BiochemicalGenetics, Vol.30, Nos, 1/2, 1992
Evidence for a Genetic Duplication Involving Alcohol Dehydrogenase Genes in Ceratitis capitata A n n a Malacrida, 1'4 Giuliano Gasperi, 1 Antigoni Z a c h a r o p o u l o u , 2 Cristina Torti, 1 Elvira Riva Francos, 3 and Riccardo Milani I
Received24 July 1991 Final10 Sept. 1991
An A d h duplication is described in the medfly C e r a t i t i s c a p i t a t a . Evidence is presented for two separate A d h 1 and A d h 2 structural loci mapping at a distance of 0.49 recombination un# from each other. By deletion mapping the A d h region has been cytologically located near the free end of the left arm of the second chromosome within an area between 2C;3A segments of the polytene chromosome. The genetic analysis of the region around A d h has identified seven neighboring genes (ACOnl, Mpi, E s t 6, Aox, X d h , Mdh2, Lspl) which identify the linkage group D. The orientation of loci with regard to the centromere sets the origin of the map of the left arm of the second chromosome close to the two A d h loci. KEYWORDS: Ceratitiscapitata (medfly); alcohol dehydrogenase; gene duplication; genetic and cytogenetic mapping.
Research supported by National Research Council of Italy, Special Project RAISA, Subproject N.2, Paper N.200. Grants from I.A.E.A. (International Atomic Energy Agency, Vienna, Austria) and from European Communities Commission, Second R&D Programme "Science and Technology for Development" also contributed to this work. 1Dipartimento di Biologia Animale, Universith di Pavia, Piazza Botta, 9, 1-27100 Pavia, Italy. 2Department of Biology, Division of Genetics, Cell and Development Biology, University of Patras, Patras, Greece. 3Departamento de Pioteccion Vegetal CIT-INIA, Apdo 8111, 28080 Madrid, Spain. 4To whom correspondence should be addressed.
35 0006-2928/92/0200-0035506.50/0 © 1992 Plenum Publishing Corporation
36
M a l a c r i d a et al.
INTRODUCTION
Among the insect species of economic importance that have recently successfully entered the genetic scene, the Mediterranean fruit fly (medfly), Ceratitis capitata, has received special attention [for a comprehensive review see Robinson and Hooper (1989)]. It is probably one of the major fruit crop pest in the world. In the last 20 years the medfly has become a target for systematic studies aimed at accumulating basic genetic information on this species to develop genetic control methods by the "Sterile Insect Technique" (Joint FAO/IAEA Division, 1990). In recent years the use of biochemical mutations (Milani et aL, 1989) has broadened available knowledge on the genetic features of this species. These mutations have provided reliable tools for the study of population structure (Gasperi et al., 1990, 1991), taxonomic relations (Malacrida et aL, 1991), and applied genetics (Malacrida et al., 1988) and have improved the accuracy and usefulness of genetic maps. Polytene chromosome maps of the five autosomes of this species are now available from trichogen cells (Bedo, 1986, 1987) and salivary glands (Zacharopoulou, 1987, 1990), thus providing a tool for a correlation of chromosomes and linkage maps (Malacrida et aL, 1987). The biochemical genetic characterization of the alcohol dehydrogenase (ADH) system in C. capitata is of relevance both for the biology of this pest species and for applied purposes. Studies on the ADH enzyme in C. capitata were carried out within the framework of the development of genetic sexing methods for the control of this pest species, as have been developed in Drosophila melanogaster (Robinson, 1983; Riva and Robinson, 1986). Initial studies on the medfly revealed a different system from that existing in D. melanogaster, as it was clear that in the medfly two independent genes are coding ADH (Gasperi et al., 1987). The two genesAdh I and Adh2 code for two well-differentiated isozymes which show differences in their physicochemical features and present a complex, temporal- and tissuespecific, pattern (Gasperi et al., 1989). It was suggested that the two Adh genes of the medfly are the product of a gene duplication. The Adhl gene was previously established to be autosomal (Gasperi et al., 1989). This locus was found to map to chromosome 2 (Zapater and Robinson, 1986), in association with the Mpi, Est6, Aox, Mdh2, and Lspi loci (Malacrida et aL, 1987; Milani et al., 1989). This work presents evidence that Adhl and Adh2 are tighly linked, supporting the hypothesis thatAdhl a n d A d h 2 of C. capitata are the products of a gene duplication. We have also begun a genetic and cytogenetic analysis of the region around the Adh genes in an effort to identity neighboring genes, to determine their order and distance fromAdh genes, and to localize theAdh genes cytologically.
ADH Genetic Duplication
37 MATERIALS AND M E T H O D S
Ceratitis capitata Strains (a) Medfly strains for rare alleles at t h e A d h 1 andAdh 2,Xdh, ACOnl, and Mpi loci were derived from population samples collected in Kenya (Africa). Homozygous lines were established for the following allelic combinations: (1) Adh~/B, A. .F. l.h.A2/ A , MP ic/c, XdhB/B; (2) Adh~/D, •~xahu/u" ~uu 2 , (3) a d h A/A,Adh~/B, MpiB/B; (4) AdhlI)/D, adh~ zB,acon~/B; ( 5 ) A c l hD/D . . . . . 1 ,Adh2B/B,Xdh A/A ;and (6) Mpi B/B,Acon~/B. (b) Strain T128, selected by Robinson et al., (1986) and cytologically characterized by Zacharopoulou et aL (1991), is a strain having the following features: (1) a reciprocal translocation between the second and the fourth chromosome; (2) the Y chromosome inserted into the long arm of the second chromosome near the free end; and (3) a deletion near the free end of the 2L chromosome at Df(2)2C; 3A. This strain, which carries Adh~. Adh A alleles has been crossed with stocks with normal second-chromosome arrangements havingAdh~AdhB2 genes. Linkage Tests
The linkage relationships analyzed in the present work were based on standard backcrosses of homozygous females to multiply heterozygous males. All recombination values were based on testcrosses of heterozygous females to multiply homozygous males. Biochemical Techniques
Cellogel Electrophoresis. Sample preparations and electrophoretic separations were performed according to the method described by Malacrida et aL (1982). Isoelectric Focusing. Isoelectric focusing was performed according to LKB Inc. Manual 1804. Individual flies were homogenized in 100 ~1 of buffer and the extracts focused in thin-layer 0.5-mm acrylamide gels containing 5% ampholine, pH gradient 3-10 or 4.0-6.5. Focusing conditions were maintained at constant power (10 W) for 3 hr. The gels were stained for
38
Malacrida et al.
