Metabolomics of early blight (Alternaria solani) susceptible tomato (Solanum lycopersicum) unfolds key biomarker metabolites and involved metabolic pathways.

Tomato (Solanum lycopersicum) is among the most important commercial horticultural crops worldwide. The crop quality and production is largely hampered due to the fungal pathogen Alternaria solani causing necrotrophic foliage early blight disease. Crop plants usually respond to the biotic challenges with altered metabolic composition and physiological perturbations. We have deciphered altered metabolite composition, modulated metabolic pathways and identified metabolite biomarkers in A. solani-challenged susceptible tomato variety Kashi Aman using Liquid Chromatography-Mass Spectrometry (LC-MS) based metabolomics. Alteration in the metabolite feature composition of pathogen-challenged (m/z 9405) and non-challenged (m/z 9667) plant leaves including 8487 infection-exclusive and 8742 non-infection exclusive features was observed. Functional annotation revealed putatively annotated metabolites and pathway mapping indicated their enrichment in metabolic pathways, biosynthesis of secondary metabolites, ubiquinone and terpenoid-quinones, brassinosteroids, steroids, terpenoids, phenylpropanoids, carotenoids, oxy/sphingolipids and metabolism of biotin and porphyrin. PCA, multivariate PLS-DA and OPLS-DA analysis showed sample discrimination. Significantly up regulated 481 and down regulated 548 metabolite features were identified based on the fold change (threshold ≥ 2.0). OPLS-DA model based on variable importance in projection (VIP scores) and FC threshold (> 2.0) revealed 41 up regulated discriminant metabolite features annotated as sphingosine, fecosterol, melatonin, serotonin, glucose 6-phosphate, zeatin, dihydrozeatin and zeatin-ß-D-glucoside. Similarly, 23 down regulated discriminant metabolites included histidinol, 4-aminobutyraldehyde, propanoate, tyramine and linalool. Melatonin and serotonin in the leaves were the two indoleamines being reported for the first time in tomato in response to the early blight pathogen. Receiver operating characteristic (ROC)-based biomarker analysis identified apigenin-7-glucoside, uridine, adenosyl-homocysteine, cGMP, tyrosine, pantothenic acid, riboflavin (as up regulated) and adenosine, homocyctine and azmaline (as down regulated) biomarkers. These results could aid in the development of metabolite-quantitative trait loci (mQTL). Furthermore, stress-induced biosynthetic pathways may be the potential targets for modifications through breeding programs or genetic engineering for improving crop performance in the fields.

Genome-wide identification and characterization of Puccinia striiformis-responsive lncRNAs in Triticum aestivum.

Wheat stripe rust (yellow rust) caused by Puccinia striiformis f. sp. tritici (Pst) is a serious biotic stress factor limiting wheat production worldwide. Emerging evidence demonstrates that long non-coding RNAs (lncRNAs) participate in various developmental processes in plants via post-transcription regulation. In this study, RNA sequencing (RNA-seq) was performed on a pair of near-isogenic lines-rust resistance line FLW29 and rust susceptible line PBW343-which differed only in the rust susceptibility trait. A total of 6,807 lncRNA transcripts were identified using bioinformatics analyses, among which 10 lncRNAs were found to be differentially expressed between resistance and susceptible lines. In order to find the target genes of the identified lncRNAs, their interactions with wheat microRNA (miRNAs) were predicted. A total of 199 lncRNAs showed interactions with 65 miRNAs, which further target 757 distinct mRNA transcripts. Moreover, detailed functional annotations of the target genes were used to identify the candidate genes, pathways, domains, families, and transcription factors that may be related to stripe rust resistance response in wheat plants. The NAC domain protein, disease resistance proteins RPP13 and RPM1, At1g58400, monodehydroascorbate reductase, NBS-LRR-like protein, rust resistance kinase Lr10-like, LRR receptor, serine/threonine-protein kinase, and cysteine proteinase are among the identified targets that are crucial for wheat stripe rust resistance. Semiquantitative PCR analysis of some of the differentially expressed lncRNAs revealed variations in expression profiles of two lncRNAs between the Pst-resistant and Pst-susceptible genotypes at least under one condition. Additionally, simple sequence repeats (SSRs) were also identified from wheat lncRNA sequences, which may be very useful for conducting targeted gene mapping studies of stripe rust resistance in wheat. These findings improved our understanding of the molecular mechanism responsible for the stripe rust disease that can be further utilized to develop wheat varieties with durable resistance to this disease.

