Publications

Long-term genetic studies of wild populations are very scarce, but are essential for connecting ecological and population genetics models, and for understanding the dynamics of biodiversity. We present a study of a wild wheat population sampled over a 36-year period at high spatial resolution. We genotyped 832 individuals from regular sampling along transects during the course of the experiment. Genotypes were clustered into ecological microhabitats over scales of tens of metres, and this clustering was remarkably stable over the 36 generations of the study. Simulations show that it is difficult to determine whether this spatial and temporal stability reflects extremely limited dispersal or fine-scale local adaptation to ecological parameters. Using a common-garden experiment, we showed that the genotypes found in distinct microhabitats differ phenotypically. Our results provide a rare insight into the population genetics of a natural population over a long monitoring period.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole-genome sequencing and molecular and cytological analyses supports a model in which a breakagefusionbridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

Bread wheat (Triticum aestivum, genome BBAADD) is a young hexaploid species formed only 8,500-9,000 years ago through hybridization between a domesticated free-Threshing tetraploid progenitor, genome BBAA, and Aegilops tauschii, the diploid donor of the D subgenome. Very soon after its formation, it spread globally from its cradle in the fertile crescent into new habitats and climates, to become a staple food of humanity. This extraordinary global expansion was probably enabled by allopolyploidy that accelerated genetic novelty through the acquisition of new traits, new intergenomic interactions, and buffering of mutations, and by the attractiveness of bread wheat's large, tasty, and nutritious grain with high baking quality. New genome sequences suggest that the elusive donor of the B subgenome is a distinct (unknown or extinct) species rather than a mosaic genome. We discuss the origin of the diploid and tetraploid progenitors of bread wheat and the conflicting genetic and archaeological evidence on where it was formed and which species was its free-Threshing tetraploid progenitor. Wheat experienced many environmental changes throughout its evolution, therefore, while it might adapt to current climatic changes, efforts are needed to better use and conserve the vast gene pool of wheat biodiversity on which our food security depends.

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium \u201cPlant Genome Engineering: From Lab to Field\u201d to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.

Recent developments in high-throughput sequencing and genome editing enable breeders to use a new tool kit for precision breeding. DNA double strand breaks can be targeted to desired genomic loci by custom-designed nucleases such as Zinc-finger nucleases, TALEN and CRISPR-Cas9. The repair products of induced breaks vary depending on the repair mechanisms and the repair template. These may be targeted small insertions or deletions, gene conversions or crossovers as well as gene replacement. In this chapter we review the concepts, achievements, opportunities and challenges of these new technologies.

Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of- function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: Three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans. Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.

Fruit-tree breeding is a lengthy process with many limitations. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. Precise genome editing could be a very useful supplementary tool for the improvement of crop plants. Various genome editing techniques including ZFNs, TALENs, and most recently CRISPR/Cas9 based approaches have been successfully employed for various crop plants including fruit trees. Previously, we used a ZFN-based approach to efficiently induce precise genome editing at specific genomic loci in fig. Furthermore, CRISPR/Cas9-based approaches hold great potential in genome editing due to their simplicity and were efficiently used in the last few years in several fruit trees. Efficient regeneration and transformation systems, a prerequisite for genome editing, were developed in several Ficus carica cultivars. Here we describe efficient CRISPR/Cas9 genome editing in fig. Transgenic fig lines carrying a mutated GUS construct (mGUS), were developed. mGUS editing, using the CRISPR/Cas9 system was confirmed by GUS staining and PCR. Figs are very attractive due to their nutritive and antioxidant properties. Yet, ripened fresh figs are highly perishable and require delicate postharvest handling. Characterizing the transcriptome of ripening fig fruits we found several genes that display altered expression during ripening and related to the ABA and ethylene hormonal processes. Currently we are using the CRISPR/Cas9 methodology to generate non-GM fig fruits that exhibit delayed ripening. The different application of CRISPR/Cas9 methodologies in fruit trees will be discussed.

This Letter of Intent describes LUXE (Laser Und XFEL Experiment), an experiment that aims to use the high-quality and high-energy electron beam of the European XFEL and a powerful laser. The scientific objective of the experiment is to study quantum electrodynamics processes in the regime of strong fields. High-energy electrons, accelerated by the European XFEL linear accelerator, and high-energy photons, produced via Bremsstrahlung of those beam electrons, colliding with a laser beam shall experience an electric field up to three times larger than the Schwinger critical field (the field at which the vacuum itself is expected to become unstable and spark with spontaneous creation of electron-positron pairs) and access a new regime of quantum physics. The processes to be investigated, which include nonlinear Compton scattering and nonlinear Breit-Wheeler pair production, are relevant to a variety of phenomena in Nature, e.g. in the areas of astrophysics and collider physics and complement recent results in atomic physics. The setup requires in particular the extraction of a minute fraction of the electron bunches from the European XFEL accelerator, the installation of a powerful laser with sophisticated diagnostics, and an array of precision detectors optimised to measure electrons, positrons and photons. Physics sensitivity projections based on simulations are also provided.

AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene-targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52-1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over-expression of this variant increased HR in the nucleus, compared with the native AtRad52-1A: it increased intra-chromosomal recombination and synthesis-dependent strand-annealing HR repair rates; but conversely, it repressed the single-strand annealing pathway. The effect of AtRad52-1A over-expression on gene-targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene-targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52-1A over-expression and target/donor-specific RNAi. This suggests that sequence-specific small RNAs might be involved in AtRad52-1A-mediated HR.

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA.

