Agronomic assessment of 32 sour passionfruit genotypes in federal district

The production of passion fruit is important in Brazil. In order to contribute to the development of the most promising cultivars of passion fruit, this study aimed to evaluate the agronomic performance of 32 genotypes of passion fruit in Federal District of Brazil, and to estimate genetic parameters for use in breeding programs. Thirty-two genotypes were used in a randomized block design, with eight plants per plot and four replications. The experiment was conducted in field. Twenty-eight harvests were performed, and the variables analyzed were: productivity estimated, total number of fruits per hectare, average fruit weight and these characteristics following classification of fruits in five categories. The genotypes that presented the highest total yield estimated were MAR20 # 23, AR 01 and PLANTA 7. For industrial purposes, genotypes MAR 20 # 21 and BRS Gigante Amarelo were superior. For fresh consumption, the genotypes with the best performance were PLANT 7, AR 01 and MSC. Total productivity estimated and total number of fruits per hectare in the first-class classification showed high values of heritability and CVg/CVe ratio. These results indicate a favorable condition for selection.

Short-range template switching in great ape genomes explored using pair hidden Markov models.

Many complex genomic rearrangements arise through template switch errors, which occur in DNA replication when there is a transient polymerase switch to an alternate template nearby in three-dimensional space. While typically investigated at kilobase-to-megabase scales, the genomic and evolutionary consequences of this mutational process are not well characterised at smaller scales, where they are often interpreted as clusters of independent substitutions, insertions and deletions. Here we present an improved statistical approach using pair hidden Markov models, and use it to detect and describe short-range template switches underlying clusters of mutations in the multi-way alignment of hominid genomes. Using robust statistics derived from evolutionary genomic simulations, we show that template switch events have been widespread in the evolution of the great apes' genomes and provide a parsimonious explanation for the presence of many complex mutation clusters in their phylogenetic context. Larger-scale mechanisms of genome rearrangement are typically associated with structural features around breakpoints, and accordingly we show that atypical patterns of secondary structure formation and DNA bending are present at the initial template switch loci. Our methods improve on previous non-probabilistic approaches for computational detection of template switch mutations, allowing the statistical significance of events to be assessed. By specifying realistic evolutionary parameters based on the genomes and taxa involved, our methods can be readily adapted to other intra- or inter-species comparisons.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

Missing the Match Might Not Cost You the Game: Primer-Template Mismatches Studied in Different Hepatitis A Virus Variants.

Mismatches between template sequences and reverse transcription (RT) or polymerase chain reaction (PCR) primers can lead to underestimation or false negative results during detection and quantification of sequence-diverse viruses. We performed an in silico inclusivity analysis of a widely used RT-PCR assay for detection of hepatitis A virus (HAV) in food, described in ISO 15216-1. One of the most common mismatches found was a single G (primer) to U (template) mismatch located at the terminal 3'-end of the reverse primer region. This mismatch was present in all genotype III sequences available in GenBank. Partial HAV genomes with common or potentially severe mismatches were produced by in vitro transcription and analysed using RT-ddPCR and RT-qPCR. When using standard conditions for RT-qPCR, the mismatch identified resulted in underestimation of the template concentration by a factor of 1.7-1.8 and an increase in 95% limit of detection from 8.6 to 19 copies/reaction. The effect of this mismatch was verified using full-length viral genomes. Here, the same mismatch resulted in underestimation of the template concentration by a factor of 2.8. For the partial genomes, the presence of additional mismatches resulted in underestimation of the template concentration by up to a factor of 232. Quantification by RT-ddPCR and RT-qPCR was equally affected during analysis of RNA templates with mismatches within the reverse primer region. However, on analysing DNA templates with the same mismatches, we found that ddPCR quantification was less affected by mismatches than qPCR due to the end-point detection technique.

RNA 3D Structure Modeling by Combination of Template-Based Method ModeRNA, Template-Free Folding with SimRNA, and Refinement with QRNAS.

