Assigning Brassica microsatellite markers to the nine C-genome chromosomes using Brassica rapa var. trilocularis–B. oleracea var. alboglabra monosomic alien addition lines

Brassica rapa var. trilocularis-B. oleracea var. alboglabra monosomic alien addition lines (MAALs) were used to assign simple sequence repeat (SSR) markers to the nine C-genome chromosomes. A total of 64 SSR markers specific to single C-chromosomes were identified. The number of specific markers for each chromosome varied from two (C3) to ten (C4, C7 and C9), where the designation of the chromosomes was according to Cheng et al. (Genome 38:313-319, 1995). Seventeen additional SSRs, which were duplicated on 2-5 C-chromosomes, were also identified. Using the SSR markers assigned to the previously developed eight MAALs and recently obtained aneuploid plants, a new Brassica rapa-B. oleracea var. alboglabra MAAL carrying the alien chromosome C7 was identified and developed. The application of reported genetically mapped SSR markers on the nine MAALs contributed to the determination of the correspondence between numerical C-genome cytological (Cheng et al. in Genome 38:313-319, 1995) and linkage group designations. This correspondence facilitates the integration of C-genome genetic information that has been generated based on the two designation systems and accordingly increases our knowledge about each chromosome. The present study is a significant contribution to genetic linkage analysis of SSR markers and important agronomic traits in B. oleracea and to the potential use of the MAALs in plant breeding.     

Cloning of cDNA and chromosomal location of genes encoding the three types of subunits of the wheat tetrameric inhibitor of insect α-amylase

We have characterized three cDNA clones corresponding to proteins CM1, CM3 and CM16, which represent the three types of subunits of the wheat tetrameric inhibitor of insect -amylases. The deduced amino acid sequences of the mature polypeptides are homologous to those of the dimeric and monomeric -amylase inhibitors and of the trypsin inhibitors. The mature polypeptides are preceded by typical signal peptides. Southern blot analysis of appropriate aneuploids, using the cloned cDNAs as probes, has revealed the location of genes for subunits of the CM3 and of the CM16 type within a few kb of each other in chromosomes 4A, 4B and 4D, and those for the CM1 type of subunit in chromosomes 7A, 7B and 7D. Known subunits of the tetrameric inhibitor corresponding to genes from the B and D genomes have been previously characterized. No proteins of this class have been found to be encoded by the A genome in hexaploid wheat (genomes AA, BB, DD) or in diploid wheats (AA) and no anti -amylase activity has been detected in the latter, so that the A-genome genes must be either silent (pseudogenes) or expressed at a much lower level.     

Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and φ80 transducing phages

φ80 transducing phages for the proC², phoA and phoB genes of Escherichia coli have been obtained. Two mutants have been isolated, in which the brnQ, phoA, proC, phoB (and possibly phoR) genes have been deleted. Derivatives of a phoA, phoB deletion strain which are lysogenic for a φ80phoA transducing phage make only very low levels of alkaline phosphatase activity. These results are in agreement with a positive control mechanism for the regulation of alkaline phosphatase synthesis.     

Identification of sterility-inducing cytoplasms in rye using the plasmotype–genotype interaction test and newly developed SCAR markers

A series of 25 rye (Secale cereale L.) inbred lines was tested with respect to three mitochondrial sequence-characterized amplified region (SCAR) polymorphisms. The analysis revealed a close association between the marker-determined mitotypes and plasmotypes (cytoplasm types known from breeding data) represented by the inbreds. The mitochondrial markers also confirmed normal (N-) cytoplasmic character of three wild rye species: Secale montanum, S. vavilovii and S. kuprijanovii. For 186 plants from open-pollinated cultivars of Turkish and South American origin, cytoplasm identification was performed with the use of crossing with double non-restoring tester. In 77 plants the normal (N) cytoplasm was detected, and for 63 of these the PCR analysis was performed producing results which were consistent with the genetic data based on testcrossing and phenotype assessment. The mitochondrial markers also confirmed a presence of sterility-inducing cytoplasm in the remaining 109 plants. Moreover, the markers allowed for differentiation between Pampa (P-) and Vavilovii (V-) cytoplasmic individuals. For 60 plants the latter results were verified using crosses with a line maintaining P-cytoplasmic sterility and acting as a restorer of the V-cytoplasm. For two of these plants contradicting results were produced with the applied methods of cytoplasm identification and the basis of this discrepancy is discussed. Regardless of the identification method, widespread occurrence of a sterility-inducing cytoplasm was revealed, especially in South American populations.     

