Allele segregation analysis of F1 hybrids between independent Brassica allohexaploid lineages.

In the Brassica genus, we find both diploid species (one genome) and allotetraploid species (two different genomes) but no naturally occurring hexaploid species (three different genomes, AABBCC). Although hexaploids can be produced via human intervention, these neo-polyploids have quite unstable genomes and usually suffer from severe genome reshuffling. Whether these genome rearrangements continue in later generations and whether genomic arrangements follow similar, reproducible patterns between different lineages is still unknown. We crossed Brassica hexaploids resulting from different species combinations to produce five F1 hybrids and analyzed the karyotypes of the parents and the F1 hybrids, as well as allele segregation in a resulting test-cross population via molecular karyotyping using SNP array genotyping. Although some genomic regions were found to be more likely to be duplicated, deleted, or rearranged, a consensus pattern was not shared between genotypes. Brassica hexaploids had a high tolerance for fixed structural rearrangements, but which rearrangements occur and become fixed over many generations does not seem to show either strong reproducibility or to indicate selection for stability. On average, we observed 10 de novo chromosome rearrangements contributed almost equally from both parents to the F1 hybrids. At the same time, the F1 hybrid meiosis produced on average 8.6 new rearrangements. Hence, the increased heterozygosity in the F1 hybrid did not significantly improve genome stability in our hexaploid hybrids and might have had the opposite effect. However, hybridization between lineages was readily achieved and may be exploited for future genetics and breeding purposes.

Meiotic synapsis of homeologous chromosomes and mismatch repair protein detection in the parthenogenetic rock lizard Darevskia unisexualis.

Parthenogenetic species of Caucasian rock lizards of the genus Darevksia are important evidence for reticulate evolution and speciation by hybridization in vertebrates. Female-only lineages formed through interspecific hybridization have been discovered in many groups. Nevertheless, critical mechanisms of oogenesis and specifics of meiosis that provide long-term stability of parthenogenetic species are still unknown. Here we report cytogenetic characteristics of somatic karyotypes and meiotic prophase I nuclei in the diploid parthenogenetic species Darevskia unisexualis from the new population "Keti" in Armenia which contains an odd number of chromosomes 2n = 37, instead of the usual 2n = 38. We revealed 36 acrocentric chromosomes and a single metacentric autosomal chromosome, resulting from Robertsonian translocation. Comparative genomic hybridization revealed that chromosome fusion occurred between two chromosomes inherited from the maternal species, similar to another parthenogenetic species D. rostombekowi. To trace the chromosome behaviour in meiosis, we performed an immunocytochemical study of primary oocytes' spread nuclei and studied chromosome synapsis during meiotic prophase I in D. unisexualis based on analysis of synaptonemal complexes (SCs). We found meiotic SC-trivalent composed of one metacentric and two acrocentric chromosomes. We confirmed that the SC was assembled between homeologous chromosomes inherited from two parental species. Immunostaining of the pachytene and diplotene nuclei revealed a mismatch repair protein MLH1 loaded to all autosomal SC bivalents. Possible mechanisms of meiotic recombination between homeologous chromosomes are discussed.

Introgression lines of Solanum sitiens, a wild nightshade of the Atacama Desert, in the genome of cultivated tomato.

The wild tomato relative Solanum sitiens is a xerophyte endemic to the Atacama Desert of Chile and a potential source of genes for tolerance to drought, salinity and low-temperature stresses. However, until recently, strong breeding barriers prevented its hybridization and introgression with cultivated tomato, Solanum lycopersicum L. We overcame these barriers using embryo rescue, bridging lines and allopolyploid hybrids, and synthesized a library of introgression lines (ILs) that captures the genome of S. sitiens in the background of cultivated tomato. The IL library consists of 56 overlapping introgressions that together represent about 93% of the S. sitiens genome: 65% in homozygous and 28% in heterozygous (segregating) ILs. The breakpoints of each segment and the gaps in genome coverage were mapped by single nucleotide polymorphism (SNP) genotyping using the SolCAP SNP array. Marker-assisted selection was used to backcross selected introgressions into tomato, to recover a uniform genetic background, to isolate recombinant sub-lines with shorter introgressions and to select homozygous genotypes. Each IL contains a single S. sitiens chromosome segment, defined by markers, in the genetic background of cv. NC 84173, a fresh market inbred line. Large differences were observed between the lines for both qualitative and quantitative morphological traits, suggesting that the ILs contain highly divergent allelic variation. Several loci contributing to unilateral incompatibility or hybrid necrosis were mapped with the lines. This IL population will facilitate studies of the S. sitiens genome and expands the range of genetic variation available for tomato breeding and research.

Mismatch repair protein hMSH2-hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate.

High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.

