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A Genetic Mapping System in Caenorhabditis elegansBased on Polymorphic Sequence-Tagged Sites

Benjamin D. Williams,* Bertold Schrank,* Chau Huynh,* Ratna Shownkeent and Robert H. Waterston*

*Department of Genetics, Washington University School ofMedicine, St. Louis, Missouri631 IO, and ?Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom Manuscript received December 26, 1991 Accepted for publication March 9, 1992

ABSTRACT We devised an efficient genetic mapping system in the nematode Caenorhabditis elegans which is based upon the differences in number and location of the transposable element Tcl between the Bristol and Bergerac strains. Using the nearly completed physical map of theC. elegans genome, we selected 40 widely distributed sites which contain a Tcl element in the Bergerac strain, but not in the Bristol strain. For each site a polymerase chain reaction assay was designed that can distinguish between the Bergerac Tcl-containing site and the Bristol “empty” site. By combining appropriate assays in a single reaction, one can score multiple sites within single worms. This permits a mutation to be rapidly mapped,first to a linkage group and thento a chromosomal subregion, through analysis of only a small number of progeny from a single interstrain cross.

EQUENCE-tagged sites (STSs), which havere- Previously, Tcls have been used in efforts to mo- S cently been proposed as common landmarks for lecularly clone specific , both by direct transpo- genomic physical mapping (OLSONet al. 1989), are son tagging(MOERMAN, BENIAN and WATERSTON short, unique genomic regions that areeasily detected 1986) andby genetic mappingof the Bristol/Bergerac by polymerase chain reaction(PCR) amplification Tcl polymorphisms nearest a to identify molec- (SAIKIet al. 1988). A primary virtue of STSs is that ular clones fromthe region (RUVKUN et al. 1989). they can be distributed among laboratoriesas a DNA Conversely, cloned genes have been placed on the sequence database, obviatingthe transfer and storage linkage map by using Tcl polymorphisms as genetic of biological materials. tags (FILES,CARR and HIRSH1983). Polymorphic genomic regions have also been con- To develop a genome-wide genetic mapping strat- verted into STSs by appropriately designed PCR as- egy, individual randomly cloned Bergerac Tcls were says. These arevaluable as easily scored genetic mark- positioned within the nearly completed C. elegans ge- ers andas points of correspondence between physical nomic physical map (COULSONet al. 1986,1988, and genetic maps. Variable numbers of repetitive 1991). PCR assays were then developed for a subset mono-, di- and trinucleotide motifs (LITT and LUTY of the Tcls, selected to give markers well distributed 1989; TAUTZ1989; WEBERand MAY 1989), referred throughout the genome. The assays yield a product to as microsatellites, and variable lengths of the poly- when the Tcl is present (Bergerac DNA) but give no deoxyadenylate tracts within individual Alu elements product when the Tcl is absent (Bristol DNA). The (ECONOMOUet al. 1990) are examples of polymor- resulting STS map has sufficient density to allow a phisms that have been rapidly scored through appro- new mutation to be mapped to a chromosomal subre- priatelydesigned PCRassays. Alarge number of gion through analysis of a limited number of progeny microsatellites in the mouse genome have been sys- from a single interstrain cross. Since the STS markers tematically cloned and converted into polymorphic do nothave an associated phenotype and can be STSs with the goal of producing a polymorphic STS scored directly by PCR with DNA released from single map (CORNALLet al. 1991). animals or embryos, they offer advantages beyond a Here we report the systematic development of a simple reduction in the number of crosses needed to polymorphic STS map in the nematodeCaenorhabditis map new mutations. These are demonstrated herein elegans. The polymorphisms exploited in this scheme mapping experiments with lethal and X-linked muta- are copies of the transposable element Tcl which are tions. present at dispersed loci in the high Tcl-copy number Bergerac strain (approximately 500 copies), but ab- MATERIALS AND METHODS sent from these same locations in the low Tcl-copy Strains,genes and mutations: All mutations used in number Bristol strain (30copies) (EMMONSet a2. 1983; mappingexperiments were generated in theBristol N2 LIAO,ROSENZWEIG and HIRSH 1983). background. Most have been described by BRENNER(1 974)

