Characterization of a Meiotic Crossover in Maize Identified by a Restriction Fragment Length Polymorphism-Based Method

Marja C. P. Timmermans,’ 0. Prem Das* and Joachim Messing Waksman Institute, Rutgers university, Piscataway, New Jersey 08855-0759 Manuscript received March 6, 1996 Accepted for publication May 3, 1996

ABSTRACT Genetic map lengths do not correlate directlywith genome size, suggesting that meiotic recombination is not uniform throughout the genome. Further, the abundanceof repeated sequences in plant genomes requires that crossing over is restricted to particular genomic regions. Weused a physical mapping approach to identify these regions without the bias introducedby phenotypic selection. This approach is based on the detectionof nonparental polymorphisms formedby recombination between polymorphic alleles. In an F2 population of 48 maize plants, we identified a crossover at two of the seven restriction fragment length polymorphism loci tested. Characterization of one recombination event revealedthat the crossover mapped withina 534bp region of perfect homology between the parental alleles embedded in a 2773-bp unique sequence.No transcripts from this region could detected.be Sequences immediately surrounding thecrossover site were not detectably methylated, except SstIfor sitean probably methylated via non-CpC or CpXpG cytosine methylation. Parental methylation patterns at this SstI site and at the flanking repetitive sequences were faithfully inherited by the recombinant allele. Our observations suggest that meiotic recombination in maize occurs between perfectly homologous sequences, within unmethylated, nonrepetitive regionsof the genome.

ECOMBINATION isan importantaspect of meiosis The currentunderstanding of recombination mecha- R in most eukaryotic organisms. It ensures proper nisms stems largely from geneticand molecular studies chromosome disjunction during the reductional divi- in bacteria and fungi (PETESet al. 1991). Little is known sion of meiosis I by providing a physical connection about the recombinationprocess in plants. Several stud- between homologues. Meiotic recombination also plays ies have utilized transgenic approaches to determine an essential role in generating geneticdiversity and thus mitotic recombination frequencies between repeated contributes to genome evolution. In plants, where re- sequences in plants (PETERHANSet al. 1990; GAL et al. productive tissue is often derived from somatic lineages 1991; ASSAAD and SIGNER1992; TOVARand LICH- late in development, mitotic recombination can also TENSTEIN 1992; SWOBODAet al. 1993, 1994). The fre- contribute to genome diversity (DM et al. 1990b). quency of such events varies between and lo-’ Higher plant genomesdiffer in size over three orders per cell and appears to be affected by repeat length of magnitude, largely due to variation in repetitive DNA (PETERHANSet al. 1990), degree of homology (GAL et sequences such as multigene families and repetitive al. 1991),and position inthe genome (ASSAAD and transposable elements (FLAVELL1985). Repeated se- SINGER1992; SWOBODAet al. 1993). Similar to mitotic quences can potentially affect genome plasticity by their recombination in yeast, gene conversion-like exchange effects on homologous recombination. Unequal cross- seems to occurmore frequently than crossing over, ing over between repeats can give rise to deletions and since many ofthe recombinationalevents are notassoci- amplifications, andother recombinational processes ated with exchange of flanking markers (A~SAADand can give rise to inversions and translocations. To main- SIGNER1992; TOVARand LICHTENSTEIN1992). tain genome stability in plants, homologous recombina- Mitotic recombination between tandemly duplicated tion via crossing over needs to be tightly controlled and sequences at the p locus (ATHMAand PETERSON1991) restricted to particular genomic segments, whether in and theknl locus (LOWEet al. 1992) in maize was found somatic or meiotic tissue. to be strongly enhanced (between several hundred- to several thousand-fold) by thepresence of an active Corresponding authm:Joachim Messing, WaksmanInstitute, Rutgers transposable element between the repeats. Molecular University, P.O. Box 759, Piscataway, NJ 088550759. analysis of Mu1 excision footprints at the bzl locus also E-mail: messingQmbcl.rutgers.edu suggested theoccurrence of recombinational events ‘Present address: Department of Biology, YaleUniversity, New Haven, CT 06520-8104. upon transposon excision (DOSEFFet al. 1991). It is ‘Present address: Heartland BioTechnologies, Davenport, IA 5280& likely that transposase-induced double-strand breaks 1340. near the endsof the Mu element stimulate recombina-

