Amar Kumar

Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing

BackgroundMicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing.ResultsSmall RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAM (HAIRY MERISTEM) respectively, were experimentally validated using 5′ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions.ConclusionsWe report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage-specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development.

Global insights into high temperature and drought stress regulated genes by RNA-Seq in economically important oilseed crop Brassica juncea

BackgroundBrassica juncea var. Varuna is an economically important oilseed crop of family Brassicaceae which is vulnerable to abiotic stresses at specific stages in its life cycle. Till date no attempts have been made to elucidate genome-wide changes in its transcriptome against high temperature or drought stress. To gain global insights into genes, transcription factors and kinases regulated by these stresses and to explore information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de-novo assembly to discover B. juncea transcriptome associated with high temperature and drought stresses.ResultsWe constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS). More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPdenovo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Many of these were responsive to high temperature, drought or both stresses. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families, respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes.ConclusionsOur study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resource generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity.

A Genome-Wide Perspective of miRNAome in Response to High Temperature, Salinity and Drought Stresses in Brassica juncea (Czern) L

Micro RNAs (miRNAs) are involved in diverse biological processes including adaptive response towards abiotic stresses. To unravel small RNAs and more specifically miRNAs that can potentially regulate determinants of abiotic stress tolerance, next generation sequencing of B. juncea seedlings subjected to high temperature, high salt and drought conditions was carried out. With the help of UEA sRNA workbench software package, 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions.

Assessment of Genetic Diversity and Population Structure in a Global Reference Collection of 531 Accessions of Carthamus tinctorius L. (Safflower) Using AFLP Markers

Carthamus tinctorius L. (safflower) is an important oilseed crop that is cultivated in several countries. The present study investigates the genetic diversity and population structure of 531 safflower accessions from 43 countries representing all safflower growing regions of the world. Diversity analysis was performed using ten informative EcoRI/MseI amplified fragment length polymorphism primer pairs that were identified by screening 150 primer combinations. The selected primer pairs generated 381 fragments of which 157 were polymorphic among the analyzed accessions. The genetic diversity indices obtained for the entire collection (I = 0.4536, H = 0.2955) indicated high levels of molecular variability. The distance-based, neighbor-joining method classified the accessions into six clusters with internal subgroupings that were in consonance with 19 clusters obtained using Bayesian model-based BAPS analysis. Clusters obtained through STRUCTURE analysis (at K = 4) could not be correlated with their geographically diverse origins, while BAPS analysis (at K = 19) revealed geographical delineation with low admixture levels among most of the studied accessions. Accessions from Far East and Egypt clustered in distinct groups, indicating conserved nature of their gene pools. The Near East and Iran–Afghanistan regions were collectively found to harbor maximum diversity in accordance with earlier reports. Accessions from the Indian subcontinent showed substantial diversity that was previously undetected. The American accessions showed low molecular variability in contrast to earlier studies. Genetic sub-structuring within gene pools and inter-relationships between accessions belonging to different regional pools was also observed. To the best of our knowledge, this is the first comprehensive study of existing genetic variability in a large collection of safflower germplasm with a global distribution, which provides a more accurate representation of genetic structuring in the crop. This information will facilitate selection of elite genotypes for broadening the genetic base of various breeding programs in safflower.

Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction

Root-knot nematodes (RKNs, Meloidogyne spp.) are the most damaging plant parasites causing severe losses to crop production. The present study reports genome-wide identification and characterization of both tomato and RKN miRNAs simultaneously from RKN-infected susceptible tomato roots using high-throughput sequencing technique. RNAseq data from 11 small RNA libraries derived from 5 disease development stages identified 281 novel miRNAs of tomato in addition to 52 conserved and 4 variants of conserved miRNAs. Additionally, the same set of RNAseq data identified 38 conserved and 290 novel RKN miRNAs. Both tomato and RKN miRNAs showed differential expression at 5 stages of disease development based on digital expression profiles. In tomato, further validation through qRT-PCR confirmed that majority of miRNAs were significantly upregulated during susceptible response whereas downregulated during resistance response. The predicted targets of 8 conserved and 1 novel miRNAs were validated through 5’RLM-RACE. A negative correlation between expression profiles of a few conserved miRNAs (miR156, miR159, miR164 and miR396) and their targets (SBP, GAMYB-like, NAC and GRF1 transcription factor) was confirmed. A novel Sly_miRNA996 also showed a negative correlation with its target MYB-like transcription factor. These results indicate that the conserved and novel tomato miRNAs are involved in regulating developmental changes in host root during RKN infection. In RKN, the targets of conserved miRNAs were also predicted and a few of their predicted target genes are known to be involved in nematode parasitism. Further, the potential roles of both tomato and RKN miRNAs have been discussed.

