Regular Article
Identifying a major gene on Chromosome 2 that controls the XY pairing in Drosophila ananassae Doleschall, 1858
2025 Volume 90 Issue 3 Pages 165-171
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2025 Volume 90 Issue 3 Pages 165-171
Drosophila ananassae males have a diploid chromosome number of 2n=8. During the first meiotic division, the X–Y and 4th chromosomes pair to form a stable heterologous chromosome association that ensures normal chromosome segregation. The pairing sites have been extensively studied in D. melanogaster; however, the genetic controlling of the pairing is elusive. This study aimed to identify the gene controlling the X–Y pairing in D. ananassae. In the somatic cell division of the L8 strain of D. ananassae obtained from Sri Lanka, numerical variation in the number of Y and the chromosome 4 was observed among individuals. Five chromosomal types (XX44, XXY44, XXYY44, XXY444, and XXY4) were observed in the females and four (XY44, XO44, XYY44, and XY444) in the males. The chromosomal variation in the L8 strain was attributed to chromosome non-disjunction, resulting from an incomplete pairing of the X and Y chromosomes during male meiosis. The genetic and cytological analyses revealed a major gene controlling to X–Y paring on the right arm of the chromosome 2 in D. ananassae.
Like humans, Drosophila females have two X chromosomes (XX), whereas males have one X and one Y chromosome (XY). However, the mechanism of sex determination differs from that in humans and is based on the number of X chromosomes. Two X chromosomes produce females, whereas one X and one Y chromosome produce males. A crucial factor in Mendelian genetics is understanding how homologous chromosomes accurately recognize each other and pair during meiosis. Equally important is understanding how non-homologous X and Y chromosomes pair and are distributed in roughly equal proportions in sperm cells
Studies on male meiosis by McKee and colleagues (McKee et al. 1992; reviewed by McKee et al. 2012; Adams et al. 2020) showed that X–Y chromosome pairing during meiosis in male Drosophila melanogaster is mediated by the nucleolar organizing region of X heterochromatin (Xh). Deletion of Xh disrupts X–Y pairing, resulting in frequent X–Y nondisjunction. Inserting a cloned rRNA gene into Xh partially rescued normal pairing. Notably, the complete deletion of the rRNA transcription unit did not prevent X–Y pairing as long as most of the intergenic spacer region remained intact. Although progress has been made in identifying the pairing sites, reports on their genetic control are lacking.
It is known that homologous chromosomes do not recombine during meiosis in male D. melanogaster (Morgan, 1912, 1914) and that synaptonemal complexes are absent (Grell et al. 1972). Conversely, in male Drosophila ananassae, the first meiotic division is characterized by (1) chiasmata formation (Matsuda et al. 1983; Goñi et al. 2006), with recombination occurring in males, as reported in laboratory studies (Moriwaki 1937a; Kikkawa 1938; Hinton 1970; Moriwaki et al. 1970; Moriwaki and Tobari 1973) and observations of natural populations (Goñi et al. 2012, 2016), and (2) the rDNA region corresponding to the bobbed (bb) locus is located on the short arm of the Y chromosome and the distal region of the long arm of chromosome 4 (Moriwaki 1937b; Kikkawa 1938; Roy et al. 2005). Thus, the bb locus is absent on the X chromosome, but is present on the X and Y chromosome pair and on the rDNA region of chromosome 4, which forms a tetravalent chromosome in male meiosis (Shibusawa unpublished). The aim of this study is to identify the gene that controls X–Y paring in D ananassae. Chromosomal compositions such as XXY and XYY were commonly observed in the L8 strain from Sri Lanka. The study revealed a complete absence of X–Y pairing in the males of this strain and identified a potential gene responsible for X–Y chromosome non-disjunction in D. ananassae.
