Amplification Profiles Research Articles

Characterization of Non-coding Regions in B Mating Loci of Agrocybe salicacola Groups: Target Sites for B Mating Type Identification.

Agrocybe salicacola is a delicious and cultivable mushroom. It is important to understand this species' inherent characteristics, especially to elucidate the constitution and segregation of mating genes. In this study, two compatible B mating loci in strain YAASM0711 of A. salicacola were cloned from the monokaryons, and sequence and phylogeny analyses showed two conserved genes encoding pheromone receptors maybe lost mating activity, which determined by comparing with those of other mushrooms. In the conserved regions of mating loci, partial insertion/deletion fragments made non-coding regions posses polymorphisms, and monokaryotic strains of different mating types were distinguished from each other according to the amplification profile of variable regions, which suggested mating loci were integrally assigned to offspring strains during mitosis in A. salicacola. As our known, it is the first to develop molecular markers for B mating-type identification using variable non-coding fragments of mating loci in basidiomycetes.

Virulence genes and genetic relationship of L. monocytogenes isolated from human and food sources in Brazil

The herein presented assay provided a bacteriological and molecular characterization of 100 samples of L. monocytogenes isolated from human (43) and food (57) sources, from several regions of Brazil, and collected between 1975 and 2013. Antigenic characterization defined 49% of serotype 4b samples, followed by 28% of serotype 1/2b, 14% of serotype 1/2c, 8% of serotype 1/2a, and 1% of serotype 3b. Both type of samples from human and food origin express the same serotype distribution. Multiplex PCR analysis showed 13 strains of type 4b with the amplification profile 4b-VI (Variant I). Virulence genes hly, inlA, inlB, inlC, inlJ, actA, plcA, and prfA were detected in all samples, highlighting a deletion of 105pb on the actA gene in 23% of serotype 4b samples. Macrorestriction profile with ApaI at PFGE showed 55 pulsotypes, with the occurrence of the same pulsotype in hospitalized patients in São Paulo in 1992 and 1997, and two other highly related pulsotypes in patients hospitalized in Rio de Janeiro in 2008. Recognized pulsotypes in listeriosis cases have also been detected in food. Thus, the prevalence of a serotype and the persistence of certain pulsotypes herald future problems.

Dactylopius opuntiae: control by the Fusarium incarnatum–equiseti species complex and confirmation of mortality by DNA fingerprinting

Cultivation of the prickly pear cactus, Opuntia ficus-indica, an important cattle food in the semiarid region of Brazil, has been increasingly compromised by predation by the cochineal scale Dactylopius opuntiae. Entomopathogenic fungi represent a low environmental impact alternative to controlling this insect pest, and the genus Fusarium has been described as a promising agent for that task. As such, we selected isolates of the Fusarium incarnatum–equiseti species complex (FIESC) to evaluate their potential biological control of D. opuntiae and confirmed their efficiency/presence in the field through morphological and molecular characteristics. The pathogenicities of 25 isolates of FIESC obtained from D. opuntiae were evaluated. The isolates URM6782, URM6778, and URM6811 demonstrated kill rates above 45%, and good sporulation characteristics, and were thus selected for field testing. The isolate URM6782 showed the highest kill rate in the field, and amplification profiles obtained using the ISSR UBC834 marker confirmed that the isolates released in the field were in fact the causal agents of the high mortality of D. opuntiae. We also observed natural biological control by native Fusarium species present in the field. Our results suggest that the isolates URM6782 and URM6778 show significant promise for controlling D. opuntiae and that the ISSR primer UBC834 can be used to monitor those isolates when released into the field. This work represents an initial study directed toward the biological control of D. opuntiae using novel isolates from the FIESC.

TipMT: Identification of PCR-based taxon-specific markers

BackgroundMolecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity.ResultsHere, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species.ConclusionsThe TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/.

Capture, amplification, and global profiling of microRNAs from low quantities of whole cell lysate.

