Transmission of male recombination, segregation distortion and sex-ratio imbalance in Drosophila melanogaster

Transcription

1 J. Biosci., Vol. 10, Number 1, March 1986, pp Printed in India. Transmission of male recombination, segregation distortion and sex-ratio imbalance in Drosophila melanogaster GURBACHAN S. MIGLANI and VINDHYA MOHINDRA Department of Genetics, Punjab Agricultural University, Ludhiana , India MS received 18 May 1985; revised 30 December 1985 Abstract. From the first test cross progenies of control (no larval transfers; no ethyl methanesulphonate), physical stress (two larval transfers; no ethyl methanesulphonate) and 0 75% ethyl methanesulphonate (two larval transfers; 0 75% ethyl methanesulphonate)- treated F 1 (Oregon Κ + /dumpy black cinnabar, dp b cn) males of Drosophila melanogaster, respectively, 6,10 and 52 wild-looking first test cross males were again test crossed to obtain second generation. The overall percentages of male recombination detected in the second test cross progenies, in the three sets of experiments, were statistically the same as those in the first test cross progenies. Thus the enhanced male recombination caused by physical stress (with or without ethyl methanesulphonate) was transmitted to next generation. Non-reciprocal male recombination was observed in dp b but not in b cn region in both first and second test cross progenies. Three abnormalities, (i) production of wild-type flies in majority over dp b cn type, (ii) Non-Mendelian segregation at dp b and cn loci and (iii) sex-ratio differences for dp b cn and + b cn types observed in test cross progenies of F 1 males of Drosophila melanogaster were transmitted to next generation when induced with 0 75 % ethyl methanesulphonate but not when these abnormalities were induced with physical stress. The data suggest possible association of non-reciprocal male recombination, segregation distortion and sex-ratio imbalance in Drosophila melanogaster. In fact these may be representing different aspects of the same phenomenon. Keywords. Drosophila melanogaster, non-reciprocal recombination; male recombination; segregation distortion; sex-ratio. Introduction Recombination, normally considered to be absent in males of Drosophila melanogaster (Morgan and Bridges, 1919), can nevertheless be induced with a wide variety of physical and chemical agents (Thapar, 1982; Mohindra, 1984). With ethyl methanesulphonate (EMS) and chloroquine phosphate, using 3 genetic markers -dumpy (dp) black(b) and cinnabar(cn) in D. melanogaster, Miglani and Thapar (1983a) reported induction of non-reciprocal male recombination in dp b but not in b cn region. In addition, negative chromosomal interference and segregation distortions were also noted. In the present experiments, the nature and distribution of spontaneous, physical stress and EMSinduced male recombination events, segregation patterns and sex-ratio have been studied for two consecutive generations to provide information on the transmission of these phenomenon. Abbreviations used: EMS, Ethyl methanesulphonate; dp, dumpy; b, black; cn, cinnabar; TC 1, first test cross; TC 2, second test cross; CD, coefficients of dispersion; SD, segregation distortion. 153

2 154 Miglani and Vindhya Mohindra Materials and methods Two stocks of D. melanogaster, a standard wild-type laboratory strain (Oregon-K) and a 'mutant' stock homozygous for 3 second chromosome recessive markers, dumpy (dp: ) black (b: ) and cinnabar (cn ) were used. EMS (Sigma Chemical Co., Batch No. 89C-0439) was used as a probe. Miglani and Thapar (1983b) determined 0 75 % EMS to be LD 50 in second one-third part of larval life of D. melanogaster at 25 C. This dose of EMS was found to be most efficient in inducing recombination in males of D. melanogaster (Miglani and Thapar, 1983c). It was, therefore, decided to treat the developing F 1 (Oregon-K + /dp b cn) individuals with 0 75% EMS, mixed with food in ratio 1:9 in the second one-third part of larval life following the method of Miglani and Thapar (1983b). Accordingly, after 54 h of egg deposition at 25 C, the developing F 1 larvae were flushed out with distilled water and then physically transferred with a camel hair brush on to the medium with or without probe solution. After allowing the larvae to feed for 32 h, they were again flushed out and transferred on to the standard food medium. Each untreated or EMS-treated larva, thus, underwent two transfers referred to as 'physical stress' in this report. A control experiment was run simultaneously with the above experiments where neither EMS was added to the food nor usual two transfers of larvae were done. In the present investigation, the following 3 sets of experiments were performed: Set 1: No transfers, no EMS (control). Set 2: Two transfers; no EMS (physical stress). Set 3: Two transfers; 0 75 % EMS (0 75 % EMS). A two-day old wild-looking F 1 (having probable genotype (Oregon-K + /dp b cn)) male was crossed with 3 dp b cn females to get the first test cross progeny (TC 1 ). Every TC 1 adult was screened for sex and phenotype. A few of the wild-looking (probably + /dp b cn) TC 1 males were again test crossed to obtain the second test cross progenies (TC 2 ) on standard food medium. All the TC 2 adults were studied for sex and phenotype. In order to test the difference in frequencies of recombination recorded in the progenies of F 1 and TC 1 males from control, physical stress and 0 75% EMS experiments, z-test was used (Gupta, 1980). Chi-square test was used to determine whether or not any two complementary products or males and females in a particular phenotype fitted into the desired ratio (Gupta, 1980). The χ-square values based on poisson distribution of the number of recombinants observed, were calculated following the Snedecor-Irwin method (Snedecor, 1956). The coefficient of dispersion values were calculated using the method of Sokal and Rohlf (1969). Results The TC 1 progenies of 14,15 and 39 randomly selected D. melanogaster F 1 (Oregon-K + /dp b cn) males from control, physical stress and 0 75 % EMS experiments respectively, comprised of 3375, 5952 and individuals. Number of males and females recovered for each phenotype in TC 1 progenies is given in table 1. From these TC 1