An isofemale strain (L8) obtained from Sri Lanka in 1971 was used. It has been maintained in the laboratory for over than thirty years. The following marker strains, listed in Table 1, were used to map the genes involved in X–Y pairing. Since chromosome 4 is largely heterochromatin and does not undergo meiotic recombination, the spa strain was used. As there is no balancer strain for chromosome 3 in D. ananassae, the T(Y;3) translocation strain was used instead. The eD,Ins(2L+2R)NG2 balancer chromosome was used for chromosome 2, as it suppresses meiotic recombination with its homologs. To map a recessive gene(s) controlling X–Y pairing on chromosome 2, experiments were carried out with three strains carrying the dominant genes Oms, Arc and Pr. For gene symbols and chromosomal locations, see Hinton and Downs (1975), Matsubayashi et al. (1992), Moriwaki and Tobari (1993), Tobari et al. (1993), and Schaeffer et al. (2008). All cultures were grown at 25°C on a cornmeal-yeast-glucose agar medium.
| Strain | Chromosome | Features described | Reference |
|---|---|---|---|
| f65; spa82 | X;4 | f65 shows twisted and gnarled bristles on the X chromosome, spa82 exhibits rough and mottled eye surfaces on the chromosome 4 | Moriwaki and Tobari 1993 |
| T(Y;3L)C, e65se; ru | Y;3 | The translocation between the Y and chromosome 3, Break point at 79A. Aabbreviated as T(Y;3). | Moriwaki and Tobari 1993 |
| eD, Ins(2L+2R)NG2/ca. M(2)665 | 2 | The Ins(2L+2R) NG2 is balancer of chromosome 2, and abbereviated as eD,NG2, eD displays a semi-dominant shining black body color. | Matsuda 1991 |
| Om(2H)59/ Ins(2L+2R)NG2 | 2L | Om(2H)59 is on 41B cytological position of the left arm of chromosome 2, and shows a semi-dominant optic morphology mutation | Moriwaki and Tobari 1993 |
| Om(2F)27a/ Ins(2L+2R)NG2 | 2R | Om(2F)27a is on the 55A cytological position of the right arm of chromosome 2, and shows a semi-dominant optic morphology mutation | Moriwaki and Tobari 1993 |
| Arc Om(2E)9h Pr/Ins(2L+2R)NG2 | 2R | Om(2E)9h is on the 51A cytological position and shows a semi-dominant optic morphology mutation, Arc displays a semi-dominant wing bent downward, and Pr shows a semi-dominant very short bristles. | Moriwaki and Tobari 1993 |
To investigate the role of chromosome 4 in X–Y parings, females of the L8 strain were crossed with the males of the f65;spa82 strain. After mating, the chromosome configuration during meiosis was observed in the F2 generation of [spa] males.
To investigate the effect of chromosomes 3 and 2 on X–Y paring, females of the L8 strain were crossed with males of T(Y;3)C, e65 se; ru, and eD,Ins(2L+2R)NG2/ca. M(2)665 strains. The F1 males were crossed with L8 females and the chromosome configuration during meiosis was observed in the BC1 (backcrossed first generation) males.
Finally, to map the gene(s) controlling of X–Y paring on chromosome 2, females of the L8 strain were crossed with males of the strains Om(2H)59/ Ins(2L+2R)NG 2, Om(2F)27a/ Ins(2L+2R)NG2, and Arc Om(2E)9h Pr/ Ins(2L+2R)NG2. The F1 females were crossed with L8 males and the chromosome configuration during meiosis was observed in the recombinant BC1 males.
Chromosome preparationsChromosomes in mitotic metaphase were prepared from the ganglia and brains of third-instar larvae, whereas chromosomes in meiotic prophase were prepared from adult testes immediately after exclusion. The organs were dissected in a hypotonic solution (1% sodium citrate) containing 0.1 mg/mL colchicine. Chromosomes were fixed in an ethanol-acetic acid solution, air-dried on slides and stained with 4% Giemsa solution (Matsuda et al. 1983).
The compositions of the X, Y, and 4th chromosomes in the somatic cells of the D. ananassae L8 strain showed high inter-individual variation, with the numbers of each type listed (Fig. 1; Table 2). Five types of karyotypes were observed in females: XX44, XXY44, XXYY44, XXY444, and XXY4, in males: XY44, XO44, XYY44, and XY444.
