Sarika Jaiswal

Discovery of Putative Herbicide Resistance Genes and Its Regulatory Network in Chickpea Using Transcriptome Sequencing

Background: Chickpea (Cicer arietinum L.) contributes 75% of total pulse production. Being cheaper than animal protein, makes it important in dietary requirement of developing countries. Weed not only competes with chickpea resulting into drastic yield reduction but also creates problem of harboring fungi, bacterial diseases and insect pests. Chemical approach having new herbicide discovery has constraint of limited lead molecule options, statutory regulations and environmental clearance. Through genetic approach, transgenic herbicide tolerant crop has given successful result but led to serious concern over ecological safety thus non-transgenic approach like marker assisted selection is desirable. Since large variability in tolerance limit of herbicide already exists in chickpea varieties, thus the genes offering herbicide tolerance can be introgressed in variety improvement programme. Transcriptome studies can discover such associated key genes with herbicide tolerance in chickpea. Results: This is first transcriptomic studies of chickpea or even any legume crop using two herbicide susceptible and tolerant genotypes exposed to imidazoline (Imazethapyr). Approximately 90 million paired-end reads generated from four samples were processed and assembled into 30,803 contigs using reference based assembly. We report 6,310 differentially expressed genes (DEGs), of which 3,037 were regulated by 980 miRNAs, 1,528 transcription factors associated with 897 DEGs, 47 Hub proteins, 3,540 putative Simple Sequence Repeat-Functional Domain Marker (SSR-FDM), 13,778 genic Single Nucleotide Polymorphism (SNP) putative markers and 1,174 Indels. Randomly selected 20 DEGs were validated using qPCR. Pathway analysis suggested that xenobiotic degradation related gene, glutathione S-transferase (GST) were only up-regulated in presence of herbicide. Down-regulation of DNA replication genes and up-regulation of abscisic acid pathway genes were observed. Study further reveals the role of cytochrome P450, xyloglucan endotransglucosylase/hydrolase, glutamate dehydrogenase, methyl crotonoyl carboxylase and of thaumatin-like genes in herbicide resistance. Conclusion: Reported DEGs can be used as genomic resource for future discovery of candidate genes associated with herbicide tolerance. Reported markers can be used for future association studies in order to develop marker assisted selection (MAS) for refinement. In endeavor of chickpea variety development programme, these findings can be of immense use in improving productivity of chickpea germplasm.

Draft Genome Sequence of Two Monosporidial Lines of the Karnal Bunt Fungus Tilletia indica Mitra (PSWKBGH-1 and PSWKBGH-2)

ABSTRACT Karnal bunt disease caused by the fungus Tilletia indica Mitra is a serious concern due to strict quarantines affecting international trade of wheat. We announce here the first draft assembly of two monosporidial lines, PSWKBGH-1 and -2, of this fungus, having approximate sizes of 37.46 and 37.21 Mbp, respectively.

SBMDb: first whole genome putative microsatellite DNA marker database of sugarbeet for bioenergy and industrial applications

DNA marker plays important role as valuable tools to increase crop productivity by finding plausible answers to genetic variations and linking the Quantitative Trait Loci (QTL) of beneficial trait. Prior approaches in development of Short Tandem Repeats (STR) markers were time consuming and inefficient. Recent methods invoking the development of STR markers using whole genomic or transcriptomics data has gained wide importance with immense potential in developing breeding and cultivator improvement approaches. Availability of whole genome sequences and in silico approaches has revolutionized bulk marker discovery. We report world’s first sugarbeet whole genome marker discovery having 145 K markers along with 5 K functional domain markers unified in common platform using MySQL, Apache and PHP in SBMDb. Embedded markers and corresponding location information can be selected for desired chromosome, location/interval and primers can be generated using Primer3 core, integrated at backend. Our analyses revealed abundance of ‘mono’ repeat (76.82%) over ‘di’ repeats (13.68%). Highest density (671.05 markers/Mb) was found in chromosome 1 and lowest density (341.27 markers/Mb) in chromosome 6. Current investigation of sugarbeet genome marker density has direct implications in increasing mapping marker density. This will enable present linkage map having marker distance of ∼2 cM, i.e. from 200 to 2.6 Kb, thus facilitating QTL/gene mapping. We also report e-PCR-based detection of 2027 polymorphic markers in panel of five genotypes. These markers can be used for DUS test of variety identification and MAS/GAS in variety improvement program. The present database presents wide source of potential markers for developing and implementing new approaches for molecular breeding required to accelerate industrious use of this crop, especially for sugar, health care products, medicines and color dye. Identified markers will also help in improvement of bioenergy trait of bioethanol and biogas production along with reaping advantage of crop efficiency in terms of low water and carbon footprint especially in era of climate change. Database URL: http://webapp.cabgrid.res.in/sbmdb/

Transcriptomic signature of drought response in pearl millet ( Pennisetum glaucum (L.) and development of web-genomic resources

Pearl millet, (Pennisetum glaucum L.), an efficient (C4) crop of arid/semi-arid regions is known for hardiness. Crop is valuable for bio-fortification combating malnutrition and diabetes, higher caloric value and wider climatic resilience. Limited studies are done in pot-based experiments for drought response at gene-expression level, but field-based experiment mimicking drought by withdrawal of irrigation is still warranted. We report de novo assembly-based transcriptomic signature of drought response induced by irrigation withdrawal in pearl millet. We found 19983 differentially expressed genes, 7595 transcription factors, gene regulatory network having 45 hub genes controlling drought response. We report 34652 putative markers (4192 simple sequence repeats, 12111 SNPs and 6249 InDels). Study reveals role of purine and tryptophan metabolism in ABA accumulation mediating abiotic response in which MAPK acts as major intracellular signal sensing drought. Results were validated by qPCR of 13 randomly selected genes. We report the first web-based genomic resource (http://webtom.cabgrid.res.in/pmdtdb/) which can be used for candidate genes-based SNP discovery programs and trait-based association studies. Looking at climatic change, nutritional and pharmaceutical importance of this crop, present investigation has immense value in understanding drought response in field condition. This is important in germplasm management and improvement in endeavour of pearl millet productivity.