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level via a complex regulatory network that requires genome-wide miRNA profiling to dissect. The patterns of miRNA expression at the genome scale are rich in diagnostic and prognostic information for human diseases such as cancers. This analysis, however, requires multi-step purification of RNAs from large quantities of cells, which is not only time consuming and costly but also challenging in situations where cell numbers are limited. In this study, we report direct capture, amplification, and library preparation of miRNAs from whole cell lysate without the need of pre-purification. As a result, it enables genome-wide miRNA profiling reproducibly with low quantity of cell samples (∼500 hematopoietic cells). Specifically, we conducted a systematic investigation of two key steps - cell lysis for miRNA release and 3' adaptor ligation required for direct miRNA capture and amplification. The obtained expression profile not only distinguishes cell types but also detects individual miRNA alterations in closely related isogenic cell lines. This approach, which is substantially simple as compared to the standard methods because of elimination of the need for RNA purification, is advantageous for the measurement of low quantity samples.

A Genomic Predictive Signature for Rigosertib in Lower Risk MDS Derived By Integrating Clinical Response, Mechanism of Action Data and Simulation

Background: Lower risk non-del5q MDS patients (pts) have limited treatment options and majority pts are transfusion dependent. Erythropoiesis stimulating agents (ESA) are first line of therapy for LR-MDS pts but almost half are resistant to ESA or become resistant after responding. Hence, LR-MDS represents an area of unmet medical need for novel agents to improve hematopoiesis and reduce transfusion dependency. Rigosertib (RIG) blocks RAS-mediated activation of proteins containing a common RAS binding domain, or RBD (Athuluri-Divaker, et al; Cell 165; 643'16). Since many pathways include proteins that employ RBD (i.e. ras, raf PI3-Kinase), interference with RBD provides a novel approach to block proliferation. While RIG showed encouraging response in LR-MDS pts (Tycko, et al; Blood 2013; 122:2745), there is a need for a biomarker that can differentiate responders from non-responders. To correlate genomics with response to RIG, we employed computational tools to integrate molecular data and observed responses.Aim: To determine predictive value of a computational biology derived genomic signature in LR-MDS pts who are treated with RIG.Methods: Materials were derived from Phase I and II studies of RIG in LR-MDS pts. Efficacy was reported as transfusion independence. Bone marrow samples were collected and processed using standard methods. DNA was extracted from bone marrow mononuclear cells or T-cells (germline control) purified from peripheral blood samples. Exome sequencing was performed using Agilent SureSelect Human All Exon v4 - 51Mb kit and HiSeq 2000. Raw sequences were aligned to the human genome build hg19 using BWA software; SNPs and In/Dels analysis was performed using PICARD, SAMTOOLS and GATK.These results, entered into predictive computational biology software (Cellworks Group), generated disease-specific protein network maps using PubMed and other online resources. Digital drug simulations are conducted by measuring drug effect on a score: a composite of cell proliferation, viability and apoptosis. Each patient-specific network map was screened for RIG reduced progression in a dose-respondent manner. Computer predictions were blindly correlated with clinical outcomes.Results: Predicted response was blindly correlated with the clinical outcome for 19 pts: Results showed 16 matches and 3 mismatches; and the following predictive test statistics: PPV - 81.25%, NPV - 100%, Sensitivity - 100%: accuracy of the predictive test at 84.21%.Modeling of LR-MDS pts predicted amplification of genes MYC, FNTA on chromosome 8; amplification of genes LGALS3, AJUBA, MAX on chromosome 14; TIAM1 on chromosome 21; HMGCR, on chromosome 5; PDPK1, MAPK3, PLK1 on chromosome 16 and PIK3CA, RAF1 on chromosome 3 correlated with increased response to RIG. Aberrations that enhanced the downstream of RAS signaling via the RAF-ERK or PI3K-AKT-MTOR pathway increased the signal flow through the drug response paths. MYC amplification on chromosome 8 via increasing the Purine synthesis pathway and production of GTP increased the RAS-ERK and RHEB-MTOR signaling. MYC also enhanced CCND1 production/proliferation. Prenylation pathway genes FNTA and HMGCR amplification also affect RAS and RHEB; enhance the drug response pathways. Another pathway that was found to be significant in the drug response, reducing proliferation, was inhibition of PLK1 and AURKA. Amplification of AJUBA, PLK1 made this pathway more significant and improved drug response via inhibition of proliferation via TIAM1-RAC1-PAK1-AURKA-PLK1 and RAF-PLK1 pathway. Deletion of TP53 and NF1 on chromosome 17 did not correlate with response.MYC amplification was seen in LR and HR MDS pts and correlated with response to RIG. However, the computational biology method showed that MYC amplification alone is not a predictor for response since there were non-responder profiles with MYC amplification and responder profiles with MYC deletion that had other aberrations in genes on chromosomes 3, 14, 16 or 21.Conclusions: A predictive method that models multiple genomic abnormalities simultaneously showed greater than 80% correlation between RIG mediated protein network perturbations and clinical outcomes in LR-MDS. The method also explained lack of response and highlighted resistance pathways that could be targeted to recover sensitivity. We also established eligibility criteria for greater precision enrollment in future trials. [Display omitted] DisclosuresFruchtman:Onconova: Employment. Petrone:Onconova Therapeutics, Inc.: Employment. Zbyszewski:Onconova Therapeutics, Inc.: Employment. Hoffman:Onconova Therapeutics, Inc.: Employment. Vali:Cellworks Group: Employment. Singh:Cellworks Group: Employment. Usmani:Cellworks: Employment. Grover:Cellworks Group: Employment. Abbasi:Cellworks: Employment.

