Publications

The Periodic Table of Food Initiative addresses food biomolecular composition information gaps through a standardized, accessible and enabling platform based on analytical tools, data and capacity building. Data from 1,650 foods serve as starting point for demonstrating the capacity of this initiative to contribute to nutrition, health and food systems transformations.

This open access book covers a century of research on wheat genetics and evolution, starting with the discovery in 1918 of the accurate number of chromosomes in wheat. We re-evaluate classical studies that are pillars of the current knowledge considering recent genomic data in the wheat group comprising 31 species from the genera Amblyopyrum, Aegilops, Triticum, and other more distant relatives. For these species, we describe morphology, ecogeographical distribution, phylogeny as well as cytogenetic and genomic features. For crops, we also address evolution under human selection, namely pre-domestication cultivation and domestication. We re-examine the genetic and archeological evidence of where, when, and how domestication occurred. We discuss unique aspects of genome evolution and maintenance under polyploidization, in natural and synthetic allopolyploids of the wheat group. Finally, we propose some thoughts on the future prospects of wheat improvement. As such, it can be ofgreat interest to wheat researchers and breeders as well as to plant scientists and students interested in plant genetics, evolution, domestication, and polyploidy.

Common wheat (Triticum aestivum, BBAADD) is a major staple food crop worldwide. The diploid progenitors of the A and D subgenomes have been unequivocally identified; that of B, however, remains ambiguous and controversial but is suspected to be related to species of Aegilops, section Sitopsis. Here, we report the assembly of chromosome-level genome sequences of all five Sitopsis species, namely Aegilops bicornis, Ae. longissima, Ae. searsii, Ae. sharonensis, and Ae. speltoides, as well as the partial assembly of the Amblyopyrum muticum (synonym Aegilops mutica) genome for phylogenetic analysis. Our results reveal that the donor of the common wheat B subgenome is a distinct, and most probably extinct, diploid species that diverged from an ancestral progenitor of the B lineage to which the still extant Ae. speltoides and Am. muticum belong. In addition, we identified interspecific genetic introgressions throughout the evolution of the Triticum/Aegilops species complex. The five Sitopsis species have various assembled genome sizes (4.115.89 Gb) with high proportions of repetitive sequences (85.99%89.81%); nonetheless, they retain high collinearity with other genomes or subgenomes of species in the Triticum/Aegilops complex. Differences in genome size were primarily due to independent post-speciation amplification of transposons. We also identified a set of Sitopsis genes pertinent to important agronomic traits that can be harnessed for wheat breeding. These newly assembled genome resources provide a new roadmap for evolutionary and genetic studies of the Triticum/Aegilops complex, as well as for wheat improvement.

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both \u201chot and cold\u201d regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.

Meiotic recombination is the main driver of genetic diversity in wheat breeding. The rate and location of crossover (CO) events are regulated by genetic and epigenetic factors. In wheat, most COs occur in subtelomeric regions but are rare in centromeric and pericentric areas. The aim of this work was to increase COs in both \u201chot\u201d and \u201ccold\u201d chromosomal locations. We used Virus-Induced gene Silencing (VIGS) to downregulate the expression of recombination-suppressing genes XRCC2 and FANCM and of epigenetic maintenance genes MET1 and DDM1 during meiosis. VIGS suppresses genes in a dominant, transient and non-transgenic manner, which is convenient in wheat, a hard-to-transform polyploid. F1 hybrids of a cross between two tetraploid lines whose genome was fully sequenced (wild emmer and durum wheat), were infected with a VIGS vector ∼ 2 weeks before meiosis. Recombination was measured in F2 seedlings derived from F1-infected plants and non-infected controls. We found significant up and down-regulation of CO rates along subtelomeric regions as a result of silencing either MET1, DDM1 or XRCC2 during meiosis. In addition, we found up to 93% increase in COs in XRCC2-VIGS treatment in the pericentric regions of some chromosomes. Silencing FANCM showed no effect on CO. Overall, we show that CO distribution was affected by VIGS treatments rather than the total number of COs which did not change. We conclude that transient silencing of specific genes during meiosis can be used as a simple, fast and non-transgenic strategy to improve breeding abilities in specific chromosomal regions.

