High‐resolution insight into recombination events at the SD1 locus in rice

Recombination during meiosis plays an important role in genome evolution by reshuffling existing genetic variations into fresh combinations with the possibility of recovery of lost ancestral genotypes. While crossover (CO) events have been well studied, gene conversion events (GCs), which represent non‐reciprocal information transfer between chromosomes, are poorly documented and difficult to detect due to their relatively small converted tract size. Here, we document these GC events and their phenotypic effects at an important locus in rice containing the SD1 gene, where multiple defective alleles contributed to the semi‐dwarf phenotype of rice in the ‘Green Revolution’ of the 1960s. Here, physical separation of two defects allows recombination to generate the wild‐type SD1 gene, for which plant height can then be used as a reporter. By screening 18 000 F₂ progeny from a cross between two semi‐dwarf cultivars that carry these different defective alleles, we detected 24 GC events, indicating a conversion rate of ~3.3 × 10^?4 per marker per generation in a single meiotic cycle in rice. Furthermore, our data show that indels and single‐nucleotide polymorphisms (SNPs) do not differ significantly in GC rates, at least at the SD1 locus. Our results provide strong evidence that GC by itself can regain an ancestral phenotype that was lost through mutation. This GC detection approach is likely to be broadly applicable to natural or artificial alleles of other phenotype‐related functional genes, which are abundant in other plant genomes.     

Effect of the mouse scid mutation on meiotic recombination

The goal of this study was to determine the effect of the mouse severe combined immunodeficiency (scid) mutation on the rate of meiotic recombination, by standard backcross linkage analysis. For this purpose, we examined four crosses that involved F₁ hybrid animals heterozygous for the strain C57BL/6 and BALB/c genomes. In one set of reciprocal crosses, F₁ animals were homozygous scid/scid, and in a second set of reciprocal crosses, F₁ mice were homozygous wild-type (+/+) at the scid locus. Backcross progeny were typed for recombination between selected genetic markers on mouse Chromosomes (Chrs) 1, 4, 6, 7, 9, 15, and 17. Although some differences in recombination were observed over some intervals, the expression of the SCID phenotype did not appear to have a major or consistent effect on meiotic recombination. Received: 4 October 1995 / Accepted: 2 April 1996     

The Saccharomyces cerevisiae RDN1 locus is sequestered from interchromosomal meiotic ectopic recombination in a SIR2-dependent manner

Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching. In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites. We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence. SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1. We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^? mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Genome-Wide Control of the Distribution of Meiotic Recombination

Meiotic recombination events are not randomly distributed in the genome but occur in specific regions called recombination hotspots. Hotspots are predicted to be preferred sites for the initiation of meiotic recombination and their positions and activities are regulated by yet-unknown controls. The activity of the Psmb9 hotspot on mouse Chromosome 17 (Chr 17) varies according to genetic background. It is active in strains carrying a recombinant Chr 17 where the proximal third is derived from Mus musculus molossinus. We have identified the genetic locus required for Psmb9 activity, named Dsbc1 for Double-strand break control 1, and mapped this locus within a 6.7-Mb region on Chr 17. Based on cytological analysis of meiotic DNA double-strand breaks (DSB) and crossovers (COs), we show that Dsbc1 influences DSB and CO, not only at Psmb9, but in several other regions of Chr 17. We further show that CO distribution is also influenced by Dsbc1 on Chrs 15 and 18. Finally, we provide direct molecular evidence for the regulation in trans mediated by Dsbc1, by showing that it controls the CO activity at the Hlx1 hotspot on Chr 1. We thus propose that Dsbc1 encodes for a trans-acting factor involved in the specification of initiation sites of meiotic recombination genome wide in mice.     

