Genetic diversity of the entomopathogenic fungus Metarhizium rileyi based on de novo microsatellite markers.

Epizootics of the entomopathogenic fungus Metarhizium rileyi regulate lepidopteran populations in soybean, cotton, and peanut agroecosystems to the point that insecticide applications could be unnecessary. However, the contribution and how different strains operate during the epizootic are unknown. Several unanswered questions remain: 1. How many genotypes of M. rileyi are present during an epizootic? 2. Which genotype is the most common among them? 3. Are the genotypes involved in annual epizootics at the same location the same? Therefore, the development of molecular markers to accurately identify these genotypes is very important to answer these questions. SSR primers were designed by prospecting in silico to discriminate genotypes and infer the genetic diversity of M. rileyi isolates from the collection kept at Embrapa Soybean. We tested 13 SSR markers on 136 isolates to identify 43 clones and 12 different genetic clusters, with genetic diversity ranging from Hs = 0.15 (cluster I) to Hs = 0.41 (cluster IV) and an average diversity of 0.24. No clusters were categorically distinguished based on hosts or geographical origin using Bayesian clustering analysis. Nonetheless, some clusters comprised most of the isolates with a common geographic origin; for example, cluster VIII was mainly composed of isolates from Central-western Brazil, cluster II from Southern Brazil, and cluster XII from Quincy, Northern Florida, in the United States. Underrepresented regions (few isolates) from Pacific Island nations of Japan, the Philippines, and Indonesia (specifically from Java) were placed into clusters IX and X. Although the analyzed isolates displayed evidence of clonal structure, the genetic diversity indices suggest a potential for the species to adapt to different environmental conditions.

Bridging the Gap: Combining Genomics and Transcriptomics Approaches to Understand Stylosanthes scabra, an Orphan Legume from the Brazilian Caatinga.

Stylosanthes scabra is a scientifically orphaned legume found in the Brazilian Caatinga biome (a semi-arid environment). This work utilized omics approaches to investigate some ecophysiological aspects of stress tolerance/resistance in S. scabra, study its genomic landscape, and predict potential metabolic pathways. Considering its high-confidence conceptual proteome, 1694 (~2.6%) proteins were associated with resistance proteins, some of which were found in soybean QTL regions that confer resistance to Asian soybean rust. S. scabra was also found to be a potential source of terpenes, as biosynthetic gene clusters associated with terpene biosynthesis were identified in its genome. The analysis revealed that mobile elements comprised approximately 59% of the sequenced genome. In the remaining 41% of the sections, some of the 22,681 protein-coding gene families were categorized into two informational groups: those that were specific to S. scabra and those that expanded significantly compared to their immediate ancestor. Biological process enrichment analyses indicated that these gene families play fundamental roles in the adaptation of S. scabra to extreme environments. Additionally, phylogenomic analysis indicated a close evolutionary relationship between the genera Stylosanthes and Arachis. Finally, this study found a high number (57) of aquaporin-encoding loci in the S. scabra genome. RNA-Seq and qPCR data suggested that the PIP subfamily may play a key role in the species' adaptation to water deficit conditions. Overall, these results provide valuable insights into S. scabra biology and a wealth of gene/transcript information for future legume omics studies.

From Gene to Transcript and Peptide: A Deep Overview on Non-Specific Lipid Transfer Proteins (nsLTPs).

Non-specific lipid transfer proteins (nsLTPs) stand out among plant-specific peptide superfamilies due to their multifaceted roles in plant molecular physiology and development, including their protective functions against pathogens. These antimicrobial agents have demonstrated remarkable efficacy against bacterial and fungal pathogens. The discovery of plant-originated, cysteine-rich antimicrobial peptides such as nsLTPs has paved the way for exploring the mentioned organisms as potential biofactories for synthesizing antimicrobial compounds. Recently, nsLTPs have been the focus of a plethora of research and reviews, providing a functional overview of their potential activity. The present work compiles relevant information on nsLTP omics and evolution, and it adds meta-analysis of nsLTPs, including: (1) genome-wide mining in 12 plant genomes not studied before; (2) latest common ancestor analysis (LCA) and expansion mechanisms; (3) structural proteomics, scrutinizing nsLTPs' three-dimensional structure/physicochemical characteristics in the context of nsLTP classification; and (4) broad nsLTP spatiotemporal transcriptional analysis using soybean as a study case. Combining a critical review with original results, we aim to integrate high-quality information in a single source to clarify unexplored aspects of this important gene/peptide family.

Plant Thaumatin-like Proteins: Function, Evolution and Biotechnological Applications.

