123

Genetic recombination in plants Patrick S Schnable*, An-Ping Hsiat and Basil J Nikolauf

Meiotic recombination generates novel allelic arrays on one . Exonucleases resect the 5’ ends of the . Recent experiments have revealed an DSB, thereby generating two single-stranded regions that extraordinarily nonrandom distribution of recombination flank the position of the original DSB. Subsequently breakpoints along the lengths of plant chromosomes; for the exposed 3’ ends displace the DNA strands with the example, recombination breakpoints often resolve within same polarities from the nonsister chromatid. After the genie sequences, and thereby generate novel . invading strands basepair with the complementary strands The mechanism by which recombination breakpoints are of the nonsister , they are used as primers determined is an area of active investigation. In addition, for DNA synthesis for which the complementary DNA recent developments are providing recombination-based strand is used as the template (Figure 1). Ultimately this technologies for creating targeted alterations in the repair mechanism generates double Holliday junctions. architecture of plant . Depending upon how these junctions resolve, reciprocal crossovers or conversion events can result. Support for this model has been provided by recent experiments Addresses which demonstrate that required for meiotic re- *iDepartment of Agronomy, Iowa State University, Ames, IA 50011, combination in Saccharornyces encode proteins that USA *e-mail: [email protected] catalyze reactions predicted by the DSB repair model (see, +e-mail: [email protected] for example, a review by Haber [PI). Indeed, some of *Department of Biochemistry and Biophysics, Iowa State University, these proteins are conserved among , including Ames, IA 50011, USA; e-mail: [email protected] plants [3]. One such conserved protein is that encoded by Current Opinion in Plant 1998, 1 :123-l 29 the recA gene which is involved in strand invasion (Figure http://biomednet.com/elecref/1369526600100123 1).

0 Current Biology Ltd ISSN 1369-5266 -dependent DSB repair models are consistent Abbreviation with the data obtained from meiotic recombination DSB double-strand break experiments. Some of the data obtained from experiments using plants and other and designed to study the integration of extrachromosomal DNA into chromosomes Introduction via , or the repair of DSBs in mitotic is a consequence of the large-scale cells, cannot be explained by these types of models [4,5’]. organization of genetic material into chromosomes; in the Hence, the single-strand annealing model (see [6] and absence of recombination, all genes residing on the same Figure 2) and the one-sided invasion model (see [7] and would segregate as a unit and thus display Figure 3) have been proposed to explain recombination absolute genetic linkage. There are obvious advantages to events that occur in the absence of significant amounts an in maintaining blocks of favorable alleles as of DNA , or when this homology is a unit. This advantage, however, must be balanced with limited to a single side of a DSB, respectively. the evolutionary requirement for genetic diversity through the generation of novel allelic arrays via crossing-over, a This review summarizes recent data that expand our un- process which generates recombinant chromosomes. derstanding of the extraordinarily nonrandom distribution of recombination breakpoints within plant genomes. It Recombination events can result in either reciprocal explores those features of the recombination machinery recombination or the related process of . (and/or the structure) that may account for these Reciprocal recombination arises via the exchange of observations, and it highlights the recent development of genetic information between two nonsiscer chromatids. recombination-based tools that will facilitate the remod- Gene conversion represents the nonreciprocal transfer of elling of plant genomes. The reader is additionally di- DNA sequences from one nonsister chromatid to another. rected to other recent reviews on recombination [4,&11]. Because gene conversion and reciprocal recombination appear to share a common pathway, mechanistic models Distribution of recombination breakpoints have been sought that explain both phenomena. It has long been known that recombination does not occur randomly across the lengths of chromosomes. The Currently, the most widely accepted models for meiotic development of detailed genetic maps and major advances recombination are based upon the double-strand break in the physical mapping of plant genomes are making (DSB) repair model [l]. According to this model (which it possible to directly correlate physical and genetic is based on data from , phage and fungi), distances over large intervals. For example, yeast and recombination initiates via the formation of a DSB on bacterial artificial chromosome (YAC and BAC, respec- 124 Genome studies and molecular

Fiaure 1 Figure 2

(a)

5” 3’

a (d) ;;= + w (cl - - Cd) 77 c= -7 SW Current Opinion in plant Biology 3’1 0

The single-stranded annealing (SSA) model of recombination. (a) Double-stranded breaks are induced in both recombination partners. (b) 5’ to 3’ exonuclease attacks the exposed 5’ ends of both (e) duplexes. (c) Free 3’ ends anneal forming a chimeric molecule. (d) Only the chimeric molecule is recovered. Unpaired overhangs are removed and single-stranded gaps are repaired by DNA synthesis.

