Natural Populations of Drosophila Melanogaster: Restriction Map Variation in the Region of the Adh Locus in Populations from Two Hemispheres

Heredity68 (1992) 1—14 Received 8 October 1990 Genetical Society of Great Britain

The alcohol dehydrogenase polymorphism in natural populations of Drosophila melanogaster: restriction map variation in the region of the Adh locus in populations from two hemispheres

JAMES CHENGSHAN JIANG* & JOHN B. GIBSONI Molecular and Population Genetics Group, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra, ACT 2601, Australia

Restrictionendonuclease variation in the 12 kb region surrounding the Ad/i locus was measured in seven Australian and six Chinese populations of Drosophila melanogaster. There is a higher level of nucleotide-substitution variation in the Australian populations than in the Chinese, which is possibly a reflection of their origins. None of the restriction site polymorphisms, nor any of the insertions, showed a significant association with latitude. A 02 kb deletion varied with latitude in the Chinese populations. In accordance with previous studies, a majority of the insertions were located in a region 1.5—3.5 kb 3' from the Adh coding region, and a majority of the deletions were at a site 3 kb 5' to the Ad/i coding region. Two of the insertions shared homologies with known mobile elements. Overall, the data suggest that restriction endonuclease variation in the Ad/i region is not related to the dine in Adhs frequencies.

Keywords:alcoholdehydrogenase, D. melanogaster, polymorphism, restriction enzymes.

Lewontin (1974), Endler (1977) and others have Introduction cautioned that observed geographical distributions of Allozymevariation at the alcohol dehydrogenase locus allele frequencies do not by themselves allow for dis- in Drosophila melanogaster is geographically differen- crimination between hypotheses about the causes lead- tiated world-wide in a remarkable pattern, which is ing to particular patterns. The Ad/i dine in D. unique amongst enzyme polymorphisms in any melanogaster is no exception to this stricture and, organism. Most populations of D. melanogaster contain although selective and stochastic processes have been two electrophoretically distinguishable alleles, AdhF postulated to account for the geographical distribution, and Ad/is, with the population frequency of Adhs the causal mechanism(s) remains unknown. decreasing with distance from the equator. This pattern The available data from studies on natural popula- was first described for North American population tions do not provide any compelling evidence for selec- samples (Johnson & Schaffer, 1973) but later studies tive factors acting on any aspect of phenotypic revealed very similar distributions in Australasia, variation to which the Ad/i locus is known to contri- Europe, Africa and Asia (Wilks eta!., 1980; Oakeshott bute (Gibson eta!., 1980; Van Delden, 1983; Gibson & et a!., 1982; David, 1982; David et a!., 1989; Singh et Wilks, 1988). Nevertheless, data from experimentally as!., 1982; Jiang et al., 1989). The clinal distribution of maintained transplanted populations appear to show alleles at the Adh locus has thus been observed in both that Adh frequencies change in the direction predicted hemispheres and on all continents for which data are by the dine (Oakeshott et a!., 1988). The possibility available —thereare no published data from the sub- remains that any selection maintaining the Ad/i dine continent of South America. might be directed towards regions of DNA linked to the Adh locus, rather than to the products of the locus. *present address: Department of Zoology, Louisiana State It has been clearly shown in relation to this possi- University, Baton Rouge, Louisiana, 70803-1725, U.S.A. To whom correspondence should be addressed. bility, both in populations from the Eastern United

1 2 J. CHENGSHAN JIANG & J. B. GIBSON

States (Voelkereta!., 1978) and from Australia (Knibb, 1983),that the Adhdine is notcaused bygameticdis- equilibrium between the inversion In(2L)t (Mettleret a!.,1977) and the Adhlocus,which is located just outside the proximal breakpoint (Woodruff & Ashburner, 1979). A further class of variation which might be relevant to the Adhdine,and which can now be examined, is that revealed in the molecular landscape of the Adhlocusby restriction endonucleases. Investi- gations using restriction enzymes that recognize six- base pairs (Langley eta!.,1982; Birley, 1984; Cross & Birley, 1986; Aquadro eta!.,1986 and Jiang eta!., 1988) and four-base pairs (Kreitman & Aquadé 1986a,b and Simmons eta!.,1989)have revealed con- siderable variation in the Adhregion.The studies of Kreitman & Aquadé (1986a,b), which showed there was no differentiation between an east-coast and a west-coast North American sample, and that of Simmons eta!.(1989) on three populations from the east coast of the United States, are the only systematic analyses of restriction endonuclease variation in relation to the Adhdine.To supplement this information, we have investigated whether any restriction endonuclease variation in a 12 kb region encompassing the Adhlocusparallels the latitudinal variation in AdhF frequency,and whether there are any similarities in populations at the same distance from the equator in China and Australia. This paper describes the restriction endonuclease variation and later we will examine the effect of the variation on ADH activity.

