Development of STS markers for Verticillium wilt resistance in cotton based on RGA–AFLP analysis

Verticillium wilt (VW) is one of the most destructive diseases of cotton that decreases yield and quality. Identification of resistance gene analogs (RGAs) can provide potential candidate gene markers for marker-assisted selection (MAS) for VW resistance and cloning of VW resistance genes. The objective of this study was to convert RGA-targeted RGA–AFLP (amplified fragment length polymorphism) markers into sequence-tagged site (STS) markers. A total of 54 RGA–AFLP markers, including 28 from a backcross inbred line (BIL) population and 26 from a recombinant inbred line (RIL) population, were cloned and sequenced. Of the resulted 86 unique sequences, the majority were found to be homologous to genes, some of which showed similarity to genes encoding resistance-related products. A total of 163 STS primer pairs were designed and screened in the BIL population, 72 of which were also screened in the RIL population, resulting in 21 polymorphic STS markers in the BIL population and seven in the RIL population. Twelve STS markers were mapped onto eight chromosomes or linkage groups in the BIL population, six of which were mapped on the same chromosomes with their original RGA–AFLP markers including two STS markers on c4 flanking two VW resistance quantitative trait loci. These STS markers will be useful in MAS in breeding cotton for VW resistance. This study represents the first attempt to successfully convert RGA–AFLP markers into STS for mapping resistance genes in cotton.     

Phylogenetic relationships ofBacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase β-subunit genes

Comparative sequence analyses were performed on 14 genes encoding bacterial elongation factors EF-Tu and 7 genes encoding the -subunit of bacterial F₁F₀ type ATP-synthases. The corresponding predicted amino acid sequences were compared with published primary structures of homologous molecules. Phylogenetic trees were reconstructed from both data sets of aligned protein sequences and from an equivalent selection of 16S rRNA sequences by applying distance matrix and maximum parsimony methods. The EF-Tu data were in very good agreement with the rRNA data, although the resolution within the EF-Tu tree was reduced at certain phylogenetic levels. The resolution power of the ATPase -subunit sequence data were more reduced than those of the EF-Tu data. In comparison with the 16S rRNA tree there are minor differences in the order of adjacent branchings within the ATPase -subunit tree.     

Phylogenetic relationships of Puccinia horiana and other rust pathogens of Chrysanthemum × morifolium based on rDNA ITS sequence analysis

Isolates of the most important Puccinia species that have been reported on Chrysanthemum × morifolium were collected and the sequences of their ribosomal DNA internal transcribed spacers ITS1 and ITS2 were determined and used as phylogenetic markers. The focus of this study was on Puccinia horiana, due to its quarantine status and its impact in commercial chrysanthemum production. Three technical adjustments were needed to reliably obtain the nucleotide sequences starting from fresh or dried samples. The complete rDNA ITS nucleotide sequences of P. horiana, Puccinia chrysanthemi, and Puccinia tanaceti isolates of varying age and geographic origin were determined. We also identified an as yet undescribed Puccinia species on six old herbarium samples from chrysanthemum. This new species is morphologically similar to P. chrysanthemi and near identical to recent rust samples from Artemisia tridentata. P. tanaceti could not be confirmed as a pathogen of chrysanthemum. Different rDNA ITS sequences were present in P. horiana, with intra-isolate and inter-isolate variability in the length of three nucleotide repeat regions in the different rDNA tandem copies. We also identified three ITS types within P. horiana, with the rarer types displaying up to 67 bp nucleotide sequence differences. These rarer ITS types were detected at low copy number in all isolates. In general, very little rDNA ITS sequence variation was observed between P. horiana isolates from 1903 and 2003, and among isolates from different continents. Phylogenetic analyses using distance, Maximum Likelihood and Bayesian methods confirmed P. horiana, P. chrysanthemi, and the new Puccinia sp. as well-resolved groups, with P. horiana clustering in the clade where the economically important rust species of the Poaceae are located, and P. chrysanthemi and the new Puccinia sp. clustering in the clade where the majority of the rust fungi with hosts in the Asteraceae is located.     

Is hybridization involved in the evolution of the Chenopodium album aggregate? An analysis based on chromosome counts and genome size estimation

For our study of the Chenopodium album aggregate, we selected those species of Euroasiatic origin that represent the diploid–polyploid complex in Central Europe: C. album (6x), C. ficifolium (2x), C. opulifolium (6x), C. striatiforme (4x), C. strictum (4x) and C. suecicum (2x). We especially focused on (a) the origin of polyploid species and (b) the frequency of hybridization between species with different ploidy levels. We did not find any direct evidence of the existence of hybrids between two species with different ploidy levels within the C. album group. The sample/standard ratio of tetraploid and hexaploid species does not equal multiples of that of diploid species, which suggests that (i) tetraploids are not diploid autopolyploids and that (ii) hexaploids have not evolved from diploid species alone. Moreover, we have not found any hybrid plant either in the field or even in the offspring resulting from our experimental crosses. In view of these results, we adhere to the opinion that Chenopodium species do not hybridize freely across ploidy levels. Our analysis of DNA amounts, however, suggests that C. album is an allopolyploid that has arisen by hybridization between a diploid and a tetraploid species the identity of which is unknown.     