Genetics 131: 609-624 uuly, 1992) 610 D. B. Williams et al. and by HODCKINet al. (1988); the genes and corresponding to a sequence within the Tcl element, and primers specific mutations are listed below for each . for each Tcl insertion site. Each of the latter is complemen- Chromosome I: dpy-5(e61),unc-Z9(e1072), unc-75(e950), tary to DNA sequences that flank the Tc 1 insertion site and unc-lOl(ml), unc-95(su33), lev-1 l(x1.2). will work incombination with primer 6 18 to amplify a DNA : sqt-2(sc3), dpy-1 O(el28}, unc-4(e 1 ZO), unc- fragment of defined size from the Bergerac Tc1 -containing 52(st572) (B. WILLIAMSand R. WATERSTON,unpublished site, but not from the "empty" Bristol site. results), unc-52(e444). DNA for PCR assay wasprepared from individual animals Chromosome III unc-45(e286ts), dpy-l(el), dpy-l8(e364). using methods adapted from BARSTEAD,KLEIMAN and WA- Chromosome IV dpy-9(e12), deb-l(st385) (BARSTEADand TERSTON (1991). Singleanimals were picked up with a WATERSTON1991), unc-ZZ(s12). platinum wire and each placed in a 2.5-p1 drop of lysis buffer Chromosome V unc-60(e677), dpy-1 I(e224}, unc-Z3(e25), [60 cg/ml proteinase K in 10 mM Tris (pH 8.2), 50 mM KCl, dpy-Zi(e428), rol-9(sc148}. 2.5 mM MgC12, 0.45% Tween 20 and 0.05% gelatin) in the Chromosome X dpy-3(&7), unc-Z7(e155), unc-90(e1463), cap of a separate 0.5-ml tube suitable for PCR. The drops patfst558) (B. WILLIAMSand R. WATERSTON,unpublished were then moved to the bottom of the tubes by a brief results). microfuge spin, frozen (-70°, 15 min), and after the addi- Two Bergerac strains, RW7000 and DP13, were used to tion of a mineral oil overlay, heated (SO", 1 hr followed by generate the Bristol/Bergerac hybrid animals required for 95", 15 min). After cooling to 4", 22.5 pI of a "master mix" all polymorphic STS mapping experiments. DP13 is a deriv- were pipetted on top of the mineral oil overlay. The mix is ative of RW7000 obtained after maintenance for several formulated to bring the reaction volume to 25 pl with these years asan independent strain in another laboratory. There final conditions: 25 pmol of internal Tcl primer 618 and are only two differences between the strains with respect to 25 pmol each for the appropriateflanking sequence primers, the STS polymorphisms described here: (1) Tc 1 eP64 III is 10 mM Tris (pH 8.3), 50 mM KCI, 1.5 mM MgC12, 0.001% present in DP13 but absent from RW7000, and (2) Tcl (w/v) gelatin, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, stP128 V is present in RW7000, but absent from DP13 (see 0.2 mM dGTP and 0.6 unit Taq polymerase (Perkin Elmer RESULTS). Cetus, Norwalk, Connecticut). A brief microfuge spin was Techniques for culture and genetic analysis were as de- used to move the "master mix" through the mineral oil scribed by BRENNER(1974). All crosses were at 20" except overlay, and the reactions wererapidly heated (within 1 for those with unc-45(e286ts) homozygotes, which were set min) to 94" and cycled 30 times: 94" for 30 s, 58" for 1 up at 15"and then shifted to 20" after 2 days. min, and 72 O for 1 min. Failure to promptly bring the tubes Cloning, physical mapping and sequencingof Bergerac to 94" after the brief microfuge spin results in a significant Tcl insertion sites:Most of the Bergerac Tc1 s were isolated increase in background bands that can complicate interpre- from a library of RW7000 genomic DNA in vector X1059 tation of the data. Gel lanes were loaded with 10 pl of each by hybridization with Tcl sequences (I. MORI,D. MOERMAN reaction. and R. H. WATERSTON,unpublished results). DNAmini- Individual unhatched late-stage embryos were also used preps (HELMSet al. 1987) from positive clones were sub- for single PCR reactions. Eggs were picked up directly from jected to fingerprint analysis (COULSONet al. 1986) to place a culture plate using a 5-pl capillary tube which had been them in the C. elegans genomic physical map. Clones which pulled to a convenient inner diameter (50-100 pm) and could not be positioned by fingerprinting were radiolabeled filled with chitinase solution [20 mg/ml chitinase (Sigma C- by random priming (Prime-It kit, Strategene, La Jolla, Cal- 6137) in 50 mM NaCI, 70 mM KCI, 2.5 mM MgCh and 2.5 ifornia) and hybridized using standard procedures to a grid- mM CaCI2]. They were then immediately transferredto ded set of YAC clones that correspond to most of the C. worm lysis buffer, and processed as described above. The elegans physical map (COUUON et al. 1991). For clones same capillary tube was used for multiple transfers without mapping to advantageous positions, the sequence of DNA significant cross-contamination of DNA. flanking the Tcl was determined directly from miniprep Amplification products were separated using standard DNA using primer 61 8 (see Table l), which is complemen- nondenaturing 6% acrylamide minigels (Mighty Small 11, taryto Tcl sequence (ROSENZWEIG,LIAO and HIRSH Hoeffer Scientific Instruments, San Francisco, California). 1983a), by using a linear amplification sequencing method Mapping of new mutations: Since Bergerac males of the (MURRAY1989), as modified (CRAXTON1991). The same RW7000 strain do not mate efficiently, all crosses used to method was used directly with plasmids SS#2 (M. BEANAN generate Bristol/Bergerac hybrids had to be carried out and S. STROME,unpublished results) and peP64BO (D. beginning with Bristol males. Consequently, different pro- PILGRIM,unpublished results) to determine sequence flank- cedures were required to generate the hybrids when map- ing Tcl polymorphism TcbnZ I and eP64 III, respectively. ping autosomal and X-linked mutations. For the former, Fl DNA sequence flanking polymorphism maPi II was ob- hybrids were generated in two steps: (1) homozygous(non- tained directly from plasmid pAPP-HB4 (PAPP, ROUGVIE lethal mutations) or heterozygous (lethal mutations) Bristol and AMBROS199 1) using Tcl primer 618 and a standard hermaphrodites were crossed with Bristol males,and (2) 10- double stranded sequencing method (Sequenase Kit, USB, 15 male progeny were then mated with 20-30 Bergerac Cleveland, Ohio). Sequence flanking the Tcl polymorphism hermaphrodites. Fl hermaphrodites were then picked to bP1 V was taken directly from ROSENZWEIG,LIAO and HIRSH separate plates, and the mutant heterozygotes were recog- (1983b). For Tcl polymorphisms hP4 I, mgP2lIII and sP4 nizedwhen they produced homozygous mutant FP self- IV, sequence of flanking DNA was obtained from Bergerac progeny. The mutant F2 animals (dead embryos for the subclones pCeh50 (STARRet al. 1989), mgP21 (FINNEY, lethal mutations) wereassayed individually for Bergerac RUVKUN andHORVITZ 1988) and pCes233 (BAILLIE,BECK- Tcls by PCR. ENBACH and ROSE1985) respectively, by further subcloning For X-linked mutations, where passage through an X0 to place the EcoRV site from the Tcl inverted repeats malewould be difficult, the F1 hybrid was generated as adjacent to sequencing primer sites of vector pBS- (Strate- follows: (1) Bristol/Bergerac hybrid males, which have a gene, La Jolla, California), and double strand sequencing single X chromosome of Bergerac origin, were generated with the Sequenase kit (USB, Cleveland, Ohio). by mating Bristol males with Bergerac hermaphrodites. (2) PCR methods: The primers used for PCR are listed in These males were mated to the Bristol strain carrying the Table I. They include primer 61 8, which is complementary mutation to be mapped. FI hermaphrodite progeny were STS mapping in C. elegans 61 1

TABLE 1 Primer sequencesfor Tcl polymorphisms

Primer Polymorphism Primer Band size (bp) Sequence (5' + 3) 618 GAA CAC TGT GGTGAA GTT TC LG I 093 stP124 I15 GAC GCA GAC AGA CGA GAGT 734 hP4 130 CGG AAA TAT TATCAG CAC AGC 473 TCbn2 150 GCA TAA AAA GCC TCC AAG TCA LC I1 543 stP196 133 CCA AAA GTT TAA AGGAAA TGA AGC 363 StPIOO 198 GGA AAC CAA GAACAT TGG ACG 133 StPlOl 118 CGC CTG ATTTTT CCA GGT GC 816 stP5U 180 TCC AGA TAT CATATA GCT TGT TC 886 stP36 153 CAC TGTCTT GTC GAT ACC 437 stP98 269 AAG TAG AAA AAT TGCCTT GCG 723 maPl 234 CCA ATT TTC CGG TlTAAG TCG LC 111 570 eP64 137 TAA TAATTT GTCAGG AAA CGA G 845 stPl9 216 ACA AGC GGGTCT ACT GAA CC 587 mgP21 165 GGA ACA AAA GTG CCT TGG G 40 1 stP127 233 GCA TCG ATA CAA GTG GAA GC 548 stPl2O 149 AAT AAT CAG TGA AGC CTC ATG 800 stPl7 183 CTC GAT GTG TCT CAA TAG TTCC LC IV 815 stP13 236 CCC ACA ACCTTT TGC TAC AAC 056 stP5 I 28 1 GTT CGT TTT TACTGG GAA GG 93 1 stP44 209 CCA TCG TTT GTG TCT AGA GTC 653 sP4 179 "T CTG TTT ET GCT TAG ACG 510 stP5 26 1 GGA TTA TTA CCGTCT TACGCA 536 stP35 149 GCA GTC TCT AAT GCTAGA GC LC v 813 stP3 153 GCT GCA TTT CCA TTC ATG CAG 440 stP192 290 GCA CGC TGA GAG TAA CGTG 997 stP23 135 TTG TCA ACT ATT TTA CAGG CGA 736 bPI 119 AAC ACA TlT AGG TAA TGT AGC AC 998 stP6 170 TCA CAA TCG ATG ACT AAG TAC TGG 862 stPl8 203 TTG AAC TTC TCC CAC TCC TC 438 stPlO8 135 AAA GAT AAA CGC GCT TlT TGG 439 StPIO5 152 GGG TAG TTG TTC ATGTCT CG 652 stPI28 200 GCA ACGCTT TGTGGA TCT G LC x 930 stP4l 193 TGT CTA CTT ACC TTA ACT TACC 888 StP40 229 GTA TGAGCT AAT TGT ACC CTC 449 stP156 143 TGG AGG ATT CGG GCG G ATT 112 stP33 260 CGT CTA GTC GTGTGT TTC C 453 stPlU3 209 GAC GAA AAG AGG TAC ACG AG 454 stP129 160 CCA CTT ATT GCC ACTTTT TGG 450 stP6I 176 GAA TTG GTG TCC GGA CACA 452 stP72 112 CTT GAA AAT ACC ATG GCA TAC 887 stP2 127 CAA AAC GGT ATA CTC TGG TG