Untargeted Metabolomics of Alternaria solani-Challenged Wild Tomato Species Solanum cheesmaniae Revealed Key Metabolite Biomarkers and Insight into Altered Metabolic Pathways.

Untargeted metabolomics of moderately resistant wild tomato species Solanum cheesmaniae revealed an altered metabolite profile in plant leaves in response to Alternaria solani pathogen. Leaf metabolites were significantly differentiated in non-stressed versus stressed plants. The samples were discriminated not only by the presence/absence of specific metabolites as distinguished markers of infection, but also on the basis of their relative abundance as important concluding factors. Annotation of metabolite features using the Arabidopsis thaliana (KEGG) database revealed 3371 compounds with KEGG identifiers belonging to biosynthetic pathways including secondary metabolites, cofactors, steroids, brassinosteroids, terpernoids, and fatty acids. Annotation using the Solanum lycopersicum database in PLANTCYC PMN revealed significantly upregulated (541) and downregulated (485) features distributed in metabolite classes that appeared to play a crucial role in defense, infection prevention, signaling, plant growth, and plant homeostasis to survive under stress conditions. The orthogonal partial least squares discriminant analysis (OPLS-DA), comprising a significant fold change (≥2.0) with VIP score (≥1.0), showed 34 upregulated biomarker metabolites including 5-phosphoribosylamine, kaur-16-en-18-oic acid, pantothenate, and O-acetyl-L-homoserine, along with 41 downregulated biomarkers. Downregulated metabolite biomarkers were mapped with pathways specifically known for plant defense, suggesting their prominent role in pathogen resistance. These results hold promise for identifying key biomarker metabolites that contribute to disease resistive metabolic traits/biosynthetic routes. This approach can assist in mQTL development for the stress breeding program in tomato against pathogen interactions.

Comparative expression analysis of water buffalo (Bubalus bubalis) to identify genes associated with economically important traits.

The milk, meat, skins, and draft power of domestic water buffalo (Bubalus bubalis) provide substantial contributions to the global agricultural economy. The world's water buffalo population is primarily found in Asia, and the buffalo supports more people per capita than any other livestock species. For evaluating the workflow, output rate, and completeness of transcriptome assemblies within and between reference-free (RF) de novo transcriptome and reference-based (RB) datasets, abundant bioinformatics studies have been carried out to date. However, comprehensive documentation of the degree of consistency and variability of the data produced by comparing gene expression levels using these two separate techniques is lacking. In the present study, we assessed the variations in the number of differentially expressed genes (DEGs) attained with RF and RB approaches. In light of this, we conducted a study to identify, annotate, and analyze the genes associated with four economically important traits of buffalo, viz., milk volume, age at first calving, post-partum cyclicity, and feed conversion efficiency. A total of 14,201 and 279 DEGs were identified in RF and RB assemblies. Gene ontology (GO) terms associated with the identified genes were allocated to traits under study. Identified genes improve the knowledge of the underlying mechanism of trait expression in water buffalo which may support improved breeding plans for higher productivity. The empirical findings of this study using RNA-seq data-based assembly may improve the understanding of genetic diversity in relation to buffalo productivity and provide important contributions to answer biological issues regarding the transcriptome of non-model organisms.

Metabolomics-Driven Mining of Metabolite Resources: Applications and Prospects for Improving Vegetable Crops.

