Rare Recombination Events Research Articles

Marker-assisted Selection to Combine Alleles for Four Disease Resistance Genes of Tomato Collocated on Chromosome 11

Bacterial, fungal, and viral diseases of tomato (Solanum lycopersicum) are responsible for widespread yield losses, especially in humid growing environments. Chromosome 11 of tomato contains genes that modulate resistance to several prominent tomato pathogens, including bacterial spot caused by Xanthomonas spp., gray leaf spot caused by Stemphylium spp., Fusarium wilt caused by race 2 of Fusarium oxysporum f. sp. lycopersici, and tomato yellow leaf curl virus (TYLCV) caused by begomoviruses. Major resistance loci are quantitative trait locus 11 (QTL-11) and Xv3/Rx4 for bacterial spot, Sm for gray leaf spot, I2 for Fusarium wilt, and Ty-2 for TYLCV. Marker-assisted selection was used to select for rare recombination events that combined these resistance loci into a linked cassette that can be inherited together in future crosses. A pedigree breeding strategy was used with marker-assisted selection and used to identify a novel coupling of Xv3/Rx4 and Ty-2. Recombination between the two genes was estimated as 0.056 cM, demonstrating that effective combinations of resistance can be established using publicly available germplasm. Progeny from the recombinant plants were screened using inoculated seedling trials to confirm resistance. The recombinants identified maintained resistance levels similar to the resistant controls. Trial results suggest that the trait markers on chromosome 11 are tightly linked to the respective resistance loci and are effective for selecting plants with resistance to the target diseases.

Allelism and resistance loci of powdery mildew and leaf rust in Egyptian hexaploid bread wheat

To incorporate resistance genes of powdery mildew (Pm) and leaf rust (Lr) effectively in breeding programs allelic interaction should be explored. The response and molecular patterns of three Pm genes, Pm3, Pm8, Pm17, and three Lr genes, Lr10, Lr24, and Lr26 were investigated in 15 Egyptian wheat cultivars. Postulation of different Pm and Lr resistance genes in Egyptian wheat cultivars was carried out according to seedling phenotyping data that resulted from inoculation with a set of five pathotypes of Blumeria graminis f. sp. tritici and twelve pathotypes of Puccinia triticina. Responses and molecular patterns revealed the presence of resistance genes Pm3 and Lr10 in the cultivar Misr-1, Pm17, and Lr24 in the cultivar Giza-171, and Pm8 and Lr26 in a further ten cultivars. Molecular markers confirmed the presence of Pm8 and Lr26 in six wheat cultivars, Sakha-94, Sakha-95, Gemmeiza-10, Gemmeiza-11, Sids-13, and Shandweel-1. The presence of Pm17 in Giza-171 was confirmed by the STS marker iag95. A comparison of phenotypic and genotypic data between cultivars Giza-171 and Amigo demonstrated that Pm17 in cv Giza-171 is allelic to Pm8. The existence of Lr24 in Giza-171 was confirmed by using STS marker J09. Based on these results, Pm17 and Lr24 in Giza-171 may be located on translocated chromosomes. In cv Misr-1, the presence of Pm3 and Lr10 on the same region of 1AS chromosome could represent a rare recombination event. Field responses revealed specific resistance of Misr-1 and Giza-171, which may be attributed to the presence of Pm3 and Lr10 in Misr-1 and Pm17 and Lr24+ additional genes in Giza-171.

Multiple mechanisms drive genomic adaptation to extreme O2 levels in Drosophila melanogaster

To detect the genomic mechanisms underlying evolutionary dynamics of adaptation in sexually reproducing organisms, we analyze multigenerational whole genome sequences of Drosophila melanogaster adapting to extreme O2 conditions over an experiment conducted for nearly two decades. We develop methods to analyze time-series genomics data and predict adaptive mechanisms. Here, we report a remarkable level of synchronicity in both hard and soft selective sweeps in replicate populations as well as the arrival of favorable de novo mutations that constitute a few asynchronized sweeps. We additionally make direct experimental observations of rare recombination events that combine multiple alleles on to a single, better-adapted haplotype. Based on the analyses of the genes in genomic intervals, we provide a deeper insight into the mechanisms of genome adaptation that allow complex organisms to survive harsh environments.