Genetics 143 1771-1783 (August, 1996) 1772 P.M. C. Timmermans,Das 0. P. and J. Messing tion, as demonstrated for the P element in Drosophila MATERIALSAND METHODS (GLOORet al. 1991). Meiotic recombination has been studied in maize by Plant materials: Maize inbred lines A188, W64A and W22 were derived from stocks kindly provided byR. L. PHILLIPS selecting for wild-type intragenic recombinantsbetween (University of Minnesota, St. Paul, MN). combinations of mutant heteroalleles of phenotypic DNA gel blot analysis: Genomic DNA from leaf samples marker loci. Such analysis has led to an understanding were isolated as described (DM et al. 1990a). DNA samples of the effect of particular heteroallelic combinations on used for copy number estimations of repeated genomic sequences were further purified on CsCl density gradients. the frequency of recombination and thetype ofgenetic Southern blots onto Nytran membrane were prepared by stan- exchange (k., crossing over or gene conversion-like dard protocols and hybridized at 42" in 50% formamide,5~ exchange). For example, the presence of a single Ds SSC, 1% SDS,5X Denhardt's solution, 0.05% sodium pyro- insertion within a gene suppresses intragenic recombi- phosphate (pH8.0) and 50 pg/ml salmon sperm DNA. Mem- nation and favors crossing over (DOONER 1986), branes were washed three times with 1X SSC, 0.1% SDS at room temperature and twice with 0.1 or 0.2X SSC, 0.5% SDS whereas thepresence oftwo different Ds insertions at 65" for 20 min each. RFLP and other probes were isolated seems to favor conversion-like exchanges (DOONERand with the Gene-Clean kit (Bio 101) and labeled with d'P-dCTP KERMICLE 1986). This type of analysis alsorevealed that by random priming. Total maize genomic DNA (50-100 ng) the maximal frequencies of intragenic recombination was labeled using the same random priming method andused in hybridizations under conditions described above. at the maizeloci a1 (BROWNand SUNDARESAN1991; Construction and screening of subgenomic libraries: Ap- CWARDIet al. 1994), adhl (FREELING1978), b (PAT- proximately 75 pg genomic DNA from the inbredlines A188 TERSON et al. 1995), bzl (DOONER1986; DOONERand and W64A, and a plant homozygous for the recombinant KERMICLE 1986), gll (SALAMINIand LORENZONI 1970), allele of umcll6 was digested with EcoRI and separated by agarose gel electrophoresis. DNA in the rangeof 6- 10 kb was T (DOONERand KERMICLE 1986), andwx (NELSON1968) transferred onto NA45 membrane(Schleicher and Schuell). were approximately comparable, ranging from 0.9 to The membranewas divided into five segments, and DNA was 1.3 X lop3. Recombination frequencies at these loci eluted fromeach segment to further fractionate the DNA were about two orders of magnitude higher than the fragments. An approximately 300-ng insert DNA from the average rate of recombination per kb for the whole appropriate fractions was ligated to 500 ng EcoRI-digested EMBL4 DNA and packaged using the Gigapack Gold kit (Stra- maize genome, indicating that genesmay be more sus- tagene). The libraries were screened with probe umcl16 or ceptible to recombination compared to repetitive se- bs450 and positive plaques were plaque-purified twice. The quences that make up the majority of the genome. EcoRI inserts from two positive lambda clones of each library Based on these and similar observations in other or- were subcloned into pUCl19. The resulting plasmid clones ganisms, it has beenproposed that recombination were used for all further analysis. PCR amplifications to confirm the contiguity of the A188 lambda clones were carried might be preferentially initiated at regions in the ge- out on10 ng genomic DNA from inbred line A188 under the nome containing functional genes (THURIAUX1977). following conditions: 4 min at 94", followed by 30 cycles of 1 This viewis supported by the fact that genetic map min at 65", 2 min at 72", 1 min at 94", ending at 1 min at 72". lengths of most organisms are comparabledespite large DNA sequence analysis: The 2.4kb region encompassing the crossover site was subcloned as -1-kb BglII-SstI, -0.9-kb differences in genome size. The validity of this model SstI-BamHI, and -450- or -600-bp BamHI-SstI fragments into in plants is not easily determined, because the genetic pUC119. These subclones were sequenced at both strands approaches used to study both meiotic and mitotic re- using the Sequenase v2.0DNA Sequencing kit (USB). Se- combination have relied on phenotypic selection to quence data were compiled and analyzed using the GCG sequence analysis software package (University of Wisconsin, identify recombinants within known genes. However, Madison). The ends of the insertion elements present in the one way to address if recombination occurs preferen- A188 allele and their flanking regions were sequenced from tially within functional genes is to use physical mapping subclones that spanned a 2.3-kb PstI-SUA region near the 5' to identify unselected recombinants. end of the allele and a 1.3-kb KpnI-BgllI region near the 3' Here, we describe such a methodbased on thedetec- end of the large insertion element. Sequence information obtained from these subclones was compared to sequence tion of nonparental polymorphisms formed by recombi- data from the 450-bp PstI fragment (black box Figure 5) of nation between polymorphic restriction fragment the W64A allele using the GCG package. length polymorphism (RFLP) alleles. Using this assay, RNA analysis: Total RNA wasextracted fromimmature and two recombination events were identified in a popula- mature maize tissues from the inbred line W64A according to DAS et al. (1990a). Poly-A+RNA was isolated using the tion of 48 F2 plants that were screened for recombina- PolyATtract mRNA isolation system (Promega). RNA samples tion at seven RFLP loci. Both recombinational events were fractionated on formaldehyde-agarose gels and blotted were associated with flanking marker exchange, consis- to Nytran membranes. Northern blots were hybridized and tent with crossing over. The origin of one recombinant washed under conditions described above. First-strand cDNA allele was characterized in detail at themolecular level, synthesis for PCR was carried out using the cDNA Cycle kit (Invitrogen) according to the manufacturer's protocol. Ap- revealing that sequencehomology, level ofmethylation, proximately 0.5 pg poly-A+ RNAfrom embryoand endosperm and possibly chromatin structure may specify sites for [18 days after pollination (dap) 3, seedling leaves, and imma- meiotic crossing over. ture ears was treated with DNAse I and subsequently con- Recombination in Maize 1773

