DNA sequence variation of wild barley Hordeum spontaneum (L.) across environmental gradients in Israel.

Wild barley Hordeum spontaneum (L.) shows a wide geographic distribution and ecological diversity. A key question concerns the spatial scale at which genetic differentiation occurs and to what extent it is driven by natural selection. The Levant region exhibits a strong ecological gradient along the North-South axis, with numerous small canyons in an East-West direction and with small-scale environmental gradients on the opposing North- and South-facing slopes. We sequenced 34 short genomic regions in 54 accessions of wild barley collected throughout Israel and from the opposing slopes of two canyons. The nucleotide diversity of the total sample is 0.0042, which is about two-thirds of a sample from the whole species range (0.0060). Thirty accessions collected at 'Evolution Canyon' (EC) at Nahal Oren, close to Haifa, have a nucleotide diversity of 0.0036, and therefore harbor a large proportion of the genetic diversity. There is a high level of genetic clustering throughout Israel and within EC, which roughly differentiates the slopes. Accessions from the hot and dry South-facing slope have significantly reduced genetic diversity and are genetically more distinct from accessions from the North-facing slope, which are more similar to accessions from other regions in Northern Israel. Statistical population models indicate that wild barley within the EC consist of three separate genetic clusters with substantial gene flow. The data indicate a high level of population structure at large and small geographic scales that shows isolation-by-distance, and is also consistent with ongoing natural selection contributing to genetic differentiation at a small geographic scale.

Adaptive evolution of duplicated hsp17 genes in wild barley from microclimatically divergent sites of Israel.

Gene duplication is a major evolutionary driving force for establishing new gene functions. However, very little is known about the involvement of the structural divergence of recent duplicated genes in local adaptation. We evaluated the nucleotide variation of recent duplicated pair copies of the hsp17 locus for small heat shock proteins, namely, the older copy hsp17a and the younger copy hsp17b. Forty wild barley (Hordeum spontaneum) genotypes from 7 climatically divergent sites of "Evolution Canyon" I, Mount Carmel, Israel, were investigated for the sequence diversity of their hsp17 copies. We identified single nucleotide polymorphisms (SNPs) of the hsp17 locus and found 150 SNPs of hsp17a and 135 SNPs of hsp17b in the coding region. Total amino acid substitutions were present at 43 positions of hsp17a and 35 positions of hsp17b among the 7 populations. The genetic divergence and haplotype diversity were significantly different among the populations. The diversity was also correlated with microclimatic interslope divergence factors in the canyon. The natural microclimatic selection appears to adaptively diversify the duplicated copies of hsp17 of wild barley between the opposite warmer and cooler slopes, suggesting that the recent duplication of stress-responsive genes were subjected to adaptive evolution. The gene duplication and the resulting functional divergence enable plants to have better fitness in local climate change.

Stripe smuts of grasses: one lineage or high levels of polyphyly?

Stripe smut of grasses, Ustilago striiformis s.l., is a complex of smut fungi widely distributed over temperate and subtropical regions. The disease results in the shredding and death of leaf tissue following the rupture of elongated sori. Nearly 100 different grass species in more than 30 genera are infected by stripe smut. During the last two centuries more than 30 smut taxa have been described from members of this complex. The present study attempts to clarify the taxonomy and phylogeny of stripe smuts on grasses by analysing both morphological and molecular data. More than 200 specimens from different continents and host plants were examined. DNA was extracted from teliospores of 23 specimens from different hosts collected in Europe, Asia, and North America. The ITS and LSU regions of ribosomal DNA were amplified and used in phylogenetic analyses. The results of Maximum Parsimony and Bayesian analyses demonstrated that there are several lineages of stripe smut fungi. Analyses of morphological characters assessed with light and scanning electron microscopy showed high support for the differentiation of two clades as distinct from U. striiformis s.l., i.e., U. nunavutica sp. nov. and U. bromina. Two additional clades, U. striiformis s.str. on Holcus and a clade containing specimens from Elymus, were identified with molecular data although morphological differences were not apparent. Descriptions are given for each species.

Finding a wider horizon: experiences of being a next-of-kin of a person suffering from colorectal cancer as told after having participated in a psychoeducational program.