Background: The merging of genomes in inter-specific hybrids can result in novel phenotypes, including increased growth rate and biomass yield, a phenomenon known as heterosis. Heterosis is typically viewed as the opposite of hybrid incompatibility. In this view, the superior performance of the hybrid is attributed to heterozygote combinations that compensate for deleterious mutations accumulating in each individual genome, or lead to new, over-dominating interactions with improved performance. Still, only fragmented knowledge is available on genes and processes contributing to heterosis.Results: We describe a budding yeast hybrid that grows faster than both its parents under different environments. Phenotypically, the hybrid progresses more rapidly through cell cycle checkpoints, relieves the repression of respiration in fast growing conditions, does not slow down its growth when presented with ethanol stress, and shows increased signs of DNA damage. A systematic genetic screen identified hundreds of S. cerevisiae alleles whose deletion reduced growth of the hybrid. These growth-affecting alleles were condition-dependent, and differed greatly from alleles that reduced the growth of the S. cerevisiae parent.Conclusions: Our results define a budding yeast hybrid that is perturbed in multiple regulatory processes but still shows a clear growth heterosis. We propose that heterosis results from incompatibilities that perturb regulatory mechanisms, which evolved to protect cells against damage or prepare them for future challenges by limiting cell growth.

The mechanism for T-DNA integration, a critical step of Agrobacterium-mediated transgenesis, remains poorly understood. Now, a study based on mutant analysis shows that Pol θ controls T-DNA integration and generates error-prone footprints at integration sites.

Wheat straw is a potential source of feedstock for biofuel production that does not compete with food. We have screened 48 wheat lines from a collection representing a broad range of the biodiversity of wild and domestic wheat. Wheat straw was fractionated into water-soluble and nonsoluble fractions. In the water-soluble fraction (WSF), we found a broad variation in the concentration of free soluble sugars (FSS) and a narrow variation in starch. The FSS fraction could reach levels of reducing sugars as high as 130 g sugar/kg of straw. The analysis of the FSS by ion chromatography indicated that fructose and glucose were the major sugar monomers in this fraction. The composition of the nonsoluble cell wall fraction was determined by both pyrolysis and direct chemical analysis. These analyses showed a limited variation in the lignin or the cellulose fraction. There was a significant degree of variation among wheat lines in the enzymatic saccharification of the straw, following acid pretreatment. Interestingly, the straw from wild wheat had the highest degree of saccharification compared to domestic lines. These findings are of interest for the biofuel industry because they mean that wheat lines can be developed in which a significant amount of free soluble sugars can be easily extracted from straw without the need for costly pretreatment and enzymatic deconstruction. Moreover, the high FSS trait might be combined with the high enzymatic saccharification trait suggesting that wheat lines can be developed with a straw composition better adapted for biofuel production.

Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

We speculate that multicopy transposons, carrying both fitness and unfitness genes, can provide new positive and negative selection options to intractable weed problems. Multicopy transposons rapidly disseminate through populations, appearing in approximately 100% of progeny, unlike nuclear transgenes, which appear in a proportion of segregating populations. Different unfitness transgenes and modes of propagation will be appropriate for different cases: (1) outcrossing Amaranthus spp. (that evolved resistances to major herbicides); (2) Lolium spp., important pasture grasses, yet herbicide-resistant weeds in crops; (3) rice (Oryza sativa), often infested with feral weedy rice, which interbreeds with the crop; and (4) self-compatible sorghum (Sorghum bicolor), which readily crosses with conspecific shattercane and with allotetraploid johnsongrass (Sorghum halepense). The speculated outcome of these scenarios is to generate weed populations that contain the unfitness gene and thus are easily controllable. Unfitness genes can be under chemically or environmentally inducible promoters, activated after gene dissemination, or under constitutive promoters where the gene function is utilized only at special times (e.g. sensitivity to an herbicide). The transposons can be vectored to the weeds by introgression from the crop (in rice, sorghum, and Lolium spp.) or from planted engineered weed (Amaranthus spp.) using a gene conferring the degradation of a no longer widely used herbicide, especially in tandem with an herbicide-resistant gene that kills all nonhybrids, facilitating the rapid dissemination of the multicopy transposons in a weedy population.

Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase® reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases' cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established 'QQR' zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.

The importance of hybridization and polyploidization in wheat speciation has been recognized for close to a century (Sakamura 1918; Kihara 1919, 1924, 1954; Percival 1921; Sax 1927). Following these pioneering works, it quickly became apparent that polyploid wheats are not the sum of their constituent genomes. This is not unexpected because the nascent hybrids/polyploids are equipped with a complex set of regulatory elements and of copy number variation that originate from two or more divergent genomes and that generate novel types of interactions and dosage effects. Moreover, they have to adjust at the cytological level, at the level of gene expression, and at the protein level. They also have to maintain genome stability through the regulation of meiotic pairing and recombination, the orchestration of cell division, and the silencing of transposons. The recent studies described here provide an impressive account with regard to the extent and the rapid time course at which a new genetic variant was established upon hybridization and polyploidization. We describe here the current knowledge on the changes that occurred in the wheat genome upon allopolyploidization, starting from the early evolutionary and cytological studies to the recent genomic analyses.