3 Male recombination, distortion and sex-ratio in D. Melanogaster 155 Table 1. Pooled test cross progenies of F 1 and TC 1 wild looking males of D. melanogaster. Μ, Males; F, Females. progenies, 6, 10 and 52 wild-looking males from control, physical stress and 0 75% EMS experiments, respectively, yielded 868,1443 and 9028 flies in the TC 2 progenies on the standard food medium (table 1). The TC 1 and TC 2 progenies in the 3 experiments were compared for frequency of recombination, ratio of complementary products, segregation patterns and sex-ratio. The F 1 males of D. melanogaster in control experiment, yielded % recombination (table 2) which was non-significant statistically. The overall frequency of recombination in the F 1 males of physical stress experiment (1 125%) was higher (P < 0 001) than that in the F 1 males of control experiment A higher (P < 0 01) frequency of recombination was observed in F 1 males from 0 75 % EMS experiment (1 706%) than that in F 1 males from physical stress experiment. No recombinant appeared in the progenies of control TC 1 males. The frequency of recombination in pooled TC 2 progeny of TC 1 males from physical stress experiment (0 97 %) was significant (P < 0 01) as compared to the control. The TC 1 males from 0 75 % EMS experiment yielded % recombination in TC 2, which was not significantly different

4 156 Miglani and Vindhya Mohindra

5 Male recombination, distortion and sex-ratio in D. Melanogaster 157 from that in TC 1 males from physical stress experiment. The overall frequencies of recombination revealed in control experiments by F 1 males (in TC 1 progeny) and TC 1 males (in TC 2 progeny) were not significantly different. Similar was the situation in physical stress and 0 75 % EMS experiments. On the basis of number of F 1, or TC 1 males of D. melanogaster producing a particular phenotype (table 2), it was observed that in all the experiments all F 1 and TC 1 males produced the two parental phenotypes (+ + + and dp b cn) in their test cross progenies. The percentages of F 1 males producing a particular type of recombinant were higher than those of the TC 1 males producing the same recombinant phenotype, in all the experiments conducted. The percentages of flies producing no recombinants among F 1 males were lower than those among TC 1 males (table 2). On the contrary, the percentages of individuals producing one or more recombinant classes among F 1 males were higher than those among TC 1 males. The F 1 and TC 1 males of D. melanogaster were separately distributed in various classes on the basis of total number of recombinants recovered in the progeny of a particular male. The frequency distributions of F 1 and TC 1 males studied in control, physical stress and 0 75 % EMS experiments are given below. The figure preceding a hyphen represents the total number of recombinants recovered in test cross progeny of a particular male and the one followed by hyphen reveals the number of males in that class. F 1 males Control (14 males): 0 9; 1 1; 2 4. Physical stress (15 males): 0 3; 1 3; 3 4; 4 2; 10 1; 14 1; % EMS (39 males) 0 7; 1 5; 2 6; 3 1; 4 2; 5 7; 6 1; 8 1; 10 1; 11 1; 14 1; 15 2; 16 1; 17 1; 20 1; TC 1 males Control (6 males): 0 6 Physical stress (10 males): 0 7; 3 2; % EMS (52 males): 0 23; 1 7; 2 8; 3 2; 4 3; 5 2; 9 1; 10 2; 11 2; 12 1; Expected poisson frequencies were calculated from the above frequency distribution, including even the unrepresented classes. The χ-square values, calculated on the basis of differences between the observed and expected frequencies were significant (P < 0 001) from F 1 and TC 1 males from physical stress and 0 75 % EMS experiments (table 2). The coefficient of dispersion (CD) values, calculated from variance and mean of observed frequencies, for F 1 and TC 1 males of control, physical stress and 0 75 % EMS experiments, were greater than one, except for TC 1 males of control experiment where CD was zero (table 2). The number of D. melanogaster flies appearing in parental (+ + + versus dp b cn) and recombinant ( + b cn versus dp + +;+ + cn versus dp b + ; + b + versus dp + cn) pairs of complementary classes in F 1 and TC 1 males of control, physical stress