Thaumatin-like proteins (TLPs) are a highly complex protein family associated with host defense and developmental processes in plants, animals, and fungi. They are highly diverse in angiosperms, for which they are classified as the PR-5 (Pathogenesis-Related-5) protein family. In plants, TLPs have a variety of properties associated with their structural diversity. They are mostly associated with responses to biotic stresses, in addition to some predicted activities under drought and osmotic stresses. The present review covers aspects related to the structure, evolution, gene expression, and biotechnological potential of TLPs. The efficiency of the discovery of new TLPs is below its potential, considering the availability of omics data. Furthermore, we present an exemplary bioinformatics annotation procedure that was applied to cowpea (Vigna unguiculata) transcriptome, including libraries of two tissues (root and leaf), and two stress types (biotic/abiotic) generated using different sequencing approaches. Even without using genomic sequences, the pipeline uncovered 56 TLP candidates in both tissues and stresses. Interestingly, abiotic stress (root dehydration) was associated with a high number of modulated TLP isoforms. The nomenclature used so far for TLPs was also evaluated, considering TLP structure and possible functions identified to date. It is clear that plant TLPs are promising candidates for breeding purposes and for plant transformation aiming a better performance under biotic and abiotic stresses. The development of new therapeutic drugs against human fungal pathogens also deserves attention. Despite that, applications derived from TLP molecules are still below their potential, as it is evident in our review.

Genome Sequence of Metarhizium rileyi, a Microbial Control Agent for Lepidoptera.

Metarhizium rileyi (formerly known as Nomuraea rileyi) is a potential agent for microbial control of many insect pests from the order Lepidoptera, the damages of which can cause considerable loss of productivity in agriculture. We report the genome sequence and annotation of M. rileyi strain Cep018-CH2/ARSEF 7053.

A web-based bioinformatics interface applied to the GENOSOJA Project: Databases and pipelines.

The Genosoja consortium is an initiative to integrate different omics research approaches carried out in Brazil. Basically, the aim of the project is to improve the plant by identifying genes involved in responses against stresses that affect domestic production, like drought stress and Asian Rust fungal disease. To do so, the project generated several types of sequence data using different methodologies, most of them sequenced by next generation sequencers. The initial stage of the project is highly dependent on bioinformatics analysis, providing suitable tools and integrated databases. In this work, we describe the main features of the Genosoja web database, including the pipelines to analyze some kinds of data (ESTs, SuperSAGE, microRNAs, subtractive cDNA libraries), as well as web interfaces to access information about soybean gene annotation and expression.

An overall evaluation of the Resistance (R) and Pathogenesis-Related (PR) superfamilies in soybean, as compared with Medicago and Arabidopsis.

Plants have the ability to recognize and respond to a multitude of pathogens, resulting in a massive reprogramming of the plant to activate defense responses including Resistance (R) and Pathogenesis-Related (PR) genes. Abiotic stresses can also activate PR genes and enhance pathogen resistance, representing valuable genes for breeding purposes. The present work offers an overview of soybean R and PR genes present in the GENOSOJA (Brazilian Soybean Genome Consortium) platform, regarding their structure, abundance, evolution and role in the plant-pathogen metabolic pathway, as compared with Medicago and Arabidopsis. Searches revealed 3,065 R candidates (756 in Soybean, 1,142 in Medicago and 1,167 in Arabidopsis), and PR candidates matching to 1,261 sequences (310, 585 and 366 for the three species, respectively). The identified transcripts were also evaluated regarding their expression pattern in 65 libraries, showing prevalence in seeds and developing tissues. Upon consulting the SuperSAGE libraries, 1,072 R and 481 PR tags were identified in association with the different libraries. Multiple alignments were generated for Xa21 and PR-2 genes, allowing inferences about their evolution. The results revealed interesting insights regarding the variability and complexity of defense genes in soybean, as compared with Medicago and Arabidopsis.

Size of AT(n) insertions in promoter region modulates Gmhsp17.6-L mRNA transcript levels.

During earlier experiments, an SSR molecular marker (176 Soy HSP) showing high correlation (70%) with resistance/susceptibility to javanese root-knot nematode Meloidogyne javanica was identified in soybean. After being sequenced, results indicated that the SSR 176 Soy HSP marker was inserted in the promoter region of Gmhsp17.6-L gene. It was also detected in this region that resistant genotypes presented insertions between AT(31) and AT(33) in size and susceptible genotypes, AT(9). Gmhsp17.6-L gene coding region presented a perfect match in amino acid sequence in all soybean genotypes. A ribonuclease protection assay showed that Gmhsp17.6-L gene mRNA transcripts were present in all genotypes. A real-time relative quantification (qPCR) indicated in the resistant individuals higher mRNA transcripts levels, which presented in the sequencing more AT(n) insertions. These results suggest that the number of AT(n) insertions inside this promoter region could modulate up or down gene levels. Those findings can lead to the possibility of manipulating, between some limits, the mRNA transcripts levels using different sizes of AT(n) insertions.

