The genome and transcriptome of the snail Biomphalaria sudanica s.l.: immune gene diversification and highly polymorphic genomic regions in an important African vector of Schistosoma mansoni.

BACKGROUND: Control and elimination of schistosomiasis is an arduous task, with current strategies proving inadequate to break transmission. Exploration of genetic approaches to interrupt Schistosoma mansoni transmission, the causative agent for human intestinal schistosomiasis in sub-Saharan Africa and South America, has led to genomic research of the snail vector hosts of the genus Biomphalaria. Few complete genomic resources exist, with African Biomphalaria species being particularly underrepresented despite this being where the majority of S. mansoni infections occur. Here we generate and annotate the first genome assembly of Biomphalaria sudanica sensu lato, a species responsible for S. mansoni transmission in lake and marsh habitats of the African Rift Valley. Supported by whole-genome diversity data among five inbred lines, we describe orthologs of immune-relevant gene regions in the South American vector B. glabrata and present a bioinformatic pipeline to identify candidate novel pathogen recognition receptors (PRRs). RESULTS: De novo genome and transcriptome assembly of inbred B. sudanica originating from the shoreline of Lake Victoria (Kisumu, Kenya) resulted in a haploid genome size of ~ 944.2 Mb (6,728 fragments, N50 = 1.067 Mb), comprising 23,598 genes (BUSCO = 93.6% complete). The B. sudanica genome contains orthologues to all described immune genes/regions tied to protection against S. mansoni in B. glabrata, including the polymorphic transmembrane clusters (PTC1 and PTC2), RADres, and other loci. The B. sudanica PTC2 candidate immune genomic region contained many PRR-like genes across a much wider genomic region than has been shown in B. glabrata, as well as a large inversion between species. High levels of intra-species nucleotide diversity were seen in PTC2, as well as in regions linked to PTC1 and RADres orthologues. Immune related and putative PRR gene families were significantly over-represented in the sub-set of B. sudanica genes determined as hyperdiverse, including high extracellular diversity in transmembrane genes, which could be under pathogen-mediated balancing selection. However, no overall expansion in immunity related genes was seen in African compared to South American lineages. CONCLUSIONS: The B. sudanica genome and analyses presented here will facilitate future research in vector immune defense mechanisms against pathogens. This genomic/transcriptomic resource provides necessary data for the future development of molecular snail vector control/surveillance tools, facilitating schistosome transmission interruption mechanisms in Africa.

The genome and transcriptome of the snail Biomphalaria sudanica s.l.: Immune gene diversification and highly polymorphic genomic regions in an important African vector of Schistosoma mansoni.

Background: Control and elimination of schistosomiasis is an arduous task, with current strategies proving inadequate to break transmission. Exploration of genetic approaches to interrupt Schistosoma mansoni transmission, the causative agent for human intestinal schistosomiasis in sub-Saharan Africa and South America, has led to genomic research of the snail vector hosts of the genus Biomphalaria. Few complete genomic resources exist, with African Biomphalaria species being particularly underrepresented despite this being where the majority of S. mansoni infections occur. Here we generate and annotate the first genome assembly of Biomphalaria sudanica sensu lato, a species responsible for S. mansoni transmission in lake and marsh habitats of the African Rift Valley. Supported by whole-genome diversity data among five inbred lines, we describe orthologs of immune-relevant gene regions in the South American vector B. glabrata and present a bioinformatic pipeline to identify candidate novel pathogen recognition receptors (PRRs). Results: De novo genome and transcriptome assembly of inbred B. sudanica originating from the shoreline of Lake Victoria (Kisumu, Kenya) resulted in a haploid genome size of ~944.2 Mb (6732 fragments, N50=1.067 Mb), comprising 23,598 genes (BUSCO=93.6% complete). The B. sudanica genome contains orthologues to all described immune genes/regions tied to protection against S. mansoni in B. glabrata. The B. sudanica PTC2 candidate immune genomic region contained many PRR-like genes across a much wider genomic region than has been shown in B. glabrata, as well as a large inversion between species. High levels of intra-species nucleotide diversity were seen in PTC2, as well as in regions linked to PTC1 and RADres orthologues. Immune related and putative PRR gene families were significantly over-represented in the sub-set of B. sudanica genes determined as hyperdiverse, including high extracellular diversity in transmembrane genes, which could be under pathogen-mediated balancing selection. However, no overall expansion in immunity related genes were seen in African compared to South American lineages. Conclusions: The B. sudanica genome and analyses presented here will facilitate future research in vector immune defense mechanisms against pathogens. This genomic/transcriptomic resource provides necessary data for the future development of molecular snail vector control/surveillance tools, facilitating schistosome transmission interruption mechanisms in Africa.