Figure 3 (0 (9) -m-

-0001 boa- (a) sm - 3’-- I I - I

Current Opmon in Plant Biology

The double-strand DNA break repair model. (a) Two DNA duplexes. 03 ;:: ,_= (b) A double-strand break occurs in one duplex. (c) A 5’ to 3’ exonuclease attacks the exposed 5’ ends. (d) Strand invasion. 31 5’1 The free 3’ ends are used as primers for DNA synthesis and the 5.’ uncut homologue (white) acts as the template. (e) DNA synthesis (dashed lines) results in a four stranded intermediate with two Holliday junctions. (f) Gene conversion in the absence of reciprocal a recombination occurs if cuts and religation occur at positions 1 and W 5’- 2 respectively as indicated in (e). (g) Gene conversion in combination 3’ - with reciprocal recombination would result if cuts and religation occur 3’1 at positions 2 and 3 respectively as indicated in (e). 5,’ Current Opinion in Plant Biology tively) genomic libraries are now available or becoming The one-sided invasion (0.9) model of recombination. (a) A double-strand break is generated in one DNA duplex. (b) A 5’ available for many plant species, for example Arabidopsis, to 3’ exonuclease attacks the exposed 5’ overhangs. (c) D-loop maize, rice, tomato, soybean, wheat, and barley. In some formation. A free 3’ end is used as a primer for DNA synthesis cases, most notably Arabidopsis [l&15], tomato [16-191 and the uncut homologue (white) acts as a template. (d) The and rice [ZO], these YAC and BAC clones are being resulting intermediate. (e) The gap is filled and the break repaired via illegitimate recombination which introduces new genetic information organized into large contigs. These concigs represent a (hatched region). resource for investigating the relationship between the physical nature of chromosomes and the dynamics of recombination. For example, it is now clear that in many plant species rates of recombination can vary up to an order first hypothesis suggests that at least some of the of magnitude over relatively large intervals. Typically the recombinationally hyperactive regions of chromosomes regions surrounding the centromeres and, in at least some may have been identified as such because they contain a species, the nucleolus organizer region exhibit very low high density of genes. In contrast, the second hypothesis rates of recombination per physical length [21~,22,23*]. suggests that genes merely have a tendency to reside in recombinationally hyperactive regions. Both of these Two hypotheses can be advanced to explain the causal hypotheses depend on the facts that gene density varies basis of these diversities in recombination rates. The across chromosomal regions and that plant genes are Genetic recombination in plants Schnable, Hsia and Nikolau 125

