgan, it was determined that Mendel’s theories were not universally true for all species but they were used to create the Chromosomal Theory of Inheritance (Klug et al, 2009). This took into account that relationships of dominance and recessive were not the only factors in allelic inheritance, but some were inherited together in predictable ways depending on the locations of the genes on the chromosomes (Klug et al, 2009). The type of chromosome also plays into the inheritance patterns; affects correlated by locations on sex or autosomal chromosomes (Klug et al, 2009). Morgan’s theories originated by studying Drosophila, or fruit flies, which allowed for quicker reproductive studies through shorter lifecycles than those of Mendel’s pea plants (Klug et al, 2009).

Drosophila species are well noted in genetic studies for their similarities to humans: heterogametic, diploid and eukaryotic organisms (Klug et al, 2009; Westmont College, 2013). Drosophila have proven to be a key species in understanding human maladies: cancer, diabetes, metabolic diseases, and other genetically derived diseases due to the genetic similarities (Bier, 2005). The likeness to humans and Drosophila was seen in sex chromosomes, as males are heterogametic (XY chromosomes) and females are homogametic (XX chromosomes) (Klug et al, 2009). Morgan’s Drosophila studies displayed non-Mendelian ratios; implying the allelic inheritance was not always through independent assortment (Klug et al, 2009).  There were other influencing factors such as sex-linkage or gene-linkage that prompted different ratios in the progeny (Klug et al, 2009). When two traits were seen to be mutually inherited they were said to be linked; likewise, when one gene seems to be inherited by the heterogametic sex (males) more often than the homogametic sex (females) then that allele was considered linked to the sex chromosome (Klug et al, 2009). Morgan developed new crossbreeding protocols for discerning trait linkage through autosomal or sex chromosomes by a reciprocal cross (Klug et al, 2009). The breeding heterozygous females with homozygous males, displaying only recessive traits for the alleles being tested, prompted the idea of sex-linkage (Klug et al, 2009). This test determined which sex chromosome the trait would be found on: the Y chromosome depicted only inheritance by males, and the X chromosome depicted inheritance by males and females (Klug et al, 2009). Mendel and Morgan’s crosses were key in producing a study of gene linkage through the study of progeny created by di-hybrid crosses of pure breeding strains (P-cross), an F₁ cross, and a reciprocal cross (Westmont College, 2013).

While Mendel compared only crosses of true breeding strains, Morgan used strains that exhibited mutated traits, or phenotypes, which were typically homozygous recessive and varied in appearance from the wild type (WT) strains (Klug et al, 2013). This allowed Morgan to see two separate strains and the progeny from the various types of crossbreeding (Klug et al, 2009). Mendel’s Parental cross (P-cross), the resulting filial (F₁₎ generation, showed uniformity in progeny’s phenotype; however, Morgan observed that there were differences in the progeny and inheritance ratios depending on the species and strains crossed (Klug et al, 2009). Morgan used Mendel’s F₁-cross, using the previous cross’s progeny, to see if the F2 generation would conform to Mendel’s ideal ratio of phenotypes (Klug et al, 2013). Later, Morgan opted to for a reciprocal cross (Test cross) using virgin females from the F₁ generation and males with double recessive traits (Klug et al, 2009; Westmont College, 2013). The test cross was used to show allelic linkage to sex chromosomes by showing different inheritance ratios of male and female progeny than the P-cross or the F₁-cross (Klug et al, 2009; Westmont College, 2013).

Morgan determined that some alleles or genes are typically inherited together with specific predictable patterns (Klug et al, 2009; Westmont College, 2013). The probability of gene linkage was correlated to the genes’ proximity to each other on a single chromosome (Klug et al, 2009; Westmont College, 2013). Distant genes on the same chromosome or on separate chromosomes did not display linkage (Westmont College, 2013). Gene linkage was discovered to be the result of two genes crossing-over together during meiosis due to proximity, or gamete formation (Westmont College, 2013). Using this information and technique, genes and chromosomes have been mapped in order to further understand the patterns of inheritance and the genetics behind Drosophila and humans (Westmont College, 2013).