Cloning of cDNA,expression, and chromosomal location of genes encoding the three types of subunits of the barley tetrameric inhibitor of insect α-amylase

Three cDNA clones from barley developing endosperm, corresponding to proteins BTAI-CMa, BTAI-CMb and BTAI-CMd, which are the three types of subunits of the tetrameric inhibitor of insect -amylases, have been identified and sequenced. The deduced amino acid sequence of BTAI-CMb corresponds to the CM16/CM17 type of subunit in wheat (92/90% identical residues) and has one putative N-glycosylation site (NLT) and a possible kinase-C phosphorylation site (SCR). The BTAI-CMa sequence differs at four amino acid residues from a previously reported one from cv. Bomi and the sequence deduced for BTAI-CMd completes (11 N-terminal residues) and confirms previously available data. The gene for BTAI-CMa (Iat1) is located in the arm of barley chromosome 7H (syn.1), while genes for both BTAI-CMb (Iat2) and BTAI-CMd (Iat3) are in the long arm of chromosome 4H. The three genes are expressed in endosperm and their mRNAs are not detected in the other tissues tested, except Iat1, which seems to be expressed at a low level in coleoptile and roots, where it is switched off by 50 M methyl jasmonate.     

Direct and protective effects of single or combined addition of vincristine and ε-viniferin on human HepG2 cellular oxidative stress markers in vitro

The objective of this study is to examine the direct effects of low doses and high doses of ε-viniferin, a substance known to be an antioxidant, and vincristine sulphate, a chemotherapeutic agent, alone and in combination [ε-viniferin + vincristine] on HepG2 cell strain, as well as evaluate oxidative stress after incubation periods of 3, 6, and 24 h. Direct effect was determined right after the incubation period; however, for protective effect, antioxidant protection response was determined after the treatment for 1 h with 500 μM H₂O₂, which is an oxidative stressor. For this purpose, superoxide dismutase was determined for enzyme activity, and lipid hydroperoxide (LPO) and reduced glutathione concentrations were studied as indicators of oxidative stress. Results show that low [3.63 µM vincristine + 3.75 µM ε-viniferin] and high [11.25 µM vincristine + 15.8 µM ε-viniferin] doses of combination groups showed similar direct antioxidant effect on LPO levels as protective when compared to the H₂O₂ control group (p < 0.05). Superoxide dismutase enzyme showed a direct antioxidant effect in low and high dose combination groups. In addition, when the incubation period was increased to 24 h, a protective effect was observed in both dose groups (p < 0.05). Reduced glutathione activities showed a direct effect in the low dose combination group, and a protective effect in both the low and high doses in the 24 h. These results show that combined usage of drugs in HepG2 cell strain possesses a protective effect against exogenically produced oxidative stress conditions.     

Structure,chromosomal location and expression of a rice gene encoding the microsome ω-3 fatty acid desaturase

The -3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. A cDNA encoding the microsome -3 fatty acid desaturase (OsFAD3) and the corresponding genomic clone were isolated from rice (Oryza sativa L.). The OsFAD3 gene was composed of 8 exons and 7 introns. A microsatellite was present in the second exon of the OsFAD3 gene, showing polymorphism between Indica and Japonica rice varieties. The mapping of this microsatellite showed that the OsFAD3 gene was located on chromosome 11. Expression of the OsFAD3 cDNA in tobacco hairy root tissues and subsequent analysis of fatty acid compositions demonstrated the activity of the microsome -3 fatty acid desaturase. The OsFAD3 mRNA was abundant in root tissues, but was hardly detectable in leaves. In root tissues, a high level of the OsFAD3 mRNA was observed at 15 °C and 20 °C, with its level decreasing markedly at temperatures below 10 °C. The accumulation of the OsFAD3 mRNA in leaf tissues remained at quite low levels, both at normal growth temperatures and at chilling temperatures. Similar temperature responses of the OsFAD3 gene were observed both in chilling- tolerant and in chilling-intolerant rice cultivars.     