picked to separate plates, and mutant heterozygotes were were scored directly. This is in contrast to standard 3-factor recognized by their homozygous mutant self-progeny (F2). mapping, which requires an FS generation to permit homo- When mapping lethal mutations, each F, parent was tested zygosity of the recessive mutation that is being mapped.) for the X-linked Bergerac Tcls before the F2 progeny were The F1 hybrids were constructed as described above for assayed. In this way any nonhybrid F1 animals (self-progeny single autosomal or X-linked mutations starting with double of the original Bristol strain) were recognized and their mutant homozygotes. Hybrid F1 double heterozygotes were progeny discarded. recognized by the appearance of double mutant F2 self- Genetic mapping of Tcls: A procedure similar to stand- progeny. ard 3-factor mapping was used to confirm the genetic map Map Information was obtained in several ways from analy- positions of the STSs. In each crosstwo visible marker sis of F2 animals. First, we selected for recombination events mutations (a and b in the following examples) were used to between the visible markers; FS recombinants of genotypes mark a single Bristolchromosome of an FIBristol/Bergerac a+/ab and +blab were picked and assayed by PCR. By hybrid. The hybrid was then allowed to self, and individual comparing the subsets of Tcls inherited on the selected Fs animals were scored for Tcls by multiplex PCR. (Since recombinant (a+ or b+) of these animals, the the STSs behave as dominant genetic markers, F2 animals Tcls within the marker interval were ordered. Since the 612 B. D. Williams et al. STSs behave as dominant genetic markers, one or more TABLE 2 Tcls that were inherited on the unselected chromosome (ab), due to a recombination event outside of the visible Linkage groupI mapping data marker interval, occasionally complicated this analysis. These events were identified easily and usually directly Genotype of hybrid Detection of STS confirmed byPCR analysisof individual Fs progeny (Fp parent; phenotype of animals were picked to separate plates and allowed to self self-progeny stP124 hP4 TCbn2 n before they were lysed for PCR analysis). MultipleFs animals dpy-5 ~nc-29/++~ of the ab/ab genotype were tested in these instances, and all Dpy non-Unc + 6 were positive for the Tcl(s) inherited on the unselected - 16 chromosome. -26,~ Map information was also obtained by analysis of F2 24 double mutant progeny (ablab). The Tclsdetected in these animals were placed outside of the visible marker interval Non-Dpy Unc I and sometimes ordered relative to each other with these Id data; Tcls inside could only be inherited on a chromosome 7 that was double recombinant within the visible marker in- -3d terval. Similar mapping data was also obtained from Tcls 12 inherited on the unselected chromosomes of recombinant Dpy Unc 35 F2animals, as described above. -17 The four crosses usedto map STSs on chromosome I are 52 presented in Table 2. Tcl stP124 maps within the dpy-5 to unc-75 lev-1 unc-29 interval, as indicated by analysis of animals recom- Unc non-Lev 15 binant for these marker mutations (dpy-5 [8/36]stP124 [28/ 7" 361 [unc-29 hP41). Similarly, Tcls hP4 and TCbn2 map -4f within the unc-75 to lev-11 interval ([stPl24 unc-75][33/50] 26 [hP4 TCbnZ] [ 17/50] lev-1 l),but we failed to recover recom- Non-Unc Lev + 18 binants thatordered these Tcls relative to each other, + -6 suggesting that they are close together. Tcl hP4 maps to 24 the left of unc-95, as shownby animals recombinant for dpy- dpy-5 ~nc-95/++~ 5 and unc-95 ([dpy-5 stP1241 [3'1/45] hP4 [8/45] unc-95), Dpy non-Unc 21 and may also be left of unc-101 (dpy-5 [3/84]stP124 [79/ -26 841 hP4 [2/84] unc-101). 23 Three crosses were usedto map STSs on chromosome II (Table 3). Animals recombinant for the markers sqt-2 and Non-Dpy Unc + 16 unc-4 order stP100, stPl96, stPlOl and stP50 on the left + -6 half of the linkage group andplace allbut stPl00 within the 22 marker interval ([stPlOO sqt-21 [25/63] stP196 [21/63]st- dpy-5 unc-lOl/++a PlOl [10/63]stP50 [7/63]unc-4). These data suggest that Dpy non-Unc 1 stPl00 may be left of sqt-2, but do not rule out a position 36 close to and right of this marker. The map positions of -lb stP196 and stPl01 are left of dpy-IO, as shown by chromo- 38 somes that were nonrecombinant for dpy-10 and unc-52 but Non-Dpy Unc - 26 positive for these Tcls (recovered in Dpy Uncs). On the + 43 right half of linkage group II, chromosomes recombinant + -1 for markers dpy-10 and unc-52 order stP36, stP98and maPl, 46 positioning all within the dpy-10 to unc-52 marker interval Mutant chromosome from the Bristol background; wild-type ([stP50 dpy-lo] [4/130]stP36 [4/130]stP98 [ 16/130] map1 chromosome from the Bergerac background. [ 106/130] unc-52). The map position of stP36 is further Unlinked Tcls were detected and served as internal positive refined by chromosomes recombinant between dpy-10 and controls for these reactions (not shown). unc-4, which place it left of unc-4 (dpy-10 [4/25]stP36 [21/ The absence of hP4 may be due to a double crossover event or 251 unc-4). to Tcl excision. Table 4 shows the mapping data forSTSs on chromosome Tcl hP4 was inherited on the unselected chromosome or III. Animals recombinant for unc-45 and dpy-1 place eP64 through a double crossover on the selected chromosome. between these markers (unc-45 [ 10/64] eP64 [54/64]dpy-1, For six of these animals either detection of an unlinked Tcl in the same reaction, or failure to detect stP124, hP4 and TCbnZ in while nonrecombinant animals positive for stP19, stP120, multiple Fs animals, suggest these negative results are not simply mgP21, stP127 and stP17 place these Tcls right of dpy-1. due to failed PCR assays. Animals recombinant for unc-45 and dpy-18 order stPl9, fAnalysis of Fs animals indicates that stP124 was inherited on stP1.20, mgP.21, stP127 and stP17, and place all but stPl7 the unselected chromosome. within the unc-45 to dpy-18 interval (unc-45 [49/69]stP19 [7/69] stPl20 [2/69] mgP2l [2/69]stP127 [9/69] [dpy-18 right of stP44 (dpy-9 [39/52] stPl3 [6/52] stP51[1/52]stP44 stPl71. Tcl stPl7 is right of dpy-18, as shown by chromo- [6/52] [unc-22 sP4 stP5 stP35l). Tcls stP5 and stP35 are somes nonrecombinant for unc-45 and dpy-18, but positive right of unc-22 and sP4, as indicated by a chromosome that for this Tc 1, which were recovered in Dpy Uncs and as the was nonrecombinant for dpy-9 and unc-22, negative for sP4, unselected chromosome in Dpy non-Uncs. but positive for stP5 and stP35 (recovered as the unselected A cross with markers dpy-9 and unc-22 was used to map chromosome in one non-Dpy Unc animal). Tcl sP4 was not STSs on chromosome N (Table 5). Animals recombinant resolved from unc-22 in this cross. for these markers order stP13, stP51 and stP44 within the The chromosome V mapping data arepresented in Table dpy-9 to unc-22 interval and place sP4, stP5 and stP35 to the 6. Tcl stP3 is within the unc-60 to dpy-11 interval, as STS mapping in C. elegans 613 TABLE 3 Linkage groupII mapping data

