Differences in Crossover Frequency and ... - Semantic Scholar

Copyright 0 1996 by the Genetics Society of America

Differences in Crossover Frequency and Distribution Among Three Sibling Species of Drosophila John R. True, John M. Mercer and Cathy C. Laurie Department of Zoology, Duke University, Durham, North Carolina 27708

Manuscript received August 7, 1995 Accepted for publication November 10, 1995 ABSTRACT Comparisonsof the genetic and cytogenetic maps of three sibling speciesof Drosophila reveal marked of crossovers during meiosis. The maps for two differences inthe frequency and cumulative distribution of these species,Drosophila melanogaster and D.simulans, have previously been described, whilethis report presents new map data for D. maun'tiana, obtained using a set of P element markers. A genetic map covering nearly the entire genome was constructed by estimating the recombination fraction for each pair of adjacent inserts. The P-based genetic map of maun'tiana is "1.8 times longer than the standard mlanogastm map. It appears that mauritiana has higher recombination along the entire length of each chromosome, but the difference is greatest in centromere-proximal regions of theautosomes.The maun'tiana autosomes show little or no centromeric recombinational suppression, a characteristic that is prominent in melanogaster. D. simulans appears to be intermediate both in terms of total map length and intensity of the autosomal centromeric effect. These interspecific differences in recombination have important evolutionary implications for DNA sequence organization and variability. In particular, maun'tiana is expected to differ from mlanogaster in patterns and amounts of sequence variation and transposon insertions.

M

EIOTIC recombination is a very widespread and important componentof the hereditarymachinery of sexuallyreproducing eukaryotic organisms. Theoretical studies have suggested both advantages and disadvantages to the existence of recombination, but the evolutionary forces behind the maintenance of recombination in natural populations are still poorly understood (MICHODand LEVIN1988). Although there is a wealth of theoretical work in this area, thereis relatively little empirical data on how recombinational systems vary within and amongspecies. Such data could provide important clues about the evolutionary forces that affect recombination. Modifiers of recombination are subject to evolutionary forces such as selection, but recombination is also itself an importantevolutionary force thatcan influence patterns of genome organization and sequence variability. For example, unequal crossing over can affect the accumulation of tandemly repeated sequences and ectopic recombination between dispersed transposable elements can control their numbers and distribution in the genome (CHARLESWORTH et al. 1994). In addition, recombination plays a vital role in processes such as hitchhiking and background selection that affect levels and patterns of sequence polymorphism (AQUADRO et al. 1994). Thus, it is clear that an understanding of the growing body of data on genome-wide patterns of Corresponding autho~;Cathy C. Laurie, DCMB/Zoology, Box 91000, Duke University, Durham, NC 27708. E-mail: [email protected] Genetics 142: 507-523 (February, 1996)

sequence organization will require information about the amount and distribution of recombination. Associations between interspecific differences in recombination and sequence organization or variability are likely to provide important insights into processes of genome evolution. The rate and distribution of recombination within a genome aredifficult traits to measure, so there is rather little informationabout how they varywithin and among species. The genus Drosophila provides some important technical advantages in this area and, therefore, has been the subject of many recombination studet al. 1993). One of the ies (CARPENTER 1988; HAWLEY great advantages of Drosophila is its intricately banded polytene chromosomes, which have provided a means ofphysically mapping genetic markers, first through cytologicalanalysisof chromosomalrearrangements and laterthrough in situ hybridization with cloned genes as probes. Although polytene bands vary cytologically in width and spacing, their number is very highly correlated with DNA content (based ondata from HEINOet al. 1994), so they provide a good measure of physical distance over ranges of 2 2 0 bands. The relationship between the physical and genetic maps of Drosophila melanogaster chromosomes was described in a classic paper by LINDSLEY and SANDLER (1977) and updated recently by ISING (in ASHBURNER 1989). The coefficient of exchange (centimorgans per cytological band, CE) varies dramatically along each of the chromosomes. One of the striking features of the crossover distribution on theautosomes has been called