BACKGROUND For over a century, genetic diversity of wheat worldwide was eroded by continual selection for high yields and industrial demands. Wheat landraces cultivated in Israel and Palestine demonstrate high genetic diversity and a potentially wide repertoire of adaptive alleles. While most Israeli-Palestinian wheat landraces were lost in the transition to 'Green Revolution' semi-dwarf varieties, some germplasm collections made at the beginning of the 20th century survived in gene banks and private collections worldwide. However, fragmentation and poor conservation place this unique genetic resource at a high risk of genetic erosion. Herein, we describe a long-term initiative to restore, conserve, and characterize a collection of Israeli and Palestinian wheat landraces (IPLR). RESULTS We report on (i) the IPLR construction (n = 932), (ii) the historical and agronomic context to this collection, (iii) the characterization and assessment of the IPLR's genetic diversity, and (iv) a data comparison from two distinct subcollections within IPLR: a collection made by N. Vavilov in 1926 (IPLR-VIR) and a later one (1979-1981) made by Y. Mattatia (IPLR-M). Though conducted in the same eco-geographic space, these two collections were subjected to considerably different conservation pathways. IPLR-M, which underwent only one propagation cycle, demonstrated marked genetic and phenotypic variability (within and between accessions) in comparison with IPLR-VIR, which had been regularly regenerated over similar to 90 years. CONCLUSION We postulate that long-term ex situ conservation involving human and genotype x environment selection may significantly reduce accession heterogeneity and allelic diversity. Results are further discussed in a broader context of pre-breeding and conservation. (c) 2019 Society of Chemical Industry

Recombination between homeologous chromosomes, also known as homeologous exchange (HE), plays a significant role in shaping genome structure and gene expression in interspecific hybrids and allopolyploids of several plant species. However, the molecular mechanisms that govern HEs are not well understood. Here, we studied HE events in the progeny of a nascent allotetraploid (genome AADD) derived from two diploid progenitors of hexa- ploid bread wheat using cytological and whole-genome sequence analyses. In total, 37 HEs were identified and HE junctions were mapped precisely. HEs exhibit typical patterns of homologous re- combination hotspots, being biased toward low -copy, subtelo- meric regions of chromosome arms and showing association with known recombination hotspot motifs. But, strikingly, while homologous recombination preferentially takes place upstream and downstream of coding regions, HEs are highly enriched within gene bodies, giving rise to novel recombinant transcripts, which in turn are predicted to generate new protein fusion variants. To test whether this is a widespread phenomenon, a dataset of high - resolution HE junctions was analyzed for allopolyploid Brassica , rice, Arabidopsis suecica , banana, and peanut. Intragenic recombi- nation and formation of chimeric genes was detected in HEs of all species and was prominent in most of them. HE thus provides a mechanism for evolutionary novelty in transcript and protein se- quences in nascent allopolyploids.

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

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.

The rate of crossover, the reciprocal exchanges of homologous chromosomal segments, is not uniform along chromosomes differing between male and female meiocytes. To better understand the factors regulating this variable landscape, we performed a detailed genetic and epigenetic analysis of 737 crossover events in Arabidopsis thaliana. Crossovers were more frequent than expected in promoters. Three DNA motifs enriched in crossover regions and less abundant in crossover-poor pericentric regions were identified. One of these motifs, the CCN repeat, was previously unknown in plants. The A-rich motif was preferentially associated with promoters, while the CCN repeat and the CTT repeat motifs were preferentially associated with genes. Analysis of epigenetic modifications around the motifs showed, in most cases, a specific epigenetic architecture. For example, we show that there is a peak of nucleosome occupancy and of H3K4me3 around the CCN and CTT repeat motifs while nucleosome occupancy was lowest around the A-rich motif. Cytosine methylation levels showed a gradual decrease within 2 kb of the three motifs, being lowest at sites where crossover occurred. This landscape was conserved in the decreased DNA methylation1 mutant. In summary, the crossover motifs are associated with epigenetic landscapes corresponding to open chromatin and contributing to the nonuniformity of crossovers in Arabidopsis.

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 wheat group has evolved through allopolyploidization, namely, through hybridization among species from the plant genera Aegilops and Triticum followed by genome doubling. This speciation process has been associated with ecogeographical expansion and with domestication. In the past few decades, we have searched for explanations for this impressive success. Our studies attempted to probe the bases for the wide genetic variation characterizing these species, which accounts for their great adaptability and colonizing ability. Central to our work was the investigation of how allopolyploidization alters genome structure and expression. We found in wheat that allopolyploidy accelerated genome evolution in two ways: (1) it triggered rapid genome alterations through the instantaneous generation of a variety of cardinal genetic and epigenetic changes (which we termed "revolutionary" changes), and (2) it facilitated sporadic genomic changes throughout the species' evolution (i.e., evolutionary changes), which are not attainable at the diploid level. Our major findings in natural and synthetic allopolyploid wheat indicate that these alterations have led to the cytological and genetic diploidization of the allopolyploids. These genetic and epigenetic changes reflect the dynamic structural and functional plasticity of the allopolyploid wheat genome. The significance of this plasticity for the successful establishment of wheat allopolyploids, in nature and under domestication, is discussed.

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.

This article is a response to Wang and Luo.See correspondence article http://www.biomedcentral.com/1741-7007/10/30/ [WEBCITE] and the original research article http://www.biomedcentral.com/1741-7007/9/24 [WEBCITE].

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.