The evolvement of duplicated gene loci in allopolyploid plants has become the subject of intensive studies. Most duplicated genes remain active in neoallopolyploids contributing either to a favourable effect of an extra gene dosage or to the build-up of positive inter-genomic interactions when genes or regulation factors on homoeologous chromosomes are divergent. However, in a small number of loci (about 10%), genes of only one genome are active, while the homoeoalleles on the other genome(s) are either eliminated or partially or completely suppressed by genetic or epigenetic means. For several traits, the retention of controlling genes is not random, favouring one genome over the other(s). Such genomic asymmetry is manifested in allopolyploid wheat by the control of various morphological and agronomical traits, in the production of rRNA and storage proteins, and in interaction with pathogens. It is suggested that the process of cytological diploidization leading to exclusive intra-genomic meiotic pairing and, consequently, to complete avoidance of inter-genomic recombination, has two contrasting effects. Firstly, it provides a means for the fixation of positive heterotic inter-genomic interactions and also maintains genomic asymmetry resulting from loss or silencing of genes. The possible mechanisms and evolutionary advantages of genomic asymmetry are discussed.

Meiotic recombination is tightly regulated by cis- and trans-acting factors. Although DNA methylation and chromatin remodeling affect chromosome structure, their impact on meiotic recombination is not well understood. To study the effect of DNA methylation on the landscape of chromosomal recombination, we analyzed meiotic recombination in the decreased DNA methylation 1 (ddm1) mutant. DDM1 is a SWI2/SNF2-like chromatin-remodeling protein necessary for DNA methylation and heterochromatin maintenance in Arabidopsis thaliana. The rate of meiotic recombination between markers located in euchromatic regions was significantly higher in both heterozygous (DDM1/ddm1) and homozygous (ddm1/ddm1) backgrounds than in WT plants. The effect on recombination was similar for both male and female meiocytes. Contrary to expectations, ddm1 had no effect on the number of crossovers between markers in heterochromatic pericentric regions that underwent demethylation. These results are surprising, because the pericentromeric regions are hypermethylated and were expected to be the regions most affected by demethylation. Thus, DDM1 loss of function may trigger changes that enhance meiotic recombination in euchromatin regions but are not sufficient to induce the same events in heterochromatic segments. This work uncovers the repressive role of methylation on meiotic recombination in euchromatic regions and suggests that additional factors may have a role in controlling the suppression of recombination in heterochromatin.

Targeted modification of the genome is an important genetic tool, which can be achieved via homologous, non-homologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.

Background: Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination.Results: Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations.Conclusions: The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

Homologous recombination (HR) is a central cellular process involved in many aspects of genome maintenance such as DNA repair, replication, telomere maintenance, and meiotic chromosomal segregation. HR is highly conserved among eukaryotes, contributing to genome stability as well as to the generation of genetic diversity. It has been intensively studied, for almost a century, in plants and in other organisms. In this antireview, rather than reviewing existing knowledge, we wish to underline the many open questions in plant HR. We will discuss the following issues: how do we define homology and how the degree of homology affects HR? Are there any plant-specific HR qualities, how extensive is functional conservation and did HR proteins acquire new functions? How efficient is HR in plants and what are the cis and the trans factors that regulate it? Finally, we will give the prospects for enhancing the rates of gene targeting and meiotic HR for plant breeding purposes.

In higher plants, the plastidial NADH dehydrogenase (Ndh) complex supports nonphotochemical electron fluxes from stromal electron donors to plastoquinones. Ndh functions in chloroplasts are not clearly established; however, its activity was linked to the prevention of the overreduction of stroma, especially under stress conditions. Here, we show by the characterization of Orr^Ds, a dominant transposon-tagged tomato (Solanum lycopersicum) mutant deficient in the NDH-M subunit, that this complex is also essential for the fruit ripening process. Alteration to the NDH complex in fruit changed the climacteric, ripening-associated metabolites and transcripts as well as fruit shelf life. Metabolic processes in chromoplasts of ripening tomato fruit were affected in Orr^Ds, as mutant fruit were yellow-orange and accumulated substantially less total carotenoids, mainly β-carotene and lutein. The changes in carotenoids were largely influenced by environmental conditions and accompanied by modifications in levels of other fruit antioxidants, namely, flavonoids and tocopherols. In contrast with the pigmentation phenotype in mature mutant fruit, Orr^Ds leaves and green fruits did not display a visible phenotype but exhibited reduced Ndh complex quantity and activity. This study therefore paves the way for further studies on the role of electron transport and redox reactions in the regulation of fruit ripening and its associated metabolism

During evolution, novel phenotypes emerge through changes in gene expression, but the genetic basis is poorly understood. We compared the allele-specific expression of two yeast species and their hybrid, which allowed us to distinguish changes in regulatory sequences of the gene itself (cis) from changes in upstream regulatory factors (trans). Expression divergence between species was generally due to changes in cis. Divergence in trans reflected a differential response to the environment and explained the tendency of certain genes to diverge rapidly. Hybrid-specific expression, deviating from the parental range, occurred through novel cis-trans interactions or, more often, through modified trans regulation associated with environmental sensing. These results provide insights on the regulatory changes in cis and trans during the divergence of species and upon hybridization.

Phytoliths are abundant in many archaeological sites, and can provide information on past vegetation. Very few analyses of their chemical composition have been made. Our measurements of the δ¹³C composition of modern wheat phytoliths suggest the presence of relatively large amounts of sugars and/or proteins in the water-soluble fraction, and lipids in the insoluble fraction. Other experimental approaches demonstrate that modern wheat phytoliths contain large quantities of glyco-conjugated proteins in a degraded state. One open question is whether or not phytoliths contain original DNA of the mother plant. Extracting protected ancient DNA from phytoliths would open many opportunities for progress in archaeobotanical studies. In order to address this question, we developed a method to dissolve phytoliths under conditions that do not degrade naked DNA, and showed that only a minimal amount of DNA was lost during the procedure. A hypersensitive assay did not, however, detect any DNA in extracts of phytoliths from an unburned phytolith-rich layer in Iron-Age sediments from Tel Dor, Israel. Extractions from modern phytolith samples of wheat also failed to provide any indication of DNA. We conclude that DNA is absent or not routinely recoverable in a random assembly of siliceous phytoliths.