x Probe f Ill A1 88

W64A I I x YZ x YZ

4 + Crossoverregion

FIGURE 1.-Physical mapping as a means to identify unselected crossovers. (A) The diagram represents schematically the identification of recombinant alleles using RFLP analysis, using the two inbred lines A188 and W64A as examples. homology to the probe. Flanking restriction sites are indicated arbitrarily as X, Y, and Z. Recombination within the region indicated would generate two new alleles with altered distribution of restriction sites. One possible recombinant allele is shown, which resembles the W64A allele with enzyme X, but resembles the A188 allele with enzyme Y, and produces a nonparental restriction fragment with enzyme Z. (B) Linkage map of chromosome R.The RFLP loci used to identify unselected crossovers are shown, and the map distances (in cM) between each pair of RFLP loci are also indicated. umc168 (underlined) was not used to identify crossovers but was used to establish the mechanism of recombination at the bnZ8.44 locus. verted to first-strand cDNA using an oligo-dT primer. One- same genotype at a particular locus regardless of the tenth of the products were added directly as template DNA restriction enzyme used. However, if recombination oc- in the PCR reaction. PCR reactions with 0.05 pg poly-A+ RNA were included to control erroneous amplification from re- curs between polymorphic restriction sites at a locus, maining genomic DNA. PCR amplifications were carried out two types of aberrant results can be observed. With re- under the following conditions: 1 min at 94", 1 min at 60", 2 striction enzymes that detect polymorphisms on both min at 72", and 40 cycles. PCR primers specific for the hcfl06 sides of the crossover site (enzyme Z in Figure lA), a (R. MARTIENSSEN, unpublished data) and dtsl0 (KIRIHARA et recombinant allele will produce nonparentalrestriction al. 1988) cDNAs were used as positive controls and produced specific PCR products with seedling, and embryo and endo- fragments. With restriction enzymes that detect poly- sperm mRNA, respectively. morphisms on only one side of the crossover site (enzymes X and Y Figure lA), a recombinant allele will generate either one or the otherof the parental restric- RESULTS tion fragments, and the genotype of the progeny plant Identification of unselected recombinant alleles: Re- that inherited the recombinant allele will be scored striction analysis using Southern blots has been pro- differently depending on the enzyme used. Therefore, posed as a method for theidentification of recombinant Southern blot analysis ofgenomic DNA from a segregat- alleles without selecting for restored genefunction ing population using several different enzymes and the (MEAGHERet al. 1988). A schematic representation of same probe can identify putative recombinants in the the basis of this method, as used in the experiments progeny by nonparental hybridizing bands or genotypes reported here, is given in Figure 1A. Recombinant al- that arescored differently depending on the restriction leles resulting from a crossover event in the vicinity of enzyme used. a unique probe can be identified by comparing RFLP Such an analysis was conducted for genomic DNA patterns between parents and progeny, using multiple from 48 self-pollinated F2 progeny of A188 X W64A restriction enzymes for each probe. In the absence of hybrids. DNA was digested with eight different restric- recombination, a given progeny plant shouldreveal the tion enzymes, and Southern blots were hybridized with 1774 M. C. P. Timmermans, 0. P. Das and J. Messing

TABLE 1 Restriction site polymorphisms at RFLP loci of chromosome

Probes Restriction EnzvmesRestriction bn115.40 bn18.44umcll6bnl16.06 bn18.21 bn18.32 bn115.21

ECORI Yes Yes Yes Yes No Yes Yes Hind111 Yes No Yes Yes No Yes Yes EcoRV Yes Yes No No No Yes Yes XbuI No Yes" Yes Yes" Yes Yes Yes BumHI Yes Yes Yes Yes" Yes Yes" Yes KpnI Yes Yes No Yes" Yes Yes" Yes" SStI Yes" Yes" Yes" Yes" Yes" No Yes" AbaI Yes" Yes No Yes" No No Yes"

A total of 48 F2 progeny from A188 X W64A hybrids were analyzed, in groups of 28 and 20. " Cases where only the first group of 28 was tested. The presence or absence of polymorphisms for the relevant combination are indicated by yes or no, respectively.

10 RFLP probes from chromosome arm R.The RFLP the W64A allele upon digestion with BumHI, but hetero- probes umc45, umc91, and bn14.24 either did not reveal zygous for the A188 and W64A alleles following diges- many polymorphisms between the A188 and W64A al- tion with KpnI. A novel restriction fragment was ob leles, or yielded poorly resolved hybridization signals. servedwith EcoRI, whichis slightly smaller than the However, seven RFLP probes did reveal extensive poly- band of the W64A allele. Similar results were obtained morphisms between the parental alleles. The relative for other restriction enzymes, as summarized in Figure map positions of these seven loci are shown in Figure 2B (asterisks indicate a nonparental band). Based on lB, and the presence or absence of polymorphisms for the above model, the plant represented in lane 7 should each enzyme and probe combination is summarized in be heterozygous for theW64A allele and a recombinant Table 1. allele. By the criteria described above, two potential recom- Similar observations were made for another plant in binants were identified at the loci umcll6 (Figure 2) Southern blot analysis using bn18.44 as a probe. Figure and bn18.44 (Figure 3). Figure 2A shows the Southern 3A shows Southern blot hybridization data for a differ- blot data obtained after probing with umcl16 for 10 ent set of 10 plants. The scored genotype of the plant of the 48 F2 plants tested. The genotype of the plant represented in lane 4 depends on the restriction en- represented in lane 7 appears to be homozygous for zyme used. This plant appears to have inherited one A B 1 12 3 45678 910 Rertrlctlon Genotype .. at Enzyme umcllS W A Apal WI* BamH I WIW

Wl* A Bgnl €COR I WI+ 'W Kpnl WIA

Ncol Wl* EcoRI 9 A 7.5 W Xbal WIW

FIGUREZ.-Identification of the recombinant allele at the umcll6 locus. DNA prepared from 48 self-pollinated F2 progeny of A188 X W64A hybrids was digested with eight different restriction enzymes, and the resulting genomic blots were probed with the RFLP marker umcl16. (A) Southern blot data for 10 F2 progeny. The restriction enzyme used and the restriction fragment sizes of the parental alleles are indicated. The plant represented in lane 7 appears to be homozygous for the W64A allele with BamHI, heterozygous for the A188 and W64A alleles with KpnI, and reveals a novel restriction fragment with EcoRI. (B) The observed genotype of the plant represented in lane 7 for each restriction enzyme used in the Southern blot analysis. A, A188 allele; W, W64A allele; *, recombinant allele. Recombination in Maize 1775 B

Restrlctlon Gonotype at Enzyme bn18.44 FIGURE3.-Identification of the recom- W binant allele at the bn18.44 locus. Similar A Apnl AIA analysisas described for Figure 2. The plant represented in lane 4 appears to be BnmHl AIW homozygous for the A188 allele with EcoRV, heterozygous for the A188 and EcoR I AIW W64A alleles with HindIII, and reveals a A novel restriction fragment with KpnI. (B) EcoR V W The observed genotype of plant 4 for each Hlndlll AIW restriction enzymeused in the Southern blot analysis. A, A188 allele; W, W64A al- Kpnl AI+ lele; *, recombinant allele. Kpnl A ':: W s8n AI*