A D H activity (Harris and Hopkinson, 1976) and the resulting bands were scanned on a 2202 Ultrascan laser densitometer coupled to a 2220 Recording Integrator (LKB, Sweden). The relative areas under each A D H activity peak were computed for each sample. The activities expressed as planimetric units were inferred from the staining intensities of four replicates. RESULTS Linkage of the Two Adh Genes
The detection of linkage in (2. capitata is greatly facilitated by the severe restriction of crossing-over in males (Cladera, 1981; Rossler, 1985; Gethmann, 1988). Crosses were performed between flies having alternate alleles ofAdh 1 and Adh 2 genes. There is a well-documented polymorphism for the Adh~ locus, while only one rare allele has been discovered for the Adh 2 gene (Adh B) in a wild medfly population from Kenya (Gasperi et al., 1989). Several single-pair matings were established between a subline homozygous for Adh~ and Adh~ alleles and a laboratory strain homozygous for the most common Adh~ and Adh A genes. Backcrosses were performed between F 1 males doubly heterozygous Adh~ ID,Adh A/B and females doubly homozygous Adh~/B, Adh A/A. Progenies were analyzed as adults (Fig. la) and pupae (Fig. lb) using isoelectrofocusing and Cellogel electrophoresis, respectively. Of a total of 300 backcross progeny examined, only 2 expected phenotypic classes are found. One phenotype is identical to the backcross heterozygous male parents: ADH1-BD, ADH2-AB (lanes 1, 3, 5, 6, 8-11, 13, 14, 16-18, 21, and 22 in Fig. la and lane 4 in Fig. lb). The other is identical to the homozygous female parents: ADH1-B, ADH2-A (lanes 2, 4, 7, 12, 15, 19, 20, and 23 in Fig. la and lanes 1, 2, 3, and 5 in Fig. lb). This is adequate evidence for assessing the linkage between theAdh~ and theAdh 2 loci. Map Distance Between Adh~and A d h
2
The two Adh genes were mapped by recombination. The map distance between Adh 1 and Adh2 was determined from test crosses involving female parents which were doubly heterozygous Adh B/D1,~l~-2A'tA/B h and males which were doubly homozygousAdh~/B,Adh A/A (Table I). We have examined a total of 601 backcross progeny, as pupae, by isoelectric focusing. Only two phenotypes have been found: of these 300 were heterozygotes and 301 were male parental types. No recombinants have been observed. Therefore, within these limits, the two Adh genes are closely linked. The maximum
ADH Genetic Duplication
P
o
39
Adhl B/B, Adh2A/A
x
o"r Adhl D/D, Adh2B/B
"1"
Adhl B/D, Adh2 A/B
F1
Backcross:
(~ Adhl B/B, Adh2A/A
x
o~ AdhlB/D, Adh2A/B
ADH-1
a) ADH-2
adults 123
45
7 8
6
9 1011 1213 14151617
1 8 1 9 20 2 1 2 2 2 3
ADH-1
b)
pupae ADH-2
1
2
3
4
5
Fig. 1. Diagram and results of crosses between C. capitata individuals having alternate electrophoretic alleles at both Adh loci. Backcross progeny were analyzed as adults (a) by isoelectric focusing (pH range, 3-10) or as pupae (b) by Cellogel electrophoresis.
40
Malacrida et al. Table L Crcc~ovexRate BetweenAdh~ and Adhz Loci Provided by the Test Cross
? Adh~Adh~Adh~ Adh~ x d Adh~Adh2A/Adh~AdhA Test-cross progeny P~renlats
Recombinants
Adh~ Adh~ AdhDAdh~
Adh~ Adh~ Adh~ Adh~
Adh~ Adh~ Adh~ Adh2A
300
301
0
Adh~ Adh~ AdhBAdhA 0
Recombination fraction = 0/601 = 0.0%
genetic distance b e ~ e e n A d h a a n d A d h 2 compatible with this observation is 0.497 cM at the 5% probability level; at the 1% level the maximum compatible distance is 0.763 cM. M a p p i n g o f B i o c h e m i c a l Loci A r o u n d
Adh~,Adh2
The pair-by-pair checking of the linkage between A d h 1 and A d h 2 and other biochemical loci has pointed out some of the linkage relationships which link the two A d h loci with other markers such as ACOnl and Xdh loci. Table II shows the results of joint segregation of Adh~, A d h 2 with Acon 1 and Xdh, respectively, obtained in two different test-cross progenies of doubly heterozygous males to doubly homozygous females. In the two test-cross progenies, only the two expected parental phenotypes were observed. The gene cluster so far identified ~or the second chromosome includes Mpi, Est6, Aox, Mdh2, Lsp~, Xdh, a n d A c o n r The linkage was measured between Adhl, A d h 2 and the markers Mpi a n d A c o n r The recombination values between adjacent loci are given in Table III. To evaluate the recombination rate in the map interval Adhl, Adh~Mpi, triply heterozygous females Adh~?/B Adh~/B Mpi wc Table II. Joint Segregation ofAdhl, Adh2Lociwith Xdh andAconI in TestCross Progeny
Parental combination
No. of Progeny
9 ADH1-B,ADH2-A,XDH-B × d ADH1-BD,ADH2-AB,XDH-AB Total 2 ADH1-B,ADH2-A, ACON1-A× ADH1-BD,ADI:I2-AB,ACON1-AB Total
150 139 289 125 134 259
Phenotype of Progeny
X2hyp.l:l
ADH1-B,ADH2-A, XDH-B ADH1-BD,ADH2-AB,XDH-AB
0.42
ADH1-B,ADH2-A,ACON1-A ADH1-BD,ADH2-AB,ACON1-AB
0.31
ADH Genetic Duplication
41
Table III. RecombinationValues BetweenAdhl,Adheand the LociAcon~and Mpi on Chromosome 2 of Ceratitiscapitata Map interval
Adhl,Adhg-AconI Adhl,Adhz-Mpi Acon~-Mpi
No. of individuals 304 164 200
Recombinants (% _+SE) 41.11 _+0.0282 43.90 _+0.0386 33.00 + 0.0332
were crossed with homozygous Adh~, Adh A, Mpi c males. Of a total of 164 flies, 72 showed recombination between the Adhl, Adh2 and the Mpi loci, corresponding to 43.90% recombination. Such a very high interval value indicates a very large map interval, but it is too large to be accepted as a reliable measure of the actual map distance. The crossover rate in the interval between the Adh loci and Mpi was slightly higher than the estimate in the interval betweenAdh~,Adh2 andAcon~ (41.11 _+ 0.02 cM) as observed in the test-cross progeny of triply heterozygous female AdhA! B, adhA/B Acon~ lB. These data suggest thatMpi is to the right ofAconp A two-point test provided the recombination rate between Mpi and Acon~ (33.00 _+ 0,033 cM), which confirms the sequence Adhl, Adhi-Aconl-Mpi within this linkage group. Cytogenetic Mapping of Adh~, Adh z The two Adh loci have been localized cytogenetically by means of deletion mapping. Since a deficiency exists in C. capitata for the second chromosome in the Y-autosome translocated strain T128 (Zacharopoulou et al., 1991), the possibility that this deficiency might include Adh~ and Adh2 loci was tested. The T128 males, heterozygous for a second chromosome carrying the deletion Df(2L)2C;3A on the left arm, were found to be defective for both ADH1 and A D H 2 activities. Figure 2 shows males with lower band staining intensities for both A D H isozymes in comparison to their sib females, which possess the normal second chromosome karyotype. The activity values densitometrically determined ( _ S E ) for ADH-1 and ADH-2 are, respectively, 0.077 (___0.012) and 2.170 (__+0.46) in the males, while the corresponding estimates in the females are 0.202 (_+0.046) and 5.125 (+_0.22). The very low ADH1 and A D H 2 activities in the males indicate that theAdh I andAdh2 genes are included within the area of the deletion on the left arm of the second chromosome. Confirmation of the cytological localization of Adhl and Adh2 inside the deletion comes from crosses between heterozygous females ADH1-AB, ADH2-AB and T128 males, which are presumed to be hemizygousAdh~,AdhA. The results are summarised in Table IV and Fig. 3. Females progeny from such crosses were either homozygous for the allele
42
Malacrida et al.