Materials and methods The geographical locations of the seven Australian and six Chinese natural populations from which the D. melanogaster usedin the present study were sampled are shown in Fig. 1. Details of the Chinese populations have been given previously (Jiang eta!.,1989); the Australian samples were collected in 1986 and 1987 and, like the Chinese material, were maintained in the laboratory with between 75 and 125 single female lines fromeachpopulation. A single male from each of a number of single female lines was mated with a virgin female from a second—third chromosome translocation stock, T(2;3) ap,whichcontains the second chromosome balancer CyO,and the thirdchromosome balancer TM6 (Lindsley& Grell, 1968). A single Cymalefrom the progeny was backcrossed to the translocation stock females, and the Cyoffspringwere mated together to

Fig. I The locationofthe Chinese andAustralianpopula- tions of D. melanogaster. ALCOHOL DEHYDROGENASE POLYMORPHISM IN 0. MELANOGASTER 3 produce a line homozygous for a single second were screened by plaque hybridization using the sAC 1 chromosome. Any homozygous lethal chromosomes clone as a probe (which contains a 4.7 kb Eco RI frag- were maintained heterozygous with CyO. About 15 ment encompassing the Ad/i gene) labelled with second chromosomes were extracted from each of the [a-32P]-dCTP. Positive clones were plaque-purified and 13 populations, to give a total of 104 chromosomes DNA from each was transferred to mtrocellulose from the Australian populations and 90 from the membranes which were then probed with 32P-labelled Chinese populations. clones of mobile elements. The cloned mobile elements DNA was extracted from adult flies from the 194 used as probes were: p2r2S. 1, a Barn HI fragment con- lines either by the two methods previously described taining a complete 2.9 kb P element cloned into the (Jiang etal.,1988) or by the following short method. pBR 322 vector (O'Hare & Rubin, 1983); Copia, a Fifty adult flies were homogenized in 50 ul cold TE Barn HI fragment containing a 5.0 kb copia element in buffer (100 mM Tris pH 9.0, 100 mivt EDTA), then 500 pBR 322 (Rubin et a!. 1981); B1O4B, an Eco RI frag- Aul of 2 per cent SDS were added to the homogenate ment containing a 2.8 kb B1O4B, which is part of the which was then incubated at 65°C for 1 h. After the B104 element, in the PACYC 184 vector (Scherer et a!., homogenate had cooled, 120 ,ul of 8 M potassium 1982); pl4O7, a Sal I fragment containing a 5.4 kb I acetate were added and DNA was precipitated from factor in the PAT153 vector (Boucheton et a!., 1984); the supernatant with 500 p1 of isopropanol. The DNA cDm4l2, a Hind III fragment containing a 7 kb 412 pellet was washed twice with cold 70 per cent ethanol, copia-like element in pBR322 (Finnegan et a!., 1978; dried under vacuum and resuspended in 50 ul TE pPW297, a Hind III fragment containing a 6.5 kb 297 buffer containing 20 pg/ml boiled RNAse. copia-like element in pBR322 (Finnegan et a!., 1978; The DNA extracted from each of the single second Rubin et a!., 1982); pDMI-101, an Eco RI fragment chromosome lines was digested with eight hexanucleo- containing a 4.3 kb F element in pBR322 (Dawid etal., tide restriction endonucleases, Barn HI, Eco RI, Hind 1981, and I. B. Dawid personal communication). III, Hpa I, Pst I, Sal I, Xba I and Xho I, under the buffer conditions recommended by the manufacturer Results (Amersham). Double and triple digestions were made in order to construct restriction maps; agarose gels of 2 Therestriction endonuclease variation observed in the and 0.5 per cent were used for electrophoresis of the region of the Ad/i gene, using sAF-2 as a probe, in fragments but we were unable to obtain a reliable reso- seven Australian and six Chinese populations, is listed lution of less than 50 bp. in Table 1. Some of the data for the two Tasmanian The methods used to transfer the DNA fragments populations have already been published (Jiang et a!., from agarose gels to mtrocellulose filters and the pre- 1988), but they are included here for completeness. To hybridization and hybridization conditions have been facilitate comparisons with previous work on the Adh described previously (Jiang et a!., 1988). The nitro- gene region, the Barn HI site in the second exon of the cellulose filters were probed with the sAF-2 plasmid Adh transcription unit is given the co-ordinate of 0.0. (Goldberg, 1980), which includes an 11-kb insert Overall in the seven Australian populations, 13 spanning the region of the Adh gene, labelled with polymorphic restriction sites, seven insertions and one [a-32P}-dCTP by nick translocation (Rigby et al., 1977). deletion were observed (Fig. 2a). In the Chinese popu- Autoradiography was performed at —70°Con Kodak lations there were 12 polymorphic restriction sites, five film (XRP-1) using intensifying screens. insertions and one deletion (Fig. 2b). Ten of the poly- The sizes of the restriction fragments which hybrid- morphic restriction sites were shared by populations ized to the labelled probe were determined graphically on both continents but only three of the 11 different from a semi-logarithmical plot of the sizes of .DNA structural changes were shared. A 0.2 kb deletion and a fragments digested with Hind III. 0.4 kb insertion were present in most of the popula- A number of the insertions found in the Australian tions but each of the insertions larger than 1 kb was and Chinese populations were cloned and probed with unique to a particular population, although sometimes sequences from known mobile elements to testfor found in more than one single chromosome line. One homologies. Ten single second chromosome lines, each chromosome extracted from the Tamar population of which had been shown to contain a different sized contained a complex duplication of parts of the Adh insertion (one line, H2, contained two insertions), had coding region (Jiang et a!., 1988). their DNA digested with Bgl II and the fragments Estimates of nucleotide substitution variation were ligated to ) EMBL3 arms with Barn HI ends (Frischauf derived using equations for the proportion of poly- et al., 1983). Recombinant molecules were packaged morphic nucleotides (Ewens et a!., 1981; Hudson, and the 10 libraries, in Escherichia coli strain LE392, 1982), the heterozygosity per nucleotide pair, O(Ewens 4 J. CHENGSHAN JIANG & J. B. GIBSON