AIM: The aim of this study was twofold: first, to describe the experience of being the next-of-kin of a person suffering from colorectal cancer (CRC), and second, to describe how a psychoeducational program (PEP) might contribute to the next-of-kin's life experience. Psychosocial interventions for next-of-kin to cancer patients are effective in improving quality of life issues, although there are conflicting results in previous studies. Most studies have evaluated the effects of PEP for mixed cancer groups but there is little knowledge about how next-of-kin to a person treated for CRC describe their experience of life and their experience of participating in a PEP. METHODS: The study used a qualitative descriptive design. Individual, open-ended interviews with each of the 18 next-of-kin, of persons receiving treatment for CRC, who participated in a PEP. Data were analyzed using content analysis. RESULTS: Based on the experiences described by the next-of-kin to a person treated for CRC, the subtheme; Facing a personal challenge was developed. From their described experiences of participating in a PEP, the subtheme Obtaining New Insights and Perspectives emerged. One main theme was finally identified; Finding a Wider Horizon. The study illuminates the importance of integrating the next-of-kin/family in the colorectal cancer care. CONCLUSION: The findings from this study can be used to plan future interventions for next-of-kin to patients with CRC as it offers possibilities to understand the next-of-kin's situation and experience from participating in a PEP.

The mode of reproduction in natural populations of ascomycetous fungus, Emericella nidulans, from Israel.

The mode of reproduction of the soil ascomycetous fungus Emericella nidulans of Israeli populations was studied using 15 microsatellite (simple sequence repeats or SSR) trinucleotide markers. The study was performed in three canyons: two located in the northern part of Israel (Mount Carmel and western Upper Galilee) and one in the southern Negev desert. In each canyon, E. nidulans strains were isolated from the opposite slopes and (in the desert canyon) the valley bottom. Testing the reproductive structure of the populations indicated the presence of sexuality in the northern population and predominant clonality in the desert population. The predominantly clonal character of the desert population of E. nidulans was explained by the assumption that for relevant multilocus systems of a fungus, only several haplotypes can survive in the rather constant, extremely stressful desert conditions. Additionally, the very low density of E. nidulans populations in the soil of the desert canyon, which reduces the probability of finding a sexual partner, might favour predominant clonality via selfing. Increasing sexuality in E. nidulans populations on the north-facing slopes of the northern canyons may be a result of biotic stress (pressure of competitive fungal species), due to the more mild ecological conditions in these canyons.

Genome-wide analysis of DNA turnover and gene expression in stationary-phase Saccharomyces cerevisiae.

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2'-deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdU-labelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes.

Chromosomes in a hybrid zone of Israeli mole rars (Spalax, Rodentia).

Chromosomal novelties and the level of meiotic and mitotic abnormalities were studied in a hybrid zone between two chromosomally differentiated Spalax cytotypes of 2n = 58 and 2n = 52. These cytotypes differ by five Rb fusions, four centromeric shifts accompanied by heterochromatin deletion, one paracentric inversion, and the Y-chromosome reorganization. Among 149 specimens studied, 82 were hybrids with 64 different karyotypes ranging in diploid numbers from 2n = 50 to 2n = 60. Nine hybrid specimens were mosaics for the chromosome numbers due to occurrence of cell lines with different Robertsonian chromosome arrangements, and six specimens possessed variable number of B-chromosomes. Mosaicism of B-chromosomes was found also in meiotic cells however chromatid breaks and abnormal chromosome pairing during meiosis occurred very rarely. All these results imply some local genomic instability resulting in the spontaneous process of reversible Rb fusions.

[Peculiarities of the courtship song in the Drosophila melanogaster populations adapted to gradient of microecological conditions].

In theory of evolution, two chief scenarios of development of sexual isolation--allopatric and sympatric, which act respectively under conditions of geographic separation or without it. Differences in the courtship behavior and in the used signals can lead to reproduction isolation and prevent gene exchange between sympatric populations or species, in which postcopulational barrier are absent. The previous studies of natural Drosophila populations inhabiting the opposite, ecologically contract slopes of evolutional canyon (vicinities of the brook Open in the mountain ridge Karmel, Israel) revealed statistically significant differences between them by the complex of adaptive and behavioral parameters including courtship behavior and choice of partner for copulation. Astonishingly, differentiation of two populations has appeared in spite of a very small distance between two slopes. Here we report the statistically significant differences between males from the opposite slopes in characteristics of the courtship song that is one of signals serving for recognition by female of sexual partner of her species. We suggest that these differences can underlie the earlier revealed female discriminational behavior and can reflect different adaptive strategies in the populations inhabiting opposite canyon slopes.

Detection and quantification of Paenibacillus polymyxa in the rhizosphere of wild barley (Hordeum spontaneum) with real-time PCR.