Xbnl AIW parental A188 allele and a potential recombinant allele. lane 4)using additional enzymes. The two recombinant The new allele resembles the A188 allele upon diges- alleles also showedexpected segregation ratios in a sub tion with EcoRV, the W64A allele with several restriction sequent generation. Plants heterozygous for either one enzymes (e.g., HindIII), and produces nonparentalfrag- of the rearranged alleles were self-pollinated and three ments withKpnI and SucI. Note that the other nine out of 17, and four out of 17 progeny were found to samples in Figure 2A and 3A show proper correspon- be homozygous for the new umcl I6 allele and bnZ8.44 dence in genotype for all three restriction enzymes. allele, respectively. This was the typical result for all other samples and loci Flanking marker assortment in the recombinant chro- tested. mosomes: Recombinational events giving rise to novel Segregation analysis of the putative recombinant al- restriction patterns may occur either via crossing over leles: Segregation of the potential new alleles was ana- or via a geneconversion-like mechanism. Crossing over lyzed in outcrosses to the inbred line W22 to confirm is associatedwith exchange of flanking markers, that thenovel restriction patterns resulted from intralo- whereas the arrangement of such markers remains pa- cus recombination and not from artifacts in the South- rental in recombinants derived by a gene conversion- ern blot analysis. Bothplants carrying a putative recom- like mechanism. The type of recombinational exchange binant allele were crossed to W22, and the genotype that occurred at the umcl16 and bnZ8.44 loci was estab of several progeny was determined by Southern blot lished by determining the genotype at flanking marker hybridization. Both new alleles segregated as expected, loci in the original F2 plants. For the F2 plant carrying as shown inFigure 4A for the umcl16 allele and Figure the recombinant allele of umcl16, the genotype at the 4B for the bnZ8.44 allele. Outcross progeny 1, 3, 4, 6, flanking RFLP loci bnZ15.40 and bnZ15.21 was already and 7 inherited the W64A allele of umcl16, whereas evident from the initial Southern blot analysis. How- the progeny 2, 5, and 8 appear to have inherited the ever, because bnZ8.44 was the most distal marker on recombinant allele (Figure 4A). Digestion of genomic chromosome 7L usedin our analysis, genomic DNA DNA from plants 2, 5, and 8 with Xbd suggests inheri- from the F2 plant carrying the recombinant allele of tance of the W64A allele, whereas KpnI digests suggest bnZ8.44was probed with the RFLP probe umcl68, which inheritance of the A188 allele. A nonparental restric- maps 11 cM distal to bnZ8.44 (Figure 1B). The F2 plant tion fragment was observed with EcoRI, which migrates carrying the recombinantallele at umcll6was heterozy- as expected, slightly faster than the band of the W64A gous for the A188 and W64A alleles at bnZ15.40 and allele. The restriction pattern of the allele inherited by homozygous for the W64A allele at bnZ15.21. Similarly, these three plants appeared identical to that of the the F2 plant carrying the bnZ8.44 recombinant allele was putative recombinant allele in the F2 parent for all en- heterozygous at bnl16.06 and homozygous for the A1 88 zymes listed in Figure 2B (data not shown). allele at umc168 (data not shown). These observations The putative recombinant allele at bnZ8.44 also segre- are consistent with both recombination events being gated as expected (Figure 4B). Plants 1,2, and5 appear associated with exchange of flanking markers, but be- to have inherited the recombinant allele, whereas the cause we used a self-pollinated F2 population rather remaining progeny inherited the A188 allele. Again, than a backcross, we cannot be certain of the flanking the restriction pattern of the recombinant allele was marker assortment in the recombinant chromosomes. identical to that of the parental F2 plant (Figure 3A, However, to explain the observed genotypes without 1776 M. C. P. Timmermans, 0. P. Das and J. Messing A exchange of flanking markers, multiple recombination events at and flanking the umcll6 or tmm18.44 loci need to be invoked. Molecular analysis of the crossover at the umcllb locus: Both parental alleles and the recombinant allele Xbul of the umcllh locus were characterized molecularly to obtain information about the kind of sequences involved in this unselected meiotic crossover. Size-selected EcoRI libraries from the inbred lines A188 and Kpnl W64A, and from a plant homozygous for the recombinant allele were screened with umcl16. Positive clones were -9, 7.5, and 7 kb in size, respectively, consistent with previous Southern blot data (Figures 2 and 4A). EcoR I However, restriction mapping and cross-hybridization between the subclones indicated no homology between the A18& and W64Aderived clones outside a 1-kb frag- umcl16 ment including umcllb. A second size-selected EcoRI library from A188was, therefore,constructed and screened with the probe bs450 (Figure 5). This fragment is unique in the genome and is homologous but B polymorphic between the W64A and recombinant alleles. Moreover, the hybridization pattern of bs450 to genomic DNA from a planthomozygous for the recombinant allele resembled that of A188 (see below). A EcoR V positive clone of 7 kb was isolated from an A188 library using probe bs450. The contiguity of the AlSMerived clones identified with the umcl16 and bs450 probes was confirmed by Hlndlll PCR analysis of genomic DNA from A188. The appropriate ends of the two lambda clones were sequenced and PCR primers were designed accordingly. A PCR product of the expected size was obtained from A188 Kpnl genomic DNA. This PCR product was sequenced directly, and asequence identical to the ends of the lambda clones was obtained (data not shown). How- bn18.44 ever, because the region amplified is part of a repetitive element (see below), it is likely that the amplified and FIGURE4."Segregation analysis of the recombinant sequenced product was not derived specifically from umcllb and bn18.44 alleles. Plants carrying the recombinant allele at the umcll6 and h18.44 loci were outcrossed to the the clonedregion but ratherfrom different copies pres- inbred line W22. DNA from individual outcross progenywas ent in the genome.Nonetheless, the observed sequence digested with various enzymes and probed with umcllband identity between the amplified fragment and the re- bn18.44, respectively. DNA from the inbredlines A188, spective ends of the two lambda clones and the pres- W64A, and "22 was also included in the analysis. (A) Segre- ence of an EcoRI site at the correct location in the gation of the recombinant allele at the umcllb locus. Prog- eny 1, 3, 4, 6, and 7 are heterozygous for the W64A and amplified fragment suggests the contiguity of the two W22 alleles with all three restriction enzymes. Progeny 2,5, Al88derived lambda clones. If the two clones were not and 8 appear to be heterozygous for the W64A and W22 contiguous, one has to assume that the repetitive ele- alleles with Xbd, heterozygous for the A188 and W22 alleles ment near theumcll6 locus differs from the amplified with KpnI, and heterozygous for a new allele (a novel restric- copies precisely by the insertion of an unknown frag- tion fragment) and the W22 allele with BcoRI. This restriction pattern is expected followinginheritance of the recom- ment flanked by EcoRI sites. Such a possibility is un- binant allele. (R) Segregation analysis of the recombinant likely. allele at the bn18.44 locus. Progeny 3, 4, 6, 7, and 8 are Restriction maps of allthree umcll6alleles are shown heterozygous for the A188 and W22 alleles. Progeny 1, 2, in Figure 5. The restriction map of the recombinant and 5 reveal a hybridization pattern predicted for plants allele is identical to W64A around umcll6, but identical heterozygous for the recombinant allele and the W22 allele. Therecombinant allele resembles the A188 allele with to A188 around bs450. Assuming a simple crossover EcoRV, the W64A allele with HindIII, and produces a novel event as the mechanism of recombination, we can as- restriction fragment with Kpd. cribe a proximal and distal side to the locus based on Recombination in Maize 1777