Genotype of Genotype hybrid Detection of STS parent; phenotype of self-progeny StPlOO maPl stP196 stP98 StPlOlstP36 slP50 n sqt-2 ~nc-4/++~ Sqt non-Unc - + + + 3 - + + + 5 - - + + I - - - + -1 10 Non-Sqt Uncb + 22 + 16 + 9 + -6 53 dpy-10 unc-52/++" Dpy non-Unc + 4 + 2 - 7 -50 63 Non-Dpy Unc + + - 2 + + + 9 + + + -56 67 Dpy Unc - 23 + 2 + -1 26 dpy-10 ~nc-4/++~ Dpy non-Unc - 1 - 4 + -3c 8 Non-Dpy UncNon-Dpy + - Id + + 2 + + -14 17

a Mutant chromosome from the Bristol background, wild-type chromosome from the Bergerac background. Assays for stP98 and mnPl were performed on only 14 of these animals and map1 was detected in one of them; this Tcl was probably inherited on the unselected chromosome. Analysis of F3 animals indicates that stPIOO was inherited on the unselected chromosome. Tcl stPlOl may have been lost by excision; loss by recombination would require either a triple recombinant selected chromosome, or a double recombinant selected chromosome in combination with a single recombinant unselected chromosome. indicated by chromosomes recombinant for these markers recombinant for dpy-3 and unc-27 order stP41,stP40, (unc-60 [104/170] stP3 [66/170] dpy-11). Tcl stP192 maps stP156, stP33 and stPIO3, placing all but stP4l within the close to dpy-11; we failed to detect a recombination event marker interval ([stP4I dpy-31 [ 18/58] stP40 [ 15/58] stP156 between dpy-I I and stP192 in animals recombinant for dpy- [9/58] stP33 [5/58] stPIO3 [11/58] unc-27); stP4l was in- I I and unc-60 to the left (unc-60 [ 170/170] [dpy-I I stP1921 herited on the unselected chromosome in one Dpy non-Unc ), or dpy-I1 and unc-23 to the right([stPI92 dpy-l1][22/22] animal (see footnote b), placing it left ofdpy-3. Chromosomes unc-23). Data from the second cross place stP23 within the that were nonrecombinant for dpy-3 and unc-27, but positive dpy-1 I to unc-23 interval (dpy-I I [ 12/22] stP23 [ 10/22] unc- for one or more of the Tcls stP129, stP61, stP72 and stP2 23). Tcls bP1, stP6, stPI8, stPlO8 and stPlO5 are ordered (recovered in non-Dpy Uncs asthe unselected chromosome), place these STSs to theright of unc-27 and orderthem with and positioned within the dpy-11 to rol-9 interval by recom- one exception; a recombination event between stP6I and binants for these markers (dpy-I1 [10/54] bPI [2/54] stP6 stP72 was not recovered, so these Tcls cannot be ordered [7/54] stPI8 [8/54] stPIO8 [13/54] stPIO5 [14/54] rol-9). relative to each other. Animals recombinant for dpy-21 and rol-9 further refine the map positions of stPlO8 and stPIO5, placing them right of dpy-21; these recombinants also position stPI28 to the right RESULTS of stPIO5, but left of rol-9 (dpy-21 [2/84] stPIO8 [21/84] stPlO5 [43/84] stP128 [18/84] rol-9). Identification of Bergerac Tcls: Because the Ber- For the X-linkage group (see Table 7), chromosomes gerac strain of C. elegans has many more Tcls than 614 B. D. Williams et al.

TABLE 4 TABLE 5 Linkagegroup III mapping dataLinkage group N mapping data: dpy-9 unc-22/++

~~ Genotype of hybrid Detection of STS Detection of STS parent; phenotype of Phenotype of self-progeny eP64 stP19 stPl.20 mgP.21 stP127 stPl7 n self-progeny stPI?stP5I stP44 sP4 stP5stP35 n unc-45 dpy-l/++" Dpy non-Unc + + + + + + 16 Unc non-Dpy +++ + ++7 - +++++3 - + + + + +21 - - ++++1 -+ - + + +Ab - - -+++2 29 - - "- - -36 Non-Unc Dpy ------2c 25 -" - + + Id Non-Dpy Unc - - "_ -22 +- - - - - 18 - - --+ + 1c +-- - - + 10d +- "_ -3 ++ + + + +Q" + + +" -4- 35 30

Unc Dpy -++ + ++4 a Mutant chromosome of the parental strain from the Bristol "_ - - +7 background; wild-type chromosome from the Bergerac back- " - - - -9- ground. 20 Probably failed reactions. unc-45 dpy-l8/++. Tcls stP5 and stP35 were most likely inherited on the unse- Unc non-Dpy + + + + +30 lected chromosome. -++++3 " - ++1 the physical map. However, many of the clones had " - - +s too few fingerprint bands to allow positioning; these, 40 along with other problemclones, were also hybridized

