THE CYTOGENETIC STUDY OF CROSSING OVER IN INTERSPECIFIC HYBRIDS BETWEEN DROSOPHILA VIRILIS AND D. TEXANA

M. B. EVGENEV

Institute of Molecular Biology, Academy of Sciences of the USSR, MOSCOW, USSR

Manuscript received July 10, 1975 Revised copy received January 12, 1976

ABSTRACT

Spontaneous crossing over was studied by means of combined cytological and genetic methods in F, Drosophila vir& X D. texana females (series I) and in D. uirilis females carrying a D. texana fifth chromosome in hetero- zygous condition (series 11). The main criterion utilized to distinguish the oogonial crossovers from the meiotic ones is the identity of cytological positions of genetic exchange in crossovers constituting a cluster. Five clusters of crossovers with identical positions of exchange were found in the first series of experiments. In the second series of experiments not a single cluster of crossovers resulting from oogonial crossing over was found.

HE molecular mechanism of crossing over in eukaryotic organisms is still Tquite obscure. There is not even a generally accepted view concerning the exact stage of meiosis where crossing over occurs. In addition, from many experi- ments it becomes clear that the frequency od crossing over is under complex genetic control and may be influenced by various external factors. In Drosophila several mutations have been discovered which may increase or decrease the crossing over frequency (LINDSLEY et ~ 2 . 1968; HINTON $970; CHINNICI 1971). The absence of crossing over in Drosopkila melanogaster males is a familiar fact of genetics. The suppression of crossing over in males is a characteristic feature of all studied groups of Drosophila though several exceptions exist (PHILIP 1944; KALE 1969; HIRAIZUMI 1971). It is well known that crossing over in Drosophila males is absent both in meiosis and premeiotic stages. Having all these facts in mind it becomes interesting to investigate whether there is oogonial crossing over in Drosophila females or is in this case the appearance of rkcozhbinants due to only meiotic exchange. If crossing over in females takes place in oogonia and oocytes as well, it may be concluded that germ line cells of Drosophila males lack a nonspecific factor necessary for genetic exchange. As far as is known there is no direct evidence for the occurrence of spontaneous oogonial crossing over in Drosophila in genetic literature. It is necessary to mention, however, that WHITTINGHILL and DAVIS ( 1961 ) reported induced oogonial crossing over in Drosophila melanogaster. In the present work spontaneous oogonial crossing over in interspecific hybrids was studied by combined genetic and cytological methods. Genetics 83 : 355-360 June, 1976.

356 M. B. EVGEN'EV

MATERIALS A N D METHODS

Two species of the Drosophila virilis group - D. virilis and D. texana (PATTERSON and STONE 1952) were used in the present studies.

The species can be hybridized under experimental laboratory conditions and produce viable and fertile hybrids. The stock of D. virilis (142) carrying recessive marker genes scarlet (si) and eosinoid (es) eye colors localized by FUJII (1942) in the fifth chromosome and a wild-type stock of D. ierana (123) were used to obtain the F, hybrids ( 9 D. virilis x 8 D. terana).

By means of eleven backcrosses one D. iezam fifth chromosome in heterozygous condition was transferred into an otherwise intact D. virilis genotype. Thus stock 142(a) was established.

In a previous report (EVGEN'EV 1971) it was demonstrated that the frequency of crossing over between the si and e5 genes in the pure D. virilis species amounts to 15.4%. In stock 142(a) carrying the hybrid fifth chromosome in an otherwise D. virilis genotype, the si-e5 crossing over frequency is 2.2% and in the hybrid F, females (virilis x terana), 20% The unexpectedly high frequency of crossing over in the fifth chromosome of uirilis x terana F, hybrids results from the interchromosomal effect of heterozygous inversions contained in the hybrid genotype (EVGEN'EV 1970). In order to screen for oogonial crossing over in hybrid fifth chromosomes, 15 F, females from the interspecific cross (series I ) and 20 females from stock 142(a) (series 11) were individually mated with five homozygous recessive si e5 males. Each female was transferred to fresh food daily. In the first series, three or more recombinants found in a single day's brood were considered to represent a cluster. In the second series where the frequency of crossing over was ten times lower than the first one, two or more crossovers found in a brood were believed to constitute a cluster. I t is evident that such phenotypically observed clusters of cross- overs may be of meiotic origin or represent a mixture of meiotic and oogonial crossovers. In order to prove that a cluster of crossovers really resulted from oogonial crossing over it was necessary to demonstrate that crossovers which constituted a cluster had identical cytological positions of the genetic exchange.