The genetic basis of sex determination in grapes

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs.

Supergene Evolution: Recombination Finds a Way.

Supergenes are multiple linked genes that regulate complex, polymorphic traits, but little is known about their evolution. A new study of an ancient supergene in several ant species suggests that rare recombination events shape supergene evolution in surprising ways.

The alternative reality of plant mitochondrial DNA: One ring does not rule them all.

Plant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held view across the broad community of life scientists that circular genome-sized molecules are the primary form of plant mitochondrial DNA, despite the understanding by plant mitochondrial researchers that this is an inaccurate and outdated concept. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative arrangements (isoforms). Most mitochondrial genomes have been assembled using methods unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. We characterized a comprehensive, complex set of isoforms within each species and compared genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms in our data, we infer that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other Lactuca species can be largely explained by rare recombination events that rearranged the structure. Our data demonstrate that representations of plant mitochondrial genomes as simple, circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms.

ClickSeq: Replacing Fragmentation and Enzymatic Ligation with Click-Chemistry to Prevent Sequence Chimeras.

We recently reported a fragmentation-free method for the synthesis of Next-Generation Sequencing libraries called "ClickSeq" that uses biorthogonal click-chemistry in place of enzymes for the ligation of sequencing adaptors. We found that this approach dramatically reduces artifactual chimera formation, allowing the study of rare recombination events that include viral replication intermediates and defective-interfering viral RNAs. ClickSeq illustrates how robust, bio-orthogonal chemistry can be harnessed in vitro to capture and dissect complex biological processes. Here, we describe an updated protocol for the synthesis of "ClickSeq" libraries.

Dual loss of human POLQ and LIG4 abolishes random integration

Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. Here we show that DNA polymerase θ, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase θ and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Thus, DNA polymerase θ-mediated end joining is the sole homology-independent repair route in the absence of NHEJ and, intriguingly, their combined absence reveals rare Alu-Alu recombination events utilizing a stretch of homology. Our findings provide new insights into the mechanics of foreign DNA integration and the role of DNA polymerase θ in human genome maintenance.

Biased selection of propagation-related TUPs from phage display peptide libraries.

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus.

Defective-Interfering RNAs (DI-RNAs) have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. For short-read RNAseq, we employed ‘ClickSeq’ due to its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies’s (ONT) MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these ‘intermediate’ species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer ‘mature’ DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs.

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus.

The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.

Genetic architecture and evolution of the S locus supergene in Primula vulgaris.

Darwin's studies on heterostyly in Primula described two floral morphs, pin and thrum, with reciprocal anther and stigma heights that promote insect-mediated cross-pollination. This key innovation evolved independently in several angiosperm families. Subsequent studies on heterostyly in Primula contributed to the foundation of modern genetic theory and the neo-Darwinian synthesis. The established genetic model for Primula heterostyly involves a diallelic S locus comprising several genes, with rare recombination events that result in self-fertile homostyle flowers with anthers and stigma at the same height. Here we reveal the S locus supergene as a tightly linked cluster of thrum-specific genes that are absent in pins. We show that thrums are hemizygous not heterozygous for the S locus, which suggests that homostyles do not arise by recombination between S locus haplotypes as previously proposed. Duplication of a floral homeotic gene 51.7 million years (Myr) ago, followed by its neofunctionalization, created the current S locus assemblage which led to floral heteromorphy in Primula. Our findings provide new insights into the structure, function and evolution of this archetypal supergene.

Deep resequencing of CFTR in 762 F508del homozygotes reveals clusters of non-coding variants associated with cystic fibrosis disease traits.