Molecular cloning of α RYR hotspot region 1 from broiler chicken

Samples of Pectoralis major m. were collected, and an RT-PCR analysis of the a-Ryanodine receptor (a RYR) from chicken mRNA hotspot region spanning aminoacid residues 386 to 540, numbered according to the turkey sequence, revealed two classes of transcripts. The sequences of the first class were similar to turkey and human with 97 percent and 74 percent of identity, respectively, and included all transcripts with substitutions in the nucleotide sequence. The second class was characterized by the deletion of nucleotides, leading to a premature stop codon and coding for a truncated and nonfunctional protein. These results are to date the first report related to the sequencing of the chicken αRYR hotspot region 1, which will possibility serve as a guide for further studies regarding a solution in the poultry production chain related to the problem of pale, soft and exudative (PSE) meat.

Amostras do músculo Pectoralis major foram coletadas e uma RT-PCR foi conduzida para avaliar a sequência do mRNA do αRYR, região compreendida entre os resíduos de aminoácido 386-540, numerado de acordo com a sequência de perus. Os resultados revelaram duas classes de transcritos. O primeiro teve 97 por cento e 74 por cento de identidade com as sequências de αRyR e RyR1 de perus e humanos, respectivamente, e incluiu todos os transcritos com substituições de nucleotídeos. A segunda classe de transcritos foi caracterizada pela deleção de bases que levaram a um stop códon prematuro e a uma proteína truncada não-funcional. Esses resultados são até o momento, o primeiro relato de sequenciamento do αRYR, região hotspot1 de frangos e podem servir como guia para estudos futuros na tentativa de se encontrar uma solução para os problemas na cadeia de produção de frangos relacionados com as carnes PSE (pálida, flácida e exsudativa).

Variability of the mitochondrial SSU rDNA of Nomuraea species and other entomopathogenic fungi from hypocreales.

Hypocrealean arthropod pathogenic fungi have profound impact on the regulation of agricultural and medical pests. However, until now the genetic and phylogenetic relationships among species have not been clarified, such studies could clarify host specificity relationships and define species boundaries. Our purpose was to compare the sequences of the mitochondrial SSU rDNA fragments from several mitosporic entomopathogenic Hypocreales to infer relationships among them and to evaluate the possibility to use these sequences as species diagnostic tool in addition to the more commonly studied sequences of nuclear SSU rDNA. The SSU mt-rDNA proved to be useful to help in differentiation of species inside several genera. Clusters obtained with Parsimony, Bayesian, and Maximum Likelihood analyses were congruent with a new classification of the Clavicipitaceae (Sung et al. Stud Mycol. 2007;57:5-59) in which the anamorphic genera Nomuraea and Metarhizium species remain in the Clavicipitaceae and Isaria species sequenced here are assigned to the family Cordycipitaceae. Mitochondrial genomic information indicates the same general pattern of relationships demonstrated by nuclear gene sequences.

Differential gene expression and mitotic cell analysis of the drought tolerant soybean (Glycine max L. Merrill Fabales, Fabaceae) cultivar MG/BR46 (Conquista) under two water deficit induction systems

Drought cause serious yield losses in soybean (Glycine max), roots being the first plant organ to detect the water-stress signals triggering defense mechanisms. We used two drought induction systems to identify genes differentially expressed in the roots of the drought-tolerant soybean cultivar MG/BR46 (Conquista) and characterize their expression levels during water deficit. Soybean plants grown in nutrient solution hydroponically and in sand-pots were submitted to water stress and gene expression analysis was conducted using the differential display (DD) and real time polymerase chain reaction (PCR) techniques. Three differentially expressed mRNA transcripts showed homology to the Antirrhinum majus basic helix-loop-helix transcription factor bHLH, the Arabidopsis thaliana phosphatidylinositol transfer protein PITP and the auxin-independent growth regulator 1 (axi 1). The hydroponic experiments showed that after 100 min outside the nutrient solution photosynthesis completely stopped, stomata closed and leaf temperature rose. Both stress induction treatments produced significant decrease in the mitotic indices of root cells. Axi 1, PITP and bHLH were not only differentially expressed during dehydration in the hydroponics experiments but also during induced drought in the pot experiments. Although, there were differences between the two sets of experiments in the time at which up or down regulation occurred, the expression pattern of all three transcripts was related. Similar gene expression and cytological analysis results occurred in both systems, suggesting that hydroponics could be used to simulate drought detection by roots growing in soil and thus facilitate rapid and easy root sampling.

