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

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Characterization of a Meiotic Crossover in Maize Identified by a Restriction Fragment Length Polymorphism-Based Method Copyright 6 1996 by the Genetics Society of America 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 se- quences 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 maize loci 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 from each 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 con- firm 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 se- quence 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
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