Many plant roots acquire inorganic phosphate (Pi) from soils directly through the root-soil interface via high-affinity Pi transporters and/or through symbiotic associations between the cortical cells and arbuscular mycorrhizal fungi. In tomato, three phosphate transporters (LePT3, LePT4, and LePT5) are up-regulated upon colonization by arbuscular mycorrhizal fungi. In this study, the role of LePT4 in tomato is elucidated by molecular and physiological characterizations of a loss-of-function mutant lept4. In the absence of mycorrhizal infection and under solution-Pi concentrations (Cp) of 0.05 mM and 0.5 mM, the mutant exhibited severe Pi-deficiency symptoms which were associated with significantly lower Pi uptake as compared with that of the wild type. However, at a Cp of 5 mM, lept4 grew better than the wild type. Mycorrhizal infection at a Cp of 0.05 mM resulted in a significant increase in the transcripts of LePT4 in the wild type and a concomitant 2-fold increase in Pi uptake. Although upon mycorrhizal infection, lept4 also exhibited an increased Pi uptake, it was significantly lower than that of the wild type. Under a Cp of 1 mM and in the absence of mycorrhizal infection, LePT4 expression was suppressed in the wild type and a mutation in this gene resulted in a slight reduction in total Pi uptake. These data highlight the pivotal role of LePT4 in mycorrhizal-mediated Pi uptake in tomato, and show that this function may not be fully compensated by other members of the family. Characterization of the mycorrhiza-associated Pi transporter lept4 mutant, along with expression analysis of LePT3, provides evidence for the different routes of mycorrhiza-mediated Pi uptake in plants.

During homologous recombination (HR), a heteroduplex DNA is formed as a consequence of strand invasion. When the two homologous strands differ in sequence, a mismatch is generated. Earlier studies showed that mismatched heteroduplex often triggers abortion of recombination and that a pivotal component of this pathway is the mismatch repair Msh2 protein. In this study, we analysed the roles of AtMSH2 in suppression of recombination in Arabidopsis. We report that AtMSH2 has a broad range of anti-recombination effects: it suppresses recombination between divergent direct repeats in somatic cells or between homologues from different ecotypes during meiosis. This is the first example of a plant gene that affects HR as a function of sequence divergence and that has an anti-recombination meiotic effect. We discuss the implications of these results for plant improvement by gene transfer across species.

Gene targeting, which is homologous recombination-mediated integration of an extra-chromosomal DNA segment into a chromosomal target sequence, enables the precise disruption or replacement of any gene. Despite its value as a molecular genetic tool, gene targeting remains an inefficient technology in most species. We report that expression of the yeast RAD54 gene, a member of the SWI2/SNF2 chromatin remodeling gene family, enhances gene targeting in Arabidopsis by one to two orders of magnitude, from 10^-4 to 10^-3 in WT plants to 10^-2 to 10^-1. We show that integration events, detected with an assay based on the use of a fluorescent seed marker, are precise and germinally transmitted. These findings suggest that chromatin remodeling is rate-limiting for gene targeting in plants and improves the prospects for using gene targeting for the precise modification of plant genomes.

Solanaceous species are among the >200 000 plant species worldwide forming a mycorrhiza, that is, a root living in symbiosis with soil-borne arbuscular-mycorrhizal (AM) fungi. An important parameter of this symbiosis, which is vital for ecosystem productivity, agriculture, and horticulture, is the transfer of phosphate (Pi) from the AM fungus to the plant, facilitated by plasma membrane-spanning Pi transporter proteins. The first mycorrhiza-specific plant Pi transporter to be identified, was StPT3 from potato [Nature 414 (2004) 462]. Here, we describe novel Pi transporters from the solanaceous species tomato, LePT4, and its orthologue StPT4 from potato, both being members of the Pht1 family of plant Pi transporters. Phylogenetic tree analysis demonstrates clustering of both LePT4 and StPT4 with the mycorrhiza-specific Pi transporter from Medicago truncatula [Plant Cell, 14 (2002) 2413] and rice [Proc. Natl Acad. Sci. USA, 99 (2002) 13324], respectively, but not with StPT3, indicating that two non-orthologous mycorrhiza-responsive genes encoding Pi transporters are co-expressed in the Solanaceae. The cloned promoter regions from both genes, LePT4 and StPT4, exhibit a high degree of sequence identity and were shown to direct expression exclusively in colonized cells when fused to the GUS reporter gene, in accordance with the abundance of LePT4 and StPT4 transcripts in mycorrhized roots. Furthermore, extensive sequencing of StPT4-like clones and subsequent expression analysis in potato and tomato revealed the presence of a close paralogue of StPT4 and LePT4, named StPT5 and LePT5, respectively, representing a third Pi transport system in solanaceous species which is upregulated upon AM fungal colonization of roots. Knock out of LePT4 in the tomato cv. MicroTom indicated considerable redundancy between LePT4 and other Pi transporters in tomato.