The maize transposable element Activator (Ac) has been shown to be active in a number of dicots, including arabidopsis [Arabidopsis thaliana (L.) Heynh.], tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum Mill.), potato (Solanum tuberosum L.), and aspen (Populus tremuloides Michx.). However, no information is available on somatic transposition in any plant during several years of growth and development. It is not known how transposition affects genetic variability among vegetative parts that have developed during a long period of growth. In order to explore the possibility of using somatic Ac transposition for gene tagging and mutagenesis in fruit trees, a derivative of the maize Ac transposable element was introduced into 'Duncan' grapefruit (Citrus paradisi Macf.) by Agrobacterium tumefaciens (Smith & Towns.) Conn.-mediated stable transformation. Genetically identical 4-year-old sibling trees were established by grafting one of the transformants on Troyer citrange [Citrus sinensis (L.) Osbec. x Poncirus trifoliate (L.) Ras.] rootstocks. We demonstrated that the Ac element was active upon transformation in citrus (Citrus L.) trees and that transposition can create genetic variability among tree siblings and among leaves collected from different parts of the same tree. Ac was still active among propagated plants 4 years after transformation, clearly indicating that it is capable of maintaining itself in citrus trees for a long period of time. The observation of different integration patterns in different parts of the same tree and within tree siblings originating from the same transformant suggests that an Ac-based mutagenesis system could be very useful in creating somatic mutations in citrus trees.

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.

We analyzed the events that affect gene structure and expression in the early stages of allopolyploidy in wheat. The transcriptome response was studied by analyzing 3072 transcripts in the first generation of a synthetic allotetraploid (genome S^lS^lA^mA^m), which resembles tetraploid wheat (genome BBAA), and in its two diploid progenitors Aegilops sharonensis (S^lS^l) and Triticum monococcum ssp. aegilopoides (A^mA^m). The expression of 60 out of 3072 transcripts was reproducibly altered in the allotetraploid: 48 transcripts disappeared and 12 were activated. Transcript disappearance was caused by gene silencing or by gene loss. Gene silencing affected one or both homeologous loci and was associated in part with cytosine methylation. Gene loss or methylation had occurred already in the F₁ intergeneric hybrid or in the allotetraploid, depending on the locus. The silenced/lost genes included rRNA genes and genes involved in metabolism, disease resistance, and cell cycle regulation. The activated genes with a known function were all retroelements. These findings show that wide hybridization and chromosome doubling affect gene expression via genetic and epigenetic alterations immediately upon allopolyploid formation. These events contribute to the genetic diploidization of newly formed allopolyploids.

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.

The purpose of this study was to develop a model system for studying tomato genetics. Agronomic, genetic, and molecular data are presented which show that the miniature Lycopersicon esculentum cultivar, Micro-Tom (Micro tomato), fulfills the requirements for such a model. It grows at high density (up to 1357 plants/m(-2)); it has a short life cycle (70-90 days from sowing to fruit ripening); and it can be transformed at frequencies of up to 80% through Agrobacterium-mediated transformation of cotyledons. Moreover, it differs from standard tomato cultivars by only two major genes. Therefore, any mutation or transgene can be conveniently studied in Micro-Tom's background and, when needed, transferred into a standard background. We took advantage of Micro-Tom's features to improve the infrastructure for mutagenesis in tomato. A screening of 9000 M1 and 20 000 M2 EMS mutagenized plants is described. Mutants with altered pigmentation or modified shape of leaves, flowers and fruits were found. In addition, an enhancer trapping and a gene trapping system, based on the Ac/Ds maize transposable elements, were transformed into Micro-Tom and found to be active. In summary, Micro-Tom opens new prospects to achieve saturated mutagenesis in tomato, and facilitates the application of transposon-based technologies such as gene tagging, trapping and knockout.

The maize Ac/Ds transposable elements are thought to transpose via a cut-and-paste mechanism, but the intermediates formed during transposition are still unknown. In this work we present evidence that circular Ac molecules are formed in plants containing actively transposing elements. In these circles, transposon ends are joined head-to-head. The sequence at the ends' junction is variable, containing small deletions or insertions. Circles containing deleted Ac ends are probably unable to successfully reintegrate. To test the ability of circles with intact transposon ends to integrate into the genome, an artificial Ds circle was constructed by cloning the joined ends of Ac into a plasmid carrying a plant selectable marker. When such a circular Ds was introduced into tobacco protoplasts in the presence of Ac- transposase, no efficient transposase-mediated integration was observed. Although a circular transposition intermediate cannot be ruled out, the findings of circles with deleted transposon ends and the absence of transposase-mediated integration of the circular Ds suggest that some of the joined-ends-carrying elements are not transposition intermediates, but rather abortive excision products. The formation of Ac circles might account for the previously described phenomenon of Ac-loss. The origin of Ac circles and the implications for models of Ac transposition are discussed.

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.

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.

Specific binding of Nicotiana nuclear protein(s) to subterminal regions of the Ac transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions of Ac, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends of Mutator. Moreover, we show that DNA from Ds1 competes for protein binding to Ac termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region of Tgm1, Tpn1, En/Spm, Tam3 and Ds1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.

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.