ADH-1
ADH-2
99
.
_
crcr
=
Fig. 2. A D H isoelectric focusing (pH range, 3-10) patterns provided by females and males of the Y-autosome translocated strain T128. The males are heterozygous for a second chromosome carrying the deletion Df(2L)2C;3A.
passed by their fathers or heterozygous. Male offspring, however, were always single banded for one of the Adhl and Adh2 alleles inherited from their mothers and were, therefore, hemizygous. The appearance of Adha A d h 2 hemizygous progeny confirms the inclusion of the Adh region within
Table IV. ADH1 and ADH2 Phenotypes Observed in the Progeny of Matings Involving Flies from the Y-Autosome Translocated Strain (T128) of C. capitata Carrying the Deletion Df(2L)2C;3A F 1phenotypes ADH1-BB ADH1-AB ADH1-A~ ADH1-B ° ADH2-AA ADH2-AB ADH2-B" ADH2-A° Parental phenotypes ? (T128) ADH1-BB, ADH2-AA x d (T128) ADH1-B, a ADH2-A° Total 9 (T128) ADH1-BB, ADH2-AA x d ADH1-AB, ADH2-AB Total 9 ADH1-AB, ADH2-AB x d (T128) ADH1-B, a ADH2-A ° Total ~Hemizygous A D H phenotypes.
?
6
2
~
9
c~
150 0 150 51
54
62
0
d
0 248 148
48 54 202 54
9
x2
0.013opl;~)
46 97
0.1260p1:1) 0
62
0
50 50
0
46 46
0.0750p1:1:1:1)
ADH Genetic Duplication
43
ADH-1
ADH-2 1
2
3,=4
5
6
, 7 .
.
8
.
9
.
10
11 I
L
12
1
Fig. 3. ADH isoelectric focusing (pH range, 4-6.5) patterns in the progeny from a backcross between heterozygousAdh)/BAdh~/Bfemales and hemizygousAdh~Adh~ males. The pH range used in the isoelectric focusingpermits a good resolution only for ADH-1phenotypes. Female progenyare homozygousADH-1BB(lanes 1, 2, 3, 9, 12) or heterozygousADH-1AB(lanes 7, 8). Male progeny are alwayssingle-banded hemizygousADH1-A (lanes 6, 10, 11) or ADH1-B (lanes 4, 5). the deletion Df(2L)2C;3A and maps the loci to the left arm of the second chromosome between segments 2C;3A of the polytene chromosome. Orientation of Loci Around the Chromosomal Adh Region A m o n g the loci which are associated with theAdh~,Adh2 loci, we have tested Est6,Aox, Mdh 2,Mpi, andAcon~ and we can state that they are not within the limits of the Df(2L)2C;3A deficiency on the basis of biochemical genetic tests. The cytological localization of the two Adh loci inside the deletion of the 2L free end and the recombination analyses carried out in this work suggest that the loci Adh~, Adh2, Acon~, and Mpi are located on the map of the left arm of the second chromosome in this order. The orientation of the loci with respect to the centromere sets the origin of such a map close to the two Adh loci (Fig. 4). Chromosome 2 corresponds to section 1-20 of the polytene chromosome (Zacharopoulou, 1990). Consequently the data here Adh 1, Adh2
Acon I 41
Mpi 33
2L Fig. 4. Linkagemap of the left arm of chromosome2 of Ceratitiscapitata.
44
M a l a c r i d a et ai.
presented are evidence that this linkage group corresponds to the polytene element 1-20. DISCUSSION
The results presented in this paper give information both on the nature of the genetic control of the ADH system in Ceratitis capitata and on the genomic localization of the two A d h structural genes. Two closely linked structural genes are responsible for the ADH system of C. capitata. The close linkage between the two A d h loci supports the hypothesis of a gene duplication of the structural locus. Definitive proof of a duplication will be provided by molecular experiments already in progress. No recombination has been observed in a total of 601 backcross progeny. The maximum distance compatible with this observation is 0.49 cM. This distance is in general agreement with other studies of gene duplication in Drosophila. The estimated recombination distance between the duplicate loci of the LapALapD system in D. melanogaster is 0.3 cM (Beckman and Johnson, 1964). In D. montana the distance between Est 1 and Est 4 is 0.37 cM (Roberts and Baker, 1973) and in D. arizonensis the recombination distance between Est 4 and Est 5 is 0.16 cM (Zouros et al., 1982). For the two-locusAdh system of D. buzzati of the muUeri subgroup, distances of 0.4 +- 0.3 cM (Oakeshott et al., 1982) and 0.3 +_ 0.1 cM (Batterham et al., 1983) have been reported. Given the linkage of the t w o A d h loci in C. capitata, it is of some interest that the regulation of their expression has diverged considerably. In fact for these two loci there is developmental specificity and independent expression which involves the complete absence of ADH-1 isozyme in the first days of larval stage and, also, a clear-cut distribution of tissue expression (Gasperi et al., 1987). Ohno (1970) and Zuckerkandl (1978) suggested that one mode of achieving new patterns of gene expression during development is gene duplication followed by divergence in the expression of the two copies. An A d h duplication which differs in developmental expression within and among species of the mulleri subgroup of Drosophila has been documented (Batterham et al., 1984). In these cactophilic species of Drosophila it has been suggested that the presence of versatile multiple forms of ADH may be of adaptative significance (Oakeshott et al., 1982; Batterham et al., 1984; AIberola et al., 1987). In fact the originofAdh duplication is coincident with the adaptative radiation of these species and the utilization of the alcoholrich decaying cactus tissue as a habitat. A parallelism between the duplication of the A d h system in C. capitata and that in Drosophila of the mulleri subgroup remains to be determined. A testable hypothesis is that, in the medfly, the presence of two well-differentiated isozymes may be suited to temporary life requirements of this insect. A question of evolutionary
ADH Genetic Duplication
45