Analysis of TaqMan Array Cards Data by an Assumption-Free Improvement of the maxRatio Algorithm Is More Accurate than the Cycle-Threshold Method

Quantitative PCR diagnostic platforms are moving towards increased sample throughput, with instruments capable of carrying out thousands of reactions at once already in use. The need for a computational tool to reliably assist in the validation of the results is therefore compelling. In the present study, 328 residual clinical samples provided by the Public Health England at Addenbrooke's Hospital (Cambridge, UK) were processed by TaqMan Array Card assay, generating 15 744 reactions from 54 targets. The amplification data were analysed by the conventional cycle-threshold (CT) method and an improvement of the maxRatio (MR) algorithm developed to filter out the reactions with irregular amplification profiles. The reactions were also independently validated by three raters and a consensus was generated from their classification. The inter-rater agreement by Fleiss' kappa was 0.885; the agreement between either CT or MR with the raters gave Fleiss' kappa 0.884 and 0.902, respectively. Based on the consensus classification, the CT and MR methods achieved an assay accuracy of 0.979 and 0.987, respectively. These results suggested that the assumption-free MR algorithm was more reliable than the CT method, with clear advantages for the diagnostic settings.

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis.

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosis strains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

A population-wide applicable HLA-DQ2 and DQ8 genotyping using DNA from dried blood spots and duplex allele-specific qPCR amplification

Genotyping of HLA-DQ2 and DQ8 haplotypes is important for diagnosis or for screening of early risk detection of celiac disease or type 1 diabetes. Usually, venous blood DNA extraction and expensive and time consuming amplification are used, that hinder population-wide studies. We assayed a friendly HLA-DQ2 and DQ8 genotyping procedure using a combination of DNA from dried blood spot (DBS) and duplex allele-specific qPCR amplification using SYBR Green. DNA was extracted using home-made buffers and compared to an extraction commercial kit. Duplex reactions by qPCR were designed using each Tm allele amplicon for reference samples (positive HLA-DQ2 or DQ8) with allele-specific primers. DBS samples from 558 children (7.99 ± 2.47 y) were collected. The DNA final yield obtained by the home-made extractive procedure was higher than from the commercial kit (1.11 ± 0.56 vs 0.23 ± 0.14 μg), while the quality was similar for both DNA samples. There was concordance in the amplification profiles for DNA samples obtained with both methods. All of four alleles from DQ2 and DQ8 haplotypes were accurately identified in duplex reactions. By using DBS samples and DNA extraction home-made procedure, the costs were reduced by 60%. The whole procedure is cost-effective for HLA-DQ2 and DQ8 genotyping.

Detection of the Diversity of Cytoplasmic Male Sterility Sources in Broccoli (Brassica Oleracea var. Italica) Using Mitochondrial Markers.

Broccoli (Brassica oleracea var. italica) is an important commercial vegetable crop. As part of an efficient pollination system, cytoplasmic male sterility (CMS) has been widely used for broccoli hybrid production. Identifying the original sources of CMS in broccoli accessions has become an important part of broccoli breeding. In this study, the diversity of the CMS sources of 39 broccoli accessions, including 19 CMS lines and 20 hybrids, were analyzed using mitochondrial markers. All CMS accessions contained the ogu orf138-related DNA fragment and the key genes of nap CMS, pol CMS, and tour CMS were not detected. The 39 CMS accessions were divided into five groups using six orf138-related and two simple sequence repeat markers. We observed that ogu CMS R3 constituted 79.49% of the CMS sources. CMS6 and CMS26 were differentiated from the other accessions using a specific primer. CMS32 was distinguished from the other accessions based on a 78-nucleotide deletion at the same locus as the orf138-related sequence. When the coefficient was about 0.90, five CMS accessions (13CMS6, 13CMS23, 13CMS24, 13CMS37, and 13CMS39) exhibiting abnormal floral organs with poor seed setting were grouped together. The polymerase chain reaction amplification profiles for these five accessions differed from those of the other accessions. We identified eight useful molecular markers that can be used to detect CMS types during broccoli breeding. Our data also provide important information relevant to future studies on the possible origins and molecular mechanisms of CMS in broccoli.