* 80 *+ + 0 +sa m. E P PBaH PXSp Sa SseS X BsPKSDSsSsBePP EH SnBe K SpXSaBo Ss Ss Be SsSaBaE ~~1. e-" I I Lh.. ?..."d "_ + A188 e, uncl16

0 *sa E EgBaSDepP PEgP Ss Ba Ss SsSeBaE -..- I .. . .I . . .IJ . I 'Ir e", H mll6 bs4s

e-, Crossover region

FIGURE5.-Restriction maps of the A188, W64A, and the recombinant allele of the umclI6 locus. Ba, RnmHI; Bg, RgnI; E, EcoRI; H, HindIII; K, Kpnl; P, PstI; Sa, Sun; Sp, SpM; Ss, SsfI; X, XbuI. The centromeric and telomeric ends of the alleles are indicated. Single lines represent restriction fragments hybridizing to a repetitive genomic element, whereas boxes represent regions of the umcllb locus hybridizing to unique sequences in the genome. Regions of homology between the alleles are indicated by identical highlighting. PstI and SulI sites marked with an asterisk were found to be methylated in genomic DNA prepared from leaf tissue. The SsrI site marked with 0 is subjected to & novo methylation. The restriction fragments hybridizing to the umcll6, bgsl.O, bs900, and bs450 probes are indicated. The region of each allele that was sequenced to determine the crossover site is indicated, as well as the crossover region. these restriction maps and the flanking marker assort- between insertions, deletions, or substitutions is made. ment mentioned above (Figure 5).Regions of homol- The W64A and A188 alleles share a 534bp fragment ogy between the threeclones were determined by cross- with perfect homology. The flanking regions are each hybridization with various fragments derived from each about 900 bp in size and are only 93% (near the BgflI clone. These regions are indicated by differently high- site) and 95% homologous. Sequence comparisons with lighted boxes in Figure 5. The proximal ends of the the recombinant allele located the crossover site within parental alleles were not homologous. In addition, the the 534bp fragment. No polymorphisms unique to the A188 allele has two insertions of approximately 10 kb recombinant allele were observed, suggesting that the and 860 bp relative to theW64A allele. Southern hybrid- crossover was conservative. Results from two studies us- ization and sequence analysis revealed that the IO-kb ing transgenic plants suggest that mitotic intrachromo- insertion is a long terminal repeat (LTR)-retrotranspo- soma1 recombination occurs also with high fidelity (PE- son found at -1500 copies per haploid genome. The TERHANS et al. 1990; GAL el al. 1991). 860-bp insertion appears to be a solo LTR with a copy Repetitive DNA sequences in each genomic clone number of -500 copies per haploid genome. Neither were identified by Southern hybridization of the of these insertions is present in the recovered recombi- lambda clones with total maize DNA as a probe (data nant allele due to theposition of the crossover site. The not shown). Individual restriction fragments derived presence of different repetitive elements could explain from regions of the umrll6 locus that hybridized to the lack of homology and the unusually high degree of genomic maize DNA in this analysis were subsequently restriction site polymorphism between these alleles. used as probes on genomic blots to confirm that they Both the restriction maps and thehomology between contained repetitive DNA sequences. However, at this the clones placed the crossover site within a -2.4kb level of analysis,the presence of unique sequences em- BgflI-SstI fragment. This region was sequenced for all bedded in these repetitive elements cannot be ruled three genomic clones, to locate the crossover site more out. Restriction fragments that did not hybridize to ge- precisely. Figure 6 shows a schematic representation of nomic maize DNA were also used as probes ongenomic the sequence comparisons of this region between the blots to confirm that they indeed corresponded to sin- different alleles. Vertical lines indicate sequence poly- gle copy regions in the genome. Interestingly, the ho- morphisms. The width of each line correlates with the mologous regions between the A188 and W64A alleles extent of the polymorphic region, but no distinction were found to correspond to unique sequences in the 1778 M. C. P. Timmermans,Das 0. P. and J. Messing