Genome-wide single nucleotide polymorphisms and insertion–deletions of Oryza sativa L. subsp. japonica cultivars grown near the northern limit of rice cultivation

Single nucleotide polymorphisms (SNPs) and insertions–deletions (InDels) are valuable molecular markers for molecular breeding among genetically closely related cultivars. Rice (Oryza sativa L. subsp. japonica) cultivars grown in Hokkaido (45–42°N), the northernmost region of rice paddy cultivation in Japan, have been bred for over 100 years for adaptation to low summer temperatures together with high yield and good eating quality. In this study, for 10 closely related rice cultivars released in Hokkaido and cultivar Koshihikari, we identified genome-wide SNPs and InDels by next-generation sequencing. More than 29 million reads from the Hokkaido cultivars, each 101 nucleotides long, were uniquely mapped to the Nipponbare reference genome. The average of the total nucleotide length of all uniquely mapped reads corresponded to 10.9 times (3,978 Mb with genome coverage of 90.7 %) the Nipponbare reference genome. An average of 99,955 putative SNPs (1.8 times the number in Koshihikari) and 14,617 putative InDels (also 1.8 times the number in Koshihikari) were detected in Hokkaido cultivars relative to the Nipponbare genome, which enabled analyses of the inheritance of pedigree haplotypes of four cultivars, SNPs and InDels among closely related Hokkaido cultivars, and haplotype blocks unique to Hokkaido cultivars. The comprehensive SNP and InDel data provide DNA marker resources and will facilitate quantitative trait locus analysis of biparental mapping of very closely related Hokkaido cultivars. Furthermore, the haplotype blocks unique to Hokkaido cultivars represent ideal genetic regions for improvement of cultivars to be grown near the northern and southern limits of rice cultivation.     

Direct and indirect effects of ant–trophobiont interactions on the reproduction of a hummingbird-pollinated mistletoe

Plant Ecology - Fluid-feeding herbivores directly affect host plants through sap consumption. Moreover, they establish mutualistic relationships with ants, which might generate additional...     

Development and validation of a quantitative method for the determination of 12 endocannabinoids and related compounds in human plasma using liquid chromatography–tandem mass spectrometry

A sensitive and specific LC–MS/MS method for the quantification of the endocannabinoids and related structures anandamide, 2-arachidonoyl glycerol, 2-arachidonyl glycerol ether, O-arachidonoyl ethanolamide, dihomo-γ-linolenoyl ethanolamide, docosatetraenoyl ethanolamide, N-arachidonoyl dopamine, N-arachidonyl glycine, N-oleoyl dopamine, oleoyl ethanolamide, palmitoyl ethanolamide, and stearoyl ethanolamide in human plasma was developed and validated. Compounds were extracted using acetonitrile followed by solid-phase extraction. Separation was performed on a Xterra C8 column using gradient elution coupled to a triple-quadrupole MS. LLOQ levels ranged from 0.02 to 1.75 μg/mL, LODs ranged from 0.0002 to 0.1266 ng/mL, and accuracies were >80% (except stearoyl ethanolamide at lowest spike level) at all spike levels.     

Rapid detection of single nucleotide deletions: application to the β 6 (-A) mutation of the β-globin gene and to cystic fibrosis

The formation of heteroduplexes from the amplified products of homologous alleles has been shown to be useful in the identification of heterozygotes carrying deletion or insertion mutations. Here, we describe an improved procedure that allows the detection of single base pair (bp) deletions on nondenaturing polyacrylamide gels. Carriers for a common Mediterranean -thalassemic mutation, 6 (-A), could be easily detected by use of this method, as could carriers of a 1-bp deletion in the cystic fibrosis gene.     

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography–electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC™) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 μmol/L (UMP and AMP) to 1.3 μmol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.     

Molecularly-imprinted microspheres for selective extraction and determination of melamine in milk and feed using gas chromatography–mass spectrometry

Molecularly-imprinted polymers in the form of microspheres were synthesized using the dispersion polymerization protocol; cyromazine was used as dummy template, while methacrylic acid, ethylene glycol dimethacrylate and acetonitrile (MeCN) were used as functional monomer, cross-linker, and porogen, respectively. When compared with the non-imprinted polymer, the molecularly-imprinted polymers (MIPs) showed outstanding affinity toward melamine in MeCN with a maximum binding concentration (B_max) of 53.20 nmol mg⁻¹ MIPs, imprinting effect of 4.6, and a dissociation constant (K_d) of 90.45 μM. After optimization of the molecularly-imprinted solid-phase extraction conditions, a new method was developed to determine the melamine in milk and feed with gas chromatography–mass spectrometry. The performance of this method has been evaluated in the tainted milk and feed in terms of recovery, precision, linearity, the limit of detection (LOD) and limit of quantitation (LOQ). Recovery ranged in samples from 93.1 to 101.3% with intra-day and inter-day relative standard deviation values below 5.34%. The LOD and LOQ of melamine in milk and feed were 0.01 μg mL⁻¹ (μg g⁻¹) and 0.05 μg mL⁻¹ (μg g⁻¹), respectively.     