AIM: To detect and quantify the plant drought tolerance enhancing bacterium Paenibacillus polymyxa in a collection of 160 Hordeum spontaneum rhizosphere samples at the 'Evolution Canyon' ('EC'), Israel. METHODS AND RESULTS: PCR primers and a FAM-TAMRA probe (6-carboxyfluorescein, 6-carboxy-tetramethyl-rhodamine) targeting 16S rRNA genes were designed and used to detect and quantify the target strain. Two commercial kits, Bio101 Fast Spin and Mo Bio Ultra Clean Soil DNA, were tested for DNA isolation from the rhizosphere and surrounding soil. Population densities of P. polymyxa were studied in the rhizosphere of wild barley and surrounding soil from the contrasting climatic slopes at the 'EC' using the real-time PCR and culture based methods. CONCLUSION: Paenibacillus polymyxa is one of the best established species in wild barley rhizosphere at the 'EC' slopes. With the real-time PCR assay we are able to detect 1 pg of DNA per PCR corresponding to 100 cells per ml. The results at the 'EC' correlate well to bacterial estimations by culture based methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Significantly higher P. polymyxa cell number was detected in the rhizosphere of arid 'African' microclimate indicating possible role of adaptive co-evolution with plants.

Disposal of agro-industrial by-products by organic cultivation of the culinary and medicinal mushroom Hypsizygus marmoreus.

Organic mushroom cultivation is one of the fastest growing segments of agriculture. At the core of the organic philosophy lies a ban on the use of synthetic fertilizers, pesticides and herbicides, in addition to such tenets as animal welfare, energy efficiency, and social justice. Hypsizygus marmoreus (HM) is a highly praised cultivated culinary and medicinal mushroom. The objective of this paper was to assess the suitability of different spawn media and then the potential of various cultivation substrates to support HM mushroom production compatible with organic standards. This objective was met through the setup of a low-cost cultivation infrastructure. First, seven types of spawn media were tested; then we tested 24 substrates made from organic by-products for their biological efficiency (BE) with strain HM 830, using the liquid inoculation method. The best substrate in terms of BE was corn cob with bran and olive press cake, with a BE of 85.6%. The BE of the same composition but without olive press cake was only 67.5%. The next best substrates were cotton straw combinations with a BE of 31.5-53%. The spent mushroom substrate provides a good method for the disposal of solid waste. The guidance provided in this research complies with organic mushroom cultivation standards and can be used to produce certified organic mushrooms. In addition, it allows responsible and beneficial disposal of a large amount of solid agro-industrial waste.

Deletions in the desaturase2 gene in Israeli populations of Drosophila melanogaster: possible role in mate recognition.

Molecular analysis of the desaturase2 (desat2) gene responsible for female cuticular hydrocarbon synthesis, revealed that all tested Israeli populations of Drosophila melanogaster belong to the 'Low' type (low ratio of 5,9-/7,11-heptacosadiene) with a 16 bp deletion in the promoter region. Additional deletion leading to the appearance of the stop-codon TGA was found in exon 1 in populations inhabiting opposite slopes at Nahal Oren canyon. Mating experiments between carriers of various desat2 alleles estimated the putative effect of desat2 status on courtship behaviour. The allele with an additional deletion in exon 1 seems to play some role in mating success, which is associated with shorter courtship latency and courtship duration. The appearance and maintenance of this mutant allele in populations inhabiting Nahal Oren canyon may reflect flies' adaptation to peculiar microscale climatic conditions and may be associated with incipient sympatric differentiation.

Discrimination of 2n = 60 Spalax leucodon cytotypes (Spalacidae, Rodentia) in Turkey by means of classical and molecular cytogenetic techniques.

Comparative studies among seven populations of 2n = 60 S. leucodon employing classic cytogenetics (G- bands, C-bands, AgNOR-staining), fluorochrome staining, and fluorescence in situ hybridization of telomeric and rDNA probes are reported here for the first time. The studied specimens were assigned to two cytotypes: 2n = 60W and 2n = 60R. The basic karyotype of both cytotypes consisted of eight pairs of subtelocentric and 21 pairs of acrocentric autosomes, subtelocentric X and acrocentric Y chromosomes. Both cytotypes had variable numbers of B-chromosomes (1-3) and variable numbers of autosomal arms (NFa = 74-76) caused by amplification (deletion) of heterochromatin short arms in the second pair. The short arms of subtelocentric chromosomes were comprised of heterochromatin in both cytotypes. Nucleolar organizer regions (NORs) and rDNA clusters were detected at telomeric sites of the short arms in pairs Nos. 3, 5, 6, 9, and 13 in cytotype W, and in the short arms of pair No. 6, 8, 12, 13, and 16 in cytotype R. Different locations of rDNA clusters allowed unambiguous discrimination between two S. leucodon cytotypes possessing the same 2n = 60 and similar NFa (74-76) variability. Our findings suggest a high level of chromosomal divergence, which means that it is possible to consider these cytotypes as a well-differentiated, chromosomal lineage within the leucodon group.