Table 1aRestrictionendonuclease variation in the Adh gene region of the Australian natural populations of Drosophila melanogaster. I =insertion,D= deletion.Restriction sites are BarnHI (B), EcoRI(E), Hind111(H), Hpa I(Hp), Pst1(P), Xba I (B), XhoI (Xh). The size of the insertions and deletions and the position of the restriction sites are indicated in parentheses. The Adh allotype is indicated with F(AdhF ),S(Ad/is) and C/iD (Adh'3). The populations are Cygnet (C), Tamar (T), Chateau Yarrinya(CY), All Saints (AS), Araluen(Ar), Coffs Harbour (CH) andCardwell(Cd)

X X B I Hp X H H D I P Xh P B 1 I 1 1 1 B E (—8.0) (—7.4) (—7.2) (0.28) (—6.9) ('-5.3) (—3.7) (—3.0) (0.2) (5.0) (1.1) (1.2) (1.3) (1.4) (0.4)(0,7) (3.0) (4.5) (1.0) (3.9) (9.0) C3 - - + + — S — + C5 - - + - — - - + + — F — + C8 - - - - - S - — + cii — — + — — F — + C12 - — + - - S - + C20 - - - - — F — + C25 — — + — — S — + C28 — — — — + S — + C29 - - - - — — - + + — F - + C33 - - - - — — — - + — F — + — — + C35 - - - - — — - + + — F — + C45 — — - - - S — + C51 - - - - — F — + + C61 — — + — — S C72 - - — - — S — + Ti - — — — + S — + + — + — — — — + — T12 — — — — — F — + T17 - — + — — S - + + — T19 — — + — — S T29 - — — + + F — + T30 — — — — — F — + + T32 — — — — - F - + T33 — — + — — S — + + T44 - - — — — F + + — - + T50 - - + — - S — + T54 - — — — - F - + T56 - — + - - F - + T60 — — — — - F — + T73 - — + - — F - + — CY1 + .f - — - — + — — S — + + — + + - CY2 + - - - - + - - S - - + + CY3 - - + — - + — CY4 + + - — - — + — - S — - + + - — - — — — — CY5 - + + — — S - — CY6 + + — + + — + CY7 ------+ - — F - + CY8 - - - - cyg - - + - F - Cylo - — — — — - + - — — cY11 - + - F — + — + CY12 - — + — - + - — — CY13 + - F CY14 ------+ - - - F - + - — — CY15 - — + — AS1 + + + — — - - + — — — + — — AS2 + — — — — — — — - — — A53 + + — — — — + AS4 — — + — + — — AS5 + — — + ASS .. - - - - AS7 - - + — AS8 - - - - — — — — — + AS9 - - - - - + AS10 — — — — - + ASh - + — — + + AS12 — - + — AS13 — — — — F - + AS14 — — — — F - + A515 — — — — Ar? - - + - S Ar2 - — + — — — — Ar3 — + — — — - - — Ar4 - - — — + Ar5 - — + — — Ar6 - - - Ar7 — — + — F - + ArS - - - - — + + - + ALCOHOL DEHYDROGENASE POLYMORPHISM IN D. MELANOGASTER 5

Table la Continued

X X B I Hp X H H D I P Xh P E I I I I I B E (—8.0) (—74) (—7.2) (0.28) (—6.9) (—5.3) (—37) (—3.0) (0.2) (5.0) (1.1)(1.2) (1.3) (1.4) (0.4) (0.7) (3.0) (4.5) (1.0) (19) (9.0)