Non-Unc Dpy " - - - 19 against a griddedset of YAC clones representing most +- - "1 of the physically mappedgenome (COULSONet al. - +- - + 3f 1991). The 200 clones identified 33 independent sites ++- - -2 +++--1 (Figure l), indicated as vertical lines below the physi- ++++-2 cal map of each chromosome. Precise positions are + + + + +_If available within the ACEDB/CEMAP database (R. 29 DURBINand J. THIERRY-MIEG,personal communica- Unc Dpy " - - -7 tion). " - - +s Because much of the physical map has been corre- 13 lated with the genetic map through markers mapped " Mutant chromosome from the Bristol background; wild-type both physically and genetically, the physical map po- chromosome from the Bergerac background. Absence of stPl20 is most likely due to Tcl excision; loss by sition allowed us to infer the genetic map position of recombination would require a triple recombinant selected chro- most clones. Those with relatively dispersed genetic no some, or the combination of a double recombinant selected map positions were selected for furtherstudy, as were chromosome and a single recombinant unselected chromosome. One of these animals produced several Fs progeny, and each all the Tcl-containing clones that mapped to contigs tested negative for all of the chromosome Ill STSs. of unknown genetic map location. The latter were * Tcls stP127 and/or stPl7 were probably inherited on the chosen since they were likely to be located in the less unselected chromosome. Test of FJ progeny from two of these animals confirms that well characterized regions of the genome. STS mark- stP19,stPl20, mgP21, stP127 and stP17 were inheritedon the ers stP3 V, stP128 V, and stP41 X (see below) were unselected chromosome. developed from this second set of clones. f Since stPl7 maps to the right of dpy-18, this Tcl was probably inherited on the unselected chromosome. Conversion to STSs: For each clone of interest, sequence of the DNA flanking the Tc 1 site was deter- the standardBristol strain, almost every Bergerac Tc1 mined, usually by priming from a site within the Tcl should be apolymorphic site between the two strains. (see MATERIALS AND METHODS for details). From the We chose seven previously cloned Bergerac Tcls for sequence, an oligonucleotide primer was designed to development as polymorphic STSs because of their work in combination with a constant primersite within well dispersed positions on theC. elegans physical map. the Tc 1. Theprimer pairwas tested in the polymerase To increase this number, we undertook to position chainreaction to ensure that the pair gave only a 200 Tcl-hybridizing clones which hadbeen previ- single major band fromthe phage clone and Bergerac ously isolated from a Bergerac genomic library. genomic DNA, and no product fromBristol genomic Initially, these clones were subjected to restriction DNA. These primersthen served to assay forthe fragment length fingerprint analysis (COULSON et al. Bergerac, Tcl-containing site. These sites are high- 1986) andthis information was used to place them on lighted by boxes in the Figure 1, whichshows a TABLE 6 Linkage group V mapping data

~~ ~~~~ ~~ ~~ ~ ~~ ~~ ~~ Genotype of hybrid Detection of STS parent; phenotype of self-progeny stP3 stPl92 stP23stP6 bP1 stP18 stP108 stPlO5 stP128 n unc-60 dpy-1I/++a U nc non-Dpy Unc + + 55 + - Ib + + 1C - + 26 - + -Id 84 Non-Unc Dpy - - 40 - + le - + 3e - + 2e - + le + - 37 + + -2e 86 dpy-1 I ~nc-23/++~ Dpy non-Unc + 2f - 4 - 4 - -lg 11 Non-Dpy Unc + 6 + 4 + -Ih 11 dpy-11 rol-9/++' Dpy non-Rol + 5 + 2 + 5 - 4 - 6 - -4 26 Non-Dpy Rol - 5 - 2 + 4 + 7 + -10 28 dpy-21 rol-9/++' Dpy non-Rol 1' 5 1' 11 3 1' -2' 24 Non-Dpy Rol 2 15 31 -12 60 Dpy Rol 15 1 -1 17

a Mutant chromosome from the Bristol background; wild-type chromosome from the Bergerac background. The absence of stP18 in this recombinant may be due toa double crossover event or Tclexcision. The absence of stP23 is probably due to Tclexcision. The absence of bPI is probably due to Tclexcision. e Tcls stP23 and/or bP1 and/or stP6 and/or stPl8 were inherited on the unselected chromosome or a double recombinant selected chromosome. fTcl stP3 was inherited on the unselected chromosome or on a double recombinant selected chromosome. g The absence of bP1 is probably due to Tcl excision. h Tcl stP6 was inherited on the unselected chromosome or on a double recombinant selected chromosome. ' Tcls stP23 and/or stP192 and stP3 were inherited on the unselected chromosome or on a doublerecombinant selected chromosome. 615 616 B. D. Williams et al.

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genetic map scale LJ715 map units STS mapping in C. elegans 617 TABLE 7 Linkage group X mapping data:dpp3 unc-27/++

Detection of STS Phenotype of stP41 stP40 stP156 stP33 StP103 stP129 stP61 stP129 StP103 self-progenfstP33 stP156 stP40 stP41 stP72 stP2 n

~ ~~~~ ~~~~ ~ ~ ~ ~ ~~ ~~ ~ ~~~ ~ ~~ ~ a Mutant chromosome of the parental strain from the Bristol background; wild-type chromosome from the Bergerac background. Test of F5 progeny shows that stP4l was inherited on the unselected chromosome; this places stP41 to the left of dpy-3. Analysis of FS progeny were performed for three out of seven of these animals testing positive for stP61, stP72 and/or stP2; the results show that these Tcls were inherited on the unselected chromosome. The absence of stP40 is probably due to Tclexcision; loss by recombination would require a triple crossover event. Analysis ofifibgeny suggests that stPI29, stP61, stP72 and stP2 were inherited on the unselected chromosome. physical (top) and genetic (bottom) map of eachchro- PCR artefacts: PCR artefacts that might complicate mosome. mapping experiments include false negatives due to Multiplex PCR To implement an efficient genetic reaction failure and false positives due either to con- mapping strategy, STS band sizes were chosen which tamination ofsamples or to amplification ofback- permit appropriate multiplexPCR assays. Various ground bands that happen to comigrate with one or assay sets have been developed which either detect a more STS bands. single Tcl on each autosome (Figure 2, Chromosome False negatives could be detected by including an Assignment Set) or multiple Tc 1s on individual chro- assay for a nonpolymorphic site. This kind of internal mosomes (Figure 2, linkage group (LG) I, LG ZI, LG positive control wouldallow failed reactions to be ZZZ, LG N,LG V-right, LG V-left, and LG X). easily distinguished from animals that simply did not Each multiplex assay can be used witheither of the inherit anyof the Tcls normally detected by the related Bergerac strains RW7000 or DP13, with the multiplex assay. In practice, this information was not following caveat.Tc 1 eP64 ZZZ is present only in DP13 necessary for efficientmapping (see below), so we (see Figure 2, LG ZZZ), and stPI28 V is present only in decided that it was better to minimize the number of RW7000 (STS stP128 failed to amplify from DP13 primers in each reaction. DNA, data not shown); all of the other Tcls shown Although we did not see false positive bandsattrib- in Figure 1 have been detected in both strains. Control utable to cross contamination of DNA samples, am- reactions with Bristol DNA showthat all of theseTcls plification ofbackground bands comigrating with one are absent from the Bristol N2 strain (Figure 2, right- or more of the STS bands was observed on occasion most lane of each panel). in early experiments (not shown). These bands were