Scale bar=5 µm.
| X, Y, 4th chromosome set | |||
|---|---|---|---|
| In females | N | In males | N |
| XX44 | 13 | XY44 | 14 |
| XXY44 | 22 | XO44 | 10 |
| XXYY44 | 4 | XYY44 | 10 |
| XXY444 | 3 | XY444 | 2 |
| XXY4 | 1 | ||
| Total | 43 | Total | 36 |
To study the origin of the highly variable karyotype in the L8 strain, single males from the L8 strain were crossed with three virgin females from the X and chromosome 4 marker strain, f65; spa82. In this strain the X and Y chromosomes pair during the diplotene stage of meiosis to form a tetravalent chromosome association (see Fig. 2). To determine the karyotype of each L8 male parent, the somatic mitotic cells in the F1 generation from each cross were analyzed. The results showed two types of male karyotypes: XY44 and XYY44, seven and four individuals respectively (Table 3). In all the F1 progeny examined, the number of chromosome 4 was consistently two. However, the numbers of X and Y chromosomes varied among the F1 progeny, suggesting that sex chromosomes segregated randomly in the parental L8 male.
Scale bar=5 µm.
| X, Y, 4th chromosome set | No. of individuals in F1 progeny | |
|---|---|---|
| XY44 | XYY44 | |
| XX44 | 24 | 12 |
| XY44 | 28 | 12 |
| XXY44 | 11 | 11 |
| XO44 | 28 | 8 |
| XYY44 | 0 | 6 |
| XXYY44 | 0 | 2 |
| Total | 91 | 51 |
Each of the 7 XY males and 4 XYY males, was individually crossed with f65;spa82 females.
Meiosis was examined cytologically in the males of the L8 strain and in F1 males from reciprocal crosses between the L8 and the f65; spa82 strains (Table 4; Fig. 3). In all L8 male strains, the sex chromosomes failed to pair, whereas the chromosome 4 paired normally. However, in all F1 males from reciprocal crosses with the f65; spa82 strain, X, Y, and chromosome 4 paired normally (Table 4). This indicates that the mechanism controlling X–Y chromosome pairing operates independently of the mechanism for chromosome 4 pairing. Since F1 males from the reciprocal crosses exhibited the same results, the gene(s) responsible for X–Y chromosome pairing are autosomal. I tentatively name the recessive gene(s) for X–Y paring as ‘poxy’.
| Origin of males | X, Y, 4th chromosome set | No. of males with X, Y, and 4th | Sum | |
|---|---|---|---|---|
| tetravalent chromosome association | ||||
| Present | Absent | |||
| L8 | XY44 | 0 | 49 | 49 |
| XYY44 | 0 | 15 | 15 | |
| XO44 | 0 | 14 | 14 | |
| XYY444 | 0 | 2 | 2 | |
| Total | 0 | 80 | 80 | |
| F1 from L8 male parent | XY44 | 94 | 0 | 94 |
| F1 from L8 female parent | XY44 | 77 | 0 | 77 |
Scale bar=5 µm.
To investigate the role of autosomes (chromosomes 4, 3, 2) in X–Y paring, females from the L8 strains were crossed with males from the f65;spa82 strain for chromosome 4, the T(Y;3) strain for chromosome 3, and the Sb/eD, NG2 strain for chromosome 2. The results of the cytogenetic analysis (Table 5) indicate that the poxy gene(s) is not linked chromosomes 4 or 3, but on chromosome 2.
| Tested chromosome | Male genotype | No. of males with X–Y paring | Sum | |
|---|---|---|---|---|
| Present | Absent | |||
| chromosome 4 | F2 spa82/spa82 | 29 | 3 | 32 |
| chromosome 3 | BC1* X;+(L8) /T(Y;3) | 19 | 12 | 31 |
| BC1 XY;+(L8)/+ | 11 | 6 | 17 | |
| Total | 30 | 18 | 48 | |
| chromosome 2 | BC1+(L8)/eD, NG2 | 20 | 0 | 20 |
| BC1+(L8)/+ | 0 | 25 | 25 | |
| Total | 20 | 25 | 45 | |
Chromosome 4: tested in F2 males from crosses between L8 females and f65; spa82 males, chromosome 3: tested in BC1 males from crosses between the L8 strain and the T(Y;3) strain, and chromosome 2: tested in BC1 males from crosses between the L8 and the Sb/eD, NG2 strain. *BC1 (first backcrossed generation).