508

J. R. True, J. M. Mercer, and C . C . Laurie

centromeric suppression because the CEisverylow lated species in the genus. For example, the genetic in euchromatin proximal to the centromere, but rises and EVGEN'EV map of D. uirilis (-975 cm) (GUBENKO gradually as distance from the centromere increases. 1984) is approximately twice aslong as that of D. pseudoThis featureis typical of manydifferent kinds of organobscura (-450 cm) (ANDERSON 1993), whichitself is isms, based on cytological observations of chiasma disnearly twiceas long as that of D. melanogaster (-280 tribution (JONES 1987). In addition, the Xchromosome cm) (LINDSLEY ZIMM and 1992). Thesedifferences have of D. melanogaster showsa strong telomeric suppression evolved over very long time periods and may be associof crossing over, which may also be present in reduced ated with major changes in karyotype and in genome form on theautosomes. There arealso manylocal variasize and organization. D. uirilis diverged from pseudoobsctions in recombination rate, but it is not clear whether ura and melanogaster 30-60 mya and its euchromatic those variations are characteristic of the species or genome is -150 Mb compared with 110 Mb for melawhether they may be due to measurement errors or to nogaster (HARTLand LOZOVSIWYA 1995, p. 142-143). intraspecific variation in recombination rate. The karyotypes of all three species (melanogaster, uirilis, Genetic control of the pattern of recombination in pseudoobscura) differ in the numbers of metacentric and D. melanogaster is not well understood, but appears to acrocentric chromosomes (PATTERSON and STONE be due to the combined action of local cisacting factors 1952). Because few genetically mapped markers have that affect exchange frequency, transactingfactors that been cytologically localized in uirilis or pseudoobscura, control both the level and distribution of exchanges comparisons of the distribution of recombination along and chromosomal structures such as the centromere the chromosome have not been made. and telomere that seem to exert a polar influenceover This report deals with divergence in recombination et al. exchange frequency (CARPENTER 1988; HAWLEY frequency and distribution among three sibling species 1993). A clear example of cisacting control is the lack of the melanoguster subgroup: the cosmopolitan species, of recombination in heterochromatin regardless of its D. melanogasterand D. simulans, and an island endemic, chromosomal position (BAKER1958; ROBERTS 1965). In D. mauritiana. It is estimated that melanogaster diverged contrast, the polareffect of the centromere on euchrofrom the ancestor of simulans and mcmritiana -2.5-3.4 mya, whereas simulans and mauritiana diverged from matic recombination is not due to the intrinsic properoneanotherabout 0.6-0.9 mya (HEYand KLIW ties ofsequences located close to the centromere.Stud1993). All three species readily hybridize with one aniesof translocation and inversion homozygotes have other,but all hybrids with melanogaster are sterile, shown that recombination in an interval changes sigwhereas mauritiana/simulans hybrids are male sterile but nificantly when its distance to a centromere is altered female fertile (ASHBURNER1989). The karyotypes of (BEADLE1932; SZAUTER 1984). In addition, several meithese species are the same and the polytene chromootic mutants have been described that alter the distribusome banding patterns are nearly identical except for tion of exchange by making recombination rate more a large paracentric inversion on ?R and a few other nearly proportional to physical distance along the chrovery small rearrangements that distinguish melanogaster mosome and thereby reducing the centromere effect and from the othertwo species (LEMEUNIER ASHBURNER (BAKERand CARPENTER 1972). 1976). The recombination system of melanogasler is, of Although the standard genetic map of D. melanogaster course, well studied, but there has been relatively little (LINDSLEY and ZIMM 1992) is often regarded as characpublished information about recombination in its sibteristic of this species, considerable intraspecific varialing species. Some comparisons between melanogaster tion in recombination rates has been documented (reand simulans are possible because a fairly large number viewedby BROOKS 1988). For example, BROOKSand of mutants with homology to corresponding melunogasMARKS (1986) studied variation due to second chromoter genes have been isolated and mapped in simulans some modifiers of recombination. They found that the VOELKER1979). Here (STURTEVANT 1929; OHNISHI and total amount of crossing over on the second chromowe report a fine-scale genetic and cytogenetic map of some varied by 12- 14%,with larger variations for speL ) . mauritiana, which provides a basis for detailed comcific intervals. The distribution of exchange was also parison with melanogaster in the amount and distribuaffected, with intervals in the proximal euchromatin tion of recombination. showing more variation than other regions. In addition, The original motivation for producing a genetic map artificial selection for changes in recombinationin speof D. mauritiana was to provide a set of genetic markers cific intervals has produced significant responses, prothat could be used for analyzing the genetic basis of viding further evidence for variation of recombination modifiers in natural populations (e.g., CHARLESWORTH morphological divergence between mauritiana and its sibling species. Because we wished to used these markand CHARLESWORTH 1985). ers for introgression and for localization of quantitative Drosophila is one of the few genera in which several trait loci, we required that they be easily scored and species are genetically well mapped so that interspecific well distributed across the genome. For this purpose, comparisons of some of the properties of the recombiwe produced a set of P-element insertions into a whitenation system can be made. Large differences in total strain of maun'tiana, each bearing theeasily scored minigenetic map length are observed between distantly re-