I

FIGURE l.-The typical picture of fifth chromosome conjugation in D. virilis X D. tezuna F1 hybrids. (The position of the gene markers is indicated.)

CROSSING OVER IN DROSOPHILA HYBRIDS 357

FIGURE L?.-The cytologically observed crossover in FB, resulted from exchange in Cab region. The arrow indicates the place of exchange.

Since it was shown in the earlier studies that the frequency of polytene chromosome conju- gation in interspecific hybrids depends solely on the properties of the chromosome loci, it became possible to investigate the crossing over cytologically (EVGEN'EV 1970, 1971). Crossing over in the distal end of hybrid fifth chromosomes was easily observed by the change in the m i c a 1 polytene chromosome conjugation scheme in studies of salivary gland chromosomes in the larval progeny of the backcross (FB,) hybrid 9 (iexnna x virilis) by 8 uirilis. Thus if crossing over took place in region Ca-b of the hybrid fifth chromosome (Figure l), one of two complementary products of crossing over would look as shown in Figure 2. It was necessary to prove that changes in the typical polytene chromosome pairing scheme often observed in FB, really resulted from genetic exchanges in F h brid females.

To answer this question we made cytological studies of the polytene chromosomes of the 1 . y

+a progeny of phenotypically observed crossovers (* and -). The analysis indicated that si es si es

cytologically observed sites of crossing over in this series were never located more distally than Fe-f or more proximally than He-h region. These data quite coincide with FUJII (1942) results (obtained with the help of induced aberrations analysis) concerning cytological positions for genes st and es respectively. The experiments proved that cytologically observed changes in polytene chromosome conjugation in the first back-cross generation (FB,) really results from crossing over in virilis X texanu hybrid females (EVGEN'EV 1970; EVGEN'EV, in press). The positions of exchanges in the distal end of the fifth chromosome were identified using the Hsu (1952) cytological map of D. uirilis chromosomes.

Using this approach, the cytological positions of exchange of phenotypically identified cross- overs found in clusters were studied. To facilitate the cytology, apparent crossover progeny from FB, (series I and 11) showing the si or es phenotype were collected and individually backcrossed (FB,) to D. virilis flies. In these experiments the cytological positions of the genetic exchanges were identified in the polytene chromosomes of FB, larvae. Aceto-orcein smear preparations of the salivary glands of third instar larvae were made. All experiments were done at a temperature of about 23-24'.

RESULTS A N D DISCUSSION

Table 1 summarizes the results of cytological analyses performed to prove the genetic validity of phenotypically observed clusters of crossovers, i.e. the identity of the exchange position of crossovers constituting a cluster. In order to distin-

358 M. B. EVGEN’EV

TABLE 1

Cytological analysis of crossing over in the progeny of crossovers consfituiing phenoiypically observed clusters

Series

Number of clusters Total number Number of of crossovers with

of phenotypically “clusters“ identical cytologicel observed cmssovers studied studied positions of exclionge

Series I Series I1

190

75

41

27

* Females carrying hybrid fifth chromosome (st e s ) / ( + +) (series I and 11) individually mated with D. virilis males (st es ) / ( s t es) were transferred to fresh vials daily for a week. Phenotypically observed crossovers (si +)/(si es) and (+ e s ) / ( s f es) found in clusters were individually backcrossed (FB,) to D. virilis flies. All five “real” clusters of crossovers with cytologically proved identical positions of exchange were found each in one day-brood or in two subsequent day-broods of different hybrid females.

wish with confidence the oogonial crossovers from the meiotic ones, crossovers were considered to be of oogonial origin only in such cases where the phenotyp- ically observed crossovers resulted exclusively from a rare crossing over event.