Ar 9 — + — — - — + Ar10 — — + + - F - + — Ar 11 — - + + - F — + — Ar 12 — + — Ar 13 + + - — — — + Ar 14 - + - — — — + Ar 15 — + - CM 1 — + — — - - + CM 2 - — + - + — — + — — $ + + — — + — — — + — CM 3 - — + — - + — — S — — — CH 4 - — + - + + + — — + — — — + — CM 5 - - + - - F - + - CM 6 - - - + - F - + - CM 7 — — + — — + + — F — + — — — CM 8 — + — — + — — S + — — — + — CM9 - - + — — + - - F - + - CM10 - - + — - + — — $ + + — CM 11 - — + — + — CM 12 - — + — + — CM 13 - - + - + + CM 14 — - - + - null+ + — + — - — - + Cd 1 — + — + — S — - — Cd 2 — — + — + — Cd 3 - — + + + — - + — Cd 4 - - + — — + — — S — + — — - — + Cd 5 - - + — - + - - S — — + - - + Cd 6 - - + - + - + - + — S - + - — — — — — Cd 7 — — + — — + S — — Cd 8 - - + — — + — — S — — — — + Cd 9 — — + — — S — — — Cd 10 — + - Cd 11 — + + - - F — — - Cd 12 - - + - + - Cd 13 — + — — — + + Cd 15 - + + + + Cd 16 — + -

Table lb Restriction endonuclease variation in the Adh gene region of Chinese natural populations of D. melanogaster.All abbreviations are the same as in Table la. The populations are Haikou (H), Guangzhou (G), Fuzhou (F), Shanghai (S),Jinan (J) and Dalian(D)

P Xh P 1 1 1 E E X B I Hp X H H 0 1 (1.1) (1.2) (1.3) (0.48) (0.4) (0.7) (2.5) (90) (—8.6)(—7.4) (—7.2) (0.35) (—6.9) (—5.3) (—3.7) (—3.0) (0.2) (1.5) — — — — — — — — — + — F — + + Ml — — H2 - + — + - — + — ChD — + + + - - - — - - + — ChD - + H3 — — — — — — — + ÷ F — + + M4 — M5 — — — — — — — — F — + + — H6 — — — — — — + — F + M7 — — — — — — + — F — + — — — — — + M8 — — — — — + F + H9 — — — — — — + — S — + — — - — — + M 10 — — — — — + + S + + + — — — - + — H 11 — — — — — — S + + — — - — — + M 12 — — — — .- + + S + + + — — - — — + H 13 — — + — — + + S + + + — — — — — — — + H 14 — - — — + S + — — — M 15 — — — — — + + — S — + + — — — G1 -. — — — — — — — F — + + G2 - - - — - - + — Cho - + — — — — — + G3 — — — — — + + F + G4 — — — — — + + — F — + G5 — — — — — + + — F — + G6 — — — — — — + — ChD — + G7 + — — — — + + — F — + 6 J. CHENGSI-IAN JIANG & J. B. GIBSON

Tablelb Continued

F X B I Hp X H H D I P Xh P I I I E F (—8.6)(—7.4) (—7.2) (0.35) (—6.9) (—5.3) (—3.7) (—3.0) (0.2) (1.5) (1.1) (1.2) (1.3) (0.48) (0.4) (0.7) (2.5) (9.0)

08 — — — + + — F — + — — — — — — G9 — - + — — + — S — + — — — — — — 010 — — + — — + — S — + — — — — — — Gil — — — + + — S — + + — + — — — 012 — — + S — + — — — — — — G13 — - — — + — F — + — — — — — — 014 — — — — + — S — + — — — — — + Gi5 — - — — + — S — + — — — — — Fl — — + S — + — — — — — — F2 — — — — + - F — + — — — — — — F3 — — — — + — F — + — — — — — F5 — — — — + — F — + — — — — — — F7 — — — — + — F — + — — — — — — F10 — — F — + — — — — — — — — Fli — — + — S — + — — — — — — — — Fi4 — — + — F — + — — — — — — — — F16 — — + — F — + — — — — — — — F2i — — — + — F — + — — — — — — — — F22 — — + — ChD — + — + — — — — — — F23 + + — F — + — — — — — — — — F28 — — + — S — + — — — — — — — — F33 + S — + — — — — — — — — — — F39 + — S — + — — — — — — — — 52 + S — + — — — — — — — 53 + S — + — — — — — + — — 54 F + + — — — — — — — — 55 + F — + — — — — — — — + — — — 56 + F + + — — + — — — — + — — 57 + — F + + — — + — — — — + — — — 513 + F — :4 — — — — — 518 — + — — + — F + + — — + — — — — — S 24 + S — + — — — — — — O + — — — 26 + — F — + — — — O27 — + S — + + + — — — O 28 — — S — + — — — — — — O32 + — — + — F + + — + — — — — — — O 34 — — + F — + — — — — — — O39 — — + F + + — — + — — — — — J1 — F + — — — — — — — — L33 — F + — — — — — — — — J4 — — F + — — — — — — — J6 — — — — — — + F + — — — — - — J7 — — F + — — — — J8 — — - — — F + + — -. — — — + Jil — — F + — — — — — — 312 — — — + — — F ÷ 4 — — — — — — 314 + — S + — — — — — J16 - — + — S + + — + — — — — — 021 — S + — — — — — — — 026 - + — + F + — — — — 030 - — — - + — — S + — — — — — 036 — — — — F + — — — — — — 042 + + — — ChD + — — — — — — 02 — — — — + F + — — — — — — — 04 + — — — — — + F + — — — — 06 -- — — — — — + F + — — — — — 07 — — — — — + F + — — — — — — — 010 — — — — — + F + — — — — — — Dil + — — — — — + F + — — — — — — — D12 — F + — — — — — — — — 014 — — — — + F + — — — — — — 015 - — + — S + — — — — — — 016 - - — — — + F + — — — — — — — — 017 — F + — — — — — — — 020 + — F + — — + — — — + 022 + — — F + — — — — 023 - - — F + — — — — — — — + 030 — — F + — — — — + ALCOHOL DEHYDROGENASE POLYMORPHISM IN 0. MELANOGASTER 7