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Recombination in Drosophila Species

white' eye color marker. These insertions were first localized by in situ hybridization to the polytene chromosomes. Then, by estimating the recombination fraction for each pair of adjacent inserts, we produced a genetic map covering nearly the entire genome.A comparison with melanogaster reveals striking differences in overall genetic map length and in the distribution of recombination inferred from the relationship between the genetic and physical maps. MATERIALS AND METHODS Drosophilacultureconditions: Flies were raised on standard cornmeal molasses agar medium at room temperature, except where noted otherwise. Drosophila strains: D. melanogaster: Wild-type isofemale lines BWA27 (Benin, W. Africa) provided byR. SINGHand Raleigh144 (Raleigh, NC 1982) collected byP. BARNES. y w provided by G. RUBIN. fprovided by Mid-America Drosophila Stock Center. v and g provided by Bloomington Drosophila Stock Center. b cn bred from b AdhnU248 cn b u stock provided by M. ASHBURNER (bw' allele from Raleigh 144 isofemale line). D. simulans: white (No.13) provided by J. COYNE.WildP. I. type isofemale lines S. France provided by R. SINCH and Australia provided by J. BARKER. y w bred from y w mfstock provided by C-I. Wu (m+f from S. France isofemale line). v, g, a n d j provided by Bloomington DrosophilaStock Center. b cn bred from b and cn stocks provided by Bloomington Drosophila Stock Center. D. mauritiana: white provided by J. A. COYNE. Wild-type isofemale lines PetiteReviere provided by D. HICmYand Robertson providedby H. ROBERTSON. y w derived from spontaneous yellow mutation that arose in the w stock. v, g, and j provided by BloomingtonDrosophila Stock Center. b n bred from b and cn stocks provided by Bloomington Drosophila Stock Center. Interspecific homology for all mutant loci was confirmed by failure to complement in crosses with melanogaster (and/or othersimulans clade mutant alleles of known homology to melanogaster) performed in our laboratory and/or by J. A.COWE (personal communication). Polytene chromosome band index: The band index used in this paper is the number of polytene chromosome bands between a cytologically localized genetic marker and the centromere. It provides a good measure of physical distance for regions of 2 2 0 bands, because the correlation between DNA content and band number for each chromosome arm is very high ( T > 0.99 using data from HEINOet al. 1994). The band indices used here comply with the revised maps of C. B. and P. N. BRIDGES (in LINDsLEY and ZIMM1992). The band index for a given gene or Pelement insert is estimated as the midpoint of the band rangeto which it has been mapped cytologically. For mauritiana and simuluns, band indices within the large 3R inversion (relative to melanogaster) are renumbered to obtain increasing distance from the centromere. P-element transformation and characterization of transformant lines: Host embryos of D.mauritiana white- were injected with P[lac-w'] (BIERet al. 1989) and p~r25.7WC(KARESS and RUBIN 1984). Single insert lines were selected for further work by Southern hybridization, using the 4.25-kb Hind111 fragment of P[lac-w']; and digesting transformant line genomic DNA with XbaI and BglII. These P[lac-zo'] insertions appear to be very stable, as expected because rnauritiana has no endogenous P elements to provide transposase activity (BROOKFIELD et al. 1984). Many insertions have a characteristiceye color pattern andintensity that did not change over months of culture.