recombination hot spots. In fact, genes exhibit rates of can spread throughout a nucleolus organizer region recombination per kilobase that are 10 to lOO-fold higher supports the existence of such a process [30]. than the genome average (reviewed by Lichten and Goldman [ 111). For example, one-fifth of all recombination If recombination preferentially occurs within genes, one events that occurred across the 140 kb al-h? interval in should be able to find evidence for its occurrence maize resolved within a 377 bp region of the al gene [24]. by analyzing the DNA sequences of multiple alleles The a/ (anthoqaninlessl) and s/Z’ (shnken2) genes encode of a given . Indeed, the structures of for dihydroquercetin reductase (EC 1.1.1.219) and ADP- obtained from such studies often suggest an evolu- glucose pyrophosphorylase (E.C. 2.7.7.27), respectively. tionary origin dependent upon gene conversion, or closely linked reciprocal crossovers, perhaps separated by one To understand the nature of the recombinationally hyper- or more generations [31-34,35’]. Given that intragenic active regions of chromosomes it is important to determine recombination is occurring, it represents a mechanism firstly whether genes reside in recombinationally active that could generate novel chimeric alleles, that is to intervals larger than single genes, and secondly whether say, genetic variability. This mechanism would have all recombination hot spots are genes. The al--s/Z interval the capacity to combine the evolutionary advantageous of maize is being used as a model to address the first ques- domains from distinct alleles into a single novel tion, that is, do genes define discrete recombinationally upon which selection could act. Indeed, recombination active units embedded in larger recombinationally inert at the Rpl locus has generated novel resistance alleles intervals. Analyses to date support the interpretation that to the maize Puccinia sough [36]. In contrast, genes per se are recombination hot spots [24,25]. it has been proposed that structural polymorphisms at the self-incompatibility (S) locus in the Bras&-a genome An important cautionary note, however, is that, because have evolved specifically to repress recombination and most fine-scale studies of recombination have assayed thereby facilitate the maintenance of co-evolved genes recombination rates only within genes, it is not possible to within given haplotypes [37*]. conclude that all hot spots are genes. One way to settle this question would be to identify a collection of recombination Recent investigations have begun to explore the molecular hot spots and then determine whether these hot spots nature of intragenic recombination events. These exper- are genes. This process, however, is greatly complicated iments have been conducted by isolating and analyzing by the difficulty in distinguishing between genie and large numbers of intragenic recombinants from five maize non-genie regions in complex genomes which are rich in loci that are themselves recombination hot spots. In retrotransposons and other repetitive sequences [26”,27]. several instances, these analyses mapped recombination The first step in testing the hypothesis that not all hot breakpoints to a resolution of tens of base pairs. Such spots are genes comes from the identification of a single analyses of the bronze (bzl) [38**] and waxy (30)x1) meiotic crossover that occurred in an apparently nongenic [39’] loci have revealed that recombination breakpoints region of the maize genome [28’]. This crossover occurred are randomly distributed across each of these genes. in a DNA segment that exhibits a high degree of sequence In contrast, similar analyses have revealed significantly identity between the two parental homologies. In addition nonrandom distributions of recombination breakpoints this sequence is present at low copy number in the within the al [24], 61 (booster) [40}, and rl (redl) [41] maize genome. This finding suggests that recombination loci (bl and rl code for myc-like transcriptional activators. events may resolve in low-copy, highly conserved regions Specifically, most recombinants map to recombination hot of chromosomes. spots at the 5’ end of the al and 61 loci and at the 3’ end of the 71 locus. Consistent with this hypothesis, in wheat the P/z1 locus appears to enhance recombination between homologous The basis for these differences in the distribution of intervals at the expense of homeologous intervals, possibly recombination breakpoints has not yet been determined, via a sequence similarity searching mechanism [29). but at least three hypotheses can be considered. First, In addition, because recombination between repetitive because these experiments were conducted in different sequences in complex genomes would generate deleteri- genetic backgrounds, differences in trans-acting factors ous chromosomal rearrangements, there must be mech- may have influenced the spectrum of recombination anisms that suppress recombination between duplicated outcomes. Indeed, that affect recombination sequences on nonhomologous chromosomes; however, rates have been identified in Arabidopsis [42*]. But of although the ribosomal RNA (rRNA) spacers exhibit even more significance relative to this hypothesis, putative sequence dissimilarity among species, the members of rrans-acting factors have been identified in maize that the tandem arrays of rRNA genes within a given species appear to influence rates of recombination within specific are virtually identical. Hence, there must be mechanisms genetic intervals [43*]. that promote homogenization of these sequences during , possibly via gene conversion. The observation Second, different genes may have different patterns that variants associated with the Arabidopsis rRNA genes of recombination. This could be a consequence of 126 Genome studies and