Kilobases -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 9 I I I I I I I I I I L I

5' 3'

20—— —- — 00— Lu (a)

x Lu

5' 3' = = — —— — E — _2 28.s •j2 8 8 2 28 )CWIIII I II 0-Lu (b) •I Iil -.ilI IdlE- 8 Lu ILL

350 iso\ 000"

Fig. 2 Restriction endonuclease variation detected in the Ad/i region of (a) 104 chromosomes from seven Australian populations and (b) 90 chromosomes from six Chinese populations. The Ad/i transcription unit is boxed and the adult 5' leader sequence and the three exons are shaded. Polymorphic restriction sites are indicated below the map; triangles represent insertions (points towards the map) and deletions (points away from map). Estimated sizes (in bp) of the insertions and deletions, and the restriction sites within the insertions, are indicated. The Eco RI (+ 9.0)site was outside the probed region but it could be scored unambiguously in all populations.

eta!., 1981;Hudson, 1982) and the haplotype diversity frequency of the 0.2 kb deletion, which was present in (Nei & Tajima, 1981). These analyses (Table 2) show all the Chinese populations, and all but one of the that overall there is a higher level of nucleotide-substi- Australian populations (the exception was Tamar), tution variation in the Australian populations than in varied with latitude in the Chinese (r= —0.89, the Chinese populations. P < 0.01) but not in the Australian populations (Fig. 3). Restriction map haplotype diversity (Table 3) is Non-random associations between variants in the similar in the Australian and Chinese populations Ad/i region are listed in Table 4. Tests for linkage dis- when both forms of variation (site and sequence length) equilibria used Fisher's exact test for a 2 x 2 con- are considered. The level of haplotype diversity in tingency table; any variant with a frequency less than AdhSbearing chromosomes was higher than for AdhF 15 or more than 85 per cent was excluded from the chromosomes in both sets of populations. The few data analysis. The most common non-random association is available for chromosomes bearing a third kind of Adh the one between the Barn FIT (— 7.2)site and Ad/is: this allele [Adh'T (all in the Chinese samples)] indicate association occurred in eight of the 10 populations in that they are the least diverse (Table 3). which the frequency of Barn I-il (—7.2)is greater than There is no evidence from these data for any con- 15 per cent. Another common form of association is sistent relationship between latitude and the estimates between the presence of insertions/deletions and some of variation in nucleotide-substitution or in haplotype restriction sites, such as the non-random association diversity. Furthermore, none of the restriction site between Hind ITT (—3.0)and D (0.2), which occurred polymorphisms, nor any of the insertions, showed a in three populations. significant association with latitude. However, the Ten different insertions (insertion 0.28, 0.35, 0.4, 8 J. CHENGSHAN JIANG & J. B. GIBSON

Table 2 Estimates of the proportion of polymorphic nucleotides (p), overall heterozygosity (h) (Nei & Tajima, 1981), and estimated heterozygosity per nucleotide pair (0) (Ewens eta!., 1981). Standard errors (Hudson, 1982; Nei & Tajima, 1981) are given in parentheses and that for 0 assumes free recombination. n is the number of second chromosomes extracted from each population

Population Latitude n p h 6 Australia Cygnet 430 09'S 15 0.017 (0.007) 0.95 (0.01) 0.006 (0.002) Tamar 41°02'S 15 0.029(0.007) 0.86 (0.06) 0.007(0.003) ChateauYarrinya 37°36'S 15 0.022(0.011) 0.99 (0.01) 0.008(0.004) All Saints 36° 03'S 15 0.024(0.008) 0.95 (0.01) 0.009(0.005) Araluen 35° 03'S 15 0.020(0.008) 0.96 (0.01) 0.007 (0.003) CoffsHarbour 30°18'S 14 0.024(0.008) 1.00(—) 0.009(0.003) Cardwell 18°16'S 15 0.024(0.008) 0.98 (0.01) 0.009(0.003) Australian populations 104 0.021 (0.006) 0.96 (0.1) 0.008(0.002) China Haikou 20°02'N 15 0.017(0.007) 0.94 (0.02) 0.006(0.003) Guangzhou 23°08'N 15 0.012(0.006) 0.94 (0.02) Fuzhou 0.005(0.002) 26°05'N 15 0.009(0.005) 0.83 (0.07) 0.004(0.002) Shanghai 21°10'N 15 0.019(0.007) 0.89 (0.04) Jinan 0.007(0.003) 37° N 15 0.0 12 (0.006) 0.90 (0.04) 0.005 (0.002) Dalian 38°54'N 15 0.015(0.007) 0.76(0.13) 0.005(0.002) Chinese populations 90 0.014 (0.004) 0.88 (0.05) 0.005 (0.001) Overall 194 0.03(0.003) 0.904(0.018) 0.0065(0.001)