Effects of plant growth regulators on efficient plant regeneration efficiency and genetic stability analysis from two Ocimum tenuiflorum L. morphotypes

An effective, rapid and improved protocol was established for in vitro plant regeneration from nodal explants of two Ocimum tenuiflorum L. morphotypes, an medicinally important species of immense industrial value. The treatment medium used was Murashige and Skoog (MS) medium supplemented with different concentrations and combination of cytokinins (BA/KN/2-iP) and auxins (NAA/IBA/IAA). The adventitious shoot buds formation and plant regeneration capacity were significantly different (p < 0.05) between two morphotypes. The highest number of shoots (5.01 ± 0.11) in O. tenuiflorum purple leaves was obtained in the medium containing 0.5 mg l−1 BA, while in O. tenuiflorum green leaves the highest number of shoots was 7.03 ± 0.10 on a medium containing 1.0 mg l−1 BA. Regenerated shoots were rooted in vitro on low-salt MS derived media. Media were supplemented with different auxins that affected roots formation of particular morphotypes. The acclimatization of micropropagated plantlets of two morphotypes showed the best results with the sand:soil:compost (2:2:1). Genetic stability of regenerated plants as well as mother plants of two morphotypes of O. tenuiflorum L. was assessed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both RAPD and ISSR displayed a homogenous amplification profile for all the micropropagated plants of O. tenuiflorum L. when compared to their mother plants. Gas chromatography results showed that the essential oils extracted from in vitro-derived plants and their mother stock plants have similar profiles. Rapid and high multiplication frequency, molecular genetic and essential oil content stability ensures the efficacy of the protocol developed for the production of these industrially important medicinal plants.

Determination of DNA profiling of siwak and toothbrush samples used in Kingdom of Saudi Arabia

BackgroundDNA profiling is an integral part of forensic work. Enough and good samples for DNA quantification and profiling are mandatory. Aim of the studyTo quantify and profile DNA from siwak and toothbrushes and study the effect of time on this process. MethodologyThe present study included DNA profiling from siwak and effect of time on extracted DNA. Then; obtained data were compared to those extracted from toothbrushes. For this purpose, 25 uncovered siwaks, 4 covered siwaks, and 25 toothbrushes were used. Samples were divided into 8 groups; first group (4months; 4 volunteers); second group (3months, 5 volunteers); third group (2months, 4 volunteers); fourth group (1month, 5 volunteers); fifth group (1week, 3 volunteers); sixth group (the same day, 4 volunteers and covered siwak was added); seventh group (reference samples) and group 8 (positive and negative control samples). Extraction of DNA was done using Promega kit and then PCR was used for amplification and DNA profiling was done. ResultsConsiderable quantity of human genomic DNA was successfully extracted from siwaks. There was no proof of the existence of any substance in siwak or its components that may interfere with amplification of DNA by PCR or interfere with obtaining DNA profiles. Siwak proved to be a good and reliable source of human genomic DNA that is enough for DNA analysis. There has been no effect of time on DNA analysis and DNA profiling in this study (within the targeted period which is 4months). ConclusionSiwak contains enough quantity of DNA, and retained good DNA profiling; and when compared to toothbrushes, siwak is a reasonable source of DNA profiling when found at the scene of crime. In addition, time of storage up to 4months had no or little effects on results.