gene-specific factors or may be influenced by structural In addition, although apparent gene conversions have features larger than genes. Indeed, the genes included in been observed in maize [24,44,48], the ratio of gene these studies exhibit rates of recombination that differ by conversions to crossovers appears to be low relative to an order of magnitude. The reasons for these differences that found in Saccharomyces. On the basis of a small in rates are as yet unknown. In fact, the reason genes sample of maize conversion events [38**], however, the themselves are recombination hotspots has not yet been sizes of conversion tracts appear to be similar in maize, determined. Deletions of the reel gene promoter of Saccharomyces and Drosophda (i.e. 1 kb). maize, however, do not affect recombination rates in this genie , so in this case promoter Recombination-based tools activity must not be a requirement for high levels of An efficient and reliable system to target transgenes to recombination [39*]. defined locations within plant genomes would have great utility not only in conducting gene replacement proce- Third, structural features of the allele combinations used dures on endogenous genes (for example, gene knockouts in these experiments may have influenced the spectrum of and allele substitutions), but also in ensuring the stable recombination products that were obtained. Specifically, it expression of novel transgenes. Current transformation has been suggested [38**] that the transposon insertions systems rely on the apparently random integration of present in some of the allele combinations used in certain foreign DNA. This leads to several problems, including of these experiments may be responsible for the nonran- the inability to inactivate endogenous genes and the dom distribution of recombination hot spots. Indeed, it is unwanted silencing of transgenes due to position effects. known that transposon insertions can influence the rates These disadvantages can be overcome via the use at which recombination occurs in the bzl and al genes of a system in which an [24,44]. In addition, Ds (Dissociator) transposons appear to endogenous copy of a given DNA sequence recombines affect the distribution of recombination breakpoints in the with an ectopic donor copy with a similar sequence. bzl locus. In contrast, during experiments involving al Depending upon the nature of the donor sequence, such a alleles that are sequence identical but for the presence or recombination event can result in either a gene knockout absence of the Mu1 (Mutatorl) transposon, no differences or replacement. in the distribution, of recombination breakpoints were observed [24]. Homologous recombination occurs readily in bacteria and yeast, where it is used for gene replacement experiments. Another structural feature that may affect the distribution More recently it has been developed as a tool for gene of recombination breakpoints is the presence of small replacement in mammals. For example, the introduction base pair heterologies between allelic combinations. For of DSBs in yeast and mammalian genomes has been example, fewer than expected numbers of recombination used to increase recombination rates. This approach events resolve in regions of the bzl locus that have high usually involves the introduction into the genome of densities of heterologies [38”]. The impact of heterolo- a target sequence containing a rare-cutting restriction gies, however, on recombination in plants appears to be recognition site (for a review, see 181). significantly less than is observed in Sacdzaromyces [45] and that have been used for this purpose are I-&e1 (the other fungi. For example, in Ascobohs, heterozygotes at the -encoded endonuclease I from the intron hom- 62 locus that exhibit 10% DNA sequence polymorphism in ing system of Sacdzaromyces) and the HO (homothallic their 5’ ends experience gene conversion rates one to two switching) endonuclease (which is involved in -type orders of magnitude lower than heterozygotes with fewer gene rearrangements in Sacdaromyces). Subsequently, the sequence polymorphisms [46]. restriction enzyme and a donor sequence that shares homology with the target sequence are introduced into the There are other differences in the outcomes of recom- . Because these enzymes have recognition sequences bination between plants and other taxa. For example, of 18 and 24 bp, respectively, their recognition sites polarity of gene conversion is commonly observed in occur infrequently in a genome. Hence, in most cases, yeast, fungi and Drosophda. Polarity can be defined as the introduced restriction enzyme creates a DSB only the gradient across a gene in the frequencies at which at the target sequence, which can then be repaired sequence polymorphisms are converted via recombination. via a homologous recombination event between the In addition, conversions of polymorphisms at the end donor and target sequences. Such a strategy has proven with the lower conversion rate are often accompanied successful in plants. For example, in tobacco protoplasts, by co-conversions of polymorphisms at the other end of the I-&e1 system enhances recombination rates between the same gene. Polarity is thought to be a consequence extrachromosomal donor sequences and target sequences of the fact that the DSBs that initiate recombination that are either extrachromosomal or chromosomal [5’,49]. occur at discrete positions [11,47]. As discussed above, Similarly, the HO endonuclease increases by IO-fold the the distribution of recombination breakpoints is consistent rate of intrachromosomal recombination in somatic cells of with polarity in only some experiments conducted in Arabidopsis [50]. To be most useful as a tool for genome maize. rearrangements, it would be desirable to recover germinal Genetic recombination in plants Schnable, Hsia and Nikolau 127