6 158 Miglani and Vindhya Mohindra and 0 75% EMS experiments were tested for 1:1 ratio (table 3). Only those complementary products that deviated from 1:1 ratio in a particular experiment are mentioned. The pooled TC 1 progenies from control, physical stress and 0 75 % EMS experiments produced flies in overwhelming majority (P < 0 001) over dp b cn type. The pooled TC 2 progeny from 0 75 % EMS experiment revealed the same trend. The TC 1 and TC 2 progenies from physical stress and 0 75% EMS experiments produced + b cn flies in convincing majority (P < 0 001) over its complementary type dp + +. Table 3. Comparison ofcomplementary products, segregation pattern and sex-ratio in test cross progenies of F 1 and TC 1 (wild-looking) males of D. melanogaster. <, former in minority over latter; >, former in majority over latter; n, non significant; NR, No recombination; M, males; d, distorted segregation; m, Mendelian segregation. a Ρ < 0 05; b Ρ < 0 01; c Ρ < Distorted (Ρ < 0 001) pattern of segregation was revealed in F 1 males of D. melanogaster from control, physical stress and 0 75 % EMS experiment at dp, b and cn loci (table 3). The TC 1 males from 0 75% EMS experiment also exhibited distorted (P < 0 001) pattern of segregation. The TC 1 males from control and physical stress experiments, however, revealed segregation at all the 3 loci in a Mendelian fashion. The number of males and females of D. melanogaster appearing in a particular phenotypic class of TC 1 and TC 2 progenies of control, physical stress and 0 75 % EMS experiments were tested for 1:1 ratio. The cases revealing deviation from this sex-ratio

7 Male recombination, distortion and sex-ratio in D. Melanogaster 159 are mentioned. In dp b cn class, the females of pooled TC 1 progenies of physical stress and 0 75 % EMS experiments and in pooled TC 2 progeny of 0 75 % EMS experiments were in majority over the males (P < 0 001). Wildtype (+ + + ) males appeared in lesser (P < 0 001) frequency than the females of pooled TC 1 progeny of the 0 75 % EMS experiment. Discussion Appearance of very high frequencies of recombination in dp b region in test cross progenies of TC 1 males from physical stress and 0 75% EMS experiments suggested that induced male recombination in D. melanogaster was transmitted to next generation. Woodruff and Thompson (1977) reported that even after keeping a male recombination line, extracted from a natural population, for five and a halfyears in the laboratory, a high level of recombination was observed. Cronmiller and Cline (1983) observed that by injecting unfractionated DNA into M-strain eggs, a small recombination was observed for more than one generation. In one of the males which produced 3 7% recombinants in F 1, recombination after increasing upto 13 2% in F 2 of the original injected male, suddenly stopped in F 3. The frequency of recombination, induced in the present study with physical stress and 0 75 % EMS in dp b cn region of D. melanogaster (in all the phenotypes collectively), remained statistically the same in TC 1 and TC 2 generations. Thus the enhanced male recombination caused by physical stress (with or without EMS) was transmissible. The test cross progenies of F 1 males from all the experiments revealed the superiority of wild type over the triple recessive type and that of + b cn over dp + + type. This imbalance in 1:1 ratio of complementary products is also reflected, in all the TC 1 progenies, in the form of distorted segregation patterns at the dp, b and cn loci. In all cases where distorted segregation was observed, wild-type alleles were present in higher frequency over mutant alleles. Woodruff and Layman (1980) concluded that recombination and meiotic drive in natural populations of D. melanogaster were due to elements on the second chromosome region between b ( ) and cn (2R 57 5), containing segregation distortion (SD) system. According to Peacock and Erickson (1965), only half of the sperm of Drosophila males were capable of fertilizing female eggs in the case of SD; this half included mainly those gametes that carried wild-type second chromosome. SD observed with physical stress and 0 75 % EMS in the present study may be the result of meiotic drive caused by effect(s) on the SD system. The distorted segregation patterns observed in the TC 1 progenies of 0 75 % EMStreated F 1 males were transmitted to next generation whereas the distorted segregation patterns exhibited by F 1 males from control and physical stress experiments were not. This may be attributed to the chemical, EMS. The present data suggested that a major portion of recombinants detected in TC 1 and TC 2 progenies of F 1 and TC 1 males of D. melanogaster from physical stress and 0 75 % EMS experiments appeared in clusters. For example, one cluster in TC 1 progeny of one 0 75% EMS-treated F1 male comprised of as many as 28 (19 males and 9 females) + b cn recombinants. These clusters might have lead to the appearance of two complementary products ( + b cn and dp + +) in unequal frequency. Furthermore,