Vegetable crops possess a prominent nutri-metabolite pool that not only contributes to the crop performance in the fields, but also offers nutritional security for humans. In the pursuit of identifying, quantifying and functionally characterizing the cellular metabolome pool, biomolecule separation technologies, data acquisition platforms, chemical libraries, bioinformatics tools, databases and visualization techniques have come to play significant role. High-throughput metabolomics unravels structurally diverse nutrition-rich metabolites and their entangled interactions in vegetable plants. It has helped to link identified phytometabolites with unique phenotypic traits, nutri-functional characters, defense mechanisms and crop productivity. In this study, we explore mining diverse metabolites, localizing cellular metabolic pathways, classifying functional biomolecules and establishing linkages between metabolic fluxes and genomic regulations, using comprehensive metabolomics deciphers of the plant's performance in the environment. We discuss exemplary reports covering the implications of metabolomics, addressing metabolic changes in vegetable plants during crop domestication, stage-dependent growth, fruit development, nutri-metabolic capabilities, climatic impacts, plant-microbe-pest interactions and anthropogenic activities. Efforts leading to identify biomarker metabolites, candidate proteins and the genes responsible for plant health, defense mechanisms and nutri-rich crop produce are documented. With the insights on metabolite-QTL (mQTL) driven genetic architecture, molecular breeding in vegetable crops can be revolutionized for developing better nutritional capabilities, improved tolerance against diseases/pests and enhanced climate resilience in plants.

CerealESTDb: A Comprehensive Resource for Abiotic Stress-Responsive Annotated ESTs With Predicted Genes, Gene Ontology, and Metabolic Pathways in Major Cereal Crops.

Cereals are the most important food crops and are considered key contributors to global food security. Loss due to abiotic stresses in cereal crops is limiting potential productivity in a significant manner. The primary reasons for abiotic stresses are abrupt temperature, variable rainfall, and declining nutrient status of the soil. Varietal development is the key to sustaining productivity under influence of multiple abiotic stresses and must be studied in context with genomics and molecular breeding. Recently, advances in a plethora of Next Generation Sequencing (NGS) based methods have accelerated the enormous genomic data generation associated with stress-induced transcripts such as microarray, RNAseq, Expressed Sequenced Tag (ESTs), etc. Many databases related to microarray and RNA-seq based transcripts have been developed and profusely utilized. However, an abundant amount of transcripts related to abiotic stresses in various cereal crops arising from EST technology are available but still remain underutilized in absence of a consolidated database. In this study, an attempt has been made with a primary goal to integrate, analyse, and characterise the available resources of ESTs responsive to abiotic stresses in major cereals. The developed CerealESTdb presents a customisable search in two different ways in the form of searchable content for easy access and potential use. This database comprises ESTs from four major cereal crops, namely rice (Oryza sativa L.), wheat (Triticum aestivum L.), sorghum (Sorghum bicolour L.), and maize (Zea mays L.), under a set of abiotic stresses. The current statistics of this cohesive database consists of 55,826 assembled EST sequences, 51,791 predicted genes models, and their 254,609 gene ontology terms including extensive information on 1,746 associated metabolic pathways. We anticipate that developed CerealESTdb will be helpful in deciphering the knowledge of complex biological phenomena under abiotic stresses to accelerate the molecular breeding programs towards the development of crop cultivars resilient to abiotic stresses. The CerealESTdb is publically available with the URL http://cabgrid.res.in/CerealESTDb.

Machine learning in the estimation of CRISPR-Cas9 cleavage sites for plant system.