significance concerns the origin of the duplication. The Adh locus is duplicated in other Tephritidae flies, such as in 22 species of Rhagoletis (Berlocher and Bush, 1982), in Anastrepha species (Matioli et al., 1986), and in Ceratitis rosa and Capparimyia savastanoi (Malacrida et al., 1988). It is reasonable to consider that theAdh duplication in C. capitata is an old event. In this respect it is noteworthy to mention that the twoAdh loci of the medfly differ significantly because none of the electrophoretic variants at either locus overlaps the mobility of any variants at the other and no interlocus heterodimer has been found. Our second aim was to characterize the genomic localization of theAdh region in the medfly and to assign a relative position to the biochemical markers known to be linked toAdh genes. By deletion mapping the Adh region of C. capitata has been cytologically located near the free end of the left arm of the second chromosome within an area between the 2C;3A segments of the polytene chromosome. We are confident of this localization because of the agreement between the genetic data and the cytological results related to the Df(2L)2C;3A deletion (Zacharopoulou et al., 1991). Genetic analysis of the region aroundAdh has identified seven neighboring genes: Aconl, Mpi, Est6, Aox, Xdh, Mdh2, and Lspi. This gene cluster identifies the linkage group D (Malacrida et al., 1987; Milani et al., 1989). Two fundamental facts for the basic genetics of C. capitata derive from these data: (1) the gene composition of linkage group D, which includes the Adh region and (2) the unambigous correlation between this linkage group and the second chromosome. Since this chromosome (the longest of C. capitata) corresponds to section 1-20 of the polytene chromosome (Zacharopoulou, 1990), the linkage group D correlates with the polytene element 1-20. The localization of a gene in a particular linkage group can be considered an additional criterion for homology between enzyme loci (Bohm et al., 1987). The data reported here provide evidence that the composition of the gene cluster, which in the medfly includes the two Adh loci, is completely different from the one that in Drosophila melanogaster comprises the single Adh locus on chromosome 2L (O'Brien, 1987). In this connection the absence of regions of synteny between the two chromosomal Adh regions in these two species may be an additional criterion to propose the genetic differentiation between theAdh system of C. capitata and that of D. melanogaster. Biochemical analysis of the two A D H systems confirm this hypothesis (Gasperi et al., 1989). Five genes (Aconl, Mpi, Est,, Aox, Mdh2) from linkage group D are spread on the linkage map of this chromosome arm, far from theAdh region as deduced by recombination data and deficiency mapping. No information is available on the positions of the Xdh and Lspi loci relative to theAdh loci. The cytological localization of the Adh region represents a reliable starting
46
Malacrida et al.
point for the ordering of this gene cluster with respect to the c e n t r o m e r e and for the analysis of the recombinational events in the proximal and distal part of this c h r o m o s o m e arm. In this respect the first unambigous available data are summarized in Fig. 4, in which the origin of the linkage m a p of 2L appears close to the A d h region. Reliable information on the position of genes on this c h r o m o s o m e m a y be derived f r o m a comparison of the cytological m a p with the u p d a t e d linkage map. Finally, one o f our efforts in past years (Gasperi et al., 1987, 1989; Riva Francos, 1990) has b e e n to develop a genetic sexing m e t h o d for this pest species based on the A D H m o d e l of Drosophila ( R o b i n s o n et al., 1983). In this p a p e r substantial genetic evidence has b e e n obtained to show that in the D f ( 2 L ) 2 C ; 3 A deletion of the strain T128, no A D H polypeptide is synthesized. Thus we a p p e a r to have stable A d h 1 A d h 2 null mutants for further work.
ACKNOWLEDGMENT W e thank M. A s h b u r n e r for critical reading of and c o m m e n t s on the manuscript.
REFERENCES
Alberola, J., Sanchez, A., and Fontdevila, A. (1987). Adh expression in species of the mulled subgroup of Drosophila. Biochem. Genet. 25:729. Batterham, P., Gritz, E., Starmer, W. T., and Sullivan, D. T. (1983). Biochemical characterization of the products of theAdh loci of Drosophila mojavensis. Biochern. Genet. 21:871. Batterham, P., Chambers, G. K., Starmer, W. T., and Sullivan, D. T. (1984). Origin and expression of an alcohol dehydrogenase gene duplication in the genus Drosophila. Evolution 38(3):644. Beckman, L., and Johnson, F. M. (1964). Genetic control of aminopeptidases in Drosophila melanogaster. Hereditas 51:221. Bedo, D. G. (1986). Polytene and mitotic chromosome analysis in Ceratitis capitata (Diptera, Tephritidae). Can. J. Gen. Cytol. 28:180. Bedo, D. G. (1987). Polytene chromosome mapping in Ceratitis capitata (Diptera, Tephritidae). Genome 29:598. Berlocher, S. H., and Bush, G. L. (1982). An electrophoretic analysis of Rhagoletis (Diptera, Tephritidae) phylogeny. Syst. Zool. 31:136. Bohm, I., Pinsker, W., and Sperlich, D. (1987). Cytogenetic mapping of marker genes on the chromosome elements C and E of Drosophila pseudobscura and D. subobscura. Genetica 75:89. Cladera, J. L. (1981). Absence of recombination in the male of Ceratitis capitata. Experientia 37:342. Gasperi, G., Malacrida, A., Milani, R., and Robinson, A. S. (1987). Unique features of the ADH system in the medfly Ceratitis capitata Wied.AttiAss. Genet. Ital. 33:155. Gasperi, G., Malacrida, A. R., Cesti, A. F., and Milani, R. (1989). The alcohol dehydrogenase system of Ceratitis capitata: Differential expression of two ADH loci. In Cavalloro, R. (ed.), Fruit Flies of Economic Importance 87, A. A. Balkema, Rotterdam, Brookfield, pp. 287-293.