Draft whole genome sequence of groundnut stem rot fungus Athelia rolfsii revealing genetic architect of its pathogenicity and virulence

Groundnut (Arachis hypogaea L.) is an important oil seed crop having major biotic constraint in production due to stem rot disease caused by fungus, Athelia rolfsii causing 25–80% loss in productivity. As chemical and biological combating strategies of this fungus are not very effective, thus genome sequencing can reveal virulence and pathogenicity related genes for better understanding of the host-parasite interaction. We report draft assembly of Athelia rolfsii genome of ~73 Mb having 8919 contigs. Annotation analysis revealed 16830 genes which are involved in fungicide resistance, virulence and pathogenicity along with putative effector and lethal genes. Secretome analysis revealed CAZY genes representing 1085 enzymatic genes, glycoside hydrolases, carbohydrate esterases, carbohydrate-binding modules, auxillary activities, glycosyl transferases and polysaccharide lyases. Repeat analysis revealed 11171 SSRs, LTR, GYPSY and COPIA elements. Comparative analysis with other existing ascomycotina genome predicted conserved domain family of WD40, CYP450, Pkinase and ABC transporter revealing insight of evolution of pathogenicity and virulence. This study would help in understanding pathogenicity and virulence at molecular level and development of new combating strategies. Such approach is imperative in endeavour of genome based solution in stem rot disease management leading to better productivity of groundnut crop in tropical region of world.

Applications of Bioinformatics in Plant and Agriculture

The high-throughput technologies generating large-scale biological data, as well as the development of related computational tools, have united global efforts and brought revolutionary changes to the research of biology during the last decade. Today, biologists work in association with scientists from a broad spectrum of disciplines to unravel how complex biological systems work. Bioinformatics is a multidisciplinary field that makes use of computers to store and analyse molecular biology information with integration of statistical algorithms. The genome sequencing of a number of organisms has led to the discovery of many fascinating things. Today, the world feels the need of this discipline to save resources and time. This chapter emphasises on a number of applications of bioinformatics in agriculture in view of functional genomics, data mining techniques, genome-wide association studies, high-performance computing facilities in agriculture and various bioinformatics tools/databases important for breeders, biotechnologists and pathologists. Agricultural genomics leads to the global understanding of plant/animal and pathogen biology, and its application would be beneficial for agriculture.

Author Correction: Transcriptomic signature of drought response in pearl millet ( Pennisetum glaucum (L.) and development of web-genomic resources

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

MiSNPDb : a web-based genomic resources of tropical ecology fruit mango ( Mangifera indica L.) for phylogeography and varietal differentiation

Mango is one of the most important fruits of tropical ecological region of the world, well known for its nutritive value, aroma and taste. Its world production is >45MT worth >200 billion US dollars. Genomic resources are required for improvement in productivity and management of mango germplasm. There is no web-based genomic resources available for mango. Hence rapid and cost-effective high throughput putative marker discovery is required to develop such resources. RAD-based marker discovery can cater this urgent need till whole genome sequence of mango becomes available. Using a panel of 84 mango varieties, a total of 28.6 Gb data was generated by ddRAD-Seq approach on Illumina HiSeq 2000 platform. A total of 1.25 million SNPs were discovered. Phylogenetic tree using 749 common SNPs across these varieties revealed three major lineages which was compared with geographical locations. A web genomic resources MiSNPDb, available at http://webtom.cabgrid.res.in/mangosnps/ is based on 3-tier architecture, developed using PHP, MySQL and Javascript. This web genomic resources can be of immense use in the development of high density linkage map, QTL discovery, varietal differentiation, traceability, genome finishing and SNP chip development for future GWAS in genomic selection program. We report here world’s first web-based genomic resources for genetic improvement and germplasm management of mango.

Low-depth shotgun sequencing resolves complete mitochondrial genome sequence of Labeo rohita

Abstract Labeo rohita, popularly known as rohu, is a widely cultured species in whole Indian subcontinent. In the present study, we used in-silico approach to resolve complete mitochondrial genome of rohu. Low-depth shotgun sequencing using Roche 454 GS FLX (Branford, Connecticut, USA) followed by de novo assembly in CLC Genomics Workbench version 7.0.4 (Aarhus, Denmark) revealed the complete mitogenome of L. rohita to be 16 606 bp long (accession No. KR185963). It comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 putative control region. The gene order and organization are similar to most vertebrates. The mitogenome in the present investigation has 99% similarity with that of previously reported mitogenomes of rohu and this is also evident from the phylogenetic study using maximum-likelihood (ML) tree method. This study was done to determine the feasibility, accuracy and reliability of low-depth sequence data obtained from NGS platform as compared to the Sanger sequencing. Thus, NGS technology has proven to be competent and a rapid in-silico alternative to resolve the complete mitochondrial genome sequence, thereby reducing labors and time.