509

In Situ hybridizations: Each mauritiam insert was localized cytologically by in situ hybridization to the polytene chromosomes. Thephotographic maps of melanogaster (LEFEVRE 1976) were used for mapping, because the banding patterns of these two species are nearly identical. However, the polytene chromosomes of mauritiana are smaller than melanogaster, and it is much moredifficult to obtain high-resolution preparations. Therefore, inserts generally were localized only to the nearest lettered division (or border between two lettered divisions). Larvae for cytology were raised on the medium of WHEELER and CLAWON(1965) and in situ hybridization was performed according to LIM (1993). Construction of a D. mauritiana genetic map using 41acw'] inserts: All of the inserts mapping to the same cytological position were pooled and a single representative of each pool was used for constructing a genetic map. The recombination fractions between adjacent pairs of insert locations were estimated as follows.Two single-insert homozygous lines were crossed and the doubly heterozygous female progeny were then crossed to w- males. In many cases, it was difficult to distinguish P[lac-w+] heterozygotes from homozygotes, so the recombination fraction was estimated as twice the frequency of w- recombinants. Using this procedure, theexpected variance of the recombination fraction estimate is 4[x( 1 - x ) ] / n , where x is the frequency of w- recombinants and n is the total number of progeny. The average sample size was 3458 progeny. For crosses to estimate recombination fraction, 515 vials were set up with 4-5 virgin F, females (5-6 days old) and 45 w- males (of varying age). These parents were transferred to new vials after 5 days and the transfer set was cleared after another 5 days. Progeny of both sets of vials were scored on the 13th, 15th, and 17th days of culture. Due to the large scope of this experiment, nine separate sets of crosses were performed during the course of 1 yr. Room temperature varied somewhat during these experiments; ranges for each set were: ( I ) 21-24", (2) 21-24", (3) 22-26", (4) 23-26", (5) 2122", (6) 21-22", (7) 20-22", (8) 20-22", (9) 25" (incubator). Nevertheless, crosses repeated in more than one experiment gave very similar results. For example, one interval (19AB/ 19BC) gave independent estimates of6.1 (21.4)and 6.4 (?1.3) and another interval (24CD/26B) gave estimates of 11.5 ( ? 1.7) and 11.2 ( ? 1.7),where the ? figures in parentheses are 95% confidence intervals. Recombination fractions were converted to map distance using the mapping function of FOSSet al. (1993; with nt = 4), which accounts very well for the pattern of interference on the X chromosome of melanogaster. This mapping function has very little effect on recombination fractions less than 15 cM (changing only the second decimal place). Because nearly all of the recombination fractions used inthe mauritiana map construction are < I 5 cM, the map is influenced very little by this conversion. Map distances fromeachautosomal centromere to the nearest flanking Pelement markers were estimated by interpolation. A coefficient of exchange (centimorgans per band) was calculated from the map distance between those flanking markers and thenmultiplied by the numberof bands between each marker and the centromere. For the X chromosome, the centimorganbetween the centromere andnearest marker at 19EF was estimated in a similar manner using the coefficient of exchange for the interval between markers at 19BC and 19EF. D. melanogaster mapdata: The melanogaster data were pooled from two sources: FlyBase (1994), which provided genetic map and cytological positions for a set of loci mapped genetically to within 0.1 cM and cytologically to aninterval less than six bands (identified by E. KINDAHL and C. AQUADRO, personal communication) and G. ISING,who provided data