Recent studies have shown that allopolyploidy accelerates genome evolution in wheat in two ways: (1) allopolyploidization triggers rapid genome changes (revolutionary changes) through the instantaneous generation of a variety of cardinal genetic and epigenetic alterations, and (2) the allopolyploid condition facilitates sporadic genomic changes during the life of the species (evolutionary changes) that are not attainable at the diploid level. The revolutionary changes comprise (1) non-random elimination of coding and non-coding DNA sequences, (2) epigenetic changes such as DNA methylation of coding and non-coding DNA leading, among others, to gene silencing, (3) activation of genes and retroelements which in tuni alters the expression of adjacent genes. These highly reproducible changes occur in the F₁ hybrids or in the first generation(s) of the nascent allopolyploids and were similar to those that occurred twice in nature: first in the formation of allotetraploid wheat (∼ 0.5 million years ago) and second in the formation of hexaploid wheat (∼ 10,000 years ago). Elimination of non-coding sequences from one of the two homoeologous pairs in tetraploids and from two homoeologous pairs in hexaploids, augments the differentiation of homoeologous chromosomes at the polyploid level, thus providing the physical basis for the diploid-like meiotic behavior of allopolyploid wheat. Regulation of gene expression may lead to improved inter-genomic interactions. Gene inactivation brings about rapid diploidization while activation of genes through demethylation or through transcriptional activation of retroelements altering the expression of adjacent genes, leads to novel expression patterns. The evolutionary changes comprise (1) horizontal intergenomic transfer of chromosome segments between the constituent genomes, (2) production of recombinant genomes through hybridization and introgression between different allopolyploid species or, more seldom, between allopolyploids and diploids, and (3) mutations. These phenomena, emphasizing the plasticity of the genome with regards to both structure and function, might improve the adaptability of the newly formed allopolyploids and facilitate their rapid and successful establishment in nature.

It is well established that sequence divergence has an inhibitory effect on homologous recombination. However, a detailed analysis of this relationship is missing for most higher eukaryotes. We have measured the rate of somatic recombination between direct repeats as a function of the number, type, and position of divergent nucleotides in Arabidopsis. We show that a minor divergence level of 0.16% (one mutation in otherwise identical 618 bp) has a profound effect, decreasing the recombination rate approximately threefold. A further increase in the divergence level affects the recombination rate to a smaller extent until a "divergence saturation" effect is reached at relatively low levels of divergence (∼0.5%). The type of mismatched nucleotide does not affect recombination rates. The decrease in the rate of recombination caused by a single mismatch was not affected by the position of the mismatch along the repeat. This suggests that most recombination intermediate tracts contain a mismatch and thus are as long as the full length of the 618-bp repeats. Finally, we could deduce an antirecombination efficiency of ∼66% for the first mismatch in the repeat. Altogether, this work shows some degree of conservation across kingdoms when compared to previous reports in yeast; it also provides new insight into the effect of sequence divergence on homologous recombination.

Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition-function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase β-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C₃₀, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.

Two tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 μM gibberellin_{4 + 7} were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin_{4 + 7} did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N₂. The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.

Retrotransposons are a principal component of most eukaryotic genomes, representing roughly 40% of the human genome and 50-80% of some grass genomes. They are usually transcriptionally silent but can be activated under certain stresses. Despite their considerable contribution to genome structure, their impact on the expression of adjacent genes is not well understood. The steady-state transcript levels originating from Wis 2-1A retrotransposons are much higher in newly synthesized wheat amphiploids (two or more diverged genomes in the same nucleus). On activation, both Wis 2-1A long terminal repeats drive the readout synthesis of new transcripts from adjacent sequences including the antisense or sense strands of known genes. Here we report that activation of these antisense or sense transcripts is associated with silencing or activation of the corresponding genes, respectively. These data, together with the abundance of retrotransposons in genomes and their ability to be activated by various signals, support the view of transposons as potential controlling elements.Errata: In the version of this article initially published,
Figure 2 was inappropriately enhanced. The original data have been restored to
the figure. The authors state that all the display items in this publication
are previously unpublished work and accurate representations of the experiments
described. The error has been corrected in the HTML and PDF versions of the
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Recent studies have shown that speciation through allopolyploidy, i.e., inter-specific or inter-generic hybridization followed by chromosome doubling, is accompanied by a variety of rapid cardinal genetic and epigenetic changes. This paper reviews our studies on the effect of allopolyploidization on several low-copy, non-coding sequences that exist in all the diploid species of the tribe Triticeae, including the progenitors of polyploid wheat, but in polyploid wheat they occur in only one genome, either in one homologous pair (chromosome-specific sequences) or in several pairs of the same genome (genome-specific sequences). Rapid elimination of these sequences from one genome is a general phenomenon in newly synthesized allopolyploids. Elimination was a nonrandom, reproducible event whose direction was determined by the genomic combination of the amphiploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. This elimination augmented the differentiation of homeologous chromosomes (partially homologous chromosomes of the different genomes) at the polyploid level, thus providing the physical basis for the diploid-like meiotic behavior characterizing polyploid wheat. This pattern of pairing prevents intergenomic recombination, and consequently, ensures full fertility, disomic inheritance, and permanent heterosis between alleles of different genomes (homeoalleles). Accordingly, rapid elimination of these sequences improves the fitness of newly formed allopolyploids, facilitating their rapid establishment in nature as new successful species.

Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields, Regimes to achieve their efficient elimination. mostly through site-specific recombifiation or transposition, are being developed.

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc^- phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc^- characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.