Identification of functional lox sites in the plastid genome

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Extreme Recombination Frequencies Shape Genome Variation and Evolution in the Honeybee,Apis mellifera

Meiotic recombination is a fundamental cellular process, with important consequences for evolution and genome integrity. However, we know little about how recombination rates vary across the genomes of most species and the molecular and evolutionary determinants of this variation. The honeybee, Apis mellifera, has extremely high rates of meiotic recombination, although the evolutionary causes and consequences of this are unclear. Here we use patterns of linkage disequilibrium in whole genome resequencing data from 30 diploid honeybees to construct a fine-scale map of rates of crossing over in the genome. We find that, in contrast to vertebrate genomes, the recombination landscape is not strongly punctate. Crossover rates strongly correlate with levels of genetic variation, but not divergence, which indicates a pervasive impact of selection on the genome. Germ-line methylated genes have reduced crossover rate, which could indicate a role of methylation in suppressing recombination. Controlling for the effects of methylation, we do not infer a strong association between gene expression patterns and recombination. The site frequency spectrum is strongly skewed from neutral expectations in honeybees: rare variants are dominated by AT-biased mutations, whereas GC-biased mutations are found at higher frequencies, indicative of a major influence of GC-biased gene conversion (gBGC), which we infer to generate an allele fixation bias 5 – 50 times the genomic average estimated in humans. We uncover further evidence that this repair bias specifically affects transitions and favours fixation of CpG sites. Recombination, via gBGC, therefore appears to have profound consequences on genome evolution in honeybees and interferes with the process of natural selection. These findings have important implications for our understanding of the forces driving molecular evolution.     

Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome

Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes, and, as a consequence, genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex-determining regions of the genome. Here, we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100-kilobase mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be ~20% between MAT and a flanking marker at 5, 10, 50, or 100 kilobases from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted ~10- to 50-fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. MAT heterozygosity was not required for enhanced recombination, implying that this process is not due to a physical distortion from the two non-paired alleles and could also occur during same-sex mating. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and pathogenic yeasts.     

High-resolution radiation hybrid map of wheat chromosome 1D

Physical mapping methods that do not rely on meiotic recombination are necessary for complex polyploid genomes such as wheat (Triticum aestivum L.). This need is due to the uneven distribution of recombination and significant variation in genetic to physical distance ratios. One method that has proven valuable in a number of nonplant and plant systems is radiation hybrid (RH) mapping. This work presents, for the first time, a high-resolution radiation hybrid map of wheat chromosome 1D (D genome) in a tetraploid durum wheat (T. turgidum L., AB genomes) background. An RH panel of 87 lines was used to map 378 molecular markers, which detected 2312 chromosome breaks. The total map distance ranged from ~3,341 cR_35,000 for five major linkage groups to 11,773 cR_35,000 for a comprehensive map. The mapping resolution was estimated to be ~199 kb/break and provided the starting point for BAC contig alignment. To date, this is the highest resolution that has been obtained by plant RH mapping and serves as a first step for the development of RH resources in wheat.     

The impressionistic landscape of meiotic recombination

Two high-resolution maps of meiotic recombination initiation sites across the genomes of budding yeast and mice illuminate broad similarities in the control of meiotic recombination in these diverse species but also highlight key differences. These studies offer new insights into the relationships between recombination, chromosome structure, and genome evolution.     

Different foreign genes incidentally integrated into the same locus of the Streptococcus suis genome

Some strains of Streptococcus suis possess a type II restriction-modification (RM) system, whose genes are thought to be inserted into the genome between purH and purD from a foreign source by illegitimate recombination. In this study, we characterized the purHD locus of the S. suis genomes of 28 serotype reference strains by DNA sequencing. Four strains contained the RM genes in the locus, as described before, whereas 11 strains possessed other genetic regions of seven classes. The genetic regions contained a single gene or multiple genes that were either unknown or similar to hypothetical genes of other bacteria. The mutually exclusive localization of the genetic regions with the atypical G+C contents indicated that these regions were also acquired from foreign sources. No transposable element or long-repeat sequence was found in the neighboring regions. An alignment of the nucleotide sequences, including the RM gene regions, suggested that the foreign regions were integrated by illegitimate recombination via short stretches of nucleotide identity. By using a thermosensitive suicide plasmid, the RM genes were experimentally introduced into an S. suis strain that did not contain any foreign genes in that locus. Integration of the plasmid into the S. suis genome did not occur in the purHD locus but occurred at various chromosomal loci, where there were 2 to 10 bp of nucleotide identity between the chromosome and the plasmid. These results suggest that various foreign genes described here were incidentally integrated into the same locus of the S. suis genome.     