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Chromosome 3 FIGURE1.-Cytological positions of P[ Innu'] inserts in D. mnuritinna. Each symbol represents a separate line bearing a single Pinsertion that is homozygous viable. Black symbols represent homozygous fertile inserts; white symbols represent homozygous sterile inserts. Stacked symbols represent inserts at approximately the same position. Jagged lines on the right armof chromosome 3 represent the breakpoints of a fixed inversion difference between D. mrln,nogmterand D. mauritiana. for a set of transposable element markers cytologically localized and genetically mapped in relation to standard reference loci (summarized in Figure 11.1of A~HRURNER 1989). The two data sets agree substantially. The major features of the standardwlanognsto-genetic map were established by the MORGAN group between about 1913 and 192.5 (MORGANrt al. 1925), using a set of reference loci. The current genetic map positions of these reference loci (LINI)SI.EY and ZIMM 1992) are the same as those given by MORGAN rt al. (1925) and the current map lengths of each chromosome are only slightly longer than the 1925 maps. Over the past 70-80years, the mrlanogmter map has been built up by mapping new loci with respect to the reference loci, either directly or indirectly. Map distances between reference loci were calculated by MORGAN et nl. (1925) from a system of empirical curves based on recombination fractions for closely spaced markers. In essence, the map distance between two points on the wlnnogmtPrmap is the sum of recombination fractions for intervals small enough that the probability of double crossing over is negligible. D. simulans map data: Genetic map data for simuluns and demonstrations o f interspecific homology with wlanogmtPr are mainly from the compilations of STURTEVANT (1929) and VOELKER(1979), with additional data from OHOHNISHI and SISHI and VOELKER (1981) and P U R 0 (1971). In most cases, the cytological map positions of the simuluns genes have not been determined directly, but are assumed to be the same as the rnrlnnngnskr genes to which they are homologous. Genetic map positions of simulnns mutants were established originally by STTIIRTEVANT (1929) using a system of reference loci similar to those used in constructingthe melanogmkr maps. However, because of the relative paucity of genetic markers in simulnns, the mapdistances between reference loci were given as observed recombination fractions, uncorrected for possible multiple crossovers. This procedure can result in underestimates of map length. Thus, in calculating total map lengths for each chromosome, we have used the mapping fhction ofFOSS rt nl. (1993; m = 4) to convert observed recombination fractions reported by STURTEVANT (1929) for each pairof reference loci. This conversion increases the map distance estimates by only a small amount compared with using the sum of recombination fractions (ir., 0.2% for the X , 8.9% for the second chromosome and 2.3% for the third chromosome). For analysis of the relationship between genetic and physical map distances, only X and third chromosome loci were used because there are few second chromosomegenes with demonstrated homology to mrlunogmter

genes. In this analysis, map positions given in the literature were used without conversion. Relationship between genetic and physical map distances: The incomplete beta function, B(z, a, 0 ) . was chosen to provide a flexible but simple phenomenological representation of the relationship between cumulative genetic and physical map distances from the centromere. The observed physical map distance (band index) was rescaled to the interval 0,l to correspond with the domain of definition of B(z, a, b). The Levenburg-Marquardt algorithm with equal weighting was used to minimize the sum of squared residuals to obtain estimates of the parameters a, b and c for the distribution function c B(z, a, b). This algorithm was implemented using Mathematica 2.2 (WOLFRAM1991) supplemented with 2.3 prerelease versions of the packages RegressionCommon.m and Non1inearFit.m. In addition, the following derivatives are required by the algorithm and must be entered explicitly.