RNA encompasses an essential part of all known forms of life. The functions of many RNA molecules are dependent on their ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. To address this problem, computational structure prediction methods were developed that either utilize information derived from known structures of other RNA molecules (by way of template-based modeling) or attempt to simulate the physical process of RNA structure formation (by way of template-free modeling). All computational methods suffer from various limitations that make theoretical models less reliable than high-resolution experimentally determined structures. This chapter provides a protocol for computational modeling of RNA 3D structure that overcomes major limitations by combining two complementary approaches: template-based modeling that is capable of predicting global architectures based on similarity to other molecules but often fails to predict local unique features, and template-free modeling that can predict the local folding, but is limited to modeling the structure of relatively small molecules. Here, we combine the use of a template-based method ModeRNA with a template-free method SimRNA. ModeRNA requires a sequence alignment of the target RNA sequence to be modeled with a template of the known structure; it generates a model that predicts the structure of a conserved core and provides a starting point for modeling of variable regions. SimRNA can be used to fold small RNAs (<80 nt) without any additional structural information, and to refold parts of models for larger RNAs that have a correctly modeled core. ModeRNA can be either downloaded, compiled and run locally or run through a web interface at http://genesilico.pl/modernaserver/ . SimRNA is currently available to download for local use as a precompiled software package at http://genesilico.pl/software/stand-alone/simrna and as a web server at http://genesilico.pl/SimRNAweb . For model optimization we use QRNAS, available at http://genesilico.pl/qrnas .

Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor.

BACKGROUND: Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. METHODS: The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and ß2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. RESULTS: Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between α and ß subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. CONCLUSION: Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.

Space exploration by the promoter of a long human gene during one transcription cycle.

An RNA polymerase has been thought to transcribe by seeking out a promoter, initiating and then tracking down the template. We add tumor necrosis factor α to primary human cells, switch on transcription of a 221-kb gene and monitor promoter position during the ensuing transcription cycle (using RNA fluorescence in situ hybridization coupled to super-resolution localization, chromosome conformation capture and Monte Carlo simulations). Results are consistent with a polymerase immobilized in a 'factory' capturing a promoter and reeling in the template, as the transcript and promoter are extruded. Initially, the extruded promoter is tethered close to the factory and so likely to re-initiate; later, the tether becomes long enough to allow re-initiation in another factory. We suggest close tethering underlies enhancer function and transcriptional 'bursting'.

Global genome analysis of the shikimic acid pathway reveals greater gene loss in host-associated than in free-living bacteria.

BACKGROUND: A central tenet in biochemistry for over 50 years has held that microorganisms, plants and, more recently, certain apicomplexan parasites synthesize essential aromatic compounds via elaboration of a complete shikimic acid pathway, whereas metazoans lacking this pathway require a dietary source of these compounds. The large number of sequenced bacterial and archaean genomes now available for comparative genomic analyses allows the fundamentals of this contention to be tested in prokaryotes. Using Hidden Markov Model profiles (HMM profiles) to identify all known enzymes of the pathway, we report the presence of genes encoding shikimate pathway enzymes in the hypothetical proteomes constructed from the genomes of 488 sequenced prokaryotes. RESULTS: Amongst free-living prokaryotes most Bacteria possess, as expected, genes encoding a complete shikimic acid pathway, whereas of the culturable Archaea, only one was found to have a complete complement of recognisable enzymes in its predicted proteome. It may be that in the Archaea, the primary amino-acid sequences of enzymes of the pathway are highly divergent and so are not detected by HMM profiles. Alternatively, structurally unrelated (non-orthologous) proteins might be performing the same biochemical functions as those encoding recognized genes of the shikimate pathway. Most surprisingly, 30% of host-associated (mutualistic, commensal and pathogenic) bacteria likewise do not possess a complete shikimic acid pathway. Many of these microbes show some degree of genome reduction, suggesting that these host-associated bacteria might sequester essential aromatic compounds from a parasitised host, as a 'shared metabolic adaptation' in mutualistic symbiosis, or obtain them from other consorts having the complete biosynthetic pathway. The HMM results gave 84% agreement when compared against data in the highly curated BioCyc reference database of genomes and metabolic pathways. CONCLUSIONS: These results challenge the conventional belief that the shikimic acid pathway is universal and essential in prokaryotes. The possibilities that non-orthologous enzymes catalyse reactions in this pathway (especially in the Archaea), or that there exist specific uptake mechanisms for the acquisition of shikimate intermediates or essential pathway products, warrant further examination to better understand the precise metabolic attributes of host-beneficial and pathogenic bacteria.