FIGURE 1.-Physical (top line) and genetic (bottom line) maps of chromosomes I, lI, IIl, N, V and X. Bergerac Tcls that have been converted to polymorphic STSs are highlighted with boxes; their genetic map positions are based on physical map data and genetic data from crosses presented in MATERIALS AND METHODS. Lines extending above the physical mapsmark the positions of physically mapped genes; those which were used in this study are labeled. Lines just below the physical maps mark the positions of Tcl-hybridizing Bergerac clones. The physical map for each chromosome is scaled to chromosome size; these are estimated to be 18 megabases (mb) (I), 14 mb (N), 16 mb (III), 19 mb (IV), 22 mb (V) and 18 mb (X).Our genetic data place TCbn2 I near hP4, but the (*) indicates that it has not been ordered relative to hP4 or to unc-101 and unc-95. 618 B. D. Williams et al. Chromosome Assignment Set LG I LG I1 LG 111

sP4 IV- mgP21 - mgP21 111 Y TCbn2 - SlP120 s1P 196 - - hP4 I - bPI V- 124SIP - stP1Ol - I1 I1 II Ill 9% 3 % LG IV LG V LG V LG X Rinht

II II II II %" 2&" "4,g 9% -3 % % % % FIGURE2.-Multiplex PCR assays for Bergerac Tcls. Each PCR assay was performed on the DNA of a single animal and the products resolved on a standard 6%mini-acrylamide gel. STS band sizes and genetic map positions can be found in Table 1 and Figure 1, respectively. In reactions with DNA from Bergerac strain RW7000 the chromosome assignment assay set detects a single Tcl on each autosome, while sets LC I, LC 11, LC Ill, LC N,LC V (left), LC V (right) and LC X detect multiple Tcls on the corresponding individual linkage groups. Results were identical for the closely related Bergerac strain DP13 (not shown), with two exceptions. Tcl eP64 was detected in DP13, but not in RW7000 (see LC Ill), and Tcl stP128 was detected in RW7000, but not in DP13. It should be noted that when mapping with strain DP13 it is possible to substitute the stP18 assay for the stP128 assay within the LC V (right) assay set, due to similar band sizes. In control reactions with Bristol N2 DNA no bands were amplified by any of the multiplex assays. usually very faint and did not confuse interpretation fingerprint data; once placed genetically on chromo- of the data.To confirm that theseanimals were indeed some ZI, an alternative match was recognized as cor- negative for the STS bands in question, we tested rect. several self progeny from each,in some instances with For the STSs in less densely marked regions of the a simpler mixture of primers. The appearance of physical map, and for those on contigs not assigned a background bands is not surprisinggiven the complex chromosomal location, more extensive crosses were mixture of primers in each multiplex reaction, includ- used to establish the genetic positions (see MATERIALS ing a primer to the high copy number Tcl sequence. AND METHODS), and these data improved the conti- In subsequent experiments we found that by heating guity of the physical map. For example, on the right the PCR reactions soon after the components were end of chromosome V our mapping data for stPI08, mixed (see MATERIALS AND METHODS for details), the stPZ05 and stPI28 positioned the associated contigs intensity of background bands was reduced to the relative to each other. In several cases the genetic data point that they rarely caused difficulty. led to the recognition and correction of errors in the STS genetic map position: The STSs in regions of physical map. In particular, our genetic mapping of the physical map densely covered with markers could hP4 I, stPl00 IZ and stP196 IZ repositioned the asso- often be assigned to a relatively narrow genetic inter- ciated contigs. val. These STS map positions were tested in crosses Spontaneous Tcl excision in Bristol/Bergerac hy- by marking the appropriateBristol chromosome of an brids: Rates of Tc 1 excision in Bristol/Bergerac stains FI Bristol/Bergerac hybrid with two visible marker can be similar to or even enhanced relative to those mutations and then scoring recombinant and nonre- in Bergeracmutator strains (MORI, MOERMAN and combinant F2 animals for linked Tcls by multiplex WATERSTON1988, 1990). PCR. The data from these crosses (see MATERIALS AND In the course of verifying Tcl map positions, we METHODS) were consistent with the inferred STSmap observed possible spontaneous excision ofTcl s stPlOl positions, with one exception. stP36 had been given a IZ (Table 3, footnote d),stPl2O 111 (Table 4, footnote physical map position on chromosomeX with marginal 6) stP23 V (Table 6, footnote c), 6PI V (Table 6, STS mapping in C. elegans 619 FIGURE3.-Two-step polymor- A phic STS mapping strategy. (A) A new mutation, m, is first mapped to a chromosome by using a single STS Segregation of linked and to mark each autosome, STSs 1 and unlinked STS’s in F2 2 in this example. A Bristol/Bergerac mutant homozygotes F, hybrid (Bristol DNA = straight Multiplex PCR of line, Bergerac DNA = jagged line) LINKED individual F2 mutant that is heterozygous for the new mu- homozygotes pu.“w tation is constructed and allowed to *fah F1 BristoVBergerac produce self progeny. As diagramed, hybrid F segregation of linked and unlinked Tcls among thehomozygous mutant progeny will be different. Unlinked Chrom. I Tcl 2 is inherited by 75% of the UNLINKED animals, while linked STS 1 is par- Chrom. /I - 25% tially or completely “excluded” from L J the mutant homozygotes because it is inherited only when there is a cross- I7 over between the mutation and the 2 2 STS. Each FP mutant animal is scored 25% for the Tcls using a single multiplex 2 PCR reaction. The diagramed acryl- amide gelsshows that only 1 of 10 animals inherited Tcl 1, indicating linkage, while 7 of 10 inherited STS 2, indicating nonlinkage. (B)Second B STS mapping step. Additional F:! mu- tant homozygotes are assayed for sev- eral linked Bergerac Tcls, STSs 1, Individual F2 mutant Multiplex PCR of 2, 3, 4 and 5, by multiplex PCR. homozygotes individual F2 mutant Genotypes of some expected FP mu- homozygotes tant homozygotes (A-D) are dia- F1 BristoVBergerac m gramed along with the correspond- hybrid A= m ing results of multiplex PCR assays

m on DNA from these animals. By using BC the PCR data to infer positions of m ”Irn crossovers between the Bristol and r”rr*m m - 1234 5 Bergerac chromosomes, mutation m C- C- can be placed between STSs 2 and 4; m alternative positions are ruled out be- m D- D- cause they would require more com- IT plex patterns of recombination. footnotes d and g) and stP40 (Table 7, footnote d). In recombination would requirea triple recombinant some cases we failed to detect the Tcl in a multiplex chromosome in each instance. PCR assayof DNA from a single F2 animal, while With the possible exception of the stP40 excision Tcls to either side were detected. A trivial explana- events, Tcl excision frequency is low enough that it tion for theseresults is a partial failureof the multiplex does not pose a significant complication when using assay, but we have not seen this type of failure in any the Tcls as geneticmarkers. The method used to of numerous control reactions using single Bergerac initially assign a new mutation to a linkage group (see worms. Consequently, it is more likely that these Tcls below) would be unaffected by low levels of sponta- have either excised or been lost through recombina- neous Tcl excision. Subsequent mapping to a chro- tion. The latter is unlikely forthe stP101,stP120, mosomal subregion might be more seriously affected, stP23 and bPI events, since each would require either but in practice the level of redundant information a triple recombinant chromosome or fortuitous com- obtained from these crosses make it likely that rare binations of a double recombinant chromosome and excision events would be readily recognized and not a single recombinant homolog in which the necessary lead to a incorrect map position assignment. intervals for recombination are relatively small; loss Mappingstrategy: To mapa recessive mutation by recombination cannot be ruled out, however. In generated in the Bristol background, a Bristol/Ber- contrast, for stP40 enough events have been observed gerac hybrid strain that is heterozygous for the un- to provide compelling evidence of excision. Loss of mapped mutation is constructed and allowed to self- this marker occurred in three of the 14 animals in fertilize (see MATERIALS AND METHODS for details). which it couldhave been detected, andto invoke Approximately 25% of the progeny will be homozy- 620 B. D. Williams et al. 9 'L, deb-7 homozygotes 0 A 00 I