+ a;z)/d + In (z) B(z, a, b) d B(z, a, b ) / d b = (1 - ~)"3F2(b,b, 1 - U ; 1 + b, 1 + 6:

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where 3F2is a generalized hypergeometric function, 9 is the psi or digamma function, and B(a, b) is the beta function. The coefficient of exchange (CE, centimorgans per band) is the slope of the curve describing the relationship between genetic and physical map distances from the centromere. Thus, the derivative of the incomplete beta function that describes this relationship, which is proportional to the beta density function, provides a description of how the coefficient of exchange varies along a chromosome arm. An alternative approach to estimation of the beta function parameters is to fit the beta density function to observed values of CE corresponding to discrete marker intervals (as in MORTON d al. 1976) rather than fitting the incomplete beta functionto cumulative map distance. We chose the latter approach because estimates of CE for small intervals are subject to considerable measurement error. Furthermore, map distances between adjacent markers in the standard melunogmter map are often not estimated directly, but rather each markermay have been positioned in relation to reference loci at some distance from the interval. Recombination estimates using homologous markerloci:

Recomhination in Drosophila Species chromosome

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Direct comparisons o f recombination fractions were made for four intends lnarked hy mutants at loci holnologous i n the three species ( y r u , rkgand g -/mthe Xand b c n on the second chromosome). These fractionswcreestimated simultaneously for ?nr/crnogu.s!m,n/rtztn'/irrnrr, . s i m r / m . s and nrtrun'/icrnn/simzL/nn.~ Ilyhrids. Crosses t o determine recombination fractions for the intervals y r o and IFcn were performed using clouhle-mutant tester stocks. Tester stock females were mated with wilcl-type males from cac~l of tlvo isofemale lines anti the F, females were hackcrossed to tester stock males. No douhle mutant tester stocks 101-the vgand g-/intends were availahle. TIllls, in the first generation 71 females were mated with g males, gfemales were mated with / males, and then F, females were mated w i t h wild-type males. Progeny were scored using the same design and schedlllc as tile backcrosses wit11 p[/nc-7[J'] markers. Recar~scall parental and recomhinant genotypescould he scored unamhiguously in these crosses, the variance of the recombination fraction estimate is [ x ( 1 - x ) ] / n where x is the frequency of recombinants and n is the total numher of progeIly. The averagc ,,a~l,eof n in this experiment ,,,as 1336. Tests for Xchromosome nondisjunction: Six randomly chosen X-linked['I IUf-r/l' ] insert lines and l%'Owild-type lines (PCtite Reviere antl Robertson) o f mauritiana were tested for evidence of X chromosome nondisjunction. Four-day-old virgin females (wildtvpe o r homozygous for an X-linked insert) were mated intlivitluaIIy t o three 7(1 males and the vials were transferred, cleared an-d scored according to the same scI1edule as for crosses to estimate recombination fractions. An average of 24.5 single females per line were examined and an average of 3462 progeny per line were scored. Putative X 0 males ( r o - ) were mated t o 2-3 ru- females to testfertility. In addition, females homozygous for one second or third chromosome insert were mated t o Z ~ P - males as a control. These crosses were mass matings o f 4-5 pairs i n each of 15 vials. RESULTS

The cytological and genetic maps of D. mauritiana: A total of 114 P[lac-711+] inserts were localized by in situ hybridization to the salivary glandpolytenechromosomes (Figure 1 ) . These locations represent a total of 94 clearly distinct positions, which are well distributed across the genome (althoughsomewhat sparser on the second chromosome than the Xor third). This sample represents only the inserts isolated as single-insert, homozygous viable transformant lines,but the distribution is very similar to that found in a mdclnogastPr sample that also includedlethallines (LAURIE-AIII.RERG and STAM1987). The genetic map was constructed by estimating the recombination fraction between each pair of adjacent insert positions. The raw data areprovided in APPENDIX.

