Adaptive microclimatic structural and expressional dehydrin 1 evolution in wild barley, Hordeum spontaneum, at 'Evolution Canyon', Mount Carmel, Israel

'Evolution Canyon' (ECI) at Lower Nahal Oren, Mount Carmel, Israel, is an optimal natural microscale model for unravelling evolution in action highlighting the twin evolutionary processes of adaptation and speciation. A major model organism in ECI is wild barley, Hordeum spontaneum , the progenitor of cultivated barley, which displays dramatic interslope adaptive and speciational divergence on the 'African' dry slope (AS) and the 'European' humid slope (ES), separated on average by 200 m. Here we examined interslope single nucleotide polymorphism (SNP) sequences and the expression diversity of the drought resistant dehydrin 1 gene ( Dhn1 ) between the opposite slopes. We analysed 47 plants (genotypes), 4–10 individuals in each of seven stations (populations) in an area of 7000 m², for Dhn1 sequence diversity located in the 5' upstream flanking region of the gene. We found significant levels of Dhn1 genic diversity represented by 29 haplotypes, derived from 45 SNPs in a total of 708 bp sites. Most of the haplotypes, 25 out of 29 (= 86.2%), were represented by one genotype; hence, unique to one population. Only a single haplotype was common to both slopes. Genetic divergence of sequence and haplotype diversity was generally and significantly different among the populations and slopes. Nucleotide diversity was higher on the AS, whereas haplotype diversity was higher on the ES. Interslope divergence was significantly higher than intraslope divergence. The applied Tajima D rejected neutrality of the SNP diversity. The Dhn1 expression under dehydration indicated interslope divergent expression between AS and ES genotypes, reinforcing Dhn1 associated with drought resistance of wild barley at 'Evolution Canyon'. These results are inexplicable by mutation, gene flow, or chance effects, and support adaptive natural microclimatic selection as the major evolutionary divergent driving force.     

Differential selection of growth rate-related traits in wild barley, Hordeum spontaneum, in contrasting greenhouse nutrient environments

Across-species comparisons show that inherent variation in relative growth rate (RGR) and its underlying traits are correlated with habitat productivity. In this study, we test the hypothesis that growth rate-related traits confer differential selective effects in contrasting nutrient environments. We specifically test whether high RGR is targeted by selection in nutrient-rich environments whereas low values of traits that underlie RGR [specific leaf area (SLA), leaf mass fraction and leaf area ratio (LAR)] confer a direct fitness advantage in nutrient-poor environments, resulting in selection of low RGR as a correlated response. We measured RGR, its underlying component traits, and estimated fitness in a range of wild barley (Hordeum spontaneum) accessions grown under high and low nutrient conditions. Selection on component traits differed between the two environments, while total selection of RGR was not significant. Using multiple regression and path analysis to estimate direct fitness effects, a selective advantage of high LAR and SLA was demonstrated only under nutrient-rich conditions. While supporting the view that observed associations between habitat richness and some RGR-component traits reflect adaptation to differing nutrient regimes, our data suggest that direct selection targets component traits rather than RGR itself.     

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.     

Differentiation in populations of Hordeum spontaneum Koch along a gradient of environmental productivity and predictability: plasticity in response to water and nutrient stress