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.     

Identification of a Bdellovibrio bacteriovorus genetic locus, hit, associated with the host-independent phenotype.

Bdellovibrios invade and grow within the periplasmic space of suitable gram-negative bacteria. Wild-type bdellovibrios are obligately dependent on host cells for growth, but spontaneous host-independent (H-I) mutants that grow axenically on standard rich culture media can be isolated. Such mutants generally retain the ability to grow intraperiplasmically, although the plaques that they produce on lawns of host cells are smaller and more turbid than those produced by wild-type bdellovibrios. Here, we identify the first genetic locus associated with the H-I phenotype: hit (host interaction). We show that three individual H-I mutants suffered mutations at the hit locus and that recombination of wild-type hit sequences into the genomes of the H-I mutants greatly enhanced their plaquing ability. DNA sequence analysis localized the hit mutation in each of the H-I mutants to a 135-bp region of the genome. Mutations at hit may not fully account for the H-I phenotype, however, as recombination of wild-type hit sequences into the genomes of the H-I mutants had little effect on the axenic-growth phenotype of the mutants. Possible explanations for this result and potential roles for the hit locus are discussed.     

Looking for natural variation in chiasma frequency in Arabidopsis thaliana

Information concerning natural variation either in chiasma frequency or in the genetic basis of any such variation is a valuable tool to characterize phenotypic traits and their genetic control. Here meiotic recombination frequencies are analysed in nine geographically and ecologically diverse accessions of Arabidopsis thaliana, and a comparative study was carried out incorporating previous data from another eight accessions. Chiasma frequencies, estimated by counting rod and ring bivalents at metaphase I, varied up to 22% among accessions. However, no differences were found among plants of the same accession. There was a relationship, which does not necessarily imply direct proportionality, between the size of the chromosomes and their mean chiasma frequency. Chiasma frequency and distribution between arms and among chromosomes were not consistent over accessions. These findings indicate the existence of genetic factors controlling meiotic recombination both throughout the whole genome and at the whole chromosome level. The reliability of chiasma scoring as an indicator of reciprocal recombination events is also discussed.     

A reevaluation of rice mitochondrial evolution based on the complete sequence of male-fertile and male-sterile mitochondrial genomes

Plant mitochondrial genomes have features that distinguish them radically from their animal counterparts: a high rate of rearrangement, of uptake and loss of DNA sequences, and an extremely low point mutation rate. Perhaps the most unique structural feature of plant mitochondrial DNAs is the presence of large repeated sequences involved in intramolecular and intermolecular recombination. In addition, rare recombination events can occur across shorter repeats, creating rearrangements that result in aberrant phenotypes, including pollen abortion, which is known as cytoplasmic male sterility (CMS). Using next-generation sequencing, we pyrosequenced two rice (Oryza sativa) mitochondrial genomes that belong to the indica subspecies. One genome is normal, while the other carries the wild abortive-CMS. We find that numerous rearrangements in the rice mitochondrial genome occur even between close cytotypes during rice evolution. Unlike maize (Zea mays), a closely related species also belonging to the grass family, integration of plastid sequences did not play a role in the sequence divergence between rice cytotypes. This study also uncovered an excellent candidate for the wild abortive-CMS-encoding gene; like most of the CMS-associated open reading frames that are known in other species, this candidate was created via a rearrangement, is chimeric in structure, possesses predicted transmembrane domains, and coopted the promoter of a genuine mitochondrial gene. Our data give new insights into rice mitochondrial evolution, correcting previous reports.     

Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.