D. ananassae, carrying the dominant genes Oms, Arc and Pr were used to map the poxy gene. Table 6 indicates that the poxy gene(s) is located on the right arm of chromosome 2 and that approximately 8 (1/12) map units of the Om(2F)27a locus are located on the 55A cytological position. Furthermore, using the Arc Om(2E)9h Pr/ NG2 strain, which contains three semi-dominant genes on the right arm of chromosome 2, the poxy gene is not located in between these three markers but to the right side of these three markers, approximately 8 (4/51) map units away the Pr locus towards the tip of the 2R. This position places the poxy gene approximately halfway between Pr and Om(2F)27a on the D. ananassae linkage map and cytological map as shown in Fig. 4.
| BC1* male genotype | No. of males with X–Y paring | Sum | |
|---|---|---|---|
| Present | Absent | ||
| Om(2H)59/+(L8) | 7 | 9 | 16 |
| Om(2F)27a/+(L8) | 11 | 1 | 12 |
| Arc++/+++(L8) | 0 | 12 | 12 |
| +Om(2E)9h Pr /+++(L8) | 11 | 3 | 14 |
| Arc Om(2E)9h+/+++(L8) | 1 | 5 | 6 |
| ++Pr/+++(L8) | 17 | 0 | 17 |
| Arc+Pr/+++(L8) | 0 | 0 | 0 |
| +Om(2E)9h+/+++(L8) | 0 | 2 | 2 |
| Total | 29 | 22 | 51 |
*BC1 : First backcrossed generation.
The location of poxy gene (gray box) is shown relative to the linkage map (top), and to the salivary gland polytene chromosome cytological map (bottom). The linkage and polytene chromosome maps are adapted from Tobari (1993).
During meiosis, homologous chromosomes are paired by species-specific mechanisms. For example, protein-mediated interactions at kinetochores, kinetochore-peripheral regions, telomeres or pairing center regions have been reported (Orr-Weaver 1995; Vazques et al. 2002; Howley 2002). Following this initial pairing, a synaptonemal complex forms in which homologous chromosomes are fully paired across most of the chromosome. The synaptonemal complex is not observed in D. melanogaster males (Grell et al. 1972). An incomplete synaptonemal complex has been reported in D. ananassae (Moriwaki and Tsujita, 1974), but its presence has not been confirmed in subsequent studies.
The poxy gene identified in this study regulates X–Y pairing in the D. ananassae rDNA region but does not affect the autosome. Therefore, this gene is unlikely to be involved in the formation of the synaptonemal complex and suggests the existence of an alternative pairing mechanism. Extensive studies have focused on identifying regions associated with X–Y chromosome pairing during male meiosis in D. melanogaster (Orr-Weaver, 1995; McKee et al. 2012; Hylton et al. 2020), however, studies on the presence of key genes regulating this process are limited. This study shows for the first time that the wild-type allele of this gene, poxy, is required for X–Y paring in the rDNA region of the chromosome 2. These results provide new insights into the process of male meiosis and lay the ground work for future research.
An important point in the genetics of male meiosis is that while male meiosis in D. melanogaster does not involve crossing over (Morgan 1912, 1914), high frequencies of spontaneous crossing over are observed in this species (see Matsuda et al. 1993). Another relevant difference is that male meiotic bivalents in D. melanogaster remain compact through all meiotic division (Cooper 1950, 1964), whereas in D. ananassae, they dispersed from diplotene through the anaphase I (Hinton and Downs 1975; Tobari et al. 1993). The poxy gene identified in D. ananassae appears to be under different genetic control than in D. melanogaster males. Furthermore, the poxy gene is essential for the analysis of sex chromosome paring sites during male meiosis and the process of X–Y chromosome segregation. The poxy gene on the D. ananassae autosome is a potential candidate gene for future molecular and cytogenetic research.
In this study, haplo-4 and triple-4 configurations were observed in D. ananassae (see Tables 2 and 4, respectively). These chromosome aneuploidies are thought to have originated from the XXY44 female of the L8 strain. In addition, Kikkawa (1938) observed Minute mutations derived from haplo-4 in XXY44 D. ananassae females, suggesting that chromosome 4 and Y chromosome have a homologous sequence. However, the present analysis did not demonstrate the poxy gene’s influences on the relationship between the chromosome 4 and Y chromosomes during female meiosis with an extra Y chromosome.
I am grateful to the late Dr. Yoshiko N. Tobari for her long-term contributions to D. ananassae research, and KYORIN-Fly for providing me with flies. I also deeply appreciate the reviewers’ kind and valuable comments. In addition I would like to thank Editage (www.editage.jp) for English language editing.
The experimental design, execution, and manuscript writing were performed by Muneo Matsuda.