lol,n

258.9 ToTAL 462.9

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Fl(;lxb. 2.-Differences i n overall genetic map length hetween D. tnPlm/og(~.s/rrantl 11. nmrri/innn. Cytological rangcs for n w / r r i / i m urefer to the most distal P [ / n n o ' ] markrrs o n rach chroto mosome; those for 11. melanogasterrefer those markers on the standard map w i t h positions closest to the nrcr?o-i/innn endpoints (F1.y-

BASE 1994). The

following nw/anogm/rr loci were endpoints: . s ? r ( n m ) and o n c on the Xchromosome, oddaantl m ~ i S 3 3 2 o nsecond chromosome, and cnfcandjnnA on the third chromoused as these

sOllle.

The average recombination fraction is ().().',, Ivith a maximum of 0.20, which pro\,ides a fairlydel,se map of the entire genome, except for the termina1 regions Of the secondchromosome(seeFigure 1). Possible estimates: biases fraction in recombination The estimation o ~ r e c o l n ~ ~ n a fi-actions t ~ o l l can be biased

by unequal viabilities of parental and recombinantgenotypes. In this case, the estimation invokescomparing one recomhinant genotype with no inserts to three other genotypes having one or two inserts. The IT/(/rqa+]insert lines llave heen easy to mainmin in tIle laboratoy, illdicating generally good viability and fertility. In addition, the viabilities of six randomly chosen inserts were estimated quantitatively by crossing a single insertline to ?(,and then backcrossing to 7(ragain. F ~ eac]l ) ~ insert, 1.500-1900 progeny were scored and there were no significant departures from the expected 1:1 ratio. Thus, we have no evidence for a bias in the estimates of recombination fractions due to differential viabilihr. A potential bias involving Xchromosome recombination estimates is the occurrence Of patroclinousmales due to XX nondisjunction or chromosome loss in female gametes. Females tmnsheterozygous for X-linked inserts can produce X0 males that lack an insert and therefore have white eyes, just like male progeny with a recombinant X lacking both inserts. To determine the frequency of exceptional males, females homozygous for an X-linked insert from each of six different lines, as well as females from eachof two wildtype lines, were crossed with 70- males. All eight of these lines produced patroclinous7u- males at about thesame rate, which ranges from two of 4340 progeny (0.05%) to four of 2519 progeny (0.16%),with a pooled value of 29 of 27,696 progeny (0.10%). All but three of the 29 70males obtained in this experiment were sterile, as expected for X 0 males. Among the three fertile males, which were crossed to ?(I- females, two produced all white progeny. The third male produced male progeny that were all white and female progeny that all had the orange eyes typicalof a singleinsertgenotype. Evidently, this male had an X-linked insert with v e y low or no rnini-7uhil~expression, while the other twomay have had XXY mothers. These resultsindicatethat XX nondisjunction or chromosome loss will upwardly bias the estimated recombination fraction between each pair of X inserts by

512

J. R. True, J. M. Mercer, and C. C . Laurie

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Recombination in Drosophila Species