Sorting duplicated loci disentangles complexities of polyploid genomes masked by genotyping by sequencing.

Many plants and animals of polyploid origin are currently enjoying a genomics explosion enabled by modern sequencing and genotyping technologies. However, routine filtering of duplicated loci in most studies using genotyping by sequencing introduces an unacceptable, but often overlooked, bias when detecting selection. Retained duplicates from ancient whole-genome duplications (WGDs) may be found throughout genomes, whereas retained duplicates from recent WGDs are concentrated at distal ends of some chromosome arms. Additionally, segmental duplicates can be found at distal ends or nearly anywhere in a genome. Evidence shows that these duplications facilitate adaptation through one of two pathways: neo-functionalization or increased gene expression. Filtering duplicates removes distal ends of some chromosomes, and distal ends are especially known to harbour adaptively important genes. Thus, filtering of duplicated loci impoverishes the interpretation of genomic data as signals from contiguous duplicated genes are ignored. We review existing strategies to genotype and map duplicated loci; we focus in detail on an overlooked strategy of using gynogenetic haploids (1N) as a part of new genotyping by sequencing studies. We provide guidelines on how to use this haploid strategy for studies on polyploid-origin vertebrates including how it can be used to screen duplicated loci in natural populations. We conclude by discussing areas of research that will benefit from better inclusion of polyploid loci; we particularly stress the sometimes overlooked fact that basing genomic studies on dense maps provides value added in the form of locating and annotating outlier loci or colocating outliers into islands of divergence.

Uncovering the role of Rad51 in homologous recombination-mediated antigenic diversification in the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum maintains the chronicity of infections through antigenic variation, a well-coordinated immune evasion mechanism. The most prominent molecular determinant of antigenic variation in this parasite includes the members of the var multigene family. Homologous recombination (HR)-mediated genomic rearrangements have been implicated to play a major role in var gene diversification. However, the key molecular factors involved in the generation of diversity at var loci are less known. Here, we tested the hypothesis that PfRad51 could carry out recombination between var genes that are not homologous but homeologous in nature. We employed the whole-genome sequencing (WGS) approach to investigate recombination events among var sequences over 100 generations and compared the rate of sequence rearrangement at the var loci in both PfRad51-proficient and -deficient parasite lines. This brief report provides evidence that the loss of the key recombinase function renders the parasite with inefficient HR and results in fewer recombination events among the var sequences, thereby impacting the diversification of the var gene repertoire.

Effects of OsMSH6 Mutations on Microsatellite Stability and Homeologous Recombination in Rice.

DNA mismatch repair (MMR) system is important for maintaining DNA replication fidelity and genome stability by repairing erroneous deletions, insertions and mis-incorporation of bases. With the aim of deciphering the role of the MMR system in genome stability and recombination in rice, we investigated the function of OsMSH6 gene, an import component of the MMR system. To achieve this goal, homeologous recombination and endogenous microsatellite stability were evaluated by using rice mutants carrying a Tos17 insertion into the OsMSH6 gene. Totally 60 microsatellites were analyzed and 15 distributed on chromosome 3, 6, 8, and 10 showed instability in three OsMSH6 mutants, D6011, NF7784 and NF9010, compared with the wild type MSH6WT (the control). The disruption of OsMSH6 gene is associated with modest increases in homeologous recombination, ranging from 2.0% to 32.5% on chromosome 1, 3, 9, and 10 in the BCF2 populations of the mutant ND6011 and NF9010. Our results suggest that the OsMSH6 plays an important role in ensuring genome stability and genetic recombination, providing the first evidence for the MSH6 gene in maintaining microsatellite stability and restricting homeologous recombination in plants.

The mismatch repair protein MSH6 regulates somatic recombination in Arabidopsis thaliana.

The mismatch repair (MMR) pathway promotes genome stability by controlling the fidelity of replication and recombination. The first step of the pathway involves recognition of the mismatch by heterodimers composed of MutS homologs (MSH). Although MSH6 has been well characterized in yeasts and humans, the role of the plant protein has not been extensively studied. We first analyzed gene expression in Arabidopsis thaliana. The use of transgenic plants expressing the ß-glucuronidase (GUS) reporter gene under the control of approximately 1-kb region upstream of the start codon of the AtMSH6 gene demonstrated that MSH6 is preferentially expressed in undifferentiated cells with an intense cell division rate. We then examined protein function in meiotic and somatic recombination. Suppression of AtMSH6 did not affect the rate of meiotic recombination, but increased the frequency of recombination between two homeologous repeats of a marker gene by 3-fold relative to wild-type plants. Expression of the AtMSH6 gene under the control of its own promoter in msh6 homozygous mutant plants rescued the altered somatic recombination phenotype. We conclude that MSH6 shows a functional conservation across different biological kingdoms and a functional specificity in plants.