8 160 Miglani and Vindhya Mohindra clusters of recombinants induced with physical stress and 0 75 % EMS showed nonpoisson distribution. Clustering of recombinants may be attributed to variable gonial multiplication of products of occasional cross overs as suggested by Whittinghill (1947), Whittinghill and Lewis (1961), Meyer (1952), Mickey (1963) and Sharma and Swaminathan (1968). In the present study, the F1 larvae were treated wtih 0 75 % EMS in the middle one-third part of larval life. During this period, spermatogonia are predominantly present in larval testis (Miglani and Thapar, 1983c). Majority of the recombinants recovered seem to be of premeiotic origin. Coefficient of dispersion values indicated that the recombination events were clumped (contagious). This suggested that occurrence of one recombination event enhanced the probability of second such event (Sokal and Rohlf, 1969). Since D. melanogaster has XX-XY type of sex-determination, random union between eggs and sperm at fertilization should result approximately equal numbers in XX (female) and XY (male) zygotes. In certain cases, sex-ratio imbalance was noted in the present study. In pooled TC 1 progeny (all phenotypes combined) of physical stress experiment, sex-ratio imbalance vanished in the next (TC 2 ) generation but that exhibited in pooled TC1 progeny of 0 75 % EMS experiment, however, revealed itself again in TC 2 generation; overall, females predominated over males. Considering various phenotypes separately, wherever sex-ratio imbalance was significant, in the parental (+ + + and dp b cn) phenotypes, females in all the experiments predominated over males and in + b cn class, males in such cases predominated over females, in both TC 1 and TC 2 progenies of D. melanogaster. This suggested that male recombinant gametes carrying Y-chromosome are playing greater role in final realization of the recombinant adults as compared to those carrying X-chromosome. The data suggest possible association of non-reciprocal male recombination, segregation distortion and sex-ratio imbalance; in fact these may be representing different aspects of the same phenomenon. Acknowledgement We are thankful to Dr. Kulbir S. Gill for valuable discussions during the course of this investigation. References Cronmiller, C. and Cline, T. W. (1983) Genetics, 104 (Suppl. Part 2), 519. Gupta, S. P. (1980) Statistical Methods, (New Delhi: Sultan Chand and Sons) p Meyer, H. U. (1952) Drosophila Inf. Serv., 26, 111. Mickey, G. H. (1963) Drosophila Inf. Serv., 38, 60. Miglani, G. S. and Thapar, A. (1983a) X V International Congress of Genetics, New Delhi, Abstracts (Part I), p Miglani, G. S. and Thapar, A. (1983b) Drosophila Inf. Serv., 59, 86. Miglani, G. S. and Thapar, A. (1983c) Indian J. Exp. Biol., 21, 644. Mohindra, V. (1984) Non-reciprocal recombination studies in Drosophila melanogaster, M.Sc. Thesis, Punjab Agricultural University, Ludhiana.

9 Male recombination, distortion and sex-ratio in D. Melanogaster 161 Morgan, J. Η. and Bridges, C. B. (1919) Contribution to the Genetics of Drosophila melanogaster, (Washington: Carnegie Inst.), Publ. No. 278, p Peacock, W. J. and Erickson, J. Ε. (1965) Geneties, 51, 313. Sharma, R. P. and Swaminathan, M. S. (1968) Drosophila Inf. Serv. 43, 121. Snedecor, G. W. (1956) Statistical Methods (Amsterdam: Iowa State College Press). Sokal, R. R. and Rohlf, F. J. (1968) Biometry (San Francisco: W. H. Freeman and Company) p Thapar, A. (1982) Effect of various chemicals on the frequency of crossing over in Drosophila melanogaster, M. Sc. Thesis, Punjab Agricultural University, Ludhiana. Whittinghill, M. (1947) Genetics, 32, 608. Whittinghill, M. and Lewis, B. W. (1961) Genetics, 46, 459. Woodruff, R. C. and Thompson, J. Ν. (1977) Heredity, 38, 291. Woodruff, R. C. and Layman, R. F. (1980) Am. Nat., 116, 297.