Assessment of Genetic Fidelity Using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats Markers of Lawsonia inermis L. Plants Regenerated by Axillary Shoot Proliferation

An efficient, reliable and reproducible plant regeneration protocol was developed for Lawsonia inermis L. using mature nodal explants. Shoot proliferation (81.6 %) with 7.8 shoots/explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 1.0 mg l−1 6-benzyladenine. Shoot numbers were up-scaled by inducing multiple shoots from axenic nodal segments derived from the primary shoots on the shoot regeneration medium. Thus the authors could achieve ca. 129–134 shoots from single nodal explant. Ninety-three percent rooting of in vitro regenerated shoots was achieved on growth regulator free half-strength MS medium. Regenerated plantlets were successfully transferred to soil with 85 % survival rate. Genetic stability analyses of the in vitro regenerated plants using random amplified polymorphic DNA and inter simple sequence repeat markers revealed a homogeneous amplification profile for all micropropagated plants. This is the first report that evaluates the use of molecular markers to establish genetic fidelity of micropropagated L. inermis for the rapid clonal multiplication and true-to-type production of plant for attaining the ever increasing demand in pharmaceutical industries.

Polyphasic Approach Including MALDI-TOF MS/MS Analysis for Identification and Characterisation of Fusarium verticillioides in Brazilian Corn Kernels.

Fusarium verticillioides is considered one of the most important global sources of fumonisins contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol).

An improved DNA marker technique for genetic characterization using RAMP-PCR with high-GC primers.

Random amplified polymorphic DNA (RAPD) is a widely used molecular marker technique. As traditional RAPD has poor reproducibility and productivity, we previously developed an improved RAPD method (termed RAMP-PCR), which increased the reproducibility, number of bands, and efficiency of studies on polymorphism. To further develop the efficiency of this method, we used high-GC content primers for improved RAMP-PCR with DNA samples from Lonicera japonica. Comparison of amplification profiles obtained by standard RAPD primers with those obtained by regular PCR and RAMP-PCR, and high-GC primers with regular PCR and RAMP-PCR showed that the average number of bands and polymorphisms per primer gradually and significantly increased (from 6.4 to 15.0 and from 4.6 to 10.2, respectively). Cluster dendrograms showed similar results, indicating that this new method is consistent and reproducible. A total of 22 samples from different species, including plants, animals, and humans, were used for RAMP-PCR with high-GC primers. Multiple bands were successfully amplified from all samples, demonstrating that this method is a reliable technique with consistent results and may be of general interest in studies on different genera and species. We developed highly effective DNA markers, which can provide a more effective and potentially valuable approach than traditional RAPD for the genetic identification of various organisms, particularly of medicinal plants.

A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-generation sequencing.

BackgroundFormalin has been widely used by pathology laboratories. Its carcinogenicity has led researchers to explore formalin substitutes. Streck Cell Preservative (SCP) is a formalin-free preservative that can preserve cellular antigens. This study was undertaken to investigate the effects of cell preservation using SCP on nucleic acid amplification, hybridization, and next-generation sequencing (NGS) as compared to control frozen cells and cells fixed in the traditional cell and tissue fixative, 10 % neutral buffered formalin (NBF).FindingsThe breast cancer cell line, SKBR-3, was used as a model system. Prior to nucleic acid extraction and fluorescence in situ hybridization (FISH), cells were fixed in SCP or NBF overnight at room temperature with frozen cells in parallel. Analysis showed that similar DNA extraction yields and amplification profiles determined by PCR in SCP preserved cells and control frozen cells, whereas NBF preserved cells had decreased DNA yield and impaired PCR amplification. Molecular cytogenetic studies by FISH technique indicated that the ratios of ERBB2 (HER-2/neu) signals to the chromosome 17 centromere (CEP17) were comparable for frozen cells and SCP preserved cells. The fluorescence images of both SCP fixed and control frozen cells were also clear and comparable. On the contrary, the same analysis was unsuccessful with NBF preserved cells due to poor hybridization quality. Our data also demonstrated that SCP had negligible effect on NGS testing.ConclusionWe conclude that SCP can be used as an alternative to NBF as a preservative for maintaining the integrity of nucleic acids for nucleic acid amplification, sequencing and FISH analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1725-4) contains supplementary material, which is available to authorized users.

SSR markers: a tool for species identification in Psidium (Myrtaceae).