Drosophila, although characteristically very different from humans, shows many genetic similarities to humans (Bier, 2005; Burdett and van den Deuvel, 2004). This species of fly, although not technically categorized as a laboratory animal, has been very useful throughout genetic experimentation and the developing breadth of genetic understanding (Bier, 2005; Burdett and van den Deuvel, 2004; Yamamoto, 2010). The experimentation with Drosophila, in comparison to other species, has been very advantageous because of the fast reproduction, high quantity of progeny and overall ease of culturing and handling (Bier, 2005; Burdett and van den Deuvel, 2004; Yamamoto, 2010). Due to the many advantages of using Drosophila in studies, research using them has been ongoing for decades. This experiment was performed using two recessive mutants of Drosophila. The mutants used were yellow body (y) and sepia colored eye (se) flies. The experiment was set up similarly to Morgan’s protocols: using P-crosses, F₁-crosses, and Test crosses (Westmont, 2013). These crosses were used to determine if the mutated alleles were linked and the relative location on sex or autosomal chromosomes (Westmont College, 2013).

Experiment and Results:

This experiment was performed using two distinctly different and true breeding mutant strains of Drosophila. The strains were collected and bred to ensure that they were true breeding before being used for any crosses. The intent was to discover any gene linkage or sex linkage of the two mutations through observing the progeny of the crosses. Only virgin female flies were used in the crosses because Drosophila females only need to be inseminated once in their lifecycle and can store semen for future fertilization of eggs (Westmont College, 2013). In order to ensure that only virgin females were used, the adults of the pure strains were dumped 6 hours prior to performing the crosses to ensure only newly hatched virgin progeny would be used. This method helped to eliminate contamination of the strains and crosses by preventing the adults from breeding with the newborns and the used of impregnated females by the parents or by their brothers. The two mutants used were yellow body (y) and sepia eye (se) Drosophila. The y strain exhibited a light, creamy-yellow colored body color but iridescent red wild type (WT) eyes. The se strain exhibited a WT brown body color and eyes that were dark brown. These two strains had independent and uniquely mutated traits and WT traits for the rest of their traits. In both strains males (♂) and females (♀) exhibited these phenotypes.

The P-crosses crossed males and females of both mutants: vials #1 and #2 contained the crosses of y females and se males and vials #3 and #4 contained the crosses of se females and y males. There were no born progeny from vials #3 and #4, and vials #1 and #2 did not display uniformity of the F₁ generation. The female progeny exhibited all WT characteristics and the male progeny were all y with WT eyes (Table 1a). With the male progeny all exhibiting the y phenotype and all progeny exhibiting the WT eye phenotype it suggests that the y allele was linked to the X chromosome (Table 1). The female progeny did not display the y phenotype due to their being heterozygous for the allele. The uniformity of the F₁ generation and the corresponding phenotypes were not observed. Similarly, the fact that none of the F₁ females or males exhibited the se characteristic indicated that the allele was masked by a dominant allele.  According to Mendelian genetics this would designate the se allele as recessive (Klug et al, 2009). With the lack of progeny from vials #3 and #4, there was no other data to compare the P-cross progeny (Table 1b; Table 1d).

The F₁ progeny collected from the successful P-cross were used to create a F1-cross and a Test cross. The F₁-cross and the progeny, the F₂ generation, were not conforming to the expected values when both sexes were observed independently, but were more conforming when  observed together (Table 4a,b). When considering the likelihood of random assortment being the controlling factor of the observed F₂ generation, a χ² test was performed to determine if the observed phenotypes were simply due to chance or if there were influencing factors involved. The Test cross was another unsuccessful cross which hindered the study. Unfortunately the parents used in the Test cross did not reproduce and died which does not provide the chance to corroborate with the F₁ data.