Comparative Genomic Analysis of 31 Phytophthora Genomes Reveals Genome Plasticity and Horizontal Gene Transfer.

Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

PTC2 region genotypes counteract Biomphalaria glabrata population differences between M-line and BS90 in resistance to infection by Schistosoma mansoni.

Background: Biomphalaria glabrata is a snail intermediate host for Schistosoma mansoni, a trematode responsible for human schistosomiasis. BS90 is one of the most well studied strains of B. glabrata owing to its high resistance to infection by most strains of S. mansoni. An F2 mapping study from 1999 identified two RAPD markers that associated with what appeared to be single-locus, dominant resistance by the BS90 population relative to the susceptible M-line population. One marker cannot be mapped, but the other, OPM-04, maps to within 5 Mb of PTC2, a region we recently showed has a very large effect on resistance within another snail population challenged by the same strain of parasite (PR1). Here we tested the hypothesis that the PTC2 region contains the causal gene/s that explain the iconic resistance of BS90 snails. Methods: We used marker-assisted backcrossing to drive the BS90 version of the PTC2 region (+/-~1 Mb on either side) into an M-line (susceptible strain) genetic background, and the M-line version into a BS90 genetic background. We challenged the offspring with PR1-strain schistosomes and tested for effects of allelic variation in the PTC2 region in a common genetic background. Results: Relative to M-line haplotypes, the BS90 haplotype actually confers enhanced susceptibility. So we reject our original hypothesis. One possible explanation for our result was that the causal gene linked to OPM-04 is near, but not in the PTC2 block that we introgressed into each line. So we used an F2 cross to independently test the effects of the PTC2 and OPM-04 regions in a randomized genetic background. We confirmed that the BS90 haplotype confers increased susceptibility, and we see a similar, although non-significant effect at OPM-04. We discuss possible reasons why our results differed so dramatically from those of the 1999 study. We also present Pacbio assemblies of the PTC2 and flanking region in BS90 and M-line, compare with previously published PTC2 haplotypes, and discuss candidate genes that might be behind the enhanced susceptibility of the BS90 haplotype.

Offspring of first-generation hatchery steelhead trout (Oncorhynchus mykiss) grow faster in the hatchery than offspring of wild fish, but survive worse in the wild: Possible mechanisms for inadvertent domestication and fitness loss in hatchery salmon.

Salmonid fish raised in hatcheries often have lower fitness (number of returning adult offspring) than wild fish when both spawn in the wild. Body size at release from hatcheries is positively correlated with survival at sea. So one explanation for reduced fitness is that hatcheries inadvertently select for trait values that enhance growth rate under the unnatural environment of a hatchery, but that are maladaptive in the wild environment. A simple prediction of this hypothesis is that juveniles of hatchery origin should grow more quickly than fish of wild origin under hatchery conditions, but should have lower survival under wild conditions. We tested that hypothesis using multiple full sibling families of steelhead (Oncorhynchus mykiss) that were spawned using either two wild parents (WxW) or two first-generation hatchery (HxH) parents. Offspring from all the families were grown together under hatchery conditions and under semi-natural conditions in artificial streams. HxH families grew significantly faster in the hatchery, but had significantly lower survival in the streams. That we see this tradeoff after only a single generation of selection suggests that the traits involved are under very strong selection. We also considered one possible alteration to the hatchery environment that might reduce the intensity of selection among families in size at release. Here we tested whether reducing the fat content of hatchery feed would reduce the variance among families in body size. Although fish raised under a low-fat diet were slightly smaller, the variation among families in final size was unchanged. Thus, there is no evidence that reducing the fat content of hatchery feed would reduce the opportunity for selection among families on size at release.

RNA-Seq analysis of genes affected by Cyclophilin A/DIAGEOTROPICA (DGT) in tomato root development.

Cyclophilin A/DIAGEOTROPICA (DGT) has been linked to auxin-regulated development in tomato and appears to affect multiple developmental pathways. Loss of DGT function results in a pleiotropic phenotype that is strongest in the roots, including shortened roots with no lateral branching. Here, we present an RNA-Seq dataset comparing the gene expression profiles of wildtype ('Ailsa Craig') and dgt tissues from three spatially separated developmental stages of the tomato root tip, with three replicates for each tissue and genotype. We also identify differentially expressed genes, provide an initial comparison of genes affected in each genotype and tissue, and provide the pipeline used to analyze the data. Further analysis of this dataset can be used to gain insight into the effects of DGT on various root developmental pathways in tomato.

Clusters of polymorphic transmembrane genes control resistance to schistosomes in snail vectors.