B C

FIGURE4.-Test mapping of lethal mutation deb-l(st385). (A)Multiplex PCRassays of individual deb-I homozygotes from a Bristol/ Bergerdc heterozygous mutant parent. The chromosome assignment assay detects Tcls marking chromosomes I, II, III, Nand V. Each Tcl is detectedin approximately 75%of the animals except forsP4 IV, which was detected in none of them, indicating linkage. A control reaction with RW7000 DNA shows that the multiplex assay set detects all five of the Tcls. The (*) indicates a failed PCR reaction. (B)Additional deb-I homozygotes assayed for several Tcls on linkage group IV. Lanes A-E each show a representative reaction for each type of animal with at least one STS band. (C) Proposed recombination events between the Bristol (top line) and Bergerac (bottom line) chromosomes of the FIhybrid which produce the FP classes A-E [class A (n = 4), class B (n = l), class C (n = l), class D (n = 3) and class E (n = l)]. This analysis places deb-I between stP5l and stP5, which corresponds with its previously established genetic and physical map positions. gous for the mutation, and these animals are picked shapen L1 larvae. Individual mutant self progeny (n into separate tubes, theirDNA is released by lysis,and = 25) from the appropriate Bristol/Bergerac hybrid then each sample is used in a separate multiplex PCR were tested with the chromosome assignment assay assay that can detect several Bergerac Tcls. Mapping set(Figure 4A).Each Tcl exceptfor sP4 N was is accomplished in two successive steps, each using a detected in approximately 75% of the animals; sP4 different multiplex assay. In the first step the animals was detected in none of them, placing deb-I on chro- are assayed for five Tcls, onemarking each autosome. mosome IV near this STS marker. Itshould be noted As illustrated in Figure 3A the STSs behave as domi- thatone lane was negative for all five autosomal nant genetic markers, so the unlinked Tcls will be markers [Figure4A (*)I. probably due to a failed PCR detected in 75% of the animals. In contrast, thelinked reaction. Tcl will bedetected in significantly fewer of the In the second mapping step we assayed 33 addi- mutant homozygotes since this requires recombina- tional deb-I homozygotes with the LC N assay set. tion between themutation andthe Tcl. An STS Tcls were detected inonly 10 animals, confirming marker for theX chromosome is unnecessary because linkage to chromosome N. These recombinants fell X-linkage is detected when constructing the Bristol/ intothe five classes illustrated in Figure 4B. The Bergerac hybrid, due to hemizygosity of C. elegans simplest interpretation of these data is illustrated in males for the X chromosome. Figure 4C, which shows the likely crossover events In the second mapping step additional animals are between the Bristol and Bergerac chromosomes of the assayed for BergeracTc 1 s with dispersed locations on F1 hybrid which correspond to the FP classes A-E. the appropriatechromosome. As illustrated in Figure This interpretation places deb-I between stP5I and 3B, multiplex PCR on individual animals allows re- sP4, agreeing with its known map position. It should combination events to be located and the mutation be noted that a recombination event between deb-I mapped to a chromosomal subregion. and stP44 was not detected, so deb-I cannot be or- Mapping autosomal mutations: To illustrate the dered with respect to this marker. This failure is not strategy and demonstrateits effectiveness for mapping surprising given the limited number of animals tested lethal mutations, we mapped two autosomal genes and physical map datawhich suggest that deb-I is very whose physical and geneticmap positions are well tightly linked to stP44 (see Figure 1). established. In the first experiment we mapped the We chose the lethal mutation unc-52(st572) (arrest lethal mutation deb-l(st385); mutant animals either phenotype similar to deb-I(st385)) as a more stringent arrest in late embryogenesis or hatch to yieldmis- test of the first STS mapping step since approximately STS mapping in C. eleguns 62 1

9 "L, unc-5.2 homozygotes A 00 91 I

FIGURE5.-Test mapping of lethal mutation unc-52(st572). (A) Individual unc-52(st572) homozygotes from a Bristol/Bergerac parent assayed for Bergerac Tcls on linkage groups I, II, Ill, IV and V. Tcl maPl I/ is detected in significantly fewer than 75% of the animals, indicating linkage to this marker. In a control reaction with RW7000 DNA each of the autosomal Tcls is detected. (B) Additional unc-52 homozygotes assayed for multiple Tcls on chromosome II. Lanes A-I show representative reactions for each type of Fn animal with one or more STS bands [class A (n = 6). class B (n = 2). class C (n = 2). class D (n = 2). class E (n = I), class F (n = 9). class G (n = 2). class H (n = 1). class I (n = I)]. (C)Proposed recombination events between the F, Bristol (top line) and Bergerac (bottom line) chromosomes which produce Fs classes A-F and G-I.

20 map units separate it from the chromosome ZZ established map position, illustrate the mapping on marker maP1. Reactions with DNA from 36 of the 39 the X chromosome. An analysisof 39 pat-(st558) animals tested yielded 1 or more bands (Figure 5A, homozygotes (phenotype similar to the deb-1 and unc- three lanes with no bands are not shown and were 52 lethals) yielded 25 that were positive for X-linked assumed to be failed PCR reactions), and analysis of Tcls, and these animals fell into the six classes illus- this data clearly indicates linkage to maPl (detection trated by representative reactions in Figure 6A. The frequency R = 0.47, x2 = 12.75). Marginally signifi- simplest interpretation of these data place pat-(st558) cant linkage to sP4 ZV (R = 0.59, x2 = 3.92) and to the right of stP72 (Figure 6B). Since stP2 was not mgP21 ZZZ (R = 0.56, x2 = 5.64) was also detected in detected in any of the animals, pat-(sst558) cannot be this data set, however. ordered relative to this marker, but must be close to Since the strongest linkage was to chromosome ZZ, it. 41 additional unc-52 homozygotes were tested with A similar mapping experiment was performed with the LG ZZ multiplex assay in the second mapping step. unc-90(e1463) X, which is a semidominant mutation The 26 animals that were positive for one or more Tcls fell into the nineclasses illustrated by represent- causing uncoordinated movement. Assays of 101 ative reactions in Figure 5B. The simplest interpreta- homozygous mutant progeny from the Bristol/Ber- tion of these data places unc-52 right of maP1, con- gerac hybrid strain yielded four classesof animals sistent withits established map position; inferred testing positive for Bergerac Tcls (Figure 6A). As crossover events corresponding to each class are illus- indicated by thediagram in Figure6C, these data trated in Figure 5C. position unc-90 between stPl03 and stP61, which MappingX-linked mutations: Mapping experi- agrees with its established map position. Recombinant ments with lethal mutation pat-(st558), which has not animals which could position unc-90 with respect to been mapped before, and unc-90(e1463),which has an stP129 were not recovered in this experiment. 622 B. D. Williams et al. ping strategy permits the simultaneous use of 40 ge- netic markers and potentially many more as additional polymorphic STSs are developed. Through efficient scoring of single animals by multiplex PCR, a new mutation can be mapped to a chromosomal subregion after analysis of a small number of progeny from a single cross with only one mutantphenotypic class. At this point visible markers can be chosen for detailed 3-factor analysis. The savings in number of crosses is helpful, partic- ularly when a mutation presents special difficulties, B such as lethal, sterile, maternal effect, suppressor or X-linked mutations.