The y body mutation was deduced to be linked to the X chromosome through observation of the P-cross. The progeny from vials #1 and #2 in the P-cross dictated x-linkage to this allele. All of the male progeny from the F₁ generation inherited this trait from the true breeding y female parents, while the female offspring in the F₁ generation exhibited double heterozygosity for both mutations (Table 1). The eye mutant, se, exhibited no sex-linkage, followed Mendelian inheritance patterns, and was categorized as autosomal (Westmont College, 2013; Table 1). The F₂ generation was dissimilar to the F₁ generation in the fact that they showed greater variability with the display of one, both, or none of the mutations (Table 2). The F₂ generation displayed equal inheritance of the mutant alleles between the sexes with the greatest number of offspring exhibiting all WT alleles or only the y body mutation (Table 2).

Discussion:

The χ² test revealed p-values of greater than 0.05 in the sexes independently of the F₂ generation. These results depict that the yellow body (y) and sepia eyes (se) alleles do not follow Mendelian genetic theory and do not conform to the ideal ratios of phenotype inheritance. The y gene exhibited X-linkage as seen through the P-cross and the F₁-cross. Males tended to exhibit the y gene more often than the females of the same generation. This was confirmed through FlyBase that the y gene is found on the X chromosome and therefor is more easily manifested in the heterogametic males (McQuilton et al, 2012).

This experiment should be repeated in order to discern how or why many of the crosses were unsuccessful in living and reproducing. All of the failed crosses were created using pure breeding males that exhibited the y characteristic. Many studies have sought to determine why the males with the y gene do not successfully reproduce. Some suggested that the y males had decreased fertility, especially when placed in contact with a WT female (McQuilton et al, 2012). Other studies have noted that there may be social or behavioral differences between the strains, decreased sperm penetrance ability and even strain sterility that prevented the y males from reproducing with a WT female (Alipaz, Wu and Karr, 2001).

Materials and Methods:

The most crucial aspect of this study was to identify the physical characteristics of the Drosophila being used. The typical wild type (WT) fruit fly has iridescent red eyes and a brown body (Yamamoto, 2005). In contrast, the mutants used exhibited cream-yellow body colors or dark brown (sepia) eyes.   Sexing was equally as important. Male Drosophila tended to characteristically smaller in overall size, have a distinguished genital arch and exhibit sex combs on the fore limbs (Westmont College, 2013). Females in contrast were larger in size, had a characteristic vaginal slit and did not exhibit sex combs (Westmont College, 2013). Newborn and virgin flies tend to be more difficult to sex, as their secondary sexual characteristics many not be as well defined, but the males again tend to be smaller and the females tend to be very plump (Westmont College, 2013). Flies were always handled and observed under a well light dissection scope while maintaining sedation with Carbon Dioxide (CO₂) gas.

Approximately  6 hours prior to establishing the first cross, the adult flies from the two yellow body and sepia eye strains were removed from their cultures and transferred into new culture tubes. The existing culture then only contained eggs, larvae and pupae. This process allowed for the collection of only virgin individuals as they would have been born within the previous 6 hours. The virgins of the two mutant strains were sedated and sexed. At least 3 to 5 virgins of each sex and distinct mutation were used in each cross. In the event of too few virgin males, the adult non-virgin males were supplemented into the cultures. For the Parental cross (P-cross), the mutants were then transferred into new culture tubes for the two P-crosses: vials #1 and #2 contained ♀se X ♂y and vials #3 and #4 contained ♀y X ♂se (Table 1).  After flies were segregated and placed into culture tubes, a small amount of yeast and a lattice were added to the tubes and the cultures were placed into a controlled environment. It was very important to dump the adult flies in the cultures at least once a week to ensure that the progeny were not breeding in the culture.

After two weeks, the F₁-cross was created using a similar method to the P-cross. 6 hours prior to the cross preparation, the adults were dumped and transferred to new culture tubes. In this study, vials #3 and #4 could not be dumped because there were no live adults. The progeny from the P-cross were transferred, sedated, observed and sexed. Three to five of the virgin females from these cultures were transferred into new culture tubes prepared with yeast and lattice: vials #5, #6, #7, and #8 (Table 2). This cross could only be performed using the progeny from vials #1 and #2 due to the failure of the cultures and no progeny in vials #3 and #4. After these tubes were cultured, the progeny were transferred, sedated, sexed, segregated by phenotypes and counted.