The GIY-YIG endonuclease domain of Arabidopsis MutS homolog 1 specifically binds to branched DNA structures.

In plant organelle genomes, homeologous recombination between heteroallelic positions of repetitive sequences is increased by dysfunction of the gene encoding MutS homolog 1 (MSH1), a plant organelle-specific homolog of bacterial mismatch-binding protein MutS1. The C-terminal region of plant MSH1 contains the GIY-YIG endonuclease motif. The biochemical characteristics of plant MSH1 have not been investigated; accordingly, the molecular mechanism by which plant MSH1 suppresses homeologous recombination is unknown. Here, we characterized the recombinant GIY-YIG domain of Arabidopsis thaliana MSH1, showing that the domain possesses branched DNA-specific DNA-binding activity. Interestingly, the domain exhibited no endonuclease activity, suggesting that the mismatch-binding domain is required for DNA incision. Based on these results, we propose a possible mechanism for MSH1-dependent suppression of homeologous recombination.

Unstable Allotetraploid Tobacco Genome due to Frequent Homeologous Recombination, Segmental Deletion, and Chromosome Loss.

The types of mutations and their corresponding frequencies are difficult to measure in complex genomes. In this study, a high-throughput method was developed to identify spontaneous loss-of-function alleles for the resistance gene N and the transgenic avirulence gene P50 in allotetraploid tobacco. A total of 2134 loss-of-function alleles of the N gene were identified after screening 14 million F1 hybrids. Analysis of these mutants revealed striking evolutionary patterns for genes in polyploids. Only 14 of the loss-of-function mutations were caused by spontaneous point mutations or indels, while the others were caused by homeologous recombination (with a frequency of ∼1/12 000) or chromosome loss (∼1/15 000). Loss of the chromosome with the P50 insertion occurred at a similar frequency (∼1/13 000), and the frequency of spontaneous segmental deletion in this chromosome was ∼1/16 000. Both homeologous recombination and chromosome loss considerably decreased the viability of the mutants. Our data suggest that the high mutation rate in polyploids is probably due to the occurrence of homeologous recombination and the tolerance of large mutations such as chromosome loss in polyploid genomes. Frequent mutations tend to drive polyploids to extinction unless a novel mutation helps the polyploid to effectively compete with diploids or find a new ecological niche.

Mismatch tolerance during homologous recombination in mammalian cells.

We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the "recipient") rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a "donor" tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of "homeology" relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells.

Production and manipulation of blue copper oxidases for technological applications.

Fungal laccases are robust multicopper oxidoreductases. Perfectly amenable to synthetic evolution, the fungal laccase scaffold is a potential generic for the production of tailored biocatalysts, which, in principle, can be secreted at substantial levels in industrially relevant organisms. In this chapter, the strategy we have developed for the rapid production of hundreds of milligram of laccase variants is detailed. It is based on the use of two heterologous expression hosts: the yeast Saccharomyces cerevisiae for a rapid upstream screening and the fungus Aspergillus niger for downstream production. Methods for screening active and nonactive laccase variants, convenient setups for enzyme production in both organisms as well as a methodology for efficient purification of large amounts of recombinant enzymes are given. The general procedure for developing new materials for artificial catalysis is also described.

Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair.

Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression.

High homology is not required at the site of strand invasion during recombinational double-strand break repair in mammalian chromosomes.

We investigated the impact of sequence divergence on DNA double-strand break (DSB) repair occurring via recombination in cultured thymidine kinase deficient mouse fibroblasts. We stably transfected cells with a DNA construct harboring a herpes thymidine kinase (tk) gene (the "recipient") rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. The construct also contained a closely linked truncated "donor" tk sequence. The donor could potentially restore function to the recipient gene via recombination provoked by induction of a DSB at the I-SceI site in the recipient. Repair events were recoverable by selection for tk-positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Previous work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In the current study we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest a search for high homology prior to recombination that is not mediated by an invading DNA terminus.

The Role of Blm Helicase in Homologous Recombination, Gene Conversion Tract Length, and Recombination Between Diverged Sequences in Drosophilamelanogaster.

DNA double-strand breaks (DSBs) are a particularly deleterious class of DNA damage that threatens genome integrity. DSBs are repaired by three pathways: nonhomologous-end joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). Drosophila melanogaster Blm (DmBlm) is the ortholog of Saccharomyces cerevisiae SGS1 and human BLM, and has been shown to suppress crossovers in mitotic cells and repair mitotic DNA gaps via HR. To further elucidate the role of DmBlm in repair of a simple DSB, and in particular recombination mechanisms, we utilized the Direct Repeat of white (DR-white) and Direct Repeat of whitewith mutations (DR-white.mu) repair assays in multiple mutant allele backgrounds. DmBlm null and helicase-dead mutants both demonstrated a decrease in repair by noncrossover HR, and a concurrent increase in non-HR events, possibly including SSA, crossovers, deletions, and NHEJ, although detectable processing of the ends was not significantly impacted. Interestingly, gene conversion tract lengths of HR repair events were substantially shorter in DmBlm null but not helicase-dead mutants, compared to heterozygote controls. Using DR-white.mu, we found that, in contrast to Sgs1, DmBlm is not required for suppression of recombination between diverged sequences. Taken together, our data suggest that DmBlm helicase function plays a role in HR, and the steps that contribute to determining gene conversion tract length are helicase-independent.