Genetic relationships between Chinese, Japanese, and Brazilian soybean gene pools revealed by simple sequence repeat (SSR) markers

An understanding of the relationship of geographically different soybean gene pools, based on selectively neutral DNA markers would be useful for the selection of divergent parental cultivars for use in breeding. We assessed the relationships of 194 Chinese, 59 Japanese, and 19 Brazilian soybean cultivars (n = 272) using 12 simple sequence repeat (SSR) markers. Quantification Theory III and clustering analyses showed that the Chinese and Japanese cultivars were genetically quite distant to each other but not independent, while Brazilian cultivars were distantly related to the cultivars from the other two countries and formed a cluster that was distant from the other two gene pool clusters. Our results indicated that the Brazilian soybean gene pool is different from the Chinese and Japanese pool. Exchanges of these gene pools might be useful to increase the genetic variability in soybean breeding.

Characterization and phylogenetic analysis of Brazilian chicken anaemia virus.

Chicken anaemia virus (CAV) was detected by a Nested-PCR assay in field samples from different regions of Brazil. The 539 bp amplified fragments of vp1 gene from 44 field samples were sequenced and 10 new nucleotide sequences of CAV were observed. These sequences were phylogenetically analysed by Mega2 using neighbour joining distance methods with 1000 bootstrap replications. Phylogenetic analysis did not show correlation between CAV pathology pattern and genetic groups. The 10 nucleotide sequences of the Brazilian samples were also analysed together with 30 sequences of CAV strains previously described from other countries. The genetic variability observed was not related to the geographical distribution. Amino acid substitutions were detected at 9 positions of the Brazilian sequences and two of them had not been observed before, (65)R replacing the Q residue and (98)F replacing Y residue.

Population structure of the Brazilian southern green stink bug, Nezara viridula.

The Southern Green Stink Bug, Nezara viridula (L.) (Heteroptera: Pentatomidae), is a cosmopolitan and economically important pest to several crops. Studies on N. viridula migration and population structure have been neglected. We studied geographically distinct Brazilian N. viridula populations to assess their variability and to determine gene flow among them. DNA from specimens collected on soybean fields were subjected to RAPD analysis to determine genetic similarity and population structure parameters. All N. viridula populations studied were genetically distinct from the others. The maximum similarity occurred between populations from Londrina and Sertanópolis (Parana State). The Cruz Alta population was the most divergent from the others. Despite the short distance between Cambé and Londrina (ca. 29 km), and the absence of geographic barriers, both populations clustered in different groups and the estimated gene flow index (Nm) among them was 2.02, indicating relatively restricted migration. The estimated overall index, Nm was 1.41 suggesting that N. viridula is a better flier than the Neotropical Brown stink bug, Euschistus heros (Nm =0.83).

VSQual: a visual system to assist DNA sequencing quality control.

A lack of pliant software tools that support small- to medium-scale DNA sequencing efforts is a major hindrance for recording and using laboratory workflow information to monitor the overall quality of data production. Here we describe VSQual, a set of Perl programs intended to provide simple and powerful tools to check several quality features of the sequencing data generated by automated DNA sequencing machines. The core program of VSQual is a flexible Perl-based pipeline, designed to be accessible and useful for both programmers and non-programmers. This pipeline directs the processing steps and can be easily customized for laboratory needs. Basically, the raw DNA sequencing trace files are processed by Phred and Cross_match, then the outputs are parsed, reformatted into Web-based graphical reports, and added to a Web site structure. The result is a set of real time sequencing reports easily accessible and understood by common laboratory people. These reports facilitate the monitoring of DNA sequencing as well as the management of laboratory workflow, significantly reducing operational costs and ensuring high quality and scientifically reliable results.

VSQual: a visual system to assist DNA sequencing quality control

A lack of pliant software tools that support small- to medium-scale DNA sequencing efforts is a major hindrance for recording and using laboratory workflow information to monitor the overall quality of data production. Here we describe VSQual, a set of Perl programs intended to provide simple and powerful tools to check several quality features of the sequencing data generated by automated DNA sequencing machines. The core program of VSQual is a flexible Perl-based pipeline, designed to be accessible and useful for both programmers and non-programmers. This pipeline directs the processing steps and can be easily customized for laboratory needs. Basically, the raw DNA sequencing trace files are processed by Phred and Cross_match, then the outputs are parsed, reformatted into Web-based graphical reports, and added to a Web site structure. The result is a set of real time sequencing reports easily accessible and understood by common laboratory people. These reports facilitate the monitoring of DNA sequencing as well as the management of laboratory workflow, significantly reducing operational costs and ensuring high quality and scientifically reliable results.