Extensive phenotypic variability is commonly observed in individuals with Mendelian disorders, even among those with identical genotypes in the disease-causing gene. To determine whether variants within and surrounding CFTR contribute to phenotypic variability in cystic fibrosis (CF), we performed deep sequencing of CFTR in 762 patients homozygous for the common CF-causing variant, F508del. In phase 1, ~200 kb encompassing CFTR and extending 10 kb 5′ and 5 kb 3′ of the gene was sequenced in 486 F508del homozygotes selected from the extremes of sweat chloride concentration. In phase 2, a 510 kb region, which included the entire topologically associated domain of CFTR, was sequenced in 276 F508del homozygotes drawn from extremes of lung function. An additional 163 individuals who carried F508del and a different CF-causing variant were sequenced to inform haplotype construction. Region-based burden testing of both common and rare variants revealed seven regions of significance (α=0.01), five of which overlapped known regulatory elements or chromatin interactions. Notably, the −80 kb locus known to interact with the CFTR promoter was associated with variation in both CF traits. Haplotype analysis revealed a single rare recombination event (1.9% frequency) in intron 15 of CFTR bearing the F508del variant. Otherwise, the majority of F508del chromosomes were markedly similar, consistent with a single origin of the F508del allele. Together, these high-resolution variant analyses of the CFTR locus suggest a role for non-coding regulatory motifs in trait variation among individuals carrying the common CF allele.

Rare recombination events generate sequence diversity among balancer chromosomes in Drosophila melanogaster

Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the locations of FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (sn(X2)) on FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost sn(X2) by this mechanism on a historical timescale. Finally, we characterize the original allele of the Bar gene (B(1)) that is carried on FM7, and validate the hypothesis that the origin and subsequent reversion of the B(1) duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila.

European molecular epidemiology and strain diversity of feline calicivirus

Feline calicivirus (FCV) causes a variable syndrome of upper respiratory tract disease, mouth ulcers and lameness. A convenience-based prospective sample of oropharyngeal swabs (n=426) was obtained from five countries (France,...

Structural genomic changes underlie alternative reproductive strategies in the ruff (Philomachus pugnax).

The ruff is a Palearctic wader with a spectacular lekking behavior where highly ornamented males compete for females. This bird has one of the most remarkable mating systems in the animal kingdom, comprising three different male morphs (independents, satellites and faeders) that differ in behavior, plumage color and body size. Remarkably, the satellite and faeder morphs are controlled by dominant alleles. Here we have used whole-genome sequencing and resolved the enigma of how such complex phenotypic differences can have a simple genetic basis. The Satellite and Faeder alleles are both associated with a 4.5-Mb inversion that occurred about 3.8 million years ago. We propose an evolutionary scenario where the Satellite chromosome arose by a rare recombination event about 500,000 years ago. The ruff mating system is the result of an evolutionary process in which multiple genetic changes contributing to phenotypic differences between morphs have accumulated within the inverted region.

Paternity testing of two female siblings with Investigator Argus X-12 kit: A case with several rare mutation and recombination events

Stable haplotypes of closely associated X-STRs have proven to be a powerful tool in kinship analysis especially for cases when father/daughter relationships are to be tested. We present a presumably easy case: A woman wanted to know whether or not her daughters have the same biological father. Initial results with Argus X-12 kit showed only one mismatch at DXS10148, which could have been explained by a paternal one-step mutation. However, comparison between the maternal haplotype and the daughters showed an incompatibility in linkage group III. To explain this, a maternal 3-step mutation in HPRTB or two mutation/recombination events in linkage group III must have occurred. Analysis of 29 autosomal STRs gave a high probability for being full siblings. Analysis of 13 additional X-STRs showed no further exclusion from having the same father. Reconstruction of possible maternal haplotypes demonstrated that two recombination events in linkage group III are very unlikely. We presume that one maternal mutation in DXS10103 and one recombination event in linkage group III must have occurred together with one paternal mutation in DXS10148.This case with 3 rare events occuring within the range of the Argus X-12 kit shows that the use of X-STRs requires not only knowledge of allele and haplotype frequencies but also of mutation rates and rare recombination events of markers within linkage groups. In special cases it would be necessary to involve further X markers. Collaboration of different laboratories will help to solve such problems.