Table 3 Restriction map haplotype diversity in the Adh region. The numbers of different haplotypes for each category are given in parentheses

All Variation considered chromosomes AdhF Adh AdhFD Australian populatioiis* Restrictionsjte 0.95(48) 0.90(19) 0.97(28) — Sequencelength 0.79(16) 0.58(8) 0.54(7) — All variation 0.97(59) 0.93 (28) 0.97 (30) — Chinese populations Restriction site 0.90 (28) 0.78 (14) 0.80 (10) 0.64 (4) Sequence length 0.86 (14) 0.67(7) 0.71 (4) 0.46 (3) All variation 0.96 (48) 0.90 (31) 0.92(13) 0.64 (4) *One chromosome carried an Ad/i null allele.

0.48, 0.7, 1.0, 1.5, 3.0, 4.5 and 5.0 kb) were sub-cloned Cygnet, Tamar and All Saints populations, showed in the bacteriophage vector EMBL3 and probed with homology with B1O4B, which usually has approxi- DNA sequences from each of seven known mobile ele- mately 100 copies in the D. melanogaster genome ments. The results (Table 5) show that two of the (Scherer et a!., 1982). A second insertion, 0.4 kb, cloned insertions shared homology with known mobile showed homology to the mobile element Fbi, which elements. Insertion 0.28 kb, which was present in the usually has about 25 copies in the D. melanogaster ALCOHOL DEHYDROGENASE POLYMORPHISM IN D. MELANOGASTER 9

1.0 genome(Dawid et al., 1981). Insertion 0.4 kb occurred •• ineach of the Australian and Chinese populations at frequenciesranging from 0.07 to 0.47. 0.8 Discussion 5 0.6- Theestimates of the proportion of polymorphic nucleotides, the overall heterozygosity and the hetero- 0.4 zygosityper nucleotide pair are each higher for the 0• Australianpopulations than for the Chinese popula- U. tions but, in the total dataset, these values are not dis- 0.2 0 0 similar to those obtained in earlier studies on o populationsfrom North America, Europe and Australia(Table 6). 0 I I I I 15 20 25 30 35 40 45 Thedifferences between the Australian and Chinese populations —particularlyin p and 6 —mightbe Latitude relatedto the history of the populations on the two Fig. 3 Latitudinal variation in the frequency of the deletion continents. David & Capy (1988) have suggested that 0.2kb in populations from China •)andAustralia (0). populationsof D. melanogaster may be considered in

Table 4Significantgametic disequilibria in the Australian and Chinese populations. The abbreviations I and D represent insertionsand deletions and their sizes (kb) are given in parentheses

Populations Significant gametic disequilibria

Australia Hind ffl( —3.0):D(0.2)** Cygnet Hind III( —3.0):Adh*, Adh:D(0.2)* — Taniar BarnHI(7.2):Adh**,Hpa I( —6.9):BamHI(3.9)** Pst 1(1.1 ):I(0.4)**, Xho 1(1 .2):EcoR1(9.0)* III( — Chateau Yarrinya Hind III( —3.0):Hind 3.7)*, Hind III( —3.0):D(0.2)*, Barn HI( —7.2):Adh**,I(1.0):Xba I( —8.0)**, Xba I( —7.4):I(0.4)** I( —7.4)*, All Saints Barn HI( —7.2):Adh**,Xba I( —5.2):Xba XbaI( —5.2):PstI(1.3)*,I(0.4):XbaI( —7•4)**, I(0.4):XbaI( —5.2)*,I(0.4):PstI(1.3)* Araluen BarnHI( —7.2):Adh**,Hind Ill( —3,0):D(0.2)** Coffs Harbour Hpa I( —6.9):1(0,4)**,1(0.4):Pst 1(1.1 )*, Hpa 1( —6.9):BamH1(3.9)**, I(0.4):Bam HI(3.9)* Cardwell BarnHI( —7.2):Adh**,Hind III( —3.7):D(0.2)** China Haikou D(0.2):EcO RI(9.0)*, Pst 1(1 .3):I(0.4)* Guangzhou Barn HI( —7.2):Adh* Fuzhou None Shanghai Barn HI( —7 .2):A dh**, D(0.2):PstI( 1.1 )* I(0.48):XbaI( —74)*,I(0.48):BarnHI( —7.2)* I(0.48):PstI( 1.3)" Jinan BarnHI( —7.2):Adh** Dahlian None *p<0.05, "P<0.01. 10 J. CHENGSHAN JIANG & J. B. GIBSON

Table 5 Cloned insertions and the results of hybridization with probes derived from known mobile elements

Second Mobile element probest chromosomeInsertion line size(kb) pr25.1CopiaB1O4BP1407412297FlOl

T29 0.28 — — + — — — — CYI 0.4 — — — — — — + CY2 1.0 — — — — — — — AS13 5.0 — — — — — — — Ar13 3.0 — — — — — — — Cd5 0.7 — — — — — — — Cd15 4.5 — — — — — — — H2 0.35, 0.4 — — — — — — — H4 1.5 — — — — — — — S39 0.48 — — — — — — —

+ Denotespositive hybridization. f See Materials and Methods for details.