Mismatch repair and homeologous recombination.

DNA mismatch repair influences the outcome of recombination events between diverging DNA sequences. Here we discuss how mismatch repair proteins are active in different homologous recombination subpathways and specific reaction steps, resulting in differential modulation of these recombination events, with a focus on the mechanism of heteroduplex rejection during the inhibition of recombination between slightly diverged (homeologous) DNA sequences.

Near-absent levels of segregational variation suggest limited opportunities for the introduction of genetic variation via homeologous chromosome pairing in synthetic neoallotetraploid Mimulus.

Genetic variation is the fundamental medium of evolution. In allopolyploids, which are the product of hybridization and whole genome duplication, if homologous chromosomes always pair, then all descendants of a single diploid F1 hybrid lineage will be genetically identical. Contrarily, genetic variation among initially isogenic lineages is augmented when homeologous chromosomes pair; this added variation may contribute to phenotypic evolution. Mimulus sookensis is a naturally occurring, small-flowered allotetraploid derived from the large-flowered Mimulus guttatus and small-flowered Mimulus nasutus. Because diploid F1 hybrids between M. guttatus and M. nasutus have large flowers, phenotypic evolution post-polyploidization is implied in M. sookensis. Here, we present genetic and phenotypic analyses of synthetic neoallotetraploid Mimulus derived from a cross between M. guttatus and M. nasutus. Genetic marker data from S2 and BC1N progeny suggest that chromosomes regularly pair with their homologous counterpart. By measuring the phenotype of synthetic neoallotetraploids, we demonstrate that polyploidization per se does not induce the small flowers of M. sookensis. Moreover, phenotypic measurements of synthetic allotetraploid F2s and S4 families suggest that rare homeologous recombination events have a negligible phenotypic effect in the first few generations. In total, the results are consistent with either exceedingly rare homeologous pairing and recombination or spontaneous fragment loss. The low levels of fragment loss and phenotypic variation in neoallotetraploids suggest that homeologous recombination after polyploidization is not a major mechanism of phenotypic evolution in M. sookensis. Rather, it may be that spontaneous mutations or epigenetic changes after allopolyploidization have driven phenotypic evolution in M. sookensis.

Homoeologous GSL-ELONG gene replacement for manipulation of aliphatic glucosinolates in Brassica rapa L. by marker assisted selection.

Aliphatic glucosinolates are the predominant sulfur-rich plant secondary metabolites in economically important Brassica crops. Glucosinolates and their hydrolysis products are involved in plant-microbe, plant-insect, plant-animal, and plant-human interactions. It is, therefore, important to manipulate glucosinolate profiles and contents in Brassica species. In this study, aliphatic glucosinolates were genetically manipulated through homoeologous recombination in backcross lines followed by marker assisted selection in B. rapa. A resynthesized B. napus line, from a cross between B. rapa and B. oleracea, was backcrossed with Chinese cabbage doubled haploid line, RI16. Marker assisted selection for non-functional gene was performed in each backcross generations. Advanced backcross progenies (BC3F2) were developed to identify homoeologous gene replacement and/or introgression. Reduction in 5C aliphatic glucosinolates (gluconapoleiferin, glucoalyssin, and glucobrassicanapin) was observed in BC3F2 progenies of the recurrent parent that carried the GSL-ELONG (-) gene. The GSL-ELONG (-) positive backcross progenies were also screened by the A-genome and BraGSL-ELONG gene specific marker, which linked with 5C aliphatic glucosinolates. The A-genome specific marker was absent in the plants of advanced backcross progenies which showed reduction in 5C aliphatic glucosinolates. The results suggest that the functional allele had been replaced by the non-functional GSL-ELONG (-) allele from B. oleracea. Some advanced backcross progenies (BC3F2) positive for the GSL-ELONG (-) allele and the A-genome specific SCAR marker BraMAM1-1 did not show reduction in 5C aliphatic glucosinolates, suggesting that GSL-ELONG (-) allele is recessive. Replacement of the functional locus in the A-genome by non-functional counterpart in the C-genome reduced the content of 5C aliphatic glucosinolates in B. rapa seeds with 20 µmol/g.