De novo transcriptome profiling of cold-stressed siliques during pod filling stages in Indian mustard (Brassica juncea L.)

Low temperature is a major abiotic stress that impedes plant growth and development. Brassica juncea is an economically important oil seed crop and is sensitive to freezing stress during pod filling subsequently leading to abortion of seeds. To understand the cold stress mediated global perturbations in gene expression, whole transcriptome of B. juncea siliques that were exposed to sub-optimal temperature was sequenced. Manually self-pollinated siliques at different stages of development were subjected to either short (6 h) or long (12 h) durations of chilling stress followed by construction of RNA-seq libraries and deep sequencing using Illuminas NGS platform. De-novo assembly of B. juncea transcriptome resulted in 133,641 transcripts, whose combined length was 117 Mb and N50 value was 1428 bp. We identified 13,342 differentially regulated transcripts by pair-wise comparison of 18 transcriptome libraries. Hierarchical clustering along with Spearman correlation analysis identified that the differentially expressed genes segregated in two major clusters representing early (5–15 DAP) and late stages (20–30 DAP) of silique development. Further analysis led to the discovery of sub-clusters having similar patterns of gene expression. Two of the sub-clusters (one each from the early and late stages) comprised of genes that were inducible by both the durations of cold stress. Comparison of transcripts from these clusters led to identification of 283 transcripts that were commonly induced by cold stress, and were referred to as “core cold-inducible” transcripts. Additionally, we found that 689 and 100 transcripts were specifically up-regulated by cold stress in early and late stages, respectively. We further explored the expression patterns of gene families encoding for transcription factors (TFs), transcription regulators (TRs) and kinases, and found that cold stress induced protein kinases only during early silique development. We validated the digital gene expression profiles of selected transcripts by qPCR and found a high degree of concordance between the two analyses. To our knowledge this is the first report of transcriptome sequencing of cold-stressed B. juncea siliques. The data generated in this study would be a valuable resource for not only understanding the cold stress signaling pathway but also for introducing cold hardiness in B. juncea.

Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis

Background Safflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing. Principal Findings Low throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower. Conclusion Next generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection.

Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-infected tomato (Solanum lycopersicum) roots reveals complex gene expression profiles and metabolic networks of both host and nematode during susceptible and resistance responses

Root-knot nematodes (RKNs, Meloidogyne incognita) are economically important endoparasites with a wide host range. We used a comprehensive transcriptomic approach to investigate the expression of both tomato and RKN genes in tomato roots at five infection time intervals from susceptible plants and two infection time intervals from resistant plants, grown under soil conditions. Differentially expressed genes during susceptible (1827, tomato; 462, RKN) and resistance (25, tomato; 160, RKN) interactions were identified. In susceptible responses, tomato genes involved in cell wall structure, development, primary and secondary metabolite, and defence signalling pathways, together with RKN genes involved in host parasitism, development and defence, are discussed. In resistance responses, tomato genes involved in secondary metabolite and hormone-mediated defence responses, together with RKN genes involved in starvation stress-induced apoptosis, are discussed. In addition, 40 novel differentially expressed RKN genes encoding secretory proteins were identified. Our findings provide novel insights into the temporal regulation of genes involved in various biological processes from tomato and RKN simultaneously during susceptible and resistance responses, and reveal the involvement of a complex network of biosynthetic pathways during disease development.

Molecular aspects of plant-nematode interactions

Plants are continuously exposed to a wide range of pathogens, including parasitic nematodes. Being sessile, plant have developed elaborate defense mechanisms that allow them to recognize potential invading pathogens and to initiate successful defenses. Plant parasitic nematodes are ubiquitous, tiny and microscopic animals, which can attack nearly all parts of the plant and globally cause crop loss of

Utilization of Molecular, Phenotypic, and Geographical Diversity to Develop Compact Composite Core Collection in the Oilseed Crop, Safflower (Carthamus tinctorius L.) through Maximization Strategy.

100 billion annually. The co-evolution of plants and plant-parasitic nematodes with respect to resistance and parasitism has resulted in remarkable adaptations of the host and parasite life cycles. We review here the recent knowledge acquired on the molecular players that help in successful parasitism within the host plant roots. We then also discuss the molecular players and mechanisms underlying plant resistance against these parasitic nematodes. Understanding the molecular basis of plant-nematode interactions will be a way forward to design environmental friendly control strategies to target harmful pathogen.