Enzymatic analysis of the effect of naturally occurring Leu138Pro mutation identified in SHV β-lactamase on hydrolysis of penicillin and ampicillin

Background  

The aim of this study was to analyze the significance of leucine to proline substitution at position 138(Leu138Pro) on the hydrolysis of penicillin and ampicillin that we identified in the bla _SHV gene of clinical Escherichia coli swine isolate.     

Research on characteristics of biomass distribution in urban forests of Shanghai metropolis based on remote sensing and spatial analysis北大核心CSCD

Aims Monitoring and quantifying the biomass and its distribution in urban trees and forests are crucial to understanding the role of vegetation in an urban environment. In this paper, an estimation method for biomass of urban forests was developed for the Shanghai metropolis, China, based on spatial analysis and a wide variety of data from field inventory and remote sensing. Methods An optimal regression model between forest biomass and auxiliary variables was established by stepwise regression analysis. The residual value of regression model was computed for each of the sites sampled and interpolated by Inverse-distance weighting (IDW) to predict residual errors of other sites not subjected to sampling. Forest biomass in the study area was estimated by combining the regression model based on remote sensing image data and residual errors of spatial distribution map. According to the distribution of plantations and management practices, a total of 93 sample plots were established between June 2011 and June 2012 in the Shanghai metropolis. To determine a suitable model, several spectral vegetation indices relating to forest biomass and structure such as normalized difference vegetation index (NDVI), ratio vegetation index (RVI), difference vegetation index (DVI), soil-adjusted vegetation index (SAVI), and modified soil-adjusted vegetation index (MSAVI), and new images synthesized through band combinations such as the sum of TM2, TM3 and TM4 (denoted Band 234), and the sum of TM3, TM4 and TM5 (denoted Band 345) were used as alternative auxiliary parameters . Important findings The biomass density in urban forests of the Shanghai metropolis varied from 15 to 120 t•hm2. The higher densities of forest biomass concentrated mostly in the urban areas, e.g. in districts of Jing'an and Huangpu, mostly ranging from 35 to 70 t•hm2. Suburban localities such as the districts of Jiading and Qingpu had lower biomass densities at around 15 to 50 t•hm2. The biomass density of Cinnamomum camphora trees across the Shanghai metropolis varied between 20 and 110 t•hm2. The spatial biomass distribution of urban forests displayed a tendency of higher densities in northeastern areas and lower densities in southwestern areas. The total biomass was 3.57 million tons (Tg) for urban forests and 1.33 Tg for C. camphora trees. The overall forest biomass was also found to be distributed mostly in the suburban areas with a fraction of 93.9%, whereas the urban areas shared a fraction of only 6.1%. In terms of the areas, the suburban and urban forests accounted for 95.44% and 4.56%, respectively, of the total areas in the Shanghai metropolis. Among all the administrative districts, the Chongming county and the new district of Pudong had the highest and the second highest biomass, accounting for 20.1% and 19.18% of the total forest biomass, respectively. In contrast, the Jing'an district accounted for only 0.11% of the total forest biomass. The root-mean-square error (RMSE), mean absolute error (MAE) and mean relative error (MRE) of the model for estimating urban forest biomass in this study were 8.39, 6.86 and 24.22%, respectively, decreasing by 57.69%, 55.43% and 64.00% compared to the original simple regression model and by 62.21%, 58.50%, 65.40% compared to the spatial analysis method. Our results indicated that a more efficient way to estimate urban forest biomass in the Shanghai metropolis might be achieved by combining spatial analysis with regression analysis. In fact, the estimated results based on the proposed model are also more comparable to the up-scaled forest inventory data at a city scale than the results obtained using regression analysis or spatial analysis alone.     

First step toward the “fingerprinting” of brain tumors based on synchrotron radiation X-ray fluorescence and multiple discriminant analysis

Synchrotron-radiation-based X-ray fluorescence was applied to the elemental microimaging of neoplastic tissues in cases of various types of brain tumors. The following cases were studied: glioblastoma multiforme, gemistocytic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ganglioglioma, fibrillary astrocytoma, and atypical transitional meningioma. Apart from neoplastic tissue, the analysis included areas of tissue apparently without malignant infiltration. The masses per unit area of P, S, Cl, K, Ca, Fe, Cu, Zn, Br, and Rb were used to construct a diagnostic classifier for brain tumors using multiple discriminant analysis. It was found that S, Cl, Cu, Fe, K, Br, and Zn are the most significant elements in the general discrimination of tumor type. The highest similarity in elemental composition was between atypical transitional meningioma and fibrillary astrocytoma. The smallest differentiation was between glioblastoma multiforme and oligodendroglioma. The mean percentage of correct classifications, estimated according to the a posteriori probabilities procedure, was 99.9%, whereas the mean prediction ability of 87.6% was achieved for ten new cases excluded previously from the model construction. The results showed that multiple discriminant analysis based on elemental composition of tissue may be a potentially valuable method assisting differentiation and/or classification of brain tumors.     

Ultrastructural effects of trace elements and environmental pollution in Italian “Triangle of Death” on Pseudevernia furfuracea (L.) Zopf

Abstract

The lichen Pseudevernia furfuracea was exposed to environmental trace elements in the district of Acerra (province of Naples, southern Italy), one of the points forming Italy's “Triangle of Death”. P. furfuracea thalli were exposed in bags at different sites for 6 months, periodically collected and examined by transmission electron microscopy (TEM) to assess ultrastructural changes. An our earlier study demonstrated that these exposed lichens were strongly contaminated by trace elements (Sorbo S, Aprile G, Strumia S, Castaldo Cobianchi R, Leone A, et al. 2008. Trace element accumulation in P. furfuracea (L.) Zopf exposed in Italy's so-called Triangle of Death. Sci Total Environ 407: 647–654.). The algal cells were more affected than the fungal symbiont. Exposition at urban sites gave the most frequent changes. Four trace elements (Cd, Pb, Cu and Zn) were used to study the effects of ultrastructural trace element both in field and in vitro treatments. The lichen developed comparable ultrastructural changes when exposed to different trace elements and the changes were not specific to the treatment used (lichen bag exposition, in field and in vitro treatments). The in vitro treatment gave the highest frequency of damage at all time points. X-ray TEM microanalysis revealed trace elements inside the cell walls and the cytoplasmic vesicles of the lichens cultured with the trace elements; this localization is probably related to tolerance mechanisms.     

Organization and expression of polygalacturonase and other ripening related genes in Ailsa Craig “Neverripe” and “Ripening inhibitor” tomato mutants

The organization and expression of ripening-related genes were investigated in normal tomato (Lycopersicon esculentum cv. Ailsa Craig) and in Neverripe (Nr) and Ripening inhibitor (rin) mutants.Hybridization studies with ripening-related cDNA clones showed that the gene for polygalacturonase (PG) is barely expressed in rin and expressed at a low level in Nr fruit. Four other genes were found to be expressed at reduced levels in rin. Exogenous ethylene was able to restore higher levels of expression of all the genes showing reduced expression in rin except that for PG. However, exogenous ethylene did not restore normal ripening in rin fruit. Analysis of chromosomal DNA by Southern blotting indicated that all the genes studied, including the PG gene, and also an upstream promoter of the PG gene, are present in the rin and Nr genomes and appear to be arranged in a similar way to those in normal tomatoes. The results are discussed in the light of the suggestion that these mutations may involve part of the regulatory apparatus leading to the expression of ripening genes such as PG.     

The translation initiation complex eIF3 in trypanosomatids and other pathogenic excavates – identification of conserved and divergent features based on orthologue analysis

Background

The initiation of translation in eukaryotes is supported by the action of several eukaryotic Initiation Factors (eIFs). The largest of these is eIF3, comprising of up to thirteen polypeptides (eIF3a through eIF3m), involved in multiple stages of the initiation process. eIF3 has been better characterized from model organisms, but is poorly known from more diverged groups, including unicellular lineages represented by known human pathogens. These include the trypanosomatids (Trypanosoma and Leishmania) and other protists belonging to the taxonomic supergroup Excavata (Trichomonas and Giardia sp.).

Results

An in depth bioinformatic search was carried out to recover the full content of eIF3 subunits from the available genomes of L. major, T. brucei, T. vaginalis and G. duodenalis. The protein sequences recovered were then submitted to homology analysis and alignments comparing them with orthologues from representative eukaryotes. Eleven putative eIF3 subunits were found from both trypanosomatids whilst only five and four subunits were identified from T. vaginalis and G. duodenalis, respectively. Only three subunits were found in all eukaryotes investigated, eIF3b, eIF3c and eIF3i. The single subunit found to have a related Archaean homologue was eIF3i, the most conserved of the eIF3 subunits. The sequence alignments revealed several strongly conserved residues/region within various eIF3 subunits of possible functional relevance. Subsequent biochemical characterization of the Leishmania eIF3 complex validated the bioinformatic search and yielded a twelfth eIF3 subunit in trypanosomatids, eIF3f (the single unidentified subunit in trypanosomatids was then eIF3m). The biochemical data indicates a lack of association of the eIF3j subunit to the complex whilst highlighting the strong interaction between eIF3 and eIF1.