ANALYSIS OF GENETIC DIVERSITY IN TUNISIAN CITRUS ROOTSTOCKS

Breeding and selection of new citrus rootstocks are nowadays of the utmost importance in the Mediterranean Basin because the citrus industry faces increasing biotic and abiotic constraints. In Tunisia, citrus contributes significantly to the national economy, and its extension is favored by natural conditions and economic considerations. Sour orange, the most widespread traditional rootstock of the Mediterranean area, is also the main one in Tunisia. In addition to sour orange, other citrus rootstocks well adapted to local environmental conditions are traditionally used and should be important genetic resources for breeding. Prior to initiation of any breeding program, the exploration of Tunisian citrus rootstock diversity was a priority. Two hundred and one local accessions belonging to four facultative apomictic species (Citrus aurantium, sour orange; Citrus sinensis, sweet orange; Citrus limon, lemon; and Citrus aurantifolia, lime) were collected and genotyped using 20 nuclear SSR markers and four InDel mitochondrial markers. Sixteen distinct Multi-locus genotypes (MLGs) were identified and compared to references from French and Spanish collections. The differentiation of the four varietal groups was well-marked. Each group displayed a relatively high allelic diversity, primarily due to very high heterozygosity. The Tunisian citrus rootstock genetic diversity is predominantly due to high heterozygosity and differentiation between the four varietal groups. The phenotypic diversity within the varietal groups has resulted from multiple introductions, somatic mutations and rare sexual recombination events. This diversity study enabled the identification of a core sample of accessions for further physiological and agronomic evaluations. These core accessions will be integrated into citrus rootstock breeding programs for the Mediterranean Basin. (Resume d'auteur)

Near-Absent Levels of Segregational Variation Suggest Limited Opportunities for the Introduction of Genetic Variation Via Homeologous Chromosome Pairing in Synthetic NeoallotetraploidMimulus

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.

ENHANCING ctr1-10 ETHYLENE RESPONSE2 is a novel allele involved in CONSTITUTIVE TRIPLE-RESPONSE1-mediated ethylene receptor signaling in Arabidopsis.

BackgroundThe signal output of ethylene receptor family members is mediated by unknown mechanisms to activate the Raf-like protein CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) in negatively regulating ethylene signaling. The physical interaction between the ethylene receptor histidine kinase (HK) domain and CTR1 N terminus is essential to the CTR1-mediated receptor signal output. To advance our knowledge of the involvement of CTR1-mediated ethylene receptor signaling, we performed a genetic screen for mutations that enhanced the constitutive ethylene response in the weak ctr1-10 allele.ResultsWe isolated a loss-of-function allele of ENHANCING ctr1-10 ETHYLENE RESPONSE2 (ECR2) and found that ecr2-1 ctr1-10 and the strong allele ctr1-1 conferred a similar, typical constitutive ethylene response phenotype. Genetic analyses and transformation studies suggested that ECR2 acts downstream of the ethylene receptors and upstream of the transcription factors ETHYLENE INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1), which direct the expression of ethylene response genes. Signal output by the N terminus of the ethylene receptor ETHYLENE RESPONSE1 (ETR1) can be mediated by a pathway independent of CTR1. Expression of the N terminus of the ethylene-insensitive etr1-1 but not the full-length isoform rescued the ecr2-1 ctr1-10 phenotype, which indicates the involvement of ECR2 in CTR1-mediated but not -independent, ethylene receptor signaling. ECR2 was mapped to the centromere region on chromosome 2. With incomplete sequence and annotation information and rare chromosome recombination events in this region, the cloning of ECR2 is challenging and still in progress.ConclusionsECR2 is a novel allele involved in the ethylene receptor signaling that is mediated by CTR1. CTR1 activation by ethylene receptors may require ECR2 for suppressing the ethylene response.