Genetic characterization of spontaneous mutations in Drosophila willistoni. I. Exchange and non-disjunction of the X chromosome.

Goñi, B., C. Parada, C. Rohde*, and V.L.S. Valente*.  Sección Genética Evolutiva, Facultad de Ciencias, Instituto de Biología, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay. E-mail: bgoni@fcien.edu.uy. *Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul. Caixa Postal 15053. CEP 91501-970. Porto Alegre, RS, Brazil.

     Drosophila willistoni is a Neotropical species found in the warm and humid forest of South America and has been a model organism for evolutionary studies (reviewed by Ehrman and Powell, 1982).  D. willistoni is the only member of the willistoni subgroup that inhabits domestic and natural environments of Uruguay (Goñi et al., 1997, 1998). These populations of D. willistoni located at southern latitudes offer an invaluable opportunity to study the genetic variation and the evolutionary history of geographically marginal populations. Recent studies of inversion polymorphisms in D. willistoni populations from the southern region of Brazil and Uruguay (Valente et al., 2002a, b) revealed a higher level of chromosomal polymorphism in samples from Uruguay than from the Brazilian Southernmost State of Rio Grande do Sul. These observations encourage us to investigate on the causal agent(s) of the genesis of the genetic variability in natural populations of D. willistoni.

     Since 1998, our group has been working on the isolation and the genetic characterization of spontaneous visible mutations from isofemale lines of D. willistoni collected in the wild (data not shown), aiming to find mutant loci with excellent homology to D. melanogaster. Here we report the genetic characterization of several spontaneous X linked mutations of D. willistoni isolated from the EM1.00 hypermutable line, and we describe X chromosome non-disjunction and recombination events observed in the course of this study. The EM1.00 line was established from flies emerging from rotten fruits of the “madroño” tree, Arbutus unedo, collected at the garden of Faculty of Agronomy (34° 53´ S; 56° 16´ W), Montevideo city, in April 2000. Two mutations were promptly segregating from the F₂ progeny of the EM1.00 line in June 2000: one affecting the color of the body, the yellow mutation, and eye color mutants, referred here as dull red mutation. The yellow mutants show the body, bristles and wings yellow; it parallels the description of yellow reported by Spassky and Dobzhansky (1950). (The dull red mutation is an autosome recessive locus, unpublished data). Later on, other mutations were detected and isolated, among them, two recessive eye color mutations, white and pink mutations. White mutants have white eyes and translucent ocelli, as described by Spassky and Dobzhansky (1950) and Poulson and Counce (1960). On March 13^th, 2001, four white eye color males emerged from one vial which were segregating the dull red eye color allele. Soon after, on April 6^th 2001, a new eye color mutant was detected in several males from a cross between dull red virgin females and two white males. The eye color is translucent dull ruby in young individuals darkening with age to brownish tone. Considering that eye color in young individuals resemble the “pink” (p¹ allele) mutation in D. melanogaster, it was named pink (p). This mutant may be an allele of “peach” mutation described by Lancefield and Metz (1922). We do not exclude that the dull red allele was segregating in either the white and pink mutant lines. All these mutant lines have good viability. For the genetic analysis of spontaneous mutations we used a wild stock, 17A2, that had been kept in the laboratory since 1991. It was established from a natural population collected in a wilderness area (El Dorado do Sul, southern Brazil, 30° 05´ S; 51° 39´ W).

     Flies used in mating experiments were separated randomly from flies that emerged on the same day and collected virgin every three hours. Individuals were selected with the help of an aspirator since

Table 1. Results of linkage test of white, pink and yellow mutations in D. willistoni.

Parental cross		Progeny
Females        Males		Females +	Males w	Males p	Males y	Matroclinous (%)	Patroclinous (%)	Total
w EM1.00 x 17A2		1098	1027	---	---	8 (0.73)	3 (0.29)	2136
p  EM1.00 x 17A2		1031	---	1135	---	0	0	2166
y EM1.00 x 17A2		849	---	---	765	0	2 (0.12)	1616
w EM1.00 x y EM1.00		299	172	---	---	0	2 (0.42)	473
p EM1.00 x y EM1.00		171	---	127	---	0	0	298

Table 2. Alellism test between the white and pink X-linked mutations in D. willistoni