a very small amount (2 x 0.10% Equation 0.20 cM). We did not attempt to correct this bias in analyses of the relationshipbetween genetic and physical maps discussed below, but we did correct the estimate of total X chromosome map lengthby subtracting the product of the number of intervals (22) and the bias in the rate estimate per interval (0.20 cM), which is 4.4 cM. Inaddition to the X chromosome inserts, crosses were made between females homozygous for a second or third chromosome insert and w- males. No white progeny of either sex were obtained from a total of 5028 progeny. These X , second and third chromosome insert crosses provide evidence of the expected P insert stability, because no white females were observed in a total of 32,997 progeny examined. Two inserts were localized to thefourthchromosome, providing the possibility ofestimating a recombination fraction for this chromosome, which is normally achiasmate in D. melanogaster (HOCHMAN 1976). However, we have been unable to obtain a reliable estimate of recombination between these two fourth chromosome inserts because of complications that appear to be due to fourth chromosome nondisjunction and possibly a suppression of mini-white gene expression in one of the fourth chromosome inserts in X 0 males. Interspecific comparisons of total genetic map length: Figure 2 presents a comparison of total genetic map length between the Pbased mauritiana map and the standard melanogaster map (LINDSLEY and ZIMM 1992; FLYBASE1994). Thesecomparisons were made by finding genetic markers in the melanogaster map that are located at essentially the same cytological positions as the terminal markers in mauritiana. For the extent of the genome spannedby the P[lac-w+]markers, the mauritiana genetic mapis l .8 times longer than the standard melanogaster map. Ratios of mauritiana to melanogaster geneticmaplengthsare 1.8, 1.6, and 2.1 forthe X, second, and third chromosomes, respectively. The map lengths of melanogasterand simulunswere compared between pairs of homologous loci near the chromosomal termini. The map distances for melanogaster and simulans, respectively, are 66.0 and 66.3 between y and bb; 84.0 and 108.1between @and P U , and 81.5 and 135.9 between j v and ca. Ratios of the simulans to mlanogaster map lengths are 1.O, 1.3 and 1.7 for the X, second and third chromosomes, respectively. The total simulans map is 1.3 times longer than the melanogaster map.

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Relationshipbetweengenetic and physicalmaps: The relationships between thegenetic and physical maps for each chromosome arm in melanogaster, mauritiana, and simulans are shown in Figure 3. The abscissa representsthe physical distance between agenetic marker and its centromere in terms of the number of polytene bands, which is proportional to DNA content. Theordinate representsthegeneticmap distance, which is proportional to the expected number of crossovers per meiosis between the genetic marker and its centromere(50mapunits = crossover). The fitted curves in Figure 3 show that this relationship is described very well byan incomplete beta function, which has two shape parameters. The slope of the incomplete beta function is an estimate of the coefficient of exchange (CE), a measure of the expected number of crossovers per unit ofphysical distance (centimorgans per band). The differentiated functions (which are proportional to the beta density function) are shown in Figure 4. These plots provide a rough estimate of how the coefficient of exchange varies along each chromosome arm at least within the range of observed data, the endpoints of which are marked in Figure 4. Outside that range (near the termini of each chromosome arm), the CE curves are extrapolations that probably do not accurately represent the recombination pattern. Even within the observed range, distortions of the real situation may occur because measurement errorsof terminal data pointsoften have an undueinfluence on parameters estimated from curve fitting. Therefore, we can only interpret broad features of the CE curves, since the reliability of some of the detailed shape variation is unknown. In melanogaster, the crossover distributions differ markedly amongthechromosome arms, particularly between the Xand autosomes. On each autosomal arm, the CE (slope of the curves in Figure 3) is low near the centromere, rises gradually in the proximal half and then more steeply in the distal half. Near the telomere, there may be a decrease in the CE for 2L, ?L and X, but probably not for 2R. These data indicate a strong “centromere effect” (recombination suppression) for each of the autosomal arms, although it is somewhat less strong for 2R than for the others. A suppressing effect of the autosomal telomeres may also be present, but is not pronounced. In contrast, the Xchromosome shows onlya slight depression near the centromere, but

FIGURE3.-Relationship between genetic and physical distance for D. melanogaster, D. simulans and D. mauritiana. The X is represented twice because the melanogaster and simulans data points for this chromosome largely coincide. The brackets within the major cytological division scale for ?R indicate breakpoints of an inversion that distinguishes melanogaster from the other two species. The fitted curves are proportional to the incomplete betafunction such that genetic distance from the centromere equals cB(z, a, 6), where z is standardized physical distance (band index) from the centromere and c, a and 6 are estimated parameters (see MATERIALS AND METHODS). The parameter estimates are: melanogastm r.x = 84.3, ax = 0.86, bS = 0.73, czl. = 143.5, aZ1.= 1.37, 621. = 1.29, c213 = 67.2 a2,