Table 6 Estimates of the proportion of polymorphic nucleotides (p),haplotype diversity (h), and estimated heterozygosity per nucleotide pair (0)

Pbpulation p h 0 Australia This study 0.021 0.96 0.08 Chateau Tahbilk (Cross & Birley, 1986) 0.017 0.94 North America Texas (Birley, 1984) 0.0 12 0.86 0.004 Eastern States (Aquadro etal., 1986) — 0.97 0.006 Europe Groningen(Cross&Birley, 1986) 0.025 0.66 China 0.007 This study 0.014 0.88 0.005

terms of three categories which comprise 'ancestral' The distribution of restriction endonuclease varia- populations in tropical Africa (where D. melanogaster tion between the Australian populations differs from originated), 'ancient' populations derived from the the distribution between the Chinese populations. A x2 initial colonization of Eurasia and 'new' populations analysisof the frequencies of restriction endonuclease introduced by Homo sapiens. Contemporary Chinese variants between Chinese populations shows that most populations may well be a mixture of the 'ancient' and of the populations differ from each other and the island 'new', but it is likely that the D. melanogaster, which population of Haikou differs from each of the main- colonized Australia following European settlement land populations (Table 7). In contrast, a similaranaly- (David & Capy, 1988; Lachaise et a!., 1988), had sis of the Australian data reveals no significant diverse origins, and this may account for thehigher differences between the mainland populations levels of variation in contemporary Australian except tions. popula- between Chateau Yarrinya and Coffs Harbour, which are about 2,000 km apart. All other significant differ- ALCOHOL DEHYDROGENASE POLYMORPHISM IN D. MELANOGASTER 11

Table 7 Chi-squared analysis of the frequencies of the restriction endonuclease variants compared between populations. Degrees of freedom are given in parentheses

Australian Chateau Coffs populations Cygnet Tamar Yarrinya All Saints Araluen Harbour Cardwell

Cygnet — 18.07 23.05*(12) 17.54(14) 7,4(11) 13.19(11) 12.18(14) Tamar — — 40.13***(14) 14.57(13) 11.99(12) 15.28(11) 27.07(13) ChateauYarnnya — — — 15.56(13) 20.20(12) 24.98*(14) 17.04(15) AliSaints — — — — 10.52(13) 20.45(14) 19.62(16) Araluen — — — — — 13.04(12) 14.98(14) Coffs Harbour — — — — — 11.02(12)

Chinese populations Jinan Shanghai Fuzhou Guangzhou Haikou

Dahlian 19.63*(9) 19.64*(10) 10,79(7) 11.40(7) 22.89*(11) Jinan — 24.75**(10)12.80*(6) 3434**(14)38.07***(12) — Shanghai — 22.06**(9) 26.7**(10) 40.04***(15) Fuzhou — — — 11.49(6) 26.41**(11) — — — — Guangzhou 20.88*(11)

*p<0.05, **P< 0.01,***P<0.001.

ences involved comparisons with the two Tasmanian Chinese and Australian populations, concerns the non- populations, which themselves differ, even though random localization of insertions/deletions in the region of the Adh locus. Other than the nucleotide Tamar is only 300 km north of Cygnet. The difference Adhs alleles, between the Cygnet and Tamar populations is interest- change responsible for the Adh' and ing as it could be interpreted as evidence that the two there were no restriction endonuclease polymorphisms populations, which differ markedly in Adh' frequency in the Adh coding regions, and only one change was found in an intron (one chromosome from the All (Anderson & Gibson, 1985), are more geographically Saints population had a 5.0 kb insertion in intron I). isolated than any two Australian mainland populations separated by the same distance. Other than between The majority of the insertions occur in a region 1.5—3.5 the Tasmanian populations, the data suggest that there kb 3' to the coding region. The techniques used in this study were unable to resolve insertions ordeletions of is less migration of D. melanogaster per unit distance in the eastern part of mainland China than in a similar less than 50 bp. This level of resolution is similar to that obtained by Langley et at. (1982), Birley (1984) region of Australia. and Cross & Birley (1986), but less than that achieved A striking component of the geographical hetero- the results geneity between the Chinese populationsis the by Aquadro et al. (1986). To summarize decrease in frequency with distance from the equator derived from the five separate studies, we have collated of the deletion 0.2 kb. This deletion was present in all data on the locations of insertions/deletions of greater than 50 bp. These data (Fig. 4.) show that 51 per cent Chinese and Australian populations with the exception of Tamar. However, its frequency was relatively low in of the 85 insertions in the 336 second chromosome the Australian populations, and not related to latitude. lines investigated in the Adh region were located about 1.5-2 kb 3' from the end of the Ad/i coding region in a This deletion is in a region of the Adh landscape, which 0.23 kb Eco RI/Sal I fragment (85 per cent of the has been implicated in the control of ADH expression insertions were in the region + 1.5 kb to + 3.5 kb). Of (Goldberg et al., 1983; Posakony et at., 1985) and we show elsewhere that it does have an effect on ADFI the 70 deletions, 67 appeared to be at a site approxi- activity levels. Nevertheless, the lack of consistencyin mately 3 kb 5' to the Adh coding region, and two were its distribution in Chinese and Australian populations in the general region of the majority of the insertions. This clustering of insertions and deletions may arise would, at this stage, appear to mitigate against the idea from the susceptibilities of the two regions to the inser- that the deletion is implicated in the Adh dine. A remarkable feature of the restriction endo- tion of mobile elements (the DNA sequences of the two nuclease variation, which is highly consistent in the regions are not yet known, but they maycontain recog- 12 J. CHENGSHAN JIANG & J. B. GIBSON I 0) 0 I a 0 I Ca 0 insertions of tO 0 Number I 0 I 0 I — 0 I P0 0 I 0 Ca deletions