ADH Genetic Duplication
47
Gasperi, G., Malacrida, A. R., Guglielmino, C. R., and Milani, R. (1990). Electrophoretic multilocus analysis for the study of natural populations of the medfly Ceratitis capitata. In Genetic Sexing of the Mediterranean FruitFly, International Atomic Energy Agency, Vienna, Austria, pp. 90-94. Gasperi, G., Guglielmino, C. R., Malacrida, A. R., and Milani, R. (1991). Genetic variability and gene flow in geographic populations of the medfly Ceratitis eapitata. Heredity 64:347. Gethmann, R. C. (1988). Crossing over in males of higher Diptera (Brachycera). J. Hered. 79:344. Harris, H., and Hopkinson, D. A. (1976). Handbook of Enzyme Electrophoresis in Human Genetics, North-Holland, Amsterdam. Joint FAO/IAEA Division (1990). Genetic Sexing of the Mediterranean Fruit Fly, International Atomic Energy Agency, Vienna. Malacrida, A., Gasperi, G., and Milani, R. (1982). 6PGD in the housefly: Mapping of the Pgd locus in linkage group III ofMusca domestica. J. Hered. 73:349. Malacrida, A. R., Gasperi, G., and Milani, R. (1987). Genome organization of Ceratitis capitata: Linkage groups and evidence for sex-ratio distorters. In Economopoulos, A. P. (ed.), Fru# Flies, Elsevier, Amsterdam, pp. 169-174. Malacrida, A., Gasperi, G., Baruffi, L., Biscaldi, G. F., and Milani, R. (1988). Updating of the genetics of Ceratitis cap#am (Wied.). In Modem Insect Controk Nuclear Techniques and Biotechnology, International Atomic Energy Agency, Vienna, pp. 221-227. Malacrida, A. R., Guglielmino, C. R., Gasperi, G., Baruffi, L., Villani, P. C., and Milani, R. (1991). Genetical approach to systematics and phylogeny of Trypetinae (Diptera, Tephritidae). Boll. Zool. 58:355. Matioli, S. R., Morgante, J. S., and Malavasi, A. (1986). Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera, Tephritidae): Evidence for gene duplication. Biochem. Genet. 24:13. Milani, R., Gasperi, G., and Malacrida, A. (1989). Biochemical genetics (of Ceratitis capitata). In Robinson, A. S., and Hooper, G. (eds.), Fruit Flies: Their Biology, Natural Enemies and Control, VoL 3B, Elsevier, Amsterdam, pp. 33-56. Oakeshott, J. G., Chambers, G. K., East, P. D., Gibson, J. B., and Barker, J. S. F. (1982). Evidence for a genetic duplication involving alcohol dehydrogenase genes in Drosophila buzzati and related species.Aust. J. Biol. Sci. 53:73. O'Brien, S. (1987). Genetic Maps 1987, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Ohno, S. (1970). Evolution by Gene Duplication, Springer-Verlag, New York. Riva, M. E., and Robinson, A. S. (1986). Introduction of alcohol dehydrogenase null mutants in the mediterranean fruit fly. Ceratitis capitata. Biochem. Genet. 24:765. Riva Francos, M. E. (1990). Alcohols as discriminating agents for genetic sexing in the mediterranean fruit fly, Ceratitis capitata (Wied.). In Genetic Sexing of the Mediterranean Fruit Fly. International Atomic Energy Agency, Vienna, pp. 147-171. Roberts, R. M., and Baker, W. K. (1973). Frequency distribution and linkage disequilibrium of active and null esterase isozymes in natural populations of Drosophila montana. Am. Nat. 107:709. Robinson, A. S. (1983). Sex-ratio manipulation in relation to insect pest control. Annu. Rev. Genet. 17:191. Robinson, A. S., and Hooper, G. (eds.) (1989). Fruit Flies: Their Biology, Natural Enemies and Control, Vols. 3A, B, Elsevier, Amsterdam. Robinson, A. S., Riva, M. E., and Zapater, M. (1986). Genetic sexing in the Mediterranean fruit fly, Ceratitis capitata, using the Adh locus. Theor. AppL Genet. 72:455. Rossler, Y. (1985). Effect of genetic recombination in males of the Mediterranean fruit fly (Diptera Tephritidae) on the integrity of genetic sexing strains produced for sterile insect releases. Ann. Entomol. Soc. Am. 78:265. Zacharopoulou, A. (1987). Cytogenetic analysis of mitotic and salivary gland chromosomes in the Medfly Ceratitis capitata. Genome 29:67. Zacharopoulou, A. (1990). Polytene chromosome maps in the Medfly Ceratitis capitatu. Genome 33:184.
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Zacharopoulou, A., Riva, M., Malacrida, A., and Gasperi, G. (1991). Cytogenetic characterization of a genetic sexing strain in Ceratitis capitata. Genome 34:606. Zapater, M., and Robinson, A. S. (1986). Sex chromosome aneuploidy in male-linked translocation in Ceratitis capitata. Can. J. Genet. CytoL 28:161. Zouros, E., Van Delden, W., Odense, R., and van Dijk, H. (1982). An esterase duplication in Drosophila: Differences in expression of duplicate loci within and among related species. Biochem. Genet. 20:929. Zuckerkandl, E. (1978). Multilocus enzymes, gene regulation, and genetic sufficiency. J. Mol. Evol. 12:57.
Evidence for a Genetic Duplication Involving Alcohol Dehydrogenase Genes in Ceratitis capitata A n n a Malacrida, 1'4 Giuliano Gasperi, 1 Antigoni Z a c h a r o p o u l o u , 2 Cristina Torti, 1 Elvira Riva Francos, 3 and Riccardo Milani I
Received24 July 1991 Final10 Sept. 1991
An A d h duplication is described in the medfly C e r a t i t i s c a p i t a t a . Evidence is presented for two separate A d h 1 and A d h 2 structural loci mapping at a distance of 0.49 recombination un# from each other. By deletion mapping the A d h region has been cytologically located near the free end of the left arm of the second chromosome within an area between 2C;3A segments of the polytene chromosome. The genetic analysis of the region around A d h has identified seven neighboring genes (ACOnl, Mpi, E s t 6, Aox, X d h , Mdh2, Lspl) which identify the linkage group D. The orientation of loci with regard to the centromere sets the origin of the map of the left arm of the second chromosome close to the two A d h loci. KEYWORDS: Ceratitiscapitata (medfly); alcohol dehydrogenase; gene duplication; genetic and cytogenetic mapping.
Research supported by National Research Council of Italy, Special Project RAISA, Subproject N.2, Paper N.200. Grants from I.A.E.A. (International Atomic Energy Agency, Vienna, Austria) and from European Communities Commission, Second R&D Programme "Science and Technology for Development" also contributed to this work. 1Dipartimento di Biologia Animale, Universith di Pavia, Piazza Botta, 9, 1-27100 Pavia, Italy. 2Department of Biology, Division of Genetics, Cell and Development Biology, University of Patras, Patras, Greece. 3Departamento de Pioteccion Vegetal CIT-INIA, Apdo 8111, 28080 Madrid, Spain. 4To whom correspondence should be addressed.
35 0006-2928/92/0200-0035506.50/0 © 1992 Plenum Publishing Corporation
36
M a l a c r i d a et al.
INTRODUCTION
Among the insect species of economic importance that have recently successfully entered the genetic scene, the Mediterranean fruit fly (medfly), Ceratitis capitata, has received special attention [for a comprehensive review see Robinson and Hooper (1989)]. It is probably one of the major fruit crop pest in the world. In the last 20 years the medfly has become a target for systematic studies aimed at accumulating basic genetic information on this species to develop genetic control methods by the "Sterile Insect Technique" (Joint FAO/IAEA Division, 1990). In recent years the use of biochemical mutations (Milani et aL, 1989) has broadened available knowledge on the genetic features of this species. These mutations have provided reliable tools for the study of population structure (Gasperi et al., 1990, 1991), taxonomic relations (Malacrida et aL, 1991), and applied genetics (Malacrida et al., 1988) and have improved the accuracy and usefulness of genetic maps. Polytene chromosome maps of the five autosomes of this species are now available from trichogen cells (Bedo, 1986, 1987) and salivary glands (Zacharopoulou, 1987, 1990), thus providing a tool for a correlation of chromosomes and linkage maps (Malacrida et aL, 1987). The biochemical genetic characterization of the alcohol dehydrogenase (ADH) system in C. capitata is of relevance both for the biology of this pest species and for applied purposes. Studies on the ADH enzyme in C. capitata were carried out within the framework of the development of genetic sexing methods for the control of this pest species, as have been developed in Drosophila melanogaster (Robinson, 1983; Riva and Robinson, 1986). Initial studies on the medfly revealed a different system from that existing in D. melanogaster, as it was clear that in the medfly two independent genes are coding ADH (Gasperi et al., 1987). The two genesAdh I and Adh2 code for two well-differentiated isozymes which show differences in their physicochemical features and present a complex, temporal- and tissuespecific, pattern (Gasperi et al., 1989). It was suggested that the two Adh genes of the medfly are the product of a gene duplication. The Adhl gene was previously established to be autosomal (Gasperi et al., 1989). This locus was found to map to chromosome 2 (Zapater and Robinson, 1986), in association with the Mpi, Est6, Aox, Mdh2, and Lspi loci (Malacrida et aL, 1987; Milani et al., 1989). This work presents evidence that Adhl and Adh2 are tighly linked, supporting the hypothesis thatAdhl a n d A d h 2 of C. capitata are the products of a gene duplication. We have also begun a genetic and cytogenetic analysis of the region around the Adh genes in an effort to identity neighboring genes, to determine their order and distance fromAdh genes, and to localize theAdh genes cytologically.