CRISPR-Cas9 system is one of the recent most used genome editing techniques. Despite having a high capacity to alter the precise target genes and genomic regions that the planned guide RNA (or sgRNA) complements, the off-target effect still exists. But there are already machine learning algorithms for people, animals, and a few plant species. In this paper, an effort has been made to create models based on three machine learning-based techniques [namely, artificial neural networks (ANN), support vector machines (SVM), and random forests (RF)] for the prediction of the CRISPR-Cas9 cleavage sites that will be cleaved by a particular sgRNA. The plant dataset was the sole source of inspiration for all of these machine learning-based algorithms. 70% of the on-target and off-target dataset of various plant species that was gathered was used to train the models. The remaining 30% of the data set was used to evaluate the model's performance using a variety of evaluation metrics, including specificity, sensitivity, accuracy, precision, F1 score, F2 score, and AUC. Based on the aforementioned machine learning techniques, eleven models in all were developed. Comparative analysis of these produced models suggests that the model based on the random forest technique performs better. The accuracy of the Random Forest model is 96.27%, while the AUC value was found to be 99.21%. The SVM-Linear, SVM-Polynomial, SVM-Gaussian, and SVM-Sigmoid models were trained, making a total of six ANN-based models (ANN1-Logistic, ANN1-Tanh, ANN1-ReLU, ANN2-Logistic, ANN2-Tanh, and ANN-ReLU) and Support Vector Machine models (SVM-Linear, SVM-Polynomial, SVM-Gaussian However, the overall performance of Random Forest is better among all other ML techniques. ANN1-ReLU and SVM-Linear model performance were shown to be better among Artificial Neural Network and Support Vector Machine-based models, respectively.

MetaConClust - Unsupervised Binning of Metagenomics Data using Consensus Clustering.

Background: Binning of metagenomic reads is an active area of research, and many unsupervised machine learning-based techniques have been used for taxonomic independent binning of metagenomic reads. Objective: It is important to find the optimum number of the cluster as well as develop an efficient pipeline for deciphering the complexity of the microbial genome. Methods: Applying unsupervised clustering techniques for binning requires finding the optimal number of clusters beforehand and is observed to be a difficult task. This paper describes a novel method, MetaConClust, using coverage information for grouping of contigs and automatically finding the optimal number of clusters for binning of metagenomics data using a consensus-based clustering approach. The coverage of contigs in a metagenomics sample has been observed to be directly proportional to the abundance of species in the sample and is used for grouping of data in the first phase by MetaConClust. The Partitioning Around Medoid (PAM) method is used for clustering in the second phase for generating bins with the initial number of clusters determined automatically through a consensus-based method. Results: Finally, the quality of the obtained bins is tested using silhouette index, rand Index, recall, precision, and accuracy. Performance of MetaConClust is compared with recent methods and tools using benchmarked low complexity simulated and real metagenomic datasets and is found better for unsupervised and comparable for hybrid methods. Conclusion: This is suggestive of the proposition that the consensus-based clustering approach is a promising method for automatically finding the number of bins for metagenomics data.

Understanding Rice-Magnaporthe Oryzae Interaction in Resistant and Susceptible Cultivars of Rice under Panicle Blast Infection Using a Time-Course Transcriptome Analysis.

Rice blast is a global threat to food security with up to 50% yield losses. Panicle blast is a more severe form of rice blast and the response of rice plant to leaf and panicle blast is distinct in different genotypes. To understand the specific response of rice in panicle blast, transcriptome analysis of blast resistant cultivar Tetep, and susceptible cultivar HP2216 was carried out using RNA-Seq approach after 48, 72 and 96 h of infection with Magnaporthe oryzae along with mock inoculation. Transcriptome data analysis of infected panicle tissues revealed that 3553 genes differentially expressed in HP2216 and 2491 genes in Tetep, which must be the responsible factor behind the differential disease response. The defense responsive genes are involved mainly in defense pathways namely, hormonal regulation, synthesis of reactive oxygen species, secondary metabolites and cell wall modification. The common differentially expressed genes in both the cultivars were defense responsive transcription factors, NBS-LRR genes, kinases, pathogenesis related genes and peroxidases. In Tetep, cell wall strengthening pathway represented by PMR5, dirigent, tubulin, cell wall proteins, chitinases, and proteases was found to be specifically enriched. Additionally, many novel genes having DOMON, VWF, and PCaP1 domains which are specific to cell membrane were highly expressed only in Tetep post infection, suggesting their role in panicle blast resistance. Thus, our study shows that panicle blast resistance is a complex phenomenon contributed by early defense response through ROS production and detoxification, MAPK and LRR signaling, accumulation of antimicrobial compounds and secondary metabolites, and cell wall strengthening to prevent the entry and spread of the fungi. The present investigation provided valuable candidate genes that can unravel the mechanisms of panicle blast resistance and help in the rice blast breeding program.