ot Fig. 4 The distribution of insertions I • 0 and deletions (larger than 50 bp) in the 11.8 kb region of the Adh gene. Data I Number 0' 0 have been collated from the present study and from Langley et al. (1982), I 0) 0 Birley (1984), Cross & Birley (1986) and Aquadro etal.(1986). I —I o

nitionsequences for one or more mobile elements) or it described by Aquadro et a!. (1986). In our study this may be that insertions in the region cause no selective insertion was homologous to FlOl whilst the one found disadvantage. in the North American populations was homologous to Most of the insertions found in this and in previous cDM2161, which is an F-like element similar in struc- studies have not been identified. The exceptions are the ture to FlOl (Aquadro eta!., 1986). In the Adh region insertions 0.28 and 0.4 kb. The 0.28 kb insertion none of the insertions so far tested shared homology shared homology with the copia-like element B1O4B, with P elements. which is usually present in about 100 copies in the A further notable aspect of restriction endonuclease genome of D. melanogaster. This type of insertion was variation in the Ad/i region is the linkage disequi- found only in the Tasmanian and the All Saints popula- librium between Barn HI (—7.2)and Adh. This asso- tions. A typical B104 element is 8.7 kb and is flanked ciation is a consistent finding from studies of the by 429 bp direct terminal repeats. Finnegan et a!. molecular landscape of the Adh region (Langley et a!., (1978) found that a 2.4 kb fragment inserted in B104 1982; Aquadro et a!., 1986). The linkage, however, is was part of a cDM412 clone. Wensink et a!. (1979) not as tight in the Australian and Chinese data as it is in showed that D. melanogaster DNA contains clusters of the North American populations. It has been suggested short repetitive sequences (less than 1 kb) scattered that the mutation giving rise to Adh'' occurred in an over a large number (1000) of chromosome regions, Adh allele on a chromosome lacking the Barn HI and some of these clusters hybridize to the region of (—7.2)site and that subsequently the AdhF allele the cDM412 clone which contains the B104 fragment increased in frequency in temperate regions (Cross & with the 1.4 kb insertion. The 0.28 insertion may be Birley, 1986). The disequilibrium would take a long one of these repetitive sequences. It has been estimated time to decay in the absence of any selection maintain- that the rates of insertion and excision of some of the ing the association (Avery & Hill, 1979). It may thus be copia and copia-like elements is less than 4 x 10 per relevant that in the 'old' Chinese populations the link- element per generation (Harada eta!., 1990). age is looser than in the Australian or North American The other insertion that we identified (0.4 kb) was populations (Aquadro et a!., 1986; Kreitman & found in Australian and Chinese populations and Aquade, 1 986a). There is a higher level of hetero- appears similar in size and location to an insertion zygosity for haplotypes with Adhs than for those with ALCOHOL DEHYDROGENASE POLYMORPHISM IN D. MELANOGASTER 13

AdhF, and this supports the DNA sequence data which change in the DNA sequences coding for alcohol de- show that Adhs is the ancestral allele (Kreitman hydrogenase in sibling species of Drosophila. Nature 309 1983; Ashburner et a!., 1984; Bodmer & Ashburner, 425-430. 1984; Cohe & Moore, 1988). The few data available in BOUCHETON, A., PARO, R., SANG, H. M., PELISON, A. AND FINNEGAN the present study further show that the haplotypes with D. i. 1984. The molecular basis of I-R hybrid dysgenesis in Drosophila melanogaster Identification, cloning, and AdhFI have the lowest level of heterozygosity lending properties of the I factor. Cell, 38, 153—163. support to the argument based on amino acid and CHAMBERS, G. K., LAyER, W. G., CAMPBELL, S. AND GIBSON, j.a.1981. 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