Bg/ II sst I Barn HI I I 1073 1990 9 1073 2337

I I 111 II Ill1 I II 11111 I.IIIp Rec I W64A

Crossover Site FIGURE6.-Pairwise sequence comparisons between the recombinant, A188, and W64A alleles. A schematic representation of the sequence analysis of the crossover region is shown. The vertical lines represent sequence polymorphisms between the compared alleles. The width of the lines reflects the extent of the polymorphic region, but no distinction is made between nucleotide deletions, insertions, or substitutions. The 534bp fragmentencompassing the crossover site is indicated by the arrow. maize genome (indicatedas boxes in Figure 5),whereas combination. To determine if the crossover region is nonhomologous regions hybridized to repetitive geno- part of a transcription unit, various probes encom- mic sequences (indicated by single lines in Figure 5). passingall unique sequences of the W64A genomic The meiotic crossover occurred within a perfectly ho- clone were hybridized to RNA isolated from several im- mologous region of a single copy sequence. This obser- mature and mature tissues ofthe inbred lineW64A. No vation is probably not due to the fact that single copy transcripts were detected using Northern blots con- RFLP probes were used to identify potential recombi- taining 1 pg of poly-A+ RNAfrom roots, seedling leaves, nant alleles. The identification of crossovers depends mature leaves, apices, immature ears, embryos 18 and on the presence of polymorphic restriction sites, which 30 dap,and endosperms 18 and 30 dap(data not can be several kb outside the probed region. TheEcoRI shown). However, due to the limited sensitivity of this fragments hybridizing to the urncll6 probe included assay, it cannot be ruled out that the crossover site is several repeat sequences. Moreover, the crossover site part of a transcription unit. in the A188 allele is >10 kb removed from the urncll6 We used the “FEXA” computer program (SOLOWV sequence and separated by repetitive elements. et al. 1994) fromthe “BCM GeneFinder” to locate Gene conversions and crossovers havebeen proposed possible exons within the -2.4kb sequenced region, to reflect the search for homology necessary to allow the 450-bp PstI fragment from W64A (black box in Fig- pairing between homologous chromosomes (see PETES ure 5), andthe corresponding regions from A188. The et al. 1991). If crossovers indeed result from the homol- FEXA program predicts internal exons based on the ogy search during chromosome pairing, then the previ- presence of potential splice donor and acceptor sites, ous information suggests an organization as shown in and open readingframes. The parameters of this exon Figure 7 for the alignmentof the parental alleles during prediction program are optimized for plant sequences. meiosis I. Allelic pairing between short regions of single The FEXA program, however, did not recognize any copy sequences with high homology is interrupted by potential exons in the A188 and W64A sequences. In the presence of nonhomologous repetitive elements. addition, we used the program “Grail” (UBERBACHER Transcription of the crossover region: The high fre- and MURAL1991), which is designed to recognize pro- quency of intragenic recombination in maize suggests moter sequences, open reading frames, splice donor that genes could be the preferred sites for meiotic re- and acceptor sites, and poly-adenylationsignals and

=IOh .