Identification of the Worldwide Olive Germplasm Bank of Córdoba (Spain) using SSR and morphological markers

Olive is one of the most ancient crop plants and the World Olive Germplasm Bank of Cordoba (WOGBC), Spain, is one of the world’s largest collections of olive germplasm. We used 33 SSR (Simple Sequence Repeats) markers and 11 morphological characteristics of the endocarp to characterise, identify and authenticate 824 trees, representing 499 accessions from 21 countries of origin, from the WOGBC collection. The SSR markers exhibited high variability and information content. Of 332 cultivars identified in this study based on unique combinations of SSR genotypes and endocarp morphologies, 200 were authenticated by genotypic and morphological markers matches with authentic control samples. We found 130 SSR genotypes that we considered as molecular variants because they showed minimal molecular differences but the same morphological profile than 48 catalogued cultivars. We reported 15 previously described and 37 new cases of synonyms as well as 26 previously described and seven new cases of homonyms. We detected several errors in accession labelling, which may have occurred at any step during establishment of plants in the collection. Nested sets of 5, 10 and 17 SSRs were proposed to progressively and efficiently identify all of the genotypes studied here. The study provides a useful protocol for the characterisation, identification and authentication of any olive germplasm bank that has facilitated the establishment of a repository of true-to-type cultivars at the WOGBC.     

Modeling of the structure of protein–DNA complexes using the data from FRET and footprinting experiments

We discuss the question of constructing three-dimensional models of DNA in complex with proteins using computer modeling and indirect methods of studying the conformation of macromolecules. We consider the methods of interpreting the experimental data obtained by indirect methods of studying the three-dimensional structure of biomolecules. We discuss some aspects of integrating such data into the process of constructing the molecular models of DNA–protein complexes based on the geometric characteristics of DNA. We propose an algorithm for estimating conformations of such complexes based on the information about the local flexibility of DNA and on the experimental data obtained by Forster resonance energy transfer (FRET) and hydroxyl footprinting. Finally, we use this algorithm to predict the hypothetical configuration of DNA in a nucleosome bound with histone H1.     

On-line antioxidant activity determination: comparison of hydrophilic and lipophilic antioxidant activity using the ABTS•+ assay

Abstract

The ABTS/H₂O₂/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples – conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) – provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.     

High-frequency variation of tree-ring width of some native and alien tree species in Latvia during the period 1965–2009

The plasticity of climate-growth relationships of trees is one of the main factors determining the climate-induced changes in forest productivity and composition. In this study, high-frequency variation of tree-ring width (TRW) of four native and three alien tree species and two hybrids of Populus L. growing in Latvia (hemiboreal zone) was compared using a principal component analysis based on TRW indices for the period 1965–2009. The effect of climatic factors was assessed using a bootstrapped correlation analysis. Influence of common climatic factors related to the length of the vegetation season, winter temperature, and water regime in summer was traced in the TRW of the studied species and hybrids. The combination and effect of the identified factors differed by species (and hybrids), to a certain extent explaining the diversity of TRW patterns. Nevertheless, some similarities among the species were also observed, suggesting the plasticity of growth response. Scots pine was generally sensitive to winter temperatures, but Norway spruce was mainly sensitive to summer water regime, while black alder was sensitive to winter temperatures and precipitation in spring. In contrast, silver birch showed the lowest sensitivity to the tested climatic factors (demonstrating sensitivity to winter precipitation in a few sites), suggesting tolerance to weather fluctuations. The TRW of the alien species was primarily sensitive to climatic factors related to water regime in the summer of the year preceding the formation of tree-ring, implying differences in mechanisms regulating wood increment. Nevertheless, temperature in the dormant period was significant for European larch in a few sites, suggesting sensitivity to cold damage. The variation of TRW of Populus hybrids diverged from others, as their growth was negatively correlated with the temperature in autumn, spring, and summer and positively correlated with water balance. Although the annual water balance in Latvia is positive, the effect of water deficit on tree growth was apparent.