Habitat-specific natural selection at a flowering-time QTL is a main driver of local adaptation in two wild barley populations.

Understanding the genetic basis of local adaptation requires insight in the fitness effects of individual loci under natural field conditions. While rapid progress is made in the search for genes that control differences between plant populations, it is typically unknown whether the genes under study are in fact key targets of habitat-specific natural selection. Using a quantitative trait loci (QTL) approach, we show that a QTL associated with flowering-time variation between two locally adapted wild barley populations is an important determinant of fitness in one, but not in the other population's native habitat. The QTL mapped to the same position as a habitat-specific QTL for field fitness that affected plant reproductive output in only one of the parental habitats, indicating that the genomic region is under differential selection between the native habitats. Consistent with the QTL results, phenotypic selection of flowering time differed between the two environments, whereas other traits (growth rate and seed weight) were under selection but experienced no habitat-specific differential selection. This implies the flowering-time QTL as a driver of adaptive population divergence. Our results from phenotypic selection and QTL analysis are consistent with local adaptation without genetic trade-offs in performance across environments, i.e. without alleles or traits having opposing fitness effects in contrasting environments.

Repetitive DNA and chromosomal rearrangements: speciation-related events in plant genomes.

Chromosomal change is one of the more hotly debated potential mechanisms of speciation. It has long been argued over whether--and to what degree--changes in chromosome structure contribute to reproductive isolation and, ultimately, speciation. In this review we do not aim to completely analyze accumulated data about chromosomal speciation but wish to draw attention to several critical points of speciation-related chromosomal change, namely: (a) interrelations between chromosomal rearrangements and repetitive DNA fraction; (b) mobility of ribosomal DNA clusters; and (c) rDNA and transposable elements as perpetual generators of genome instability.

Seedling Resistance to Tan Spot and Stagonospora nodorum Leaf Blotch in Wild Emmer Wheat (Triticum dicoccoides).

Tan spot and Stagonospora nodorum blotch (SNB), caused by Pyrenophora tritici-repentis and Stagonospora nodorum, respectively, are two destructive foliar diseases of wheat, causing significant yield reduction worldwide. The objective of this study was to evaluate 172 accessions of wild emmer wheat (Triticum dicoccoides) for seedling resistance to tan spot and SNB. All accessions were inoculated with P. tritici-repentis race 1 and a mixture of three diverse isolates of S. nodorum, respectively. The accessions were also evaluated for sensitivity to host-selective toxins (HSTs), including ToxA produced by both S. nodorum and P. tritici-repentis and culture filtrate produced by S. nodorum. A total of 34 accessions were resistant to tan spot, and 136 accessions were resistant to SNB. Among these accessions, 31 were resistant to both diseases. Significant correlations between HST insensitivity and disease resistance were observed. Our results showed that T. dicoccoides is a good genetic source of resistance to tan spot and SNB in wheat.

Different clustering of genomes across life using the A-T-C-G and degenerate R-Y alphabets: early and late signaling on genome evolution?

In this study, we have calculated distances between genomes based on our previously developed compositional spectra (CS) analysis. The study was conducted using genomes of 39 species of Eukarya, Eubacteria, and Archaea. Based on CS distances, we produced two different consensus dendrograms for four- and two-letter (purine-pyrimidine) alphabets. A comparison of the obtained structure using purine-pyrimidine alphabet with the standard three-kingdom (3K) scheme reveals substantial similarity. Surprisingly, this is not the case when the same procedure is based on the four-letter alphabet. In this situation, we also found three main clusters-but different from those in the 3K scheme. In particular, one of the clusters includes Eukarya and thermophilic bacteria and a part of the considered Archaea species. We speculate that the key factor in the last classification (based on the A-T-G-C alphabet) is related to ecology: two ecological parameters, temperature and oxygen, distinctly explain the clustering revealed by compositional spectra in the four-letter alphabet. Therefore, we assume that this result reflects two interdependent processes: evolutionary divergence and superimposed ecological convergence of the genomes, albeit another process, horizontal transfer, cannot be excluded as an important contributing factor.

Looking for organization patterns of highly expressed genes: purine-pyrimidine composition of precursor mRNAs.

We analyzed precursor messenger RNAs (pre-mRNAs) of 12 eukaryotic species. In each species, three groups of highly expressed genes, ribosomal proteins, heat shock proteins, and amino-acyl tRNA synthetases, were compared with a control group (randomly selected genes). The purine-pyrimidine (R-Y) composition of pre-mRNAs of the three targeted gene groups proved to differ significantly from the control. The exons of the three groups tested have higher purine contents and R-tract abundance and lower abundance of Y-tracts compared to the control (R-tract-tract of sequential purines with Rn>or=5; Y-tract-tract of sequential pyrimidines with Yn>or=5). In species widely employing "intron definition" in the splicing process, the Y content of introns of the three targeted groups appeared to be higher compared to the control group. Furthermore, in all examined species, the introns of the targeted genes have a lower abundance of R-tracts compared to the control. We hypothesized that the R-Y composition of the targeted gene groups contributes to high rate and efficiency of both splicing and translation, in addition to the mRNA coding role. This is presumably achieved by (1) reducing the possibility of the formation of secondary structures in the mRNA, (2) using the R-tracts and R-biased sequences as exonic splicing enhancers, (3) lowering the amount of targets for pyrimidine tract binding protein in the exons, and (4) reducing the amount of target sequences for binding of serine/arginine-rich (SR) proteins in the introns, thereby allowing SR proteins to bind to proper (exonic) targets.

P53 in blind subterranean mole rats--loss-of-function versus gain-of-function activities on newly cloned Spalax target genes.

A tumor suppressor gene, p53, controls cellular responses to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Recently, we demonstrated that in blind subterranean mole rats, Spalax, a model organism for hypoxia tolerance, the p53 DNA-binding domain contains a specific Arg174Lys amino acid substitution. This substitution reduces the p53 effect on the transcription of apoptosis genes (apaf1, puma, pten and noxa) and enhances it on human cell cycle arrest and p53 stabilization/homeostasis genes (mdm2, pten, p21 and cycG). In the current study, we cloned Spalax apaf1 promoter and mdm2 intronic regions containing consensus p53-responsive elements. We compared the Spalax-responsive elements to those of human, mouse and rat and investigated the transcriptional activity of Spalax and human Arg174Lys-mutated p53 on target genes of both species. Spalax and human-mutated p53 lost induction of apaf1 transcription, and increased induction of mdm2 transcription. We conclude that Spalax evolved hypoxia-adaptive mechanisms, analogous to the alterations acquired by cancer cells during tumor development, with a bias against apoptosis while favoring cell arrest and DNA repair.

Molecular-genetic biodiversity in a natural population of the yeast Saccharomyces cerevisiae from "Evolution Canyon": microsatellite polymorphism, ploidy and controversial sexual status.

The yeast S. cerevisiae is a central model organism in eukaryotic cell studies and a major component in many food and biotechnological industrial processes. However, the wide knowledge regarding genetics and molecular biology of S. cerevisiae is based on an extremely narrow range of strains. Studies of natural populations of S. cerevisiae, not associated with human activities or industrial fermentation environments, are very few. We isolated a panel of S. cerevisiae strains from a natural microsite, "Evolution Canyon" at Mount Carmel, Israel, and studied their genomic biodiversity. Analysis of 19 microsatellite loci revealed high allelic diversity and variation in ploidy level across the panel, from diploids to tetraploids, confirmed by flow cytometry. No significant differences were found in the level of microsatellite variation between strains derived from the major localities or microniches, whereas strains of different ploidy showed low similarity in allele content. Maximum genetic diversity was observed among diploids and minimum among triploids. Phylogenetic analysis revealed clonal, rather than sexual, structure of the triploid and tetraploid subpopulations. Viability tests in tetrad analysis also suggest that clonal reproduction may predominate in the polyploid subpopulations.

Laccase and manganese peroxidase activities of Phellinus robustus and Ganoderma adspersum grown on food industry wastes in submerged fermentation.

Phellinus robustus produced both laccase (700-4,000 U l(-1)) and manganese peroxidase (MnP) (1,000-11,300 U l(-1)) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600-34,000 U l(-1)). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.