Polarized gene conversion at the bz locus of maize

Nucleotide diversity is greater in maize than in most organisms studied to date, so allelic pairs in a hybrid tend to be highly polymorphic. Most recombination events between such pairs of maize polymorphic alleles are crossovers. However, intragenic recombination events not associated with flanking marker exchange, corresponding to noncrossover gene conversions, predominate between alleles derived from the same progenitor. In these dimorphic heterozygotes, the two alleles differ only at the two mutant sites between which recombination is being measured. To investigate whether gene conversion at the bz locus is polarized, two large diallel crossing matrices involving mutant sites spread across the bz gene were performed and more than 2,500 intragenic recombinants were scored. In both diallels, around 90% of recombinants could be accounted for by gene conversion. Furthermore, conversion exhibited a striking polarity, with sites located within 150 bp of the start and stop codons converting more frequently than sites located in the middle of the gene. The implications of these findings are discussed with reference to recent data from genome-wide studies in other plants.Gene conversion in organisms where all four products of a meiotic tetrad can be recovered refers to a departure from the normal 2:2 segregation of alleles and arises from the repair of meiotic double-strand breaks (DSBs) by a homologous recombination mechanism (1). Gene conversion represents the nonreciprocal, but faithful, transfer of information between two homologous DNA sequences, usually located in homologous chromosomes. The stretch of DNA that is transferred during a gene conversion event, called the conversion tract, can vary in yeast from a few hundred bases to more than 12 kb and is composed of sequences found in only one of the parental chromosomes, i.e., it is continuous, not patchy. Conversion polarity within a gene, which is the tendency of markers located near one end of the gene to convert more frequently than those located at the opposite end, has been reported in several Ascomycete fungi: Ascobolus (2), Neurospora (3), yeast (4), and Aspergillus (5). The high conversion end is usually the 5′ end (6), but can also be the 3′ end (7). Conversion frequency gradients are generally accepted to reflect a preferential initiation site for recombination that is located at the high conversion end of the gene (8, 9).In most organisms that undergo meiosis, only one of the four meiotic products is ordinarily recovered, so it is not possible to identify gene conversion on the basis of aberrant segregation ratios. A notable exception is the Arabidospsis quartet1 (qrt1) mutant that allows pollen tetrad analysis and has been used to demonstrate gene conversion events unambiguously by their classic 3:1 segregation (10) to estimate genome-wide conversion frequencies (11, 12) and to measure the tract lengths of such conversion events (12, 13). Usually, however, gene conversions have been identified by the flanking marker arrangement of intragenic recombinants (IGRs). This convention derives from the observation that, in yeast asci displaying gene conversion of a central marker flanked by two closely linked outside markers, the convertant spore could carry a parental or a recombinant arrangement of flanking markers with about equal frequency, on average (14, 15). Therefore, geneticists studying recombination in organisms where tetrad analysis is not possible have tended to refer to IGRs bearing a parental or noncrossover (NCO) arrangement of flanking markers as convertants, a convention that we also follow in this paper. In these cases, a convenient way of detecting conversion polarity is to compare the relative frequencies with which the two parentally marked IGRs are recovered from heteroallelic combinations (5, 16).In contrast to observations in fungi, it was noted repeatedly in maize recombination experiments that the vast majority of IGRs were crossovers (COs), i.e., associated with an exchange of flanking markers (17). Most of those studies dealt with polymorphic heterozygotes in which the recombining heteroalleles were derived from progenitor alleles that differed by single nucleotide and indel polymorphisms in as much as 1.6% of their sequences (18) and often included a transposon insertion allele. This experimental setup helped to place recombination junctions, but affected the outcome of the experiment. A different picture emerged from recombination studies with dimorphic heterozygotes at the bz locus, in which the recombining heteroalleles were derived from the same progenitor and differed only at the two sites between which recombination was being measured (19). In dimorphic heterozygotes, one CO class still predominated (that expected from the relative location of the mutations in the gene), but the NCO classes occurred in much higher numbers, so that the CO/NCO ratio was often less than 1. Similar results have been obtained at the r locus (20). Thus, the CO/NCO ratio in maize can be allele dependent. The variability in the CO/NCO ratio for different recombination hotspots observed in a highly polymorphic yeast hybrid (21) may reflect the extent of allelic polymorphisms among different loci.The sharp difference in the outcome of experiments involving polymorphic and dimorphic heterozygotes was explained in terms of the dual recombination pathway proposed by Allers and Lichten (22) and supported by other work (23–25), whereby repair of the initiating DSB produces COs via a double Holliday junction (DHJ) intermediate and NCOs via a synthesis-dependent strand annealing (SDSA) pathway. In this model, the decision to repair a DSB as a CO (via a DHJ) or a NCO (via SDSA) would happen at or soon after the initial step of strand invasion. The bz and r data suggest that that decision is affected by the extent of mismatches in the heteroduplex DNA formed by the invading strand. In the absence of extensive heterologies, mismatch repair proteins would not bind to the heteroduplex and the newly synthesized strand would be displaced readily leading to the recovery of NCO products.In the studies with bz dimorphic heterozygotes, the two NCO classes occurred in roughly similar numbers, i.e., the NCO class that carried the flanking markers of the proximal (5′) allele was recovered approximately as frequently as the NCO class that carried the flanking markers of the distal (3′) allele. Thus, there was no indication of preferential conversion of the proximal or distal allele (26, 27). However, the sample of mutant sites used in those studies did not include any at either end of the gene. Because most conversion gradients in yeast show strong 5′ or 3′ polarity, we could have missed a conversion gradient at bz that was steep at one or both ends but hardly detectable thereafter. To examine at greater depth the issue of conversion polarity within a higher plant gene, we now isolated a series of new bz mutations from the Bz-McC progenitor allele and extended our analysis to include sites at both the 5′ and 3′ ends of the bz gene. Mutations covering the length of the gene were tested in all possible pairwise combinations in two large diallels, with remarkably similar results in the two experiments. We find that bz mutants derived from the Bz-McC allele, which is flanked by single-copy DNA sequences on either side (28), show a U-shaped conversion gradient, with higher conversion frequencies at both the 5′ and 3′ ends than at the center. The implications of these findings are discussed with reference to recent data from genome-wide studies in several organisms.