Plants from four populations of Hordeum spontaneum originating in distinct environments of Israel were compared for stress induced phenotypic plasticity. The environments ranged along a gradient of increasing rainfall amount and predictability from low (desert) to moderate (semisteppe batha) to high (Mediterranean grassland and mountain, the latter also experiencing frost stress). The plants were exposed to a set of four treatments: no stress (optimum water and nutrients), water, nutrient and both water and nutrient stress. Plants from the four populations (or ecotypes) exhibited different patterns of plasticity in response to the different stresses (water and nutrients) and in different trait categories (reproductive, fitness and resource allocation). The importance of plasticity in response to water stress appears to decrease, and to nutrient stress appears to increase along the increasing rainfall gradient. The mountain ecotype, growing in an area with high potential productivity (amount of rainfall) but experiencing periodic frosts, was the most plastic among ecotypes in resource allocation under both water and nutrient stress, but exhibited low plasticity in other trait categories. In contrast, the desert ecotype had low plasticity in resource allocation under water stress and the lowest plasticity among the four ecotypes in all trait categories in response to nutrient stress. The ecotype originating in Mediterranean grassland, a predictable and most favourable environment, was highly plastic in fitness and allocation traits in response to low nutrient levels which is likely to occur due to competition in productive environment. We discuss the observed differences in ecotype plasticity as part of their environmentally induced adaptive ‘strategies’. We found no support for the hypothesis that plants originating in environments with greater variation and unpredictability are more plastic. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society 2002, 75 , 301–312.     

Upregulation of DNA repair genes in active cirrhosis associated with hepatocellular carcinoma

Phenotypic changes in injured livers involve complex network of genes whose interplays may lead to fibrosis and cirrhosis, a major risk of hepatocellular carcinoma. Gene expression profiles in fibrotic livers were analyzed by using cDNA microarray, hierarchical clustering and gene ontology. Analyses of a major cluster of upregulated genes in cirrhosis identified a new set of genes involved in DNA repair and damage. The upregulation of DNA repair genes was confirmed by real-time quantitative polymerase chain reaction and associated with necroinflammatory activity (P<0.001). Increased DNA repair activity in cirrhosis with inflammatory activity may reflect increased DNA damages as a consequence of chronic liver injury.     

Differential expression of immune response genes associated with subclinical mastitis in dairy buffaloes

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.     

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.     

Chibby functions in Xenopus ciliary assembly,embryonic development,and the regulation of gene expression

Wnt signaling and ciliogenesis are core features of embryonic development in a range of metazoans. Chibby (Cby), a basal-body associated protein, regulates β-catenin-mediated Wnt signaling in the mouse but not Drosophila. Here we present an analysis of Cby?s embryonic expression and morphant phenotypes in Xenopus laevis. Cby RNA is supplied maternally, negatively regulated by Snail2 but not Twist1, preferentially expressed in the neuroectoderm, and regulates β-catenin-mediated gene expression. Reducing Cby levels reduced the density of multiciliated cells, the number of basal bodies per multiciliated cell, and the numbers of neural tube primary cilia; it also led to abnormal development of the neural crest, central nervous system, and pronephros, all defects that were rescued by a Cby-GFP chimera. Reduction of Cby led to an increase in Wnt8a and decreases in Gli2, Gli3, and Shh RNA levels. Many, but not all, morphant phenotypes were significantly reversed by the Wnt inhibitor SFRP2. These observations extend our understanding of Cby?s role in mediating the network of interactions between ciliogenesis, signaling systems and tissue patterning.     

Analysis of gene expressions associated with increased allelopathy in rice (Oryza sativa L.) induced by exogenous salicylic acid

The defense characteristics of allelopathic rice accession PI312777 and its counterpart Lemont induced by exogenous salicylic acid (SA) to suppress troublesome weed barnyardgrass (BYG) were investigated using the methods of suppression subtractive hybridization (SSH) and real-time fluorescence quantitative PCR (qRT-PCR). The results showed that exogenous SA could induce the allelopathic effect of rice on BYG and this inducible defense was SA dose-respondent and treatment time-dependent. PI312777 exhibited higher inhibitory effect than Lemont on BYG after treated with different concentrations of SA. The activities of cell protective enzymes including SOD, POD and CAT in the BYG plants co-cultured with PI312777 treated by SA were highly depressed compared with the control (co-cultured with rice without SA-treatment). Similar but lower depression on these enzymes except for CAT was also observed in the BYG plants when co-cultured with Lemont treated by SA. It is therefore suggested that allelopathic rice should be more sensitive than non-allelopathic rice to exogenous SA. Seventeen genes induced by SA were obtained by SSH analysis from PI312777. These genes encode receptor-kinase proteins, ubiquitin carrier proteins, proteins related to phenylpropanoid metabolism, antioxidant related proteins and some growth-mediating proteins. The differential expressions of these genes were validated in part by qRT-PCR in the two rice accessions. Our work elucidated that allelopathic rice possesses an active chemical defense and auto-detoxifying enzyme system such as the up-regulated enzymes involved in de novo biosynthesis of phenolic allelochemicals and the glutathione-S-transferase (GST) associated with xenobiotic detoxification.     

Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm,Bombyx mori

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth‐instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori.     

335.4 kb microduplication in chromosome band Xp11.2p11.3 associated with developmental delay, growth retardation, autistic disorder and dysmorphic features

About 10% of causative mutations for mental retardation in male patients involve X chromosome (X-linked mental retardation, XLMR). We describe a case of a 3-year-old boy presenting with developmental delay, autistic features and growth and speech delay. Array-CGH analysis detected a microduplication on the X chromosome (Xp11.2p11.3), spanning 335.4 kb and including 3 known genes (ZNF81, ZNF182 and SPACA5). Genome-wide association studies show that approximately 30% of mutations causing XLMR are located in Xp11.2p11.3, where few pathogenic genes have been identified to date (such as ZNF41, PQB1 and ZNF81). ZNF81 codifies a zinc finger protein and mutations (non-sense mutations, deletions and structural rearrangements) involving this gene have already been described in association with mental retardation. Larger duplications in the same region have also been observed in association with mental retardation, and, in one case, the over-expression of ZNF81 has also been verified by mRNA quantification. No duplications of the single gene have been identified. To our knowledge, the microduplication found in our patient is the smallest ever described in Xp11.2p11.3. This suggests that the over-expression of ZNF81 could have pathological effects.     

Evaluation of reference genes for real‐time PCR quantification of gene expression in the Australian sheep blowfly,Lucilia cuprina

The Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), is an important pest of sheep in Australia and other parts of the world. However, the paucity of information on many fundamental molecular aspects of this species limits the ability to exploit functional genomics techniques for the discovery of new drug targets for its control. The present study aimed to facilitate gene expression studies in this species by identifying the most suitable reference genes for normalization of mRNA expression data. Quantitative real‐time polymerase chain reaction (PCR) was performed with 11 genes across RNA samples from eggs, L1, L3, pupae and adult life stages, and two normalization programs, Normfinder and geNorm, were then applied to the data. The results showed an ideal set of genes (18S rRNA, 28S rRNA, GST1, β‐tubulin and RPLPO) for data normalization across all life stages. The most suitable reference genes for studies within specific life stages were also identified. GAPDH was shown to be a poor reference gene. The reference gene recommendations in this study will be of use to laboratories investigating gene expression in L. cuprina and related blowfly species     

Salicylic acid-, but not cytokinin-induced, resistance to WClMV is associated with increased expression of SA-dependent resistance genes in Phaseolus vulgaris

Two-week-old Phaseolus vulgaris plants, wick-fed with 1 mmol/L salicylic acid (SA) or 50 nmol/L dihydrozeatin (DHZ), showed partial inhibition of the accumulation of white clover mosaic virus (WClMV) in infected primary leaves. This inhibition was measured as a decrease in the accumulation of both viral mRNA and viral coat protein, especially at the early stages of infection. Salicylic acid treatment resulted in moderately increased expression of phenylalanine ammonia lyase (PAL), NPR1, PR1 and HSP70 genes that participate in resistance to pathogens in plants. In contrast, DHZ treatments did not induce significant changes in expression of these genes. The expression of the P. vulgaris alternative oxidase (AOX) gene homolog, an enzyme implicated in plant resistance to viruses, showed low constitutive expression during the first 11 days post-infection and was not affected by either SA or DHZ. It appears that, while SA induced the NPR1-PR1 pathogen defense pathway genes, both SA and DHZ may use a different pathway to induce resistance to WClMV infection in P. vulgaris plants.     

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.     

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.