Schistosomiasis is a debilitating parasitic disease infecting hundreds of millions of people. Schistosomes use aquatic snails as intermediate hosts. A promising avenue for disease control involves leveraging innate host mechanisms to reduce snail vectorial capacity. In a genome-wide association study of Biomphalaria glabrata snails, we identify genomic region PTC2 which exhibits the largest known correlation with susceptibility to parasite infection (>15 fold effect). Using new genome assemblies with substantially higher contiguity than the Biomphalaria reference genome, we show that PTC2 haplotypes are exceptionally divergent in structure and sequence. This variation includes multi-kilobase indels containing entire genes, and orthologs for which most amino acid residues are polymorphic. RNA-Seq annotation reveals that most of these genes encode single-pass transmembrane proteins, as seen in another resistance region in the same species. Such groups of hyperdiverse snail proteins may mediate host-parasite interaction at the cell surface, offering promising targets for blocking the transmission of schistosomiasis.

Schistosomiasis is a widespread parasitic disease, affecting over 200 million people in tropical countries. It is caused by schistosome worms, which are carried by freshwater snails. These snails release worm larvae into the water, where they can infect humans ­ for example, after bathing or swimming. Treatment options for schistosomiasis are limited. Eliminating the freshwater snails is one way to control the disease, but this is not always effective in the long term and the chemicals used can also harm other animals in the water. Another way to manage schistosomiasis could be to stop the worms from infecting their snail host by breaking the parasites' life cycle without killing the snails. It is already known that some snails are naturally resistant to infection by some strains of schistosomes. Since this immunity is also inherited by the offspring of resistant snails, there is likely a genetic mechanism behind it. However, very little else is known about any genes that might be involved. Tennessen et al. therefore set out to identify what genes were responsible for schistosome resistance and how they worked. The experiments used a large laboratory colony of snails, whose susceptibility to schistosome infection varied among individual animals. To determine the genes behind this variation, Tennessen et al. first searched for areas of DNA that also differed between the immune and infected snails. Comparing genetic sequences across over 1,000 snails revealed a distinct region of DNA that had a large effect on how likely they were to be infected. This section of DNA turned out to be highly diverse, with different snails carrying varying numbers and different forms of the genes within this region. Many of these genes appear to encode proteins found on the surface of snail cells, which could affect whether snails and worms can recognize each other when they come into contact. This in turn could determine whether or not the worms can infect their hosts. These results shed new light on how the snails that carry schistosomes may be able to resist infections. In the future, this knowledge could be key to controlling schistosomiasis, either by releasing genetically engineered, immune snails into the wild (thus making it harder for the parasites to reproduce) or by using the snails' mechanism of resistance to design better drug therapies.

Expansion and Divergence of Argonaute Genes in the Oomycete Genus Phytophthora.

Modulation of gene expression through RNA interference is well conserved in eukaryotes and is involved in many cellular processes. In the oomycete Phytophthora, research on the small RNA machinery and function has started to reveal potential roles in the pathogen, but much is still unknown. We examined Argonaute (AGO) homologs within oomycete genome sequences, especially among Phytophthora species, to gain a clearer understanding of the evolution of this well-conserved protein family. We identified AGO homologs across many representative oomycete and stramenopile species, and annotated representative homologs in P. sojae. Furthermore, we demonstrate variable transcript levels of all identified AGO homologs in comparison to previously identified Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) homologs. Our phylogenetic analysis further refines the relationship of the AGO homologs in oomycetes and identifies a conserved tandem duplication of AGO homologs in a subset of Phytophthora species.

A Targeted Capture Linkage Map Anchors the Genome of the Schistosomiasis Vector Snail, Biomphalaria glabrata.

The aquatic planorbid snail Biomphalaria glabrata is one of the most intensively-studied mollusks due to its role in the transmission of schistosomiasis. Its 916 Mb genome has recently been sequenced and annotated, but it remains poorly assembled. Here, we used targeted capture markers to map over 10,000 B. glabrata scaffolds in a linkage cross of 94 F1 offspring, generating 24 linkage groups (LGs). We added additional scaffolds to these LGs based on linkage disequilibrium (LD) analysis of targeted capture and whole-genome sequences of 96 unrelated snails. Our final linkage map consists of 18,613 scaffolds comprising 515 Mb, representing 56% of the genome and 75% of genic and nonrepetitive regions. There are 18 large (> 10 Mb) LGs, likely representing the expected 18 haploid chromosomes, and > 50% of the genome has been assigned to LGs of at least 17 Mb. Comparisons with other gastropod genomes reveal patterns of synteny and chromosomal rearrangements. Linkage relationships of key immune-relevant genes may help clarify snail-schistosome interactions. By focusing on linkage among genic and nonrepetitive regions, we have generated a useful resource for associating snail phenotypes with causal genes, even in the absence of a complete genome assembly. A similar approach could potentially improve numerous poorly-assembled genomes in other taxa. This map will facilitate future work on this host of a serious human parasite.