ADH Genetic Duplication
37 MATERIALS AND M E T H O D S
Ceratitis capitata Strains (a) Medfly strains for rare alleles at t h e A d h 1 andAdh 2,Xdh, ACOnl, and Mpi loci were derived from population samples collected in Kenya (Africa). Homozygous lines were established for the following allelic combinations: (1) Adh~/B, A. .F. l.h.A2/ A , MP ic/c, XdhB/B; (2) Adh~/D, •~xahu/u" ~uu 2 , (3) a d h A/A,Adh~/B, MpiB/B; (4) AdhlI)/D, adh~ zB,acon~/B; ( 5 ) A c l hD/D . . . . . 1 ,Adh2B/B,Xdh A/A ;and (6) Mpi B/B,Acon~/B. (b) Strain T128, selected by Robinson et al., (1986) and cytologically characterized by Zacharopoulou et aL (1991), is a strain having the following features: (1) a reciprocal translocation between the second and the fourth chromosome; (2) the Y chromosome inserted into the long arm of the second chromosome near the free end; and (3) a deletion near the free end of the 2L chromosome at Df(2)2C; 3A. This strain, which carries Adh~. Adh A alleles has been crossed with stocks with normal second-chromosome arrangements havingAdh~AdhB2 genes. Linkage Tests
The linkage relationships analyzed in the present work were based on standard backcrosses of homozygous females to multiply heterozygous males. All recombination values were based on testcrosses of heterozygous females to multiply homozygous males. Biochemical Techniques
Cellogel Electrophoresis. Sample preparations and electrophoretic separations were performed according to the method described by Malacrida et aL (1982). Isoelectric Focusing. Isoelectric focusing was performed according to LKB Inc. Manual 1804. Individual flies were homogenized in 100 ~1 of buffer and the extracts focused in thin-layer 0.5-mm acrylamide gels containing 5% ampholine, pH gradient 3-10 or 4.0-6.5. Focusing conditions were maintained at constant power (10 W) for 3 hr. The gels were stained for
38
Malacrida et al.
A D H activity (Harris and Hopkinson, 1976) and the resulting bands were scanned on a 2202 Ultrascan laser densitometer coupled to a 2220 Recording Integrator (LKB, Sweden). The relative areas under each A D H activity peak were computed for each sample. The activities expressed as planimetric units were inferred from the staining intensities of four replicates. RESULTS Linkage of the Two Adh Genes
The detection of linkage in (2. capitata is greatly facilitated by the severe restriction of crossing-over in males (Cladera, 1981; Rossler, 1985; Gethmann, 1988). Crosses were performed between flies having alternate alleles ofAdh 1 and Adh 2 genes. There is a well-documented polymorphism for the Adh~ locus, while only one rare allele has been discovered for the Adh 2 gene (Adh B) in a wild medfly population from Kenya (Gasperi et al., 1989). Several single-pair matings were established between a subline homozygous for Adh~ and Adh~ alleles and a laboratory strain homozygous for the most common Adh~ and Adh A genes. Backcrosses were performed between F 1 males doubly heterozygous Adh~ ID,Adh A/B and females doubly homozygous Adh~/B, Adh A/A. Progenies were analyzed as adults (Fig. la) and pupae (Fig. lb) using isoelectrofocusing and Cellogel electrophoresis, respectively. Of a total of 300 backcross progeny examined, only 2 expected phenotypic classes are found. One phenotype is identical to the backcross heterozygous male parents: ADH1-BD, ADH2-AB (lanes 1, 3, 5, 6, 8-11, 13, 14, 16-18, 21, and 22 in Fig. la and lane 4 in Fig. lb). The other is identical to the homozygous female parents: ADH1-B, ADH2-A (lanes 2, 4, 7, 12, 15, 19, 20, and 23 in Fig. la and lanes 1, 2, 3, and 5 in Fig. lb). This is adequate evidence for assessing the linkage between theAdh~ and theAdh 2 loci. Map Distance Between Adh~and A d h
2
The two Adh genes were mapped by recombination. The map distance between Adh 1 and Adh2 was determined from test crosses involving female parents which were doubly heterozygous Adh B/D1,~l~-2A'tA/B h and males which were doubly homozygousAdh~/B,Adh A/A (Table I). We have examined a total of 601 backcross progeny, as pupae, by isoelectric focusing. Only two phenotypes have been found: of these 300 were heterozygotes and 301 were male parental types. No recombinants have been observed. Therefore, within these limits, the two Adh genes are closely linked. The maximum
ADH Genetic Duplication
P
o
39
Adhl B/B, Adh2A/A
x
o"r Adhl D/D, Adh2B/B
"1"
Adhl B/D, Adh2 A/B
F1
Backcross:
(~ Adhl B/B, Adh2A/A
x
o~ AdhlB/D, Adh2A/B
ADH-1
a) ADH-2
adults 123
45
7 8
6
9 1011 1213 14151617
1 8 1 9 20 2 1 2 2 2 3
ADH-1
b)
pupae ADH-2
1
2
3
4
5
Fig. 1. Diagram and results of crosses between C. capitata individuals having alternate electrophoretic alleles at both Adh loci. Backcross progeny were analyzed as adults (a) by isoelectric focusing (pH range, 3-10) or as pupae (b) by Cellogel electrophoresis.