The maize transposon Activator (Ac) was the first mobile DNA element to be discovered. Since then, other elements were found that share similarity to Ac, suggesting that it belongs to a transposon superfamily named hAT after hobo from Drosophila, Ac from maize, and Tam3 from snapdragon. We addressed the structure and evolution of hAT elements by developing new tools for transposon mining and searching the public sequence databases for the hallmarks of hAT elements, namely the transposase and short terminal inverted repeats (TIRs) flanked by 8-bp host duplications. We found 147 hAT-related sequences in plants, animals, and fungi. Six conserved blocks could be identified in the transposase of most hAT elements. A total of 41 hAT sequences were flanked by TIRs and 8-bp host duplications and, out of these, 34 sequences had TIRs similar to the consensus determined in this work, suggesting that they are active or recently active transposons. Phylogenetic analysis and clustering of hAT sequences suggest that the hAT superfamily is very ancient, probably predating the plant-fungi-animal separation, and that, unlike previously proposed, there is no evidence that horizontal gene transfer was involved in the evolution of hAT elements.

We have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six- to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non-homologous end joining as efficiently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show significant resistance to gamma-irradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up-regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.

DNA double-strand breaks (DSBs) lead to serious genomic deficiencies if left unrepaired. Recent studies have provided new insight into the mechanisms, the mutants and the genes involved in DSB repair in plants. These studies indicate that high fidelity DSB repair via homologous recombination is less frequent than non-homologous end-joining. Interestingly, non-homologous end-joining in plants is more error-prone than in other species, being associated with various rearrangements that often include deletions and insertions (filler DNA). We discuss the mechanism of error-prone DSB repair, which is probably an important driving force in plant genome evolution.

To study genome evolution in allopolyploid plants, we analyzed polyploid wheats and their diploid progenitors for the occurrence of 16 low-copy chromosome- or genome-specific sequences isolated from hexaploid wheat. Based on their occurrence in the diploid species, we classified the sequences into two groups: group I, found in only one of the three diploid progenitors of hexaploid wheat, and group II, found in all three diploid progenitors. The absence of group II sequences from one genome of tetraploid wheat and from two genomes of hexaploid wheat indicates their specific elimination from these genomes at the polyploid level. Analysis of a newly synthesized amphiploid, having a genomic constitution analogous to that of hexaploid wheat, revealed a pattern of sequence elimination similar to the one found in hexaploid wheat. Apparently, speciation through allopolyploidy is accompanied by a rapid, nonrandom elimination of specific, low-copy, probably noncoding DNA sequences at the early stages of allopolyploidization, resulting in further divergence of homoeologous chromosomes (partially homologous chromosomes of different genomes carrying the same order of gene loci). We suggest that such genomic changes may provide the physical basis for the diploid-like meiotic behavior of polyploid wheat.

Double strand DNA breaks in plants are primarily repaired via non-homologous end joining. However, little is known about the molecular events underlying this process. We have studied non-homologous end joining of linearized plasmid DNA with different termini configurations following transformation into tobacco cells. A variety of sequences were found at novel end junctions. Joining with no sequence alterations was rare. In most cases, deletions were found at both ends, and rejoining usually occurred at short repeats. A distinct feature of plant junctions was the presence of relatively large, up to 1.2 kb long, insertions (filler DNA), in ~ 30% of the analyzed clones. The filler DNA originated either from internal regions of the plasmid or from tobacco genomic DNA. Some insertions had a complex structure consisting of several reshuffled plasmid-related regions. These data suggest that double strand break repair in plants involves extensive end degradation, DNA synthesis following invasion of ectopic templates and multiple template switches. Such a mechanism is reminiscent of the synthesis-dependent recombination in bacteriophage T4. It can also explain the frequent 'DNA scrambling' associated with illegitimate recombination in plants.

The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby β- Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5' end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5' end of the GUS open reading frame and had a deletion at the 3' end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double- strand breaks is a potentially important factor in plant genome evolution.

Post-germinative proliferation of cells was studied in cotyledons of Nicotiana tabucum L., Petunia hybrida Vilm. and Arabidopsis thaliana (L.) Heynh. Patterns of cell divisions after germination were characterized by clonal analysis in cotyledons of N. tabacum. The fate of initial cells, which are formed by the end of embryogenesis, was quite variable: cells could undergo between one to seven, and most often, between three to five anticlinal divisions after germination. Sector shape suggested that there were more divisions in length than in width, particularly at the periphery of the cotyledon. The boundaries of clones generated by irradiation of mature seeds did not intersect the midvein, and in most cases, did not intersect lateral veins. The time course of cell divisions during post- germinative development was analyzed cytologically in cotyledons of N. tabacum and P. hybrida. No divisions were detected up to the second day after sowing (DAS), when the radicle emerged. Cotyledon cells started to divide at a rapid rate between 2 and 3 DAS, reaching a mitotic index of about 2% at 3 4 DAS. A rapid decline followed the peak, and no divisions were detected 6 7 DAS. Similarities between leaf and cotyledon development are discussed. In addition, we show that divisions in cotyledons of N. tabacum and A. thaliana chlorophyll mutants can be exploited for a quick and sensitive bioassay from which the effects of various mutagens and DNA repair genes can be assessed.