Schistosome infectivity in the snail, Biomphalaria glabrata, is partially dependent on the expression of Grctm6, a Guadeloupe Resistance Complex protein.

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis.

Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora.

Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed.

Genome-Wide Scan and Test of Candidate Genes in the Snail Biomphalaria glabrata Reveal New Locus Influencing Resistance to Schistosoma mansoni.

BACKGROUND: New strategies to combat the global scourge of schistosomiasis may be revealed by increased understanding of the mechanisms by which the obligate snail host can resist the schistosome parasite. However, few molecular markers linked to resistance have been identified and characterized in snails. METHODOLOGY/PRINCIPAL FINDINGS: Here we test six independent genetic loci for their influence on resistance to Schistosoma mansoni strain PR1 in the 13-16-R1 strain of the snail Biomphalaria glabrata. We first identify a genomic region, RADres, showing the highest differentiation between susceptible and resistant inbred lines among 1611 informative restriction-site associated DNA (RAD) markers, and show that it significantly influences resistance in an independent set of 439 outbred snails. The additive effect of each RADres resistance allele is 2-fold, similar to that of the previously identified resistance gene sod1. The data fit a model in which both loci contribute independently and additively to resistance, such that the odds of infection in homozygotes for the resistance alleles at both loci (13% infected) is 16-fold lower than the odds of infection in snails without any resistance alleles (70% infected). Genome-wide linkage disequilibrium is high, with both sod1 and RADres residing on haplotype blocks >2 Mb, and with other markers in each block also showing significant effects on resistance; thus the causal genes within these blocks remain to be demonstrated. Other candidate loci had no effect on resistance, including the Guadeloupe Resistance Complex and three genes (aif, infPhox, and prx1) with immunological roles and expression patterns tied to resistance, which must therefore be trans-regulated. CONCLUSIONS/SIGNIFICANCE: The loci RADres and sod1 both have strong effects on resistance to S. mansoni. Future approaches to control schistosomiasis may benefit from further efforts to characterize and harness this natural genetic variation.

Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs.

In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.

Reversion-reporter transgenes to analyze all six base-substitution pathways in Arabidopsis.

To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the ß-glucuronidase-encoding GUS transgene. Each allele reverts to GUS+ only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS- proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C→T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C→A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT→TC and TC→TT reversions were both high. With increased transgene copy number, spontaneous G:C→T:A reversions increased but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines.

Chromosomal rearrangements may complicate construction of Arabidopsis with multiple TDNA-insertion mutations. Here, crossing two lines homozygous for insertions in AtREV3 and AtPOLH (chromosomes I and V, respectively) and selfing F1 plants yielded non-Mendelian F2 genotype distributions: frequencies of +/++/+ and 1/1 2/2 progeny were only 0.42 and 0.25%. However, the normal development and fertility of double mutants showed AtPOLH-1 and AtREV3-2 gametes and 1/1 2/2 embryos to be fully viable. F2 distributions could be quantitatively predicted by assuming that F1 selfing produced inviable (1,2) and (+,+) gametophytes 86% of the time. Some defect intrinsic to the F1 selfing process itself thus appeared responsible. In selfing AtREV3 (+/2 ) single mutants, imaging of ovules and pollen showed arrest or abortion, respectively, of half of gametophytes; however, gametogenesis was normal in AtREV3 ( 2/2 ) homozygotes. These findings, taken together, suggested that T-DNA insertion at AtREV3 on chromosome I had caused a reciprocal I-V translocation. Spreads of meiosis I chromosomes in selfing AtREV3 (+/2 ) heterozygotes revealed the predicted cruciform four-chromosome structures, which fluorescence in situ hybridization showed to invariably include both translocated and normal chromosomes I and V. Sequencing of the two junctions of T-DNA with AtREV3 DNA and the two with gene At5g59920 suggested translocation via homologous recombination between independent inverted-repeat T-DNA insertions. Thus, when crosses between TDNA-insertion mutants yield anomalous progeny distributions, TDNA-linked translocations should be considered.

Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis.

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.

Reduction of stability of arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function.

Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers. To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines. AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility. To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants. Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ([+2], [-2], [+4], or [-4] bp). For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608. Two of four AtMSH2-RNAi plants showed similar microsatellite instability. In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested. This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity. We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots. Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats. Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue.