Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates

Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)₅- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)₅ and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)₅ and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)₅ and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.     

Genetic variation in natural populations of Capparis from Turkey, as revealed by RAPD analysis

In this study, the genetic diversity of 15 Turkish natural Capparis populations was screened using the randomly amplified polymorphic DNA analysis (RAPD) technique. Ten RAPD primers produced 98 loci, 73 of which were polymorphic. The binary RAPD data were computed using the POPGENE (version 1.31), a genetic data analysis software program. According to genetic diversity analysis at locus level, the total genetic diversity (H _T) and genetic diversity within population (H _s) were detected as 0.16 and 0.12, respectively. The genetic differentiation (G _ST) and gene flow (N _m) between populations were observed as 0.22 and 1.79, respectively. The mean number of allele per locus (n _a), the mean number of effective allele (n _ea), and the mean value of genetic diversity (H _e) were determined as 2, 1.20, and 0.16, respectively. According to Pearson’s correlation analysis, the mean number of allele had a strong negative correlation with wind and a strong positive correlation with rain. According to multiple regression analysis, eco-geographical factors had a significant effect on the mean number of allele, the mean number of effective allele, and the mean value of genetic diversity. The principal component analysis revealed 87.42 % of total genetic variation. The principal coordinate analysis displayed the separation of population according to genetic distances based on dissimilarities matrix values on a scattered plot graph. Five different varieties, Capparis spinosa L. var. spinosa, var aegyptia and var. canescens, and Capparis ovata Desf. var. palaestina, and var. herbacea were identified in this study. Intermediate forms of plants were observed among the specimens.     

Molecular Taxonomic Evidence for Two Distinct Genotypes of Mycobacterium yongonense via Genome-Based Phylogenetic Analysis

Recently, we introduced a distinct Mycobacterium intracellulare INT-5 genotype, distantly related to other genotypes of M. intracellulare (INT-1 to -4). The aim of this study is to determine the exact taxonomic status of the M. intracellulare INT-5 genotype via genome-based phylogenetic analysis. To this end, genome sequences of the two INT-5 strains, MOTT-H4Y and MOTT-36Y were compared with M. intracellulare ATCC 13950^T and Mycobacterium yongonense DSM 45126^T. Our phylogenetic analysis based on complete genome sequences, multi-locus sequence typing (MLST) of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126^T than the M. intracellulare strains. These results suggest their taxonomic transfer from M. intracellulare into M. yongonense. Finally, we selected 5 target genes (argH, dnaA, deaD, hsp65, and recF) and used SNPs for the identification of M. yongonese strains from other M. avium complex (MAC) strains. The application of the SNP analysis to 14 MAC clinical isolates enabled the selective identification of 4 M. yongonense clinical isolates from the other MACs. In conclusion, our genome-based phylogenetic analysis showed that the taxonomic status of two INT-5 strains, MOTT-H4Y and MOTT-36Y should be revised into M. yongonense. Our results also suggest that M. yongonense could be divided into 2 distinct genotypes (the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene) depending on the presence of the lateral gene transfer of rpoB from M. parascrofulaceum.     

Acoustic,genetic and morphological variations within the katydid Gampsocleis sedakovii (Orthoptera,Tettigonioidea)

In an attempt to explain the variation within this species and clarify the subspecies classification, an analysis of the genetic, calling songs, and morphological variations within the species Gampsocleis sedakovii is presented from Inner Mongolia, China. Recordings were compared of the male calling songs and analysis performed of selected acoustic variables. This analysis is combined with sequencing of mtDNA - COI and examination of morphological traits to perform cluster analyses. The trees constructed from different datasets were structurally similar, bisecting the six geographical populations studied. Based on two large branches in the analysis, the species Gampsocleis sedakovii was partitioned into two subspecies, Gampsocleis sedakovii sedakovii (Fischer von Waldheim, 1846) and Gampsocleis sedakovii obscura (Walker, 1869). Comparing all the traits, the individual of Elunchun (ELC) was the intermediate type in this species according to the acoustic, genetic, and morphological characteristics. This study provides evidence for insect acoustic signal divergence and the process of subspeciation.     

Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin

Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)₅ primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis.     

Analyzing Spatial Heterogeneity in DCE- and DW-MRI Parametric Maps to Optimize Prediction of Pathologic Response to Neoadjuvant Chemotherapy in Breast Cancer

The purpose of this study is to investigate the ability of multivariate analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion-weighted MRI (DW-MRI) parametric maps, obtained early in the course of therapy, to predict which patients will achieve pathologic complete response (pCR) at the time of surgery. Thirty-three patients underwent DCE-MRI (to estimate K^trans, v_e, k_ep, and v_p) and DW-MRI [to estimate the apparent diffusion coefficient (ADC)] at baseline (t₁) and after the first cycle of neoadjuvant chemotherapy (t₂). Four analyses were performed and evaluated using receiver-operating characteristic (ROC) analysis to test their ability to predict pCR. First, a region of interest (ROI) level analysis input the mean K^trans, v_e, k_ep, v_p, and ADC into the logistic model. Second, a voxel-based analysis was performed in which a longitudinal registration algorithm aligned serial parameters to a common space for each patient. The voxels with an increase in k_ep, K^trans, and v_p or a decrease in ADC or v_e were then detected and input into the regression model. In the third analysis, both the ROI and voxel level data were included in the regression model. In the fourth analysis, the ROI and voxel level data were combined with selected clinical data in the regression model. The overfitting-corrected area under the ROC curve (AUC) with 95% confidence intervals (CIs) was then calculated to evaluate the performance of the four analyses. The combination of k_ep, ADC ROI, and voxel level data achieved the best AUC (95% CI) of 0.87 (0.77–0.98).     

Comparison of methyl eugenol metabolites, mitochondrial COI, and rDNA sequences of Bactrocera philippinensis (Diptera: Tephritidae) with those of three other major pest species within the dorsalis complex

Males of the oriental fruit fly, Bactrocera dorsalis (Hendel) and some of its sibling species have strong affinity for methyl eugenol (ME). Methyl eugenol ingested by male flies is biotransformed in the crop to two ME metabolites that eventually accumulate in the rectal gland, which is known to serve as a reservoir for B. dorsalis sex pheromones. When fed with ME, males of laboratory and wild B. philippinensis Drew and Hancock selectively accumulated two metabolites, 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol, in the rectal gland, as was seen for B. dorsalis sensu stricto, B. invadens Drew, Tsuruta and White, and B. papayae Drew and Hancock. Phylogenetic analysis of COI and rDNA sequence data of these four taxa also revealed a close relationship among B. philippinensis, B. dorsalis s.s., B. invadens, and B. papayae (all four are members of the dorsalis species complex). This result corroborates pheromone analysis. The usefulness of pheromonal analysis as a chemotaxonomy tool to complement molecular and other analysis in differentiation of closely related sibling species within the Bactrocera dorsalis complex, for which use of morphological characters had been inadequate, is highlighted.     

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.     

Phylogenetic relationships among strains of the entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) isolated from Japan

The fungus Beauveria bassiana (Balsamo) Vuillemin has previously been classified using morphological characteristics, but morphology cannot reveal the phylogenetic relationships among conventionally classified strains. High levels of homology have been found in gene sequences among various B. bassiana strains, complicating the determination of their evolutionary relationships. To elucidate phylogenetic relationships among conventionally known Beauveria species, we analyzed 57 major strains of B. bassiana and 3 strains of B. brongniartii (Saccardo) Petch isolated from Japan by analysis of internal transcribed spacer (ITS) sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The ITS sequence analysis placed the 57 conventional B. bassiana strains into two clusters, B. bassiana and Beauveria pseudobassiana Rehner et Humber. In contrast, GP analysis produced five clusters of B. bassiana strains that included B. pseudobassiana clusters. These results suggested that GP was more accurate than ITS sequence analysis for determining phylogenetic relationships within B. bassiana. In addition, our findings suggested that conventional strains of B. bassiana isolated from Japan include both B. bassiana and B. pseudobassiana groups.     

Vibrio cortegadensis sp. nov., isolated from clams

A group of four strains isolated from clams (Venerupis decussata and Venerupis philippinarum) in Galicia (NW Spain) were subjected to a polyphasic characterization, based on the phenotypic characteristics, the analysis of chemotaxonomic features, the sequencing of the 16S rRNA and five housekeeping (atpA, pyrH, recA, rpoA and rpoD) genes, as well as DNA–DNA hybridization (DDH). The analysis of the phenotypic and chemotaxonomic characteristics and the results of a phylogenetic study, based on the 16S rRNA gene sequence analysis and multilocus sequence analysis, clearly indicated that these strains belong to the genus Vibrio and were allocated between the Splendidus and Anguillarum clades showing a close relationship with the type strains of Vibrio tapetis (98.8 %), Vibrio pomeroyi (98.0 %) and Vibrio crassostreae (97.9 %). DNA–DNA hybridization results confirmed that these isolates constitute a new species. The name Vibrio cortegadensis sp. nov. is proposed with C 16.17^T (=CECT 7227^T=LMG 27474^T) as the type strain.     

Morphologic and AFLP analysis of relationships between tulip species Tulipa biebersteiniana (Liliaceae)

In populations of four species of tulips (Tulipa biebersteiniana, T. patens, T. scytica and T. riparia) from the Volgograd, Kurgansk, Orenburg, and Chelyabinsk regions and the Republic of Bashkortostan, genetic diversity was studied by means of morphological and AFLP analysis. A morphological analysis of seven quantitative and two qualitative criteria was carried out. Three selective EcoRI/MseI primer pairs allowed one to genotype 81 individuals from 13 tulip populations with 87 loci. The low level of variability by AFLP loci were revealed in all species, including T. biebersteiniana (P = 20.41%, UH _e = 0.075), T. patens (26.97%, 0.082), T. scytica (27.53%, 0.086), and T. riparia (27.72%, 0.096). According to the AMOVA results, the variability proportion that characterizes the differences between the four Tulip species was lower (F _CT = 0.235) than between populations within species (F _ST = 0.439). Tulipa patens is well differentiated by means of Nei’s distances, coordination, and analysis in the STRUCTURE program. An analysis in the STRUCTURE revealed four genetic groups of tulips that are not completely in accordance with the analyzed species. This acknowledges the presence of complicated genetic process in the tulip population.     

石榴园西花蓟马种群动态及其与气象因素的关系

2007-2008年,对云南省建水石榴园西花蓟马种群动态进行了系统调查,并采用回归分析(逐步回归分析、通径分析)、主成分分析及灰色系统分析就气象因子对该虫种群动态的影响进行了系统分析。结果表明,西花蓟马在建水石榴园常年发生,冬季较低,夏季最高,成虫全年种群消长呈单峰型,高峰期为5月份。相关性分析结果表明,西花蓟马种群数量与月相对湿度间呈极显著正相关性(P<0.01),与月均气温和月最低气温间呈显著正相关性(P<0.05),与月最高气温、月均降雨量和月均蒸发量间无相关性(P>0.05)。回归分析结果表明,石榴园西花蓟马种群动态的决定因子中影响最大的气象因素是月最低气温,而月均气温和月相对湿度是影响种群数量变动的主要因素。主成分分析表明,月最低气温是主要成分,其累积方差贡献率达73.03%。灰色系统分析结果表明,影响石榴园6种蓟马种群动态最关键的因子是月相对湿度;年度间影响最大的是年总降雨量;石榴花期各蓟马的种群数量与气象因素间关联度最大的是月最低温;果期各种蓟马的种群数量与气象因素间关联度最大的是月均降雨量。     

Taxonomic treatments of Camellia (Theaceae) species with secretory structures based on integrated leaf characters

Multivariate analysis of leaf shape, anatomy, and Fourier-transform infrared (FTIR) data of 27 Camellia species with secretory structures (sects. Archecamellia, Stereocarpus, Furfuracea, Chrysantha), together with three species from related genera, Gordonia and Tutcheria (Theacea), was conducted to clarify some taxonomic problems. Our results show that crystals occurring in adaxial epidermal cells are firstly observed in Chrysantha species, and the secretory structures described are in fact cork warts. Furthermore, we introduce a form coefficient (F _c) to assess the shape of epidermal cells, since they are usually irregular and difficult to describe. Pearson correlation analysis indicates that F _c is useful to assess epidermal cell shape. Principal component analysis (PCA) of leaf shape indicates that two species from section Archecamellia and two species from section Stereocarpus are significantly different from those in section Furfuracea. Cluster analysis of FTIR data visualizes the degree of affinity among the 30 species examined here, which is consistent with the cluster analysis (CA) of anatomical data, as illustrated in the dendrogram. Therefore, our study indicates that integrated leaf characters based on leaf shape, anatomy, and FTIR data are useful in the taxonomic treatment of Camellia species with secretory structures. Taxonomic controversies among the Camellia species with secretory structures could thus be successfully addressed using only a few intact or small portions of leaves. Moreover, our results tend to support that Chrysantha species should not be merged into section Archecamellia, and that section Heterogenea should not be recognized in taxonomic treatments of Camellia species with secretory structures.     

Study of molecular genetic diversity and evolutionary history of medicinally important endangered genus Chlorophytum using DNA barcodes

This study was aimed to authenticate and present phylogenetic relationship among 19 species of genus Chlorophytum using DNA barcoding. In all, 107 accessions were analyzed with eight plastid (matK, rbcL, trnH-psbA, rpoC1, ycf5, rpoB, atp and psbK-psbI) and six nuclear (ITS) markers. The matK and rbcL were found to be ideal markers for identification and discrimination of Chlorophytum species. Phylogenetic analysis based on matK and rbcL sequences resolved the species in two major clades. All markers, except matK and rbcL, showed ambiguous reads and paralogy in analysis. DGGE analysis showed the presence of pseudogenes and/or co-amplification in these markers, which caused poor sequence quality. Phylogeny and probable evolution of genus Chlorophytum was proposed on the basis of cytological, morphological and genetic information.     

Population structure,morphological and genetic diversity within and among melon (Cucumis melo L.) landraces in Iran

ISSR markers were applied to evaluate the genetic diversity and differentiation of 270 individuals of 27 Iranian C. melo landraces of various varietal groups include vars. inodorous, cantalupensis, reticulatus, ameri, dudaim. Genetic diversity among the studied genotypes obtained by GeneAlex analysis (H?=?0.08, I?=?0.12, Na?=?0.77, PPL?=?22.6%). Cluster analysis divided Iranian melon landraces into two main cluster. Non-sweet genotype (dudaim group) was well separated from sweet genotypes (inodorous, ameri, reticulatus, cantalupensis). The most similar genotypes were BANI and TONI (0.95) and the most dissimilar ones were GER and TS (0.58). AMOVA result showed that the percentage of genetic variation among and within Iranian melon is 69% and 31%, respectively. All landraces evaluated based on 10 morphological traits which revealed the diversity of melon varietal groups. Bayesian analysis assigned ten landraces to Pop 1, eight landraces to Pop 2 and nine melon landraces to Pop 3. Bayesian and UPGMA cluster analyses demonstrated the almost related results. Our results indicated that ISSR markers technique alongside polyacrylamide gel analysis could be helpful to discriminate varieties of melon.     

Imputation-Based Population Genetics Analysis of Plasmodium falciparum Malaria Parasites

Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r² for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r², 0.87-0.96), but the performance of IMPUTE was mixed (allelic r², 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima’s D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and association analyses, and supporting global surveillance for drug resistance markers and candidate vaccine antigens.