A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants.

High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen, ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen- or LCGreen-based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples. Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene, which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.

A simple template-dependent ligase ribozyme as the RNA replicase emerging first in the RNA world.

The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

Template proteogenomics: sequencing whole proteins using an imperfect database.

Database search algorithms are the primary workhorses for the identification of tandem mass spectra. However, these methods are limited to the identification of spectra for which peptides are present in the database, preventing the identification of peptides from mutated or alternatively spliced sequences. A variety of methods has been developed to search a spectrum against a sequence allowing for variations. Some tools determine the sequence of the homologous protein in the related species but do not report the peptide in the target organism. Other tools consider variations, including modifications and mutations, in reconstructing the target sequence. However, these tools will not work if the template (homologous peptide) is missing in the database, and they do not attempt to reconstruct the entire protein target sequence. De novo identification of peptide sequences is another possibility, because it does not require a protein database. However, the lack of database reduces the accuracy. We present a novel proteogenomic approach, GenoMS, that draws on the strengths of database and de novo peptide identification methods. Protein sequence templates (i.e. proteins or genomic sequences that are similar to the target protein) are identified using the database search tool InsPecT. The templates are then used to recruit, align, and de novo sequence regions of the target protein that have diverged from the database or are missing. We used GenoMS to reconstruct the full sequence of an antibody by using spectra acquired from multiple digests using different proteases. Antibodies are a prime example of proteins that confound standard database identification techniques. The mature antibody genes result from large-scale genome rearrangements with flexible fusion boundaries and somatic hypermutation. Using GenoMS we automatically reconstruct the complete sequences of two immunoglobulin chains with accuracy greater than 98% using a diverged protein database. Using the genome as the template, we achieve accuracy exceeding 97%.

Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification.

GMO quantification, based on real-time PCR, relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.

High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley.

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.

Cost effective DNA sequencing and template preparation from bacterial colonies and plasmids.

Cost-effective DNA sequencing and template preparation were evaluated for high-throughput screening of bacterial colonies. The rolling-circle amplification to generate template for DNA sequencing was carried out using 10-fold smaller amount of phi29 DNA polymerase and excluding two other enzymes (yeast pyrophosphatase and calf intestine alkaline phosphatase) used previously. Then, a 1/40 volume of the fluorescent terminator mix recommended by the manufacturer gave a usable sequencing result although a 1/26.7 volume of the mix was used to compare the protocol more accurately with manufacturer's protocol.

Bioinformatic flowchart and database to investigate the origins and diversity of clan AA peptidases.