It I Absence of an associated visible phenotype for STS markersoffers additional advantages. Thisfeature avoids difficulties that can arise when scoring a mutant phenotype in a background of several visible marker phenotypes (when mappinga suppressor mutation, for example) and also simplifies mapping on the X chromosome. The latter is complicated in standard mapping procedures due to the hemizygosity of C. C 3 eleguns males. Mutant X0 males are often ineffective b C 0 in mating, so visible markers on the X chromosome “a “a must usually be used in conjunction with sexual trans- formation mutations in genes such as tru-l (HODCKIN and BRENNER1977), which can produce XX males. In I I-contrast, as shown in test crosses presented here, all nine STS markersfor the X chromosome can be introduced easily into a mapping strain by using Bris- tol/Bergerac hybrid males. Aside from facilitating multifactor mapping, STS FIGURE 6.-(A) Mapping of lethal mutation pat-(st558) and sem- markers allow particularly efficient and sensitive map- idominant mutation unc-90(e1463) on the X chromosome. In sepa- rate experiments pat-(st558) and unc-90 F2 homozygotes from the ping strategies because of their behavior as genetic appropriate F, Bristol/Bergerac hybrid strains were assayed for X- dominants and theirsuitability for homozygosity map- linked Bergerac Tcls. Thosewhich tested positive at least one Tcl ping. Although some dominant visible markers are fell into six classes (A-F) in the pat-(st558) experiment [class A (n = available in C. eleguns, most mapping is accomplished 4), class B (n = 2), class C (n = l), class D (n = 4), class E (n = 1). withrecessive markers and strategies thatrequire class F (n = 13); an assay for stPl0 was not included in this experiment], and four classes (A-D) in the unc-90 experiment [class significantly more cross progeny to be scored in order A (n = 21), class B (n = 2). class C (n = 2). class D (n = 26)]; a to detect linkage. representative reaction is shown for each class. (B) Proposed recom- The advantages of the STS mapping method, in- bination events responsible for F2 classes in the pat-(st558) experi- cluding the ability to directly score dead embryos by ment; this simplest interpretation of the data placespat-(st558) right were demonstrated here forlethal alleles ofdeb- of stP72, but fails to order it with respect to stP2. (C) Proposed PCR, recombination events responsible for F2 classes A-D in the unc-90 I and unc-52. If one were to map these same mutations experiment. This simplest interpretation of the data places unc-90 by standard methods, the first step would be to con- between stP103 and stP6J, corresponding to its previously estab- struct F1 animals that are truns-heterozygotes for the lished genetic map position. lethal mutation and for several visible mutations that DISCUSSION mark individual chromosomes. Since it is not possible to score the F2 lethal homozygotes for the marker The polymorphic STS mapping strategy offers sig- phenotypes, viable F2 siblings (heterozygous for the nificant advantages over standard genetic mapping lethal mutation or homozygous wild-type at this locus) procedures in C. eleguns. In the latteronly a few visible would have to be analyzed instead by cloning them markers can be used simultaneously due to the small and scoring the phenotypes of the self progeny that number of phenotypes thatare both distinct and easily they segregate in the F3 generation. It takes 3 days to scored. Consequently, several crosses are often used produce this extra generation,including several hours to place a mutation on a linkage group, and these at the dissecting microscope cloning the F~sand then must be followed by a succession of 3-factor crosses scoring F3 phenotypes. This compares with the that refine map position. In contrast, the STS map- elapsed time of 6 hr, including approximately 2 hr of STS mapping in C. elegans 623 benchtime, it takes to pick the FP homozygotes, volved in the inheritance of quantitative traits (LAN- perform PCR, and run the gels in an STS mapping DER and BOTSTEIN1989). This has been intractable experiment. At this point the comparative elapsed by standard methods, but may be feasible using the times from the first cross are approximately 9 days STS markers, since the segregation of many of these for the standard mapping method and 6 days for the markers can be scored in a single cross. STS method. Significantly more time is saved in the We thank R. BARSTEAD,M. JOHNSTON and T. SCHEDLfor helpful subsequent mapping phase,since 3-factor crosses must discussions in the preparation of this manuscript, and D. BAILLIE, be set up for the standard mapping experiment, re- M. FINNEY,A. PAPP,A. ROSE, S. STROMEand D. PILGRIMfor gift quiring at least an additional 9-1 2 days by a very of subclones. Supported by National Institutes of Health grants conservative estimate and substantially more time for GM23883 and HG00375 to R.H.W., National lnstitutes of Health the X-linked mutation put-(st558). This contrasts with postdoctoral fellowship GM12625 to B.D.W., and a Muscular Dys- trophy Association fellowshipto B.S. Some of the stains used in this the STS mapping experiment in which the mutations work were supplied by the Caenorhabditis Genetics Center, which were mapped to a chromosomal subregion in a single is funded by the National Institutes of Health National Center for additional day; more F2 mutant homozygotes from Research Resources (NCRR). the original cross were simply scored with the appro- priate chromosome specific multiplex PCR assay. LITERATURECITED Because they are common landmarks to thegenetic and physical maps, polymorphic STSs are also very BAILLIE,D. L., K. A. BECKENBACHand A. M. ROSE, 1985 Cloning within the unc-43 to unc-31 interval (linkage group IV) of the useful when using linkage data to identify DNA clones Caenorhabditiselegans genome using Tcl linkage selection. within the physical map that are likely to contain a Can. J. Genet. Cytol. 27: 457-466. gene of interest. Systematic development of additional BARSTEAD,R. J.. and R. H. WATERSTON,1991 Vinculin is essential STS markers should enhance their usefulness for this for muscle function in the nematode. J. Cell Biol. 114 715- 724. purpose, and such polymorphic STSs might be used BARSTEAD,R. J., L. KLEIMAN and R. H. WATERSTON, in an extension of the mapping procedure presented 1991 Cloning, sequencing and mapping of an a-actinin gene here. To continue mapping with STS markers on a from the nematode Caenorhabditiselegans. CellMotil. Cyto- finer scale, it would probably be most efficient to use skeleton 20 69-78. a visible marker in cis to the mutation to be mapped. BRENNER,S., 1974The genetics of Caenorhabditiselegans. Ge- netics 77: 7 1-94. This would allow crossover events in the interval CORNALLR. J., T. J. AITMAN,C. M. HEARNEand J. A. 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