Molecular DNA markers are used for detection of polymorphisms in individuals. As they are independent of developmental stage of the plant and environmental influences, they can be useful tools in taxonomy. The alleles of simple sequence repeat (SSR) markers (or microsatellites) are traditionally used to identify taxonomic units. This application demands the laborious and costly delimitation of exclusive alleles in order to avoid homoplasy. Here, we propose a method for identification of species based on the amplification profile of groups of SSR markers obtained by a transferability study. The approach considers that the SSR are conserved among related species. In this context, using Psidium as a model, 141 SSR markers developed for Psidium guajava were transferred to 13 indigenous species of Psidium from the Atlantic Rainforest. Transferability of the markers was high and 28 SSR were conserved in all species. Four SSR groups were defined and they can help in the identification of all 13 Psidium species studied. A group of 31 SSR was genotyped, with one to six alleles each. The H0 varied from 0.0 to 0.46, and PIC from 0.0 to 0.74. Cluster analysis revealed shared alleles among species. The high percentage of SSR transferability found in Psidium evidences the narrow phylogenetic relationship existing among these species since transferability occurs by the preservation of the microsatellites and anchoring regions. The proposed method was useful for distinguishing the species of Psidium, being useful in taxonomic studies.

Genetic diversity analysis based on morphological traits and microsatellite markers in barley (Hordeum vulgare)

The experimental material consisting of 10 barley (Hordeum vulgare L.) cultivars were evaluated using biometrical technique and microsatellite markers. A set 19 morphological and 47 SSR markers revealed significant differences among the quantitative and qualitative traits. In total 166 alleles were scored from PCR based amplification profiles for selected genotypes. The number of alleles ranged from 2 to 7 with an average of 3.52 alleles per locus. The band fragment size varied from 100 bp to 400 bp with PIC values ranging from 0.286 (Bmac154) to 0.804 (KV1/KV2) with an average of 0.551. Genetic diversity was estimated using Euclidean2 distance and UPGMA methods. Euclidean clustering revealed four clusters and groups II, III and IV exhibited highest mean value for most of the traits. Genotypes JB 1, LAKHAN, KB 1201 and RD 2035 were identified as most suitable genotypes for hybrid performance for yield attributing characters like tillers/plant, grains/spike, 1 000 grain weight, grain weight/spike, grain size and days to maturity. This information would be use as selection parameters to plan crosses and maximize the use of genetic diversity and expression of heterosis in barley breeding program for yield improvement.

High-Resolution Genomic Analysis of Cribriform Neuroepithelial Tumors of the Central Nervous System.

Cribriform neuroepithelial tumors (CRINET) are one of several recently characterized entities in the broad spectrum of solid tumors with SMARCB1-INI1 loss. This neoplasm seems to be exceedingly rare and displays unique neuropathologic and clinical features. To date, only a few cases of CRINET have been characterized from a molecular point of view. In this study, we investigated the molecular features of 3 cases of CRINET using multiplex ligation-dependent probe amplification and molecular inversion profiling approaches. Along with mutations and deletions of SMARCB1-INI1, molecular inversion profiling analysis revealed a stable genomic profile without significant large chromosomal changes. Focal alterations (gains) were observed in individual cases at chromosomes 4q12 (PDGFRA), 12q15 (MDM2), 7p15.1 (NPY), and 18q11.2 (CDH2). Genomic Identification of Significant Targets in Cancer analysis highlighted focal alterations, including gains at chromosomes 16q23.2 (MAF), 17q23 (AXIN2), and 8p12 (ADAM3A). No cases showed BRAF(V600E) or CTNNB1 mutations. These data indicate that CRINET present stable genetic features and lack alterations commonly identified in other pediatric brain tumors. Further studies are required to determine whether specific alterations and specific signaling pathways, in addition to SMARCB1-INI1, may be implicated in the biology of this rare tumor and whether there are additional molecular similarities between CRINET and atypical teratoid/rhabdoid tumors.

Detuning Effect Between the Cavity Resonance and Atomic Transition Frequencies on Noise Flux Profiles of Class-C Lasers

The class-C lasers are well recognized to have very narrow atomic transition bandwidths. Therefore, a minor mismatch between the cavity resonance and atomic transition frequencies (CAF) is normally led to the large modifications in the gain and noise profiles. The motion of class-C lasers is described by the three variables of cavity electric field, atomic population inversion, and dipole moment of atoms that are coupled to each other by the optical Maxwell-Bloch equations. The fluctuating features of these three variables are completely elaborated by solving their equations of motion in the presence of the cavity Langevin force. The results peculiarly indicate that the noise profile of the cavity electric field exactly mimics the amplification profile of an input signal by the laser in both below and above threshold states. The both gain and noise profiles simultaneously tend to infinity at the normalized pumping rates and CAF mismatches determined by the laser stability theory. The noise flux profiles are finally confirmed by illustrating the flux conservation.