The Test cross was performed at the same time as the F₁-cross. This cross used the F₁ generation virgin females as well but used double mutant males, which exhibited the yellow body and the sepia eye phenotypes. Three to five of the virgin females were transferred into new culture tubes and followed by 3 to 5 of the double mutant males in vials #9 and #10 (Table 3). The progeny from the Test cross were to be transferred, sedated, sexed, segregated by phenotype and counted similarly to the F₁-cross progeny; however the Test cross failed to produce progeny so this was not performed.

Using the data collected from the P-cross, F1-cross and Test cross; statistical analysis was performed using the Chi-square (χ²) test. This test was used to determine if the observed ratios of sex and phenotype corresponded to random assortment or if there was linkage or other factors influencing in the inheritance pattern. This test was only performed using the F₂ generation due to the F₁-cross being the only successful cross with a quantity of progeny to reasonably perform the test. Expected values were calculated using the Punnett square to generate expected ratios which were applied to the total number of progeny (Table 2b, Table 4a). The statistical analysis determined whether there was linkage involved in the inheritance patterns as well as if the cross and progeny conformed to Mendel’s theory of independent assortment.

Literature Cited:

Alipaz, J.A., Wu, C.I., Karr, T.L. (2001). “Gametic incompatibilities between Races of Drosophila melanogaster.” Proceedings: Biological Sciences. 268(1469), 789-795

Bier, E. (2005). Drosophila, the golden bug emerges as a tool for human genetics. Nature Reviews, 6, 9-21.

Burdett, H. and van den Heuvel, M. (2004). “Fruits and flies: a genomic perspective of an

invertebrate model organism.” Briefing in Functional Genomics and Proteomics, 3, 257-

265.

Klug, W.S., Cummings, M.R., Spencer, C.A., and Palladino, M.A. (2009). Concepts of Genetics.

San Francisco, CA: Pearson Benjamin Cummings.

McQuilton, P., St. Pierre, S.E., and Thurmond, J. (2012). FlyBase 101 – the basics of navigating

FlyBase. FlyBase Consortium. [Online]. Available: http://www.flybase.org. (accessed

April 2013).

Westmont College. (2013). Genetics Laboratory Manual. Santa Barbara, CA.

Yamamoto, M.T. (2010). “Drosophila: Genetic Resource and Stock Center; The National BioResource Project.” Experimental Animals. 59(2), 125-138
(a)                                                              

(b)

 (d)        

(c)

Table 1: Parental Crosses

Data from the P-crosses of the F₁ generation of Drosophila: (a)quantity of progeny from the successful P-cross with the differences in phenotype and sex shown; (b)unsuccessful P-cross that had no offspring; (c)Punnett square correlates the cross in Table 1(a), genotypes of the parent and offspring; (d)Punnett Square correlates Table 1(b), genotypes of the parents and the potential offspring. Although there was no data for the P-cross shown in (b) and (d), it is useful to visualize the differences in the two crosses in the phenotypes and genotypes of the parents and offspring.

^$$(y—yellow body allele; y+—wild type allele at y locus; se—sepia eye allele;

se+— wild type allele at se locus;⌠—  Y-chromosome)

(a)   

    (b)

Table 2: F₁-cross

(a)quantity of progeny by vial, sex and phenotype; (b)Punnett square correlates the F₁-cross, the genotypes and phenotypes observed and recorded in (a).

(a)

(b)

Table 3: Test Cross

(a)  Data from unsuccessful Test cross, no data to report (b) Punnett square correlates with that cross in (a). Table included for visualization of potential progeny genotypes and phenotypes.

(a)

(b)

Table 4: Chi-square test

Data from Table 2 (a) calculation of  expected values from Punnett Square (Table 2b) for expected ratio, calculation of the actual expected, and totals of each sex and both sexes; (b) breakdown of the χ2 tests performed for females, males, and both sexes.