A Novel Way of Comparing Protein 3D Structure Using Graph Partitioning Approach.

Alignment and comparison of protein 3D structures is an important and fundamental task in structural biology to study evolutionary, functional and structural relatedness among proteins. Since two decades, the research on protein structure alignment has been taken up on priority and numbers of research articles are being published. There are incremental advances over previous efforts, and still these methods continue to improve over the time and still this is an open problem in structural biology. A novel methodology has been developed for comparing protein 3D structure by employing conversion of pair of protein 3D structures into 2D graphs (undirected weighted graph), partitioning of 2D graphs into sub-graphs, matching sub-graphs with main graphs and finally these sub-graphs matches calculates similarity between the pair of proteins. The proposed method has been implemented in MATLAB and R Package. The performance of the developed methodology is tested with four existing best methods such as CE, jFATCAT, TM_Align and Dali on 100 proteins benchmark dataset with SCOP database. The proposed method is efficient in terms of time complexity, accuracy, grouping of proteins in relevant structural groups and provides additional information towards non-bonded interactions and sub-graphs indicates the dominance of secondary structure.

Inferring Relationship of Blood Metabolic Changes and Average Daily Gain With Feed Conversion Efficiency in Murrah Heifers: Machine Learning Approach.

Machine learning algorithms were employed for predicting the feed conversion efficiency (FCE), using the blood parameters and average daily gain (ADG) as predictor variables in buffalo heifers. It was observed that isotonic regression outperformed other machine learning algorithms used in study. Further, we also achieved the best performance evaluation metrics model with additive regression as the meta learner and isotonic regression as the base learner on 10-fold cross-validation and leaving-one-out cross-validation tests. Further, we created three separate partial least square regression (PLSR) models using all 14 parameters of blood and ADG as independent (explanatory) variables and FCE as the dependent variable, to understand the interactions of blood parameters, ADG with FCE each by inclusion of all FCE values (i), only higher FCE values (negative RFI) (ii), and inclusion of only lower FCE (positive RFI) values (iii). The PLSR model including only the higher FCE values was concluded the best, based on performance evaluation metrics as compared to PLSR models developed by inclusion of the lower FCE values and all types of FCE values. IGF1 and its interactions with the other blood parameters were found highly influential for higher FCE measures. The strength of the estimated interaction effects of the blood parameter in relation to FCE may facilitate understanding of intricate dynamics of blood parameters for growth.

A Comparative Study of Single-Trait and Multi-Trait Genomic Selection.

In recent years of animal and plant breeding research, genomic selection (GS) became a choice for selection of appropriate candidate for breeding as it significantly contributes to enhance the genetic gain. Various studies related to GS have been carried out in the recent past. These studies were mostly confined to single trait. Although GS methods based on single trait have not performed very well in cases like pleiotropy, missing data and when the trait under study has low heritability. Gradually, some studies were carried out to explore the possibility of methods for GS based on multiple traits in the view of overcoming the above-mentioned problems in the method of single-trait GS (STGS). Currently, multi-trait-based GS methods are getting importance as it exploits the information of correlated structure among response. In this study, we have compared various methods related to STGS, such as stepwise regression, ridge regression, least absolute shrinkage and selection operator (LASSO), Bayesian, best linear unbiased prediction, and support vector machine, and multi-trait-based GS methods, such as multivariate regression with covariance estimation, conditional Gaussian graphical models, mixed model, and LASSO. In almost all cases, multi-trait-based methods are found to be more accurate. Based on the results of this study, it may be concluded that multi-trait-based methods have great potential to increase genetic gain as they utilize the correlation among the response variable as extra information, which contributes to estimate breeding value more precisely. This study is a comprehensive review of the methods of GS right from single trait to multiple traits and comparisons among these two classes.