=1.5kb AI88 = 2.4 kb 53kb ””” ” .- - -.- - - ” W64A t-* - m776 Crossover site

FIGURE7.-Representation of the crossover region. Repetitive and methylated genomic sequences are shown as filled lines and unique, unmethylated genomic sequences are indicated by open lines. Regions of homology between the A188 and W64A alleles are aligned. The crossover region and the region hybridizing to the umcllh probe are also indicated. Recom bination in Maize in Recombination 1779 thus predicts the position of introns and exons. Since to be methylated in mature leaves, whereas those restric- this program is optimized for the recognition of exons tion sites present in unique sequences were not. The in human sequences and because transcription recogni- only exception is the unmethylated SalI site immedi- tion signals in plants can be quite diverged from mam- ately precedingthe 2.4-kb uniquesequence encom- malian consensus sequences, the program might not passing the crossover site in A188. recognize all possible exons. Four exons were identified The methylation pattern of the recombinant allele in the sequence of the W64A allele, but only two were reflected the parental A188 origin for sites to the right found for the A188 allele. This apparent discrepancy of the crossover site. To examine potential W64A meth- results from the presence of the two insertion elements ylation sites to the left and to characterize the overall in the A188 allele. Based on the computer analysis, methylation of the locus, the unique regions immedi- both insertions occurred within an exon.The two exons ately surrounding the crossover site were analyzed in conserved between the A188 and W64A alleles were more detail for the presence of methylated sequences. located within the perfectly homologous region encom- Leaf DNA was digested with HpaII, MspI, HaeIII, HhaI, passing the crossover site. The protein sequences of the and POuII, in combination with methylation-insensitive predicted exons were not homologous to any known enzymes BglII and BamHI (the BamHI site is not methyl- protein sequence in the database. Also the DNAse- ated in its sequence context) and hybridized to both quence of the -2.4kb sequenced regions from A188 the BgZII-SstI and MI-BamHI fragments (bgsl.0and and W64A showed no homology to any knownsequence bs900 in Figure 5). The hybridization patterns obtained in the database. indicated that the 35 restriction sites tested were un- Primers were designed for the potential exons con- methylated for all three alleles (data not shown). served between the A188 and W64A alleles, and anRT- Interestingly, the SstI site of the bs450 fragment was PCR experiment using poly-A+RNA from immature found to be methylated for the A188 and recombinant ears, embryo and endosperm 18 dap,and seedling allele but notfor the W64A allele (marked with an open leaves was performed to test for splicing, indicative of circle in Figure 5). The restriction maps derived from transcription. No specificRT-PCR product was obtained the lambda clones predict a 1.4 and 1.6-kb SstI frag- from these tissue samples, but productsof the expected ment uponhybridization with the bs450 probe for A188 size were obtained using genomic DNA as a PCR tem- and W64A genomic DNA, respectively. We observed the plate (data not shown). Specific PCR products were expected 1.6-kb fragment for W64A but obtained a 2.1- obtained in control experiments using hcflodderived kb fragment for both theA188 and recombinantalleles primers (R. MARTIENSSEN, unpublished data) together (Figure 8). The restriction enzyme SstI is not inhibited with mRNAisolated from seedling leaf tissue,and dul0 by CpG or CpXpG cytosine methylation but is affected derived primers (&rumet al. 1988) in combination by methylation at the internal C in its recognition se- with mRNA from embryo and endosperm tissue. There- quence GAGCTC. Non-CpG or CpXpG cytosine methyl- fore, we have not found any evidence so far that the ation has been observed for several maize transposable crossover region is part of a transcription unit. More- elements, where it has been correlatedwith inactivation over, since the insertion elements present in the A188 of the element (MCCARTYet al. 1989; SCHWARTZ 1989; allele are located in the other two exons identified with MARTIENSSEN and BARON1994). This type of cytosine the Grail program, it seems unlikely that the crossover methylation cannot be maintained by known mainte- region in the A188 allele is part of a functional tran- nance methyltransferases but may need to be intro- scription unit. duced de novo after each round of replication. There- Methylation pattern of the crossover region: DNA fore, cis sequences containing the requiredinformation methylation has been shown to suppress recombination for the de novo methylation are inherited by the recom- in mammals (HSIEHand LIEBER1992; GOODHARTet binant allele. Together, these data are consistent with al. 1993). We, therefore, compared DNA methylation the possibility that methylation determinants of individ- patterns between the parental and recombinant alleles ual chromosomes are maintained through the recombi- using methylation-sensitive restriction enzymes. In addi- nation process, including the nonclassical determinant tion, we wished to determine whether methylation de- mentioned above. terminants were maintained after recombination. Leaf DNA from A188, W64A, and a plant homozygous for DISCUSSION the recombinant allele was digested with SalI and PstI in combination with several methylation-insensitiveen- We describe a method to identify meiotic recombina- zymes and hybridized to multiple probes derived from tion events without selecting for restoration of gene the locus (data not shown). Theresults from these anal- function. This Southern blot assay is based on the occur- yses are summarized in Figure 5. Methylated restriction rence of novel restriction patterns upon recombination sites are indicated by asterisks. In general, all SalI and between polymorphic alleles. Since it allows the identi- PstI sites located within repetitive sequences were found fication of unselected recombination events, this 1780 M. C. P. Timmermans, 0.P. Das and J. Messing Meiotic intragenic recombination frequencies have been previously determinedfor the maize genes a1 (BROWNand SUNDARESAN1991; CNARDIet al. 1994), ARWARWARWARWARWARW adhl (FREELING1978), b (PATTERSONet al. 1995), kl (DOONER1986; DOONERand KERMICLE 1986), gl1 (SA- LAMINI and LORENZONI1970), r (DOONERand KER- MICLE 1986), and wx (NELSON1968), and themaximum frequency for each of these genes varied between 0.9- 1.3 X Because we have sampled 672 potential events (sevenloci x 96 chromosomes) and observed two recombination events, the frequency we observed was 3 X lo-'. However, the two situations are notstrictly comparable. First, our method should detectcrossovers that occur anywhere between polymorphic restriction sites that can extend several kbinto neighboringrepeti- tive sequences. Incontrast, the phenotypic selection methoddetects crossovers between two mutations BS450 within the gene (and oftenwithin the coding sequence) separated by a fixed distance that is usually smaller. FIGURE8.-Dp novo methylation at the umcl16 locus in the Second, our methodis theoretically independent of the A188 and recombinant alleles. Genomic DNA was prepared fidelity of recombination since no selection is imposed. from leaf tissue of plants homozygous for either the A188, W64A, and recombinantallele of the umcl I6 locus. The DNA Phenotypic selection for restoration of gene function was digested with several enzymes and subjected to Southern from mutant heteroalleles requires that therecombina- blot analysis using the bs450 fragment as a probe. A 2.1-kb tion event occurs without introduction of any newmuta- restriction fragment was observed after digestion with SslI for tions within coding regions or otherwise critical se- the A188 and recombinantallele, instead of the expected 1.6 quences. These two reasons would indicate that the kb fragment. The molecular weight marker is the 1-kh ladder (BRL). A, A188; R, recombinant; W, W64A. RFLP method could detect higher recombination fre- quenciescompared to the phenotypic selection ap method is particularly suited to test whether crossing proach. Furthermore, the F2 population analyzed here over is confined to particular genomic segments. The may carry heritable activities that increase recombina- assay depends on the presence of polymorphic restriction frequencies along chromosome7by approximately tion sites around a probed region. Since the incidence twofold (M. TIMMERMANS,0. P. DAS, J. BRADEENand J. of polymorphism differs greatly among plant species, MESSING,unpublished results). the usefulness of this assay might vary between plants. The detailed characterization of one of the identified However, maize exhibits a relatively high frequency of unselected recombinants revealed