It is obvious that es and st crossovers may appear due to exchange at any site of the chromosome between the genes. However, as it was earlier demonstrated, the frequency of crossing over in different regions of the distal end of hybrid fifth chromosomes varies significantly. In other words, those sections of chromo- somes characterized by a high frequency of conjugation in the salivary glands of hybrids also exhibited a high frequency of crossing over in hybrid females. In those regions exhibiting poor pairing in the salivary gland chromosomes, crossing over occurs only rarely.

A double crossover with one or both breaks in the region of poor pairing is a particularly rare event. Preliminary experiments showed that the frequency of such events varies from 0.2 to 0.5% depending on the sites of exchange. In the

- - --- - c-

FIGURE 3a and b.-The cytologically seen results of exchange observed in a progeny of the genotype + es/sies crossovers (FB,). Both crossovers (a and b) resulted from a double crossing over. Arrows indicate places of exchange.

CROSSING OVER I N DROSOPHILA HYBRIDS 359

first series of experiments, investigated by means of the method described above, five clusters consisted of crossovers sharing the same places of exchange were recovered from hybrid F, females (Table 1). It is necessary to emphasize that each of these clusters consists of crossovers from a double exchange. Figures 3a and 3b illustrate two such cytologically observed double crossovers. Thus, among the five clusters of crossovers found: one case included five identical crossovers, three cases included four and one case included three. These may be considered with certainty to represent “real” clusters, i.e., the products of oogonial multiplication of a crossover chromosome. The distribution of such crossovers in the progeny of pertinent females is illustrated in Table 2. In the second series of experiments where oogonial crossovers were sought in females carrying hybrid fifth chromosome (stock 142a), not a single case of a cytolog- ically proved cluster was found. However, this fact does not rule out the possibility of oogonial crossing over in females without heterozygous inversions. Apparently the frequency of such crossing over may be too low to be found in present study. Any speculation on the role of interchromosomal effect of hetero- zygous inversions in preferential induction of oogonial crossing over seems to be premature.

It should be noted that a preliminary large scale experiment failed to reveal a single crossover in the progeny of F, uirilis x texana males. WHITTINGHILL (1947) reported that in D. melanogaster the presence of Curly inversions heterozygous for the multiple recessive third chromosome markers, rucuca, had no effect upon induced crossing over in males.

GRELL, BANK and GASSNER (1972) recently showed that meiotic exchange in D. ananassae males occurs without the synaptinemal complex. On the other hand a second chromosome of D. melanogaster isolated from a natural population was found sometimes to undergo recombination in heterozygous males ( HIRAIZUMI 1971). These data and our observations on spontaneous oogonial crossing over in Fl uirilis X texana females allow the suggestion that suppression of crossing over in Drosophila males is due not to the absence of the synaptinemal complex but rather to the lack of some nonspecific factors in the germ line of males. The

TABLE 2

The general distribution of crossovers in the progeny of the females including clusters of crossovers of postulated oogonial origin

Numbers of crossovers in subsequent day-broods Females NN 1 2 3 4 5 6

1 2 9 5 3(4) 9 4

3 5 5 8 3(1) 8(3) 7 4 5 4 3(4) 6 6 2 5 3 4 1(3) 3 4 3

2 4 4 6(4) 7(1) 5 6

Figures in parentheses represent crossovers with identical cytological positions of exchange, resulted from double crossing over.

360 M. B. EVGEN’EV

hypothetical factor( s) necessary for recombination is usually present in germ line cells of Drosophila females (with an exception of c(3)G females) and may appear in males as a result of mutations leading to derepression of pertinent genes. D. ananassae males represent a striking example of this phenomenon.

LITERATURE CITED

CHINNICI, J. P., 1971

EVGEN’EV, M. B., 1970

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Corresponding editor: B. H. JUDD

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