Parental cross		0Nº of progeny
Females        Males		Females +	Males w	Males p	Patroclinous (%)	Total
w EM1.00  x  p EM1.00		1242	1042	----	13 (0.57)	2297
p EM1.00  x  w EM1.00		804	----	809	0	1613

Table 3. Recombination value between w ~ p, w ~ y and p ~ y regions of D. willistoni

Female genotype1	Male progeny Class                 N		Region	Recombination value 2 (%)
w/p Total	[w] [p] [+]	3193 2701 180 6074	w ~ p	6.23
p/w Total	[w] [p] [+]	3030 2506 132 5668	w ~  p	5.27
w/y Total	[w] [y] [y w] [+]	580 659 60 74 1373	w ~ y	9.76
p/y Total	[p] [y] [y p] [+]	351 380 36 38 805	p ~ y	9.19

¹The female parental strain is located at the left side of the genotype column. ² Estimated values from two male class progeny (see text).

     Routine cytological method (Ashburner, 1967) was applied to examine the chromosome gene arrangement of the mutant lines. Cytological data indicate that all but one of the EM1.00 lines and the the use of triethylamine vapors, the Drosophila anesthetic substance commonly used in our lab, is known to affect the fertility of flies (Fresia, 2001). Progeny was scored until the 21st day to avoid overlapping generations. Flies were cultured at 25 ± 1ºC with standard corn-yeast-agar media.17A2 strain are polymorphic for the XR E inversion (described in Regner et al., 1996;  Rohde, 2000;  Valente et al., 2002b). Individuals from the white mutation are homozygous for the XR E inversion. The XL has no inversion. The autosomes segregate some of the paracentric inversions commonly found in segregating populations from the southern region of Brazil and Uruguay (Valente et al., 2002a, b); they are: IIL D, E, F and B inversions, and the IIIJ, B. C and H inversion.  The IIR arm has no inversion.

     Two genetic tests were performed: linkage and allelism tests. For the linkage test, single mutant females were crossed with two 17A2 wild type (or mutant) males. Two additional replicates were performed for each cross. As shown in Table 1, F₁ progeny from these crosses indicate that the white, pink, and yellow loci are located on the X chromosome. Moreover, exceptional F₁ progeny, identified as matroclinous or patroclinous flies, revealed the occurrence of X non-disjunction events at rates between 0.12 to 0.79% in some of the EM1.00 mutant lines tested. The exceptional progeny were tested for fertility.  All the females produced offspring while the males were all infertile. For the allelism test, we performed the reciprocal crosses between white and pink mutant flies. The F₁ progeny were analyzed and then mass mated, and the recombination value of F₂ male progeny then analyzed (Table 3). Table 2 shows the results of the F₁ progeny. Data indicate that the white and pink loci are distinct X-linked recessive mutations and confirm that the white EM1.00 mutant line does not mask any pink mutant allele. Once more, exceptional males at a rate of 0.57% were observed in the F₁ progeny from white parental females.

     Table 3 shows the results on the recombination value between three regions on the X chromosome. The X chromosome in D. willistoni is a metacentric chromosome (Dobzhansky, 1950; see also Santos-Colares et al., 2002) and its left and right arms are homologues to the D. melanogaster X and 3L (see Ehrman and Powell, 1982). Recombination values in the interval w~-p were estimated from the recombinant [+] and the non recombinant [p] classes, i.e., [180/2701 + 180]100 = 6.23%; therefore, the estimate should be taken cautiously. The map distance in the interval y ~ w and y ~ p are quite similar in values 9.76 cM and 9.19 cM. Our data agree with the map distances reported for the y ~ w interval by Spassky and Dobzhansky (1950). These authors map the white locus to the position 1.1 and the yellow locus to the position 12.0. However, Lancefield and Metz (1922) reported the yellow locus in the middle of the X chromosome to the position 42 (or either 0) and the peach locus to the position 44 (or +2). If the pink mutation is an allele of the peach locus, then the location of pink should be in the middle, between the yellow and white loci. Studies of linkage map of D. willistoni have faced the unavoidable difficulty of extensive nonoverlapping inversion polymorphisms, resulting in linkage maps only valid for the strains studied (Ehrman and Powell, 1982).