40
Malacrida et al. Table L Crcc~ovexRate BetweenAdh~ and Adhz Loci Provided by the Test Cross
? Adh~Adh~Adh~ Adh~ x d Adh~Adh2A/Adh~AdhA Test-cross progeny P~renlats
Recombinants
Adh~ Adh~ AdhDAdh~
Adh~ Adh~ Adh~ Adh~
Adh~ Adh~ Adh~ Adh2A
300
301
0
Adh~ Adh~ AdhBAdhA 0
Recombination fraction = 0/601 = 0.0%
genetic distance b e ~ e e n A d h a a n d A d h 2 compatible with this observation is 0.497 cM at the 5% probability level; at the 1% level the maximum compatible distance is 0.763 cM. M a p p i n g o f B i o c h e m i c a l Loci A r o u n d
Adh~,Adh2
The pair-by-pair checking of the linkage between A d h 1 and A d h 2 and other biochemical loci has pointed out some of the linkage relationships which link the two A d h loci with other markers such as ACOnl and Xdh loci. Table II shows the results of joint segregation of Adh~, A d h 2 with Acon 1 and Xdh, respectively, obtained in two different test-cross progenies of doubly heterozygous males to doubly homozygous females. In the two test-cross progenies, only the two expected parental phenotypes were observed. The gene cluster so far identified ~or the second chromosome includes Mpi, Est6, Aox, Mdh2, Lsp~, Xdh, a n d A c o n r The linkage was measured between Adhl, A d h 2 and the markers Mpi a n d A c o n r The recombination values between adjacent loci are given in Table III. To evaluate the recombination rate in the map interval Adhl, Adh~Mpi, triply heterozygous females Adh~?/B Adh~/B Mpi wc Table II. Joint Segregation ofAdhl, Adh2Lociwith Xdh andAconI in TestCross Progeny
Parental combination
No. of Progeny
9 ADH1-B,ADH2-A,XDH-B × d ADH1-BD,ADH2-AB,XDH-AB Total 2 ADH1-B,ADH2-A, ACON1-A× ADH1-BD,ADI:I2-AB,ACON1-AB Total
150 139 289 125 134 259
Phenotype of Progeny
X2hyp.l:l
ADH1-B,ADH2-A, XDH-B ADH1-BD,ADH2-AB,XDH-AB
0.42
ADH1-B,ADH2-A,ACON1-A ADH1-BD,ADH2-AB,ACON1-AB
0.31
ADH Genetic Duplication
41
Table III. RecombinationValues BetweenAdhl,Adheand the LociAcon~and Mpi on Chromosome 2 of Ceratitiscapitata Map interval
Adhl,Adhg-AconI Adhl,Adhz-Mpi Acon~-Mpi
No. of individuals 304 164 200
Recombinants (% _+SE) 41.11 _+0.0282 43.90 _+0.0386 33.00 + 0.0332
were crossed with homozygous Adh~, Adh A, Mpi c males. Of a total of 164 flies, 72 showed recombination between the Adhl, Adh2 and the Mpi loci, corresponding to 43.90% recombination. Such a very high interval value indicates a very large map interval, but it is too large to be accepted as a reliable measure of the actual map distance. The crossover rate in the interval between the Adh loci and Mpi was slightly higher than the estimate in the interval betweenAdh~,Adh2 andAcon~ (41.11 _+ 0.02 cM) as observed in the test-cross progeny of triply heterozygous female AdhA! B, adhA/B Acon~ lB. These data suggest thatMpi is to the right ofAconp A two-point test provided the recombination rate between Mpi and Acon~ (33.00 _+ 0,033 cM), which confirms the sequence Adhl, Adhi-Aconl-Mpi within this linkage group. Cytogenetic Mapping of Adh~, Adh z The two Adh loci have been localized cytogenetically by means of deletion mapping. Since a deficiency exists in C. capitata for the second chromosome in the Y-autosome translocated strain T128 (Zacharopoulou et al., 1991), the possibility that this deficiency might include Adh~ and Adh2 loci was tested. The T128 males, heterozygous for a second chromosome carrying the deletion Df(2L)2C;3A on the left arm, were found to be defective for both ADH1 and A D H 2 activities. Figure 2 shows males with lower band staining intensities for both A D H isozymes in comparison to their sib females, which possess the normal second chromosome karyotype. The activity values densitometrically determined ( _ S E ) for ADH-1 and ADH-2 are, respectively, 0.077 (___0.012) and 2.170 (__+0.46) in the males, while the corresponding estimates in the females are 0.202 (_+0.046) and 5.125 (+_0.22). The very low ADH1 and A D H 2 activities in the males indicate that theAdh I andAdh2 genes are included within the area of the deletion on the left arm of the second chromosome. Confirmation of the cytological localization of Adhl and Adh2 inside the deletion comes from crosses between heterozygous females ADH1-AB, ADH2-AB and T128 males, which are presumed to be hemizygousAdh~,AdhA. The results are summarised in Table IV and Fig. 3. Females progeny from such crosses were either homozygous for the allele
42
Malacrida et al.
ADH-1
ADH-2
99
.
_
crcr
=
Fig. 2. A D H isoelectric focusing (pH range, 3-10) patterns provided by females and males of the Y-autosome translocated strain T128. The males are heterozygous for a second chromosome carrying the deletion Df(2L)2C;3A.
passed by their fathers or heterozygous. Male offspring, however, were always single banded for one of the Adhl and Adh2 alleles inherited from their mothers and were, therefore, hemizygous. The appearance of Adha A d h 2 hemizygous progeny confirms the inclusion of the Adh region within
Table IV. ADH1 and ADH2 Phenotypes Observed in the Progeny of Matings Involving Flies from the Y-Autosome Translocated Strain (T128) of C. capitata Carrying the Deletion Df(2L)2C;3A F 1phenotypes ADH1-BB ADH1-AB ADH1-A~ ADH1-B ° ADH2-AA ADH2-AB ADH2-B" ADH2-A° Parental phenotypes ? (T128) ADH1-BB, ADH2-AA x d (T128) ADH1-B, a ADH2-A° Total 9 (T128) ADH1-BB, ADH2-AA x d ADH1-AB, ADH2-AB Total 9 ADH1-AB, ADH2-AB x d (T128) ADH1-B, a ADH2-A ° Total ~Hemizygous A D H phenotypes.
?
6
2
~
9
c~
150 0 150 51
54
62
0
d
0 248 148
48 54 202 54
9
x2
0.013opl;~)
46 97
0.1260p1:1) 0
62
0
50 50
0
46 46
0.0750p1:1:1:1)
ADH Genetic Duplication
43
ADH-1
ADH-2 1
2
3,=4
5
6
, 7 .
.
8
.
9
.
10
11 I
L
12
1
Fig. 3. ADH isoelectric focusing (pH range, 4-6.5) patterns in the progeny from a backcross between heterozygousAdh)/BAdh~/Bfemales and hemizygousAdh~Adh~ males. The pH range used in the isoelectric focusingpermits a good resolution only for ADH-1phenotypes. Female progenyare homozygousADH-1BB(lanes 1, 2, 3, 9, 12) or heterozygousADH-1AB(lanes 7, 8). Male progeny are alwayssingle-banded hemizygousADH1-A (lanes 6, 10, 11) or ADH1-B (lanes 4, 5). the deletion Df(2L)2C;3A and maps the loci to the left arm of the second chromosome between segments 2C;3A of the polytene chromosome. Orientation of Loci Around the Chromosomal Adh Region A m o n g the loci which are associated with theAdh~,Adh2 loci, we have tested Est6,Aox, Mdh 2,Mpi, andAcon~ and we can state that they are not within the limits of the Df(2L)2C;3A deficiency on the basis of biochemical genetic tests. The cytological localization of the two Adh loci inside the deletion of the 2L free end and the recombination analyses carried out in this work suggest that the loci Adh~, Adh2, Acon~, and Mpi are located on the map of the left arm of the second chromosome in this order. The orientation of the loci with respect to the centromere sets the origin of such a map close to the two Adh loci (Fig. 4). Chromosome 2 corresponds to section 1-20 of the polytene chromosome (Zacharopoulou, 1990). Consequently the data here Adh 1, Adh2
Acon I 41
Mpi 33
2L Fig. 4. Linkagemap of the left arm of chromosome2 of Ceratitiscapitata.
44
M a l a c r i d a et ai.
presented are evidence that this linkage group corresponds to the polytene element 1-20. DISCUSSION
The results presented in this paper give information both on the nature of the genetic control of the ADH system in Ceratitis capitata and on the genomic localization of the two A d h structural genes. Two closely linked structural genes are responsible for the ADH system of C. capitata. The close linkage between the two A d h loci supports the hypothesis of a gene duplication of the structural locus. Definitive proof of a duplication will be provided by molecular experiments already in progress. No recombination has been observed in a total of 601 backcross progeny. The maximum distance compatible with this observation is 0.49 cM. This distance is in general agreement with other studies of gene duplication in Drosophila. The estimated recombination distance between the duplicate loci of the LapALapD system in D. melanogaster is 0.3 cM (Beckman and Johnson, 1964). In D. montana the distance between Est 1 and Est 4 is 0.37 cM (Roberts and Baker, 1973) and in D. arizonensis the recombination distance between Est 4 and Est 5 is 0.16 cM (Zouros et al., 1982). For the two-locusAdh system of D. buzzati of the muUeri subgroup, distances of 0.4 +- 0.3 cM (Oakeshott et al., 1982) and 0.3 +_ 0.1 cM (Batterham et al., 1983) have been reported. Given the linkage of the t w o A d h loci in C. capitata, it is of some interest that the regulation of their expression has diverged considerably. In fact for these two loci there is developmental specificity and independent expression which involves the complete absence of ADH-1 isozyme in the first days of larval stage and, also, a clear-cut distribution of tissue expression (Gasperi et al., 1987). Ohno (1970) and Zuckerkandl (1978) suggested that one mode of achieving new patterns of gene expression during development is gene duplication followed by divergence in the expression of the two copies. An A d h duplication which differs in developmental expression within and among species of the mulleri subgroup of Drosophila has been documented (Batterham et al., 1984). In these cactophilic species of Drosophila it has been suggested that the presence of versatile multiple forms of ADH may be of adaptative significance (Oakeshott et al., 1982; Batterham et al., 1984; AIberola et al., 1987). In fact the originofAdh duplication is coincident with the adaptative radiation of these species and the utilization of the alcoholrich decaying cactus tissue as a habitat. A parallelism between the duplication of the A d h system in C. capitata and that in Drosophila of the mulleri subgroup remains to be determined. A testable hypothesis is that, in the medfly, the presence of two well-differentiated isozymes may be suited to temporary life requirements of this insect. A question of evolutionary
ADH Genetic Duplication
45
significance concerns the origin of the duplication. The Adh locus is duplicated in other Tephritidae flies, such as in 22 species of Rhagoletis (Berlocher and Bush, 1982), in Anastrepha species (Matioli et al., 1986), and in Ceratitis rosa and Capparimyia savastanoi (Malacrida et al., 1988). It is reasonable to consider that theAdh duplication in C. capitata is an old event. In this respect it is noteworthy to mention that the twoAdh loci of the medfly differ significantly because none of the electrophoretic variants at either locus overlaps the mobility of any variants at the other and no interlocus heterodimer has been found. Our second aim was to characterize the genomic localization of theAdh region in the medfly and to assign a relative position to the biochemical markers known to be linked toAdh genes. By deletion mapping the Adh region of C. capitata has been cytologically located near the free end of the left arm of the second chromosome within an area between the 2C;3A segments of the polytene chromosome. We are confident of this localization because of the agreement between the genetic data and the cytological results related to the Df(2L)2C;3A deletion (Zacharopoulou et al., 1991). Genetic analysis of the region aroundAdh has identified seven neighboring genes: Aconl, Mpi, Est6, Aox, Xdh, Mdh2, and Lspi. This gene cluster identifies the linkage group D (Malacrida et al., 1987; Milani et al., 1989). Two fundamental facts for the basic genetics of C. capitata derive from these data: (1) the gene composition of linkage group D, which includes the Adh region and (2) the unambigous correlation between this linkage group and the second chromosome. Since this chromosome (the longest of C. capitata) corresponds to section 1-20 of the polytene chromosome (Zacharopoulou, 1990), the linkage group D correlates with the polytene element 1-20. The localization of a gene in a particular linkage group can be considered an additional criterion for homology between enzyme loci (Bohm et al., 1987). The data reported here provide evidence that the composition of the gene cluster, which in the medfly includes the two Adh loci, is completely different from the one that in Drosophila melanogaster comprises the single Adh locus on chromosome 2L (O'Brien, 1987). In this connection the absence of regions of synteny between the two chromosomal Adh regions in these two species may be an additional criterion to propose the genetic differentiation between theAdh system of C. capitata and that of D. melanogaster. Biochemical analysis of the two A D H systems confirm this hypothesis (Gasperi et al., 1989). Five genes (Aconl, Mpi, Est,, Aox, Mdh2) from linkage group D are spread on the linkage map of this chromosome arm, far from theAdh region as deduced by recombination data and deficiency mapping. No information is available on the positions of the Xdh and Lspi loci relative to theAdh loci. The cytological localization of the Adh region represents a reliable starting
46
Malacrida et al.
point for the ordering of this gene cluster with respect to the c e n t r o m e r e and for the analysis of the recombinational events in the proximal and distal part of this c h r o m o s o m e arm. In this respect the first unambigous available data are summarized in Fig. 4, in which the origin of the linkage m a p of 2L appears close to the A d h region. Reliable information on the position of genes on this c h r o m o s o m e m a y be derived f r o m a comparison of the cytological m a p with the u p d a t e d linkage map. Finally, one o f our efforts in past years (Gasperi et al., 1987, 1989; Riva Francos, 1990) has b e e n to develop a genetic sexing m e t h o d for this pest species based on the A D H m o d e l of Drosophila ( R o b i n s o n et al., 1983). In this p a p e r substantial genetic evidence has b e e n obtained to show that in the D f ( 2 L ) 2 C ; 3 A deletion of the strain T128, no A D H polypeptide is synthesized. Thus we a p p e a r to have stable A d h 1 A d h 2 null mutants for further work.
ACKNOWLEDGMENT W e thank M. A s h b u r n e r for critical reading of and c o m m e n t s on the manuscript.
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