Prediction of miRNA and Identification of their Relationship Network Related to Late Blight Disease of Potato.

BACKGROUND: Late blight is a serious disease in potato caused by Phytophthora infestans. To date only few miRNA have been discovered which are related to late blight disease of potato during host pathogen interaction. Recent studies showed that miRNA, an important gene expression regulator, plays a very important role in host-pathogen interaction by silencing genes either by destructing or blocking of translation of mRNA. METHOD: Homology search was performed between non-redundant mature miRNA sequences from miRBase database and Solanum tuberosum EST sequences from NCBI database. Screening of the potential miRNA was done after secondary structure prediction. The target related to late blight disease of respective miRNA was functionally annotated. To identify the relationship between the predicted and mature miRNAs, multiple sequence alignment and evolutionary relationships were established. RESULTS AND CONCLUSION: 34 Candidate miRNA related to late blight disease of potato were identified which were associated to five target genes. These miRNAs were linked with Avr3a, INF1, INF2b genes which are elicitin like protein and triggers a hypersensitive response to host cell. Mapping of target sequences showed similarity with Solanum lycopersicum NRC1 gene of chr.1, which are reported as a casual protein required for Pto-mediated cell death and resistance in N. benthamiana. NRC1 are considered as a RX-CC_like domain-containing protein which shows similarity with coiledcoil domain of the potato virus X resistance protein (RX) in Solanum tuberosum. RX recognizes pathogen effector proteins and triggers a response that may be as severe as localized cell death thereby providing resistance against potato virus X.

The Halophile protein database.

Halophilic archaea/bacteria adapt to different salt concentration, namely extreme, moderate and low. These type of adaptations may occur as a result of modification of protein structure and other changes in different cell organelles. Thus proteins may play an important role in the adaptation of halophilic archaea/bacteria to saline conditions. The Halophile protein database (HProtDB) is a systematic attempt to document the biochemical and biophysical properties of proteins from halophilic archaea/bacteria which may be involved in adaptation of these organisms to saline conditions. In this database, various physicochemical properties such as molecular weight, theoretical pI, amino acid composition, atomic composition, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (Gravy) have been listed. These physicochemical properties play an important role in identifying the protein structure, bonding pattern and function of the specific proteins. This database is comprehensive, manually curated, non-redundant catalogue of proteins. The database currently contains 59 897 proteins properties extracted from 21 different strains of halophilic archaea/bacteria. The database can be accessed through link. Database URL: http://webapp.cabgrid.res.in/protein/

Design and development of portal for biological database in agriculture.

UNLABELLED: The application of novel and modern techniques in genetic engineering and genomics has resulted in information explosion in genomics. Three major genome databases under International Nucleotide Sequence Database collaboration NCBI, DDBJ and EMBL have been providing a convenient platform for submission of sequences which they share among themselves. Many institutes in India under Indian Council of Agricultural Research have scientists working on biotechnology and bioinformatics research. The various studies conducted by them, generate massive data related to biological information of plants, animals, insects, microbes and fisheries. These scientists are dependent on NCBI, EMBL, DDBJ and other portals for their sequence submissions, analysis and other data mining tasks. Due to various limitations imposed on these sites and the poor connectivity problem prevents them to conduct their studies on these open domain databases. The valued information generated by them needs to be shared by the scientific communities to eliminate the duplication of efforts and expedite their knowledge extended towards new findings. A secured common submission portal system with user-friendly interfaces, integrated help and error checking facilities has been developed in such a way that the database at the backend consists of a union of the items available on the above mentioned databases. Standard database management concepts have been employed for their systematic storage management. Extensive hardware resources in the form of high performance computing facility are being installed for deployment of this portal. AVAILABILITY: http://cabindb.iasri.res.in:8080/sequence_portal/