A mathematical model and a computer simulation were used to study PCR specificity. The model describes the occurrences of non-targeted PCR products formed through random primer-template interactions. The PCR simulation scans DNA sequence databases with primers pairs. According to the model prediction, PCR with complex templates should rarely yield non-targeted products under typical reaction conditions. This is surprising as such products are often amplified in real PCR under conditions optimized for stringency. The causes for this 'PCR paradox' were investigated by comparing the model predictions with simulation results, We found that deviations from randomness in sequences from real genomes could not explain the frequent occurrence of non-targeted products in real PCR. The most likely explanation to the 'PCR paradox' is a relatively high tolerance of PCR to mismatches. The model also predicts that mismatch tolerance has the strongest effect on the number of non-targeted products, followed by primer length, template size and product size limit. The model and the simulation can be utilized for PCR studies, primer design and probing DNA uniqueness and randomness.

Variation in the electrophoretic pattern of the high-molecular-weight (HMW) glutenin subunits was studied in the Ammiad population of wild tetraploid wheat, Triticum turgidum var. dicoccoides (genome AABB), during a 5-year period (1983-4 to 1987-8). These storage proteins were analysed following one-dimensional sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis (SDS PAGE), using seeds collected annually from individual plants at 249 defined sampling sites distributed in 11 habitats. Since plants did mt grow at all the sampling points each year, 1108 accessions were analysed altogether. The population was found to be highly polymorphic: the HMW glutenin loci of genome A, Glu-Al-1 and Glu-Al-2, had four and two alleles. respectively, and those of genome B. Glu-Bl-1 and Glu-Bl-2, had five and seven alleles, respectively. The A-genome alleles appeared in 4 combinations, and the B-genome alleles appeared in 12 combinations. There were 18 intergenomic combinations (A and B genotypes), some of which were very rare while others were abundant and distributed along transects in clusters. The spatial distribution of these genotypes was nonrandom, with each of the 11 habitats characterized by different genotype frequencies. Yearly changes in genotypes, mostly occurring in the last 2 years of the study, had little effect on the total frequencies of die various genotypes. A high affinity was found between specific HMW glutenin genotypes and certain habitats. This affinity may have resulted from a random fixation of specific genotypes in different habitats (founder effect) or, alternatively, from natural selection, thus indicating either linkage between HMW glutenin alleles and adaptive genes (hitchhiking effect) or fitness of some of these allele combinations to specific micro-environments.

Grain protein percentage (GPP) was studied in 910 accessions of the wild tetraploid wheat, Triticum turgidum var. dicoccoides, collected from 22 populations representing different ecogeographical conditions in Israel. High values of GPP were found, ranging from 19.7% to 28.0% for population means, and from 14.1% to 35.1% for single accessions. Marginal populations had usually lower GPP and smaller intra-population variation than central ones. Repeated sampling of some central populations for four consecutive years revealed relatively large intra-population fluctuations. A high and significant genetic component of variation was found within and between populations by a nested analysis of variance in two nurseries. However, the regression coefficients of parents vs. offsprings were relatively low, indicating a smaller genetic component of variation which may be accounted for by a significant genotype × environment interaction. No correlation was found between GPP and ecological factors, except for soil type: accessions growing on terra-rossa had higher GPP than those growing on basaltic soil. Accessions with black glumes, or with glabrous auricles, or with large grains exhibited high GPP values. A strategy for collecting accessions with high GPP is presented, and the potential use of high GPP genotypes in breeding programs is discussed.

The mode of inheritance, linkage groups, and chromosomal location of 23 morphological and 4 biochemical traits were characterized in the wild tetraploid emmer wheat, Triticum turgidum var. dicoccoides. These traits were described and their mode of inheritance was determined by their segregation in four F₂ populations derived from crosses between four var. dicoccoides accessions and a tetraploid durum cultivar. Linkage groups among the genes encoding for these traits were determined or postulated, and their chromosomal location was deduced by linkage to previously located genes. The genetic control of the following traits was characterized and is first reported here: black keel; hairy leaf sheath; hairy auricles; hairy rachilla; hairy kernel brush; obtuse flag leaf; and curved neck/peduncle. The linkage data indicated that developmentally-related genes tended to occur in clusters.

Polymorphism of the high molecular weight glutenin subunits was studied in 456 accessions of the wild wheat Triticum turgidum var. dicoccoides (2n = 4x = 28; genomes AABB), originating from 21 populations in Israel. A total of 50 different SDS PAGE migration patterns were observed, resulting from the combinations of 15 subunit patterns of the A genome and 24 subunit patterns of the B genome. Most migration patterns consisted of five subunits, varying between three and six. The migration patterns of the A genome had zero to three subunitstwo being most common. The apparent molecular weights (MWs) of the slowest migrating subunit (114, 000 to 103, 500) and of the next in rate of migration (106, 000 to 96, 000) were highly correlated (r = 0·97). Also, both subunits were either present (in most accessions) or absent. In 82.3 per cent of the accessions, the third subunit (MW 76, 000 to 71, 500) was absent, while in 16.9 per cent of the accessions all three subunits of the A genome were absent. The migration patterns of the B genome had one to three subunitsthree being most common. The slowest migrating subunit (99, 500 to 93, 000) was present in almost all cases (99·3 per cent). The MWs of the next two subunits (90, 500 to 82, 000 and 86, 000 to 78, 000, respectively) were highly correlated (r = 0·95). Also, either both subunits were present, as in most cases (94·4 per cent), or absent (5·6 per cent). A nomenclature for the genes encoding for the HMW glutenins is proposed based on the following model: The three subunit groups controlled by each genome are encoded by two genes. In genome A, one gene (Glu-A1-1), with 12 alleles, encodes for the two correlated subunit groups 1Ax and 1Ax; the other gene (Glu-A1-2), with 3 alleles, encodes for the fast-migrating subunit group (1Ay). In genome B, one gene (Glu-B1-1), with 8 alleles, encodes for the slow-migrating subunit group (1Bx), and the other gene (Glu-B1-2), with 10 alleles, encodes for the two correlated subunit groups, 1By and 1By. The polymorphism of the HMW glutenin genes found in var. dicoccoides is much higher than that of cultivated wheats as well as of genes coding for enzymes in var. dicoccoides.