recombination events. Although no such events were Depending upon the nature of the donor template this recovered in this experiment, subsequent modifications repair process can result in gene knockouts or allele may lead to this capacity. substitutions at the target site. Because gap repair occurs in plants carrying active Mutator and AC (Activator) transposon Site-specific (e.g. FLPIFRT, Crellox and systems [63*,64*], similar transposon-based approaches to R-R&; for a review, see [9]) are also being tested in are being tested in plants. plants. The FLPIFRT, Cm/lox and R-R& systems are derived from the 2l.r circular plasmid of Sacc/raromyces Other gene targeting technologies are also under de- cerevisiae, the bacteriophage Pl, and Zygosaccharomyces velopment. For example, it has been reported that rouxii, respectively. These enzymes catalyze reversible DNA-RNA hybrid molecules can be stably integrated recombination reactions between pairs of recognition into a plant genome following protoplast [65], sequences. If the two recognition sequences reside on as occurs in mammalian cells [66]. In addition, homo- a single DNA molecule, these enzymes can generate logous recombination events between donor sequences deletions or inversions, depending upon the relative ori- introduced via Agrobacterium-mediated transformation and entations of the two recognition sequences. Alternatively endogenous genes in Arabidopsis and tobacco have resulted if the two recognition sites are on different molecules, in successful gene disruptions [67,68] that in the most recombination can generate targeted integrations or trans- recent experiments have yielded germinally transmitted locations. Because the recognition site for each of these mutants [69”]. enzymes is large (34 bp in the case of FRT and lox and 31 bp in the case of RSs), they are unlikely to occur by Conclusions chance in a genome. Meiotic recombination has been best characterized in Saccharomyces. The on-going plant expressed sequence Both somatic and germinal recombination events have tagged projects will provide important tools to ascertain been recovered using the R/R& system in Arabidopsis whether the recombination machinery is well conserved [51]. Similarly, the FLP/FRT system is functional in a structurally between plants and Saccharomyces. It is be- number of plant species, including maize, tobacco and coming clear, however, that recombination outcomes (at Arabidopk [SZ-551. It has recently been demonstrated that least) differ between plants and J’accharomyces. Hence, it FLP-mediated recombination can be regulated via the use will be important to develop a mechanistic understanding of an inducible promoter [54). In addition, as demonstrated of recombination in plants to enhance the utility of the in Drosophila [56], FLP-mediated recombination can developing repertoire of recombination-based tools, which facilitate large-scale chromosome manipulations. A similar have the promise of being able to facilitate efforts to approach, but based on the Crellox system, has been used restructure plant genomes. to generate large-scale chromosomal rearrangements in tomato, tobacco and Arabidopsis [57-59,60’]. As a first step Two of the important questions that remain to be resolved in two of the more recent experiments, Ds transposons that are the nature of recombination hot spots (i.e. are all hot carried a fox sequence were introduced into Arabidopsis spots genes?) and the mechanisms by which the recombi- and tomato genomes via T-DNA mediated transformation. nation machinery selects recombination sites within plant Transposition events then moved the lox sequences from genomes. One significant difference between the genomes the T-DNA sites to multiple positions along of plants and Sacc/iaromyces is that the former contain chromosomes. Subsequently, Cre-mediated recombination large amounts of repetitive and polymorphic sequences generated inversions between pairs of /ox sites in both in their intergenic regions. Hence, one possibility is somatic and germinal tissues [58,60*]. In one of these ex- that recombination is restricted to regions that exhibit periments [60’], in vitro Cre-mediated recombination was relatively lower levels of DNA sequence polymorphism used to release large chromosomal fragments which were and/or have fewer large structural polymorphisms. Clearly, sized using pulse-field electrophoresis. This technology genes would meet this criterion, but so might other regions could, therefore, be used to generate detailed physical of the genome. A further explanation for why genes and genetic maps as well as to facilitate the functional are recombinationally hyperactive, which has not been analysis of chromosomal segments via the isolation and discussed in this review, might be that the recombination phenotypic characterization of deletions [60*]. In addition, machinery operates most efficiently on open chromatin. this technology may ultimately be used for site-specific gene targeting in plants, as has already been done in a Acknowledgements murine cell line [61]. The authors apologize to those colleagues whose scientific contributions could not be cited due to space limitations. The authors’ research is supported by competitive grants from the United States Department of Transposon-induced DSBs have been used in gene Agriculture-National Research Initiative (9500807 and 97-01407) and targeting experiments in Drosophila [62]. In this species, Pioneer Hi-Bred. This is Journal Paper No. J-17693 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa, Project 3334 and DSBs that occur in the vicinity of a transposon can be 3485, and supported by Hatch Act and State of Iowa funds. Assistance with repaired using a nonallelic donor template for gap repair. the figures was provided by Hong Yao. 128 Genome studies and molecular genetics

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