several interesting polymorphism (SHAITUCK-EIDENSet al. 1990), and our aspects about the site at which this crossover occurred. assay could be particularly useful in this model system. The crossover site mapped within a perfectly homolo- The high degree of polymorphism in maize is also gous region of a single copy sequence thatwas unmeth- apparent from our analysis. The W64A and A188 inbred ylated. A combination of these properties could be im- lines showed polymorphism with multiple restriction portant in targeting crossovers toparticular allelic enzymes at seven of the 10 RFLP loci tested (Table 1). sequences and preventing nonallelic repetitive ele- At the umcl16 locus, the high frequency of restriction ments from crossing over. Ectopic recombination be- site polymorphism can be largely ascribed to the pres tween repeats can result in large genomic rearrange- ence of different repeat sequences in the A188 and ments, such as inversions, deletions and translocations. W64A alleles. This type of polymorphism has also been Ectopic recombination frequencies between naturally observed at the adhl (JOHNS el al. 1983) and kl (FUR- occurring repeat sequences in yeast are suppressed in TEK et nl. 1988; RAISTON et al. 1988) loci and is likely meiosis (KUPIECand PETES1988a,b). In addition, the very common in maize. In addition, the umcl16 locus observed recombination events were rarely associated of A188 and W64A contained -5% sequence polymor- with crossing over. The high frequency of intragenic phism within the conserved region (Figure 6). This type recombination relative to the average recombination of polymorphism is also common in maize and has been rate for the whole genome also suggests the existence observed for each locus where sequence information of mechanisms involved in suppressing recombination from multiple alleles was compared (adhl, SACHSet al. frequencies between repetitive elements in maize. 1986; kl,FURTEK et al. 1988; RALSTON et al. 1988; shl, Fungi have developed several mechanisms to sup WERRet al. 1985; ZACKet al. 1986; 27 kDa zein gene, press recombination between repeats. For instance, re- DAS et al. 1991b) . duced recombination between Ty elements in Saccham Recombination in Maize 1781 mycescerevisiae is partly mediated by the transacting site in this case was not mapped to a region of high l3R1 system (ROTHSTEIN 1984;WALLIS et al. 1989). In homology but instead to a region with many sequence Neurospora crassa, repeat-induced point mutations (RIP) differences (DM et al. 1990b, 1991b). occur premeiotically between repeated sequences Most transgenic studies in tobacco, Brassica, and (SELKER1990). This reduces thehomology between the Arabidopsis used perfect repeats of 350-600 bp as re- repeats and thus inhibits recombination. In Ascobolus combination substrates. However,GAL et al. (1991) immersus, repeated sequences become methylated pre- determined crossover sites resulting from mitotic in- meiotically (MIP), which likely reduces their recombi- trachromosomal “pop-out” recombination between nation frequency (ROSSIGNOLand FAUGERON1995). Al- imperfect repeat sequences and observed that most though the cosuppression phenomena (NAPOLIet al. crossovers occurred within the longest stretch of perfect 1990;JORGENSEN 1995) suggest that plants can recog- homology, a 122-bp fragment. In another study, a 53- nize ectopic sequence homology, there is no evidence bp repeat sequenceprovided enough homology to facil- yet for the existence of an active mechanism that sup itate mitotic recombination, but differences in the ex- presses recombination or crossing overbetween re- perimental set up did not allow direct comparisons of peats. However, the degree of homology and the meth- recombination frequencies and no distinction between ylation status of particular sequences are likely to play gene conversion and crossingover was possible (PE- a role. TERHANS et al. 1990). The homology requirement for Characterization of the umcll6crossovershowed that gene conversion in yeast is only 25-60bp (HAYDEN and the crossover junction mapped to thelargest stretch of BYERS1992). perfect homology between the two parental sequences. Based on our results and the previous observations This stretch is 534 bp long, within a total homologous at the 2’7-kDa zein gene locus, we suggest that meiotic region of at least 2337 bp. A reduced frequency of re- crossing over in maize preferentially occurs between combination resulting from mismatches in the recom- sequence domains with perfect homology. Depending bining DNA molecules has been documented forbacte- on the divergence between repeats, this homology re- ria (SHENand HUANG1986), yeast (BORTSand WER quirement could reduce their frequency of crossing 1987; HAYDEN and BYERS1992; SELVAet al. 1995), Dro- over, as in N. crassa (SELKER1990). In addition, the sophila (CARPENTER 1982), and mammalian cells distribution of repetitive elements varies between in- (WALDMANand LISKAY1988). Recombination fre- bred lines, as seen here at the umcll6locus. This could quency appears to be determined by a minimal length further reduce thefrequency of crossing over between of perfect uninterrupted homology, rather than the repeats since only ectopic recombination is possible. overall percentage of sequence homology. For exam- Consistent with this is the observation that crossover ple, the minimal length of homology required forcross- frequencies between linked T-DNAs were several fold ing over in yeast (HAYDEN and BYERS 1992) and mam- higher when measured in petunia inbreds than in hy- malian cells (WALDMANand LISKAY1988) was found to brids ( ROBBINSet al. 1995). be about 200-250 nucleotides. The presence of mis- Methylation has been postulated to affect recombina- matches in the heteroduplex affects branch migration tion (HOLLIDAY1987), and site-specific recombination (SELVAet al. 1995) and possibly the resolution of recom- of methylated immunoglobulin genes is much reduced bination intermediates, such that recombination events in mammalian cells (HSIEHand LIEBER 1992;GOODH- associated with short stretches of homologous hetero- ART et al. 1993). Several observations from the r locus duplexare lesslikely to be resolvedas crossovers in maize suggestthat recombination frequencies at this (ROEDER1990; PETES et al. 1991; HAYDEN and BIERS locus are also inversely correlated with DNA methyla- 1992). tion levels ( EGGLESTONet al. 1995).Most of the unequal In plants, few recombination events have been char- recombination events at the R-rstandard or R-stippled acterized in detail and consequently the minimal ho- complexes occurred between the 3‘ ends of the rgenes, mology required for recombination is unknown. At the which are less methylated in comparison to the 5‘ ends 27-kDa zein locus in maize, two recombinant alleles of the genes. Furthermore, early studies found that re- derived from recombination between the tandemly du- combination frequencies within R-r:standard and its de- plicated zein genes havepreviously beenidentified. rivatives are reducedfollowing paramutation (Buyand One recombinant allele most likely arose from meiotic BRINK 1966). These reduced recombination frequen- unequal crossing over between the duplications (DM cies have recently been correlated with increased levels et al. 1991a). In this case also, the crossover site mapped of methylation at the r locus (see EGGLESTONet al. to an 1168-bp region of perfect homology, within a 10- 1995). Consistent with these observations, the crossover kb region of lesser homology. The other recombinant site at the umcll6 locus mapped to unmethylated se- allele appears to have arisen via a mitotic gene conver- quences. However, the methylation pattern observed in sion-like rearrangement (W. Hu, M. TIMMEWS and the leaf might not accurately reflect that present during J. MESSING,in preparation). Interestingly, the crossover sporogenesis. 1782 M. C. P. Timmermans, 0. P. Das and J. Messing

The parental methylation pattern was faithfully inher- ATHMA, P.,and T. PETERSON,1991 Acinduces homologous recombination at the maize Plocus. Genetics 128 163-173. ited by the recombinantallele, even after several gener- BORST, R.H., and J. E. HABER,1987 Meiotic recombination in yeast: ations. This includes the Sstl site that is methylated de alteration by multiple heterozygosities. Science 237: 1459-1465. novo in the A188 and recombinant alleles. Faithful BRAY,R. A., and R. A.BRINK, 1966 Mutation and paramutation at maintenance of methylation through the recombina- the R locus in maize. Genetics 54: 137-149. BROWN,J., and V. SUNDARESAN,1991 A recombination hotspot in tion process may be a significant factor in generating the maize AI intragenic region. Theor. Appl. Genet. 81: 185- allelic diversity and could be important for the mainte- 188. nance of specific epigenetic states (HOLLIDAY1987). CARPENTER,A. T. 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