     In the course of identifying the sex chromosome relationships in D. willistoni, Lancefield and Metz (1921) examined progeny of flies with recessive sex linked mutations looking for the occurrence of primary non-disjunction. Only two out of 150 cultures produced the expected exceptional progeny. A single exceptional female from one line, “line A”, produced an exceptional female, six of her daughters produced 1.14% of exceptional progeny. XXY chromosome constitution was confirmed in the daughters of exceptional females from line A. The other line, “line B” gave results essentially similar to line A (1.1%). The average frequency of secondary non-disjunction in D. willistoni observed by Lancefield and Metz (1921) was 1.7%. XXY females are indistinguishable from normal XX females (other than being matroclinous), but show abnormalities in X chromosome disjunction. These abnormalities, known as secondary non-disjunction, are quite different from primary non-disjunction.  The rates are quite a lot lower, i.e. primary non-disjunction in D. melanogaster is about 0.05-0.1%, and usually a difference in the number of matroclinous females and patroclinous males is observed, the latter being the most frequent (see references in Ashburner, 1989). Our results suggest that secondary non-disjunction in D. willistoni occurred at the average frequency of 0.43%.  Exceptional progeny was observed from XXY females of the yellow and the white EM1.00 lines. Since all these mutant lines derive from the same isofemale line, XXY females most probably were undetected during the inbred experiments and can be traced to the first broods of the isofemale line EM1.00 or the wild caught female itself (note above, the outcome of the first mutant flies).  Our data do not allow us to distinguish between “reductional” and “equational” primary exceptions, since equational exceptions are homozygous daughters for recessive markers, whose mothers are heterozygotes. Lancefield and Metz (1921) found two certain cases of equational non-disjunction in D. willistoni. How frequent non-disjunction events occur in D. willistoni populations, and how chromosome inversions and exchange in females influence the occurrence of secondary non-disjunction, are some of the biological problems in the evolutionary biology of D. willistoni.

     Acknowledgments: The authors wish to thank the Bloomington Drosophila Stock Center for sending us several D. melanogaster mutation lines. We also thank Prof. E. Scvortzoff (PEDECIBA, Universidad de la República), for the critical reading. This study was partly supported by CSIC (Uruguay), CNPq, FAPERGS and PROPESQ-UFRGS (Brazil).

     References: Ashburner, M., 1967, Chromosoma 27: 47-63; Ashburner, M., 1989, Drosophila. A Laboratory Handbook. Vol. 1. Cold Spring Harbor Laboratory Press, New York;  Dobzhansky, Th., 1950, Heredity 41: 156-158;  Ehrman, L., and J.R. Powell 1982, The Genetics and Biology of Drosophila, 3b (Ashburner, M., H.L. Carson and J.N. Thompson, jr., eds.), pp: 193-225. London, Academic Press;  Ferry, R.M., R.C. Lancefield, and C.W. Metz 1923, J. Hered. 14: 372-384; Fresia, P., J. Graneri, and B. Goñi 2002, Dros. Inf. Serv. 84: 141-142;  Goñi, B., M.E. Martínez, and P. Daguer 1997, Revta. bras. Ent. 41: 89-93;  Goñi, B., M.E. Martínez, V.L.S. Valente, and C.R. Vilela 1998, Revta. bras. Ent. 42: 131-140;  Lancefield, R.C., and C.W. Metz 1921, Proc. Natl. Acad. Sci, 7: 225-229;  Lancefield, R.C., and C.W. Metz 1922, Am. Nat. 56: 211-241;  Poulson and Counce 1960, Dros. Inf. Serv. 34: 69;  Regner, L.P., M.S.O. Pereira, C.E.V. Alonso, and V.L.S. Valente 1996, J. Heredity 87: 191-198;  Rohde, C., 2000, PhD Thesis, Universidade Federal do Rio Grande do Sul, Brasil;  Santos-Colares, M.C., B. Goñi, and V.L.S. Valente 2002, Dros. Inf. Serv. 85: (this issue);  Spassky, B., and Th. Dobzhansky 1950, Heredity 4: 201-215;  Valente, V.L.S., B. Goñi, V.H. Valiati, C. Rohde, and N.B. Morales 2001a, Genet. Mol. Biol. (in press);  Valente, V.L.S., C. Rohde, V.H. Valiati, N.B. Morales, and B. Goñi 2001b, Dros. Inf. Serv. 84: 55-59.