Polymorphism of high molecular weight (HMW) glutenin subunits in 466 accessions of the wild tetraploid wheat Triticum turgidum var. dicoccoides in Israel was characterized with regard to the ecogeographical distribution of the HMW glutenin alleles, both between and within 22 populations, and along transects in a single population. While some populations were monomorphic for all the HMW glutenin loci, namely, Glu-A1-1, Glu-A1-2, Glu-B1-1 and Glu-B1-2, others contained up to four alleles per locus. Intrapopulation variability could be predicted by the geographical distribution: marginal populations tended to be more uniform than those at the center of distribution. The various HMW glutenin alleles tended to be clustered, both at a regional level and within a single population along transects of collection. It is suggested that this clustering is due to selection pressures acting both at a regional and at a microenvironmental level. This was confirmed by the significant correlations found between the MW of subunits encoded by Glu-A1-1 and the populations' altitude, average temperature and rainfall. The possible selective values of seed storage proteins are discussed.

The possibility of the production of long-lived anomalous particles (APs), particles with anomalously short mean free paths, is explored in 147-GeV/c +p, K+p, and pp interactions. Limits are set on AP production using the noninteracting particles produced in these hadron-proton collisions and using published interaction cross sections to define nonanomalous behavior. The three-standard-deviation upper limits on the fraction of produced particles that may be considered APs decrease from less than 3% at an AP-proton cross section of 150 mb to less than 0.1% at an AP-proton cross section of 3 b.

Several aneuploid lines and one intervarietal substitution line of the hexaploid wheat Triticum aestivum (2n = 6χ = 42; genomes AABBDD) cv. Chinese Spring were used to study the effects of different doses of chromosomes 1B, 1D, or 1A on the amount of the high molecular weight ('HMW') glutenins and gliadins of endosperm. These homeologous chromosomes carry HMW glutenin and gliadin gene clusters on their long and short arms, respectively. Increasing the dosage of chromosome 1B of Chinese Spring in plants having in their 3n endosperm zero or the normal three doses of the homeologue 1D, as well as in plants carrying in their endosperm one dose of 1B of the cultivar Timstein, had a dual effect: on one hand, a nonlinear increase in the amount of each subunit encoded by the chromosome whose dosage was elevated and, on the other hand, a compensating nonspecific decrease in the amount of other HMW glutenin and gliadin subunits encoded either by the homeoalleles on 1D or by the homoalleles on 1B of Timstein, respectively. Deletion of chromosome arm 1BL, which carries only a few HMW glutenin genes, had no significant effect on the amount of HMW glutenins encoded by 1DL and HMW gliadins encoded by 1DS and 1BS. However, deletion of 1BS or 1DS, each carrying many gliadin genes, caused a significant but nonspecific increase in the HMW glutenins and gliadins encoded by the remaining arms of 1B and 1D. The possible mechanism and evolutionary implications of gene-dosage compensation in polyploid wheat are discussed.

Five lines of tetraploid wheat were tested for their grain protein content at 10 levels of fertilization ranging from 90 to 2610 mg pure nitrogen per plant. The low levels yielded, in all genotypes,the protein percentage normally obtained under agricultural practice or in the natural habitat. The five lines included: two high protein accessions of the wild wheat, 'l'riticu1n i'ltrgidmn var. dicoccoicles, one clitr1t1n cultivar (Inbar), and the F1 and F6 derivatives of a cross between one of the var. clicoccoides accessions and Inbar. Protein percentage of all genotypes was strongly affected by fertilization, although to a different degree; a significant genotype x fertilization interaction was observed. As a result of that interaction the genetic estimate of dominance ("cl") for protein percentage was found to be significantly affected by the fertilization level: at low levels of fertilization the low protein parent (Inhar) was partially dominant, whereas at high levels - the high protein parent (var. clicoccoicles). At the low levels of fertiliz,ttion, the differences between genotypes were more pronounced than at high levels. Hence, the commonly applied agricultural levels are recommended for any genotypic evaluation ofgermplasm for protein percentage. Heterosis was observed in protein weight per grain and grain weight. Protein ·weight per grain was almost unaffected by the level of fertilization and is therefore suggested as a gootl parameter for breeding wheat with high protein content.

The study was carried out in the first year on samples of random F₅ lines, uniform in height and in heading date, of three crosses between semi dwarf spring wheat cultivars (Triticum aestivum L.), differing in grain weight and in their Rht gene. In the second year only the progenies of the early heading F₅ lines were studied. All the material was grown in the absence of lodging. The culm-length genotypes of the different lines were identified by test crosses and by a seedling GA response test. No differences in grain weight were found between the two semi dwarf genotypes (Rht₁Rht₁rht₂rht₂ and rht₁rht₁Rht₂Rht₂). The tall genotype (rht₁rht₁rht₂rht₂) was significantly higher in grain weight than the two semi dwarf genotyes and the grain weight of these genotypes exceeded markedly the grain weight of the dwarf genotype (Rht₁Rht₁Rht₂Rht₂). These genotypic effects were independent of differences in plant height, heading date or number of grains per spike.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei from ³H-thymidine pulse (15-20 sec) and ¹⁴C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA.DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region.Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.