BACKGROUND: Clan AA of aspartic peptidases relates the family of pepsin monomers evolutionarily with all dimeric peptidases encoded by eukaryotic LTR retroelements. Recent findings describing various pools of single-domain nonviral host peptidases, in prokaryotes and eukaryotes, indicate that the diversity of clan AA is larger than previously thought. The ensuing approach to investigate this enzyme group is by studying its phylogeny. However, clan AA is a difficult case to study due to the low similarity and different rates of evolution. This work is an ongoing attempt to investigate the different clan AA families to understand the cause of their diversity. RESULTS: In this paper, we describe in-progress database and bioinformatic flowchart designed to characterize the clan AA protein domain based on all possible protein families through ancestral reconstructions, sequence logos, and hidden markov models (HMMs). The flowchart includes the characterization of a major consensus sequence based on 6 amino acid patterns with correspondence with Andreeva's model, the structural template describing the clan AA peptidase fold. The set of tools is work in progress we have organized in a database within the GyDB project, referred to as Clan AA Reference Database http://gydb.uv.es/gydb/phylogeny.php?tree=caard. CONCLUSION: The pre-existing classification combined with the evolutionary history of LTR retroelements permits a consistent taxonomical collection of sequence logos and HMMs. This set is useful for gene annotation but also a reference to evaluate the diversity of, and the relationships among, the different families. Comparisons among HMMs suggest a common ancestor for all dimeric clan AA peptidases that is halfway between single-domain nonviral peptidases and those coded by Ty3/Gypsy LTR retroelements. Sequence logos reveal how all clan AA families follow similar protein domain architecture related to the peptidase fold. In particular, each family nucleates a particular consensus motif in the sequence position related to the flap. The different motifs constitute a network where an alanine-asparagine-like variable motif predominates, instead of the canonical flap of the HIV-1 peptidase and closer relatives.

Preparation of yeast mitochondrial DNA for direct sequence analysis.

We describe two simple protocols for preparation of templates for direct sequencing of yeast mitochondrial DNA (mtDNA) by automatic DNA analyzers. The protocols work with a range of yeast species and yield a sufficient quantity and quality of the template DNA. In combination with primer-walking strategy, they can be used either as an alternative or a complementary approach to shot-gun sequencing of random fragment DNA libraries. We demonstrate that the templates are suitable for re-sequencing of the mtDNA for comparative analyses of intraspecific variability of yeast strains as well as for primary determination of the complete mitochondrial genome sequence.

A high-throughput, low-cost method for the preparation of "sequencing-ready" phage DNA template.

We have developed a simple and efficient protocol for the isolation of good-quality recombinant phage DNA useful for all downstream processing, including automated sequencing. The overnight-grown phage particles were effectively precipitated (without any contaminating Escherichia coli DNA and other culture media components) by adjusting the pH of the culture medium to 5.2 with sodium acetate, followed by addition of ethanol to 25%. The phage DNA was selectively precipitated with ethanol in the presence of guanidinium thiocyanate under alkaline pH, resulting in uniform quality and quantity of phage DNA. The quality of the phage DNA preparation was demonstrated by DNA sequencing that provided an average read length of >700 bases (PHRED20 quality). This protocol for plating, picking, growing, and subsequent DNA purification of individual phage clones can be completely automated using any standard robotic platform. This protocol does not require any commercial kits and can be completed within 2h.

A simplified PCR methodology for semiquantitatively analyzing dengue viruses.

The standard methodology for titrating dengue viruses, the plaque assay, is slow, time consuming and relatively expensive. Other methods require machinery that may not be routinely accessible to all researchers, particularly those in developing nations. We therefore sought to develop a rapid, simplified semiquantitative polymerase chain reaction (PCR) methodology based on the use of a template mimic. In particular, it was desired that the mimic should be applicable for use a DNA template to avoid the requirement for producing an in vitro RNA transcript. A 511 base pair fragment of the capsid-PrM junction of dengue serotype 4 was cloned into pGEM-T Easy vector and subjected to splicing overlap extension-PCR to generate a 160 base pair deletion. The deleted plasmid mimic competed competitively against the parent plasmid as well as the first strand cDNA of all four dengue viruses. The primers used are specific for the dengue virus, and no product was seen with first strand cDNA from a closely related flavivirus, Japanese encephalitis virus. Under the conditions used, accurate quantitation of the dengue viruses in the range of 10(3) to 10(6) pfu can be achieved in a single day, as opposed to the 7 days required for conventional plaque assay.