Conclusions

The presence of most eIF3 subunits in trypanosomatids is consistent with an early evolution of a fully functional complex. Simplified versions in other excavates might indicate a primordial complex or secondary loss of selected subunits, as seen for some fungal lineages. The conservation in eIF3i sequence might indicate critical functions within eIF3 which have been overlooked. The identification of eIF3 subunits from distantly related eukaryotes provides then a basis for the study of conserved/divergent aspects of eIF3 function, leading to a better understanding of eukaryotic translation initiation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1175) contains supplementary material, which is available to authorized users.     

Regression analysis of “current status” life tables on duration of breastfeeding in Sri Lanka

Abstract

This paper illustrates the application of conventional regression techniques to the analysis of breastfeeding rates derived from the birth dates of respondents’ recent children and their breastfeeding status at date of interview. Rates of this type have been found to be less biased than rates based on recall of the duration of breastfeeding, but require new analytical approaches to be fully exploited.     

On being “actionable”: clinical sequencing and the emerging contours of a regime of genomic medicine in oncology

This article explores commercial, academic, and national initiatives aimed at using sequencing technologies to generate “actionable” genomic results that can be applied to the clinical management of oncology patients. We argue that the term “actionable” is not merely a buzzword, but signals the emergence of a distinctive sociotechnical regime of genomic medicine in oncology. Unlike other regimes of genomic medicine that are organized around assessing and managing inherited risk for developing cancer (e.g. BRCA testing), actionable regimes aim to generate predictive relationships between genetic information and drug therapies, thereby generating new kinds of clinical actions. We explore how these genomic results are made actionable by articulating them with existing clinical routines, clinical trials, regulatory regimes, and health care systems; and in turn, how clinical sequencing programs have begun to reconfigure knowledge and practices in oncology. Actionability regimes confirm the emergence of bio-clinical decision-making in oncology, whereby the articulation of molecular hypotheses and experimental therapeutics become central to patient care.     

Quantitative trait analysis of resistance to plum pox virus in the apricot F1 progeny “Harlayne” × “Vestar”

Plum pox virus (PPV) is a devastating stone fruit disease of major importance, and better understanding of the genetic control of resistance to this trait would be useful for more efficient development of resistant cultivars. Previous studies have reported a locus of major effect from PPV resistance on linkage group 1. The current study confirms these results by mapping plum pox virus resistance in a F1 progeny issued from a cross between “Harlayne”, as a PPV-resistant parent, and “Vestar” as a susceptible parent. The hybrids were grafted simultaneously and subsequently inoculated with the PPV-M and D strains. The symptom scoring on leaves was performed nine times over two vegetative cycles. Marker–trait associations were analyzed using the Kruskal–Wallis (KW) non-parametric test, and the PPV resistance loci were mapped using composite interval mapping (CIM). We show that both analyses (KW and CIM) highlighted the upper part of linkage group 1 of the apricot “Harlayne” genitor.     

Comparison of the transmembrane helices of bovine rhodopsin in the crystal structure and the C α template based on cryo-electron microscopy maps and sequence analysis of the G protein-coupled receptors

G protein-coupled receptors (GPCR) are activated by a diverse array of extracellular signals, ranging from light to polypeptide molecules. The receptors propagate these signals intracellularly using G protein secondary messenger pathways. A common feature in the architecture of these receptors is their seven transmembrane domains. The first crystal structure of a GPCR, bovine rhodopsin, has recently been solved at 2.8 Å. We compared the seven membrane-spanning helices (TMH) from the crystal structure of bovine rhodopsin with those from the low-resolution model of bovine rhodopsin based on the cryo-electron microscopy structure of frog rhodopsin developed by Dr Joyce Baldwin. The model developed by Baldwin used a consensus sequence approach to predict the rotational position of each helix with respect to the other six helices. Superposition of the entire helix bundle of the Baldwin model with the crystal structure gave a RMS difference (RMSD) of 3.2 Å for the 198 C f atoms which suggests a high level of similarity in the arrangement of the helices. Except for TMH IV (RMSD of 4.0 Å), the position of corresponding helices within the helix bundle overlapped well. The superposition of individual helices showed that the RMSD values over 3 Å in the global superposition were largely due to one or more of the following: (i) differences in the unraveling and kinks for these helices, (ii) translation of TMH perpendicular to the membrane and (iii) rotation of helices up to 31°, except for TMH IV in which an additional contribution to the RMSD came from the aforementioned observation. As other crystal structures of GPCRs become available, a comparison with the Baldwin consensus model may reveal larger differences than those observed here.