Gopal Misra

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.).

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi-type chickpea genome using next-generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520-Mb assembly covers 70% of the predicted 740-Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27,571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA-Seq reads identified several tissue-specific and stress-responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole-genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.

Genome-Wide Development and Use of Microsatellite Markers for Large-Scale Genotyping Applications in Foxtail Millet [Setaria italica (L.)]

The availability of well-validated informative co-dominant microsatellite markers and saturated genetic linkage map has been limited in foxtail millet (Setaria italica L.). In view of this, we conducted a genome-wide analysis and identified 28 342 microsatellite repeat-motifs spanning 405.3 Mb of foxtail millet genome. The trinucleotide repeats (∼48%) was prevalent when compared with dinucleotide repeats (∼46%). Of the 28 342 microsatellites, 21 294 (∼75%) primer pairs were successfully designed, and a total of 15 573 markers were physically mapped on 9 chromosomes of foxtail millet. About 159 markers were validated successfully in 8 accessions of Setaria sp. with ∼67% polymorphic potential. The high percentage (89.3%) of cross-genera transferability across millet and non-millet species with higher transferability percentage in bioenergy grasses (∼79%, Switchgrass and ∼93%, Pearl millet) signifies their importance in studying the bioenergy grasses. In silico comparative mapping of 15 573 foxtail millet microsatellite markers against the mapping data of sorghum (16.9%), maize (14.5%) and rice (6.4%) indicated syntenic relationships among the chromosomes of foxtail millet and target species. The results, thus, demonstrate the immense applicability of developed microsatellite markers in germplasm characterization, phylogenetics, construction of genetic linkage map for gene/quantitative trait loci discovery, comparative mapping in foxtail millet, including other millets and bioenergy grass species.

Development of eSSR-Markers in Setaria italica and Their Applicability in Studying Genetic Diversity, Cross-Transferability and Comparative Mapping in Millet and Non-Millet Species

Foxtail millet ( Setaria italica L.) is a tractable experimental model crop for studying functional genomics of millets and bioenergy grasses. But the limited availability of genomic resources, particularly expressed sequence-based genic markers is significantly impeding its genetic improvement. Considering this, we attempted to develop EST-derived-SSR (eSSR) markers and utilize them in germplasm characterization, cross-genera transferability and in silico comparative mapping. From 66,027 foxtail millet EST sequences 24,828 non-redundant ESTs were deduced, representing ~16 Mb, which revealed 534 (~2%) eSSRs in 495 SSR containing ESTs at a frequency of 1/30 kb. A total of 447 pp were successfully designed, of which 327 were mapped physically onto nine chromosomes. About 106 selected primer pairs representing the foxtail millet genome showed high-level of cross-genera amplification at an average of ~88% in eight millets and four non-millet species. Broad range of genetic diversity (0.02–0.65) obtained in constructed phylogenetic tree using 40 eSSR markers demonstrated its utility in germplasm characterizations and phylogenetics. Comparative mapping of physically mapped eSSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~68%), maize (~61%) and rice (~42%) chromosomes. Synteny analysis of eSSRs of foxtail millet, rice, maize and sorghum suggested the nested chromosome fusion frequently observed in grass genomes. Thus, for the first time we had generated large-scale eSSR markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.

An advanced draft genome assembly of a desi type chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is an important pulse legume crop. We previously reported a draft genome assembly of the desi chickpea cultivar ICC 4958. Here we report an advanced version of the ICC 4958 genome assembly (version 2.0) generated using additional sequence data and an improved genetic map. This resulted in 2.7-fold increase in the length of the pseudomolecules and substantial reduction of sequence gaps. The genome assembly covered more than 94% of the estimated gene space and predicted the presence of 30,257 protein-coding genes including 2230 and 133 genes encoding potential transcription factors (TF) and resistance gene homologs, respectively. Gene expression analysis identified several TF and chickpea-specific genes with tissue-specific expression and displayed functional diversification of the paralogous genes. Pairwise comparison of pseudomolecules in the desi (ICC 4958) and the earlier reported kabuli (CDC Frontier) chickpea assemblies showed an extensive local collinearity with incongruity in the placement of large sequence blocks along the linkage groups, apparently due to use of different genetic maps. Single nucleotide polymorphism (SNP)-based mining of intra-specific polymorphism identified more than four thousand SNPs differentiating a desi group and a kabuli group of chickpea genotypes.

Designing climate-resilient rice with ideal grain quality suited for high-temperature stress

To ensure rice food security, the target outputs of future rice breeding programmes should focus on developing climate-resilient rice varieties with emphasis on increased head rice yield coupled with superior grain quality. This challenge is made greater by a world that is increasingly becoming warmer. Such environmental changes dramatically impact head rice and milling yield as well as increasing chalkiness because of impairment in starch accumulation and other storage biosynthetic pathways in the grain. This review highlights the knowledge gained through gene discovery via quantitative trait locus (QTL) cloning and structural-functional genomic strategies to reduce chalk, increase head rice yield, and develop stable lines with optimum grain quality in challenging environments. The newly discovered genes and the knowledge gained on the influence of specific alleles related to stability of grain quality attributes provide a robust platform for marker-assisted selection in breeding to design heat-tolerant rice varieties with superior grain quality. Using the chalkiness trait in rice as a case study, we demonstrate here that the emerging field of systems genetics can help fast-track the identification of novel alleles and gene targets that can be pyramided for the development of environmentally robust rice varieties that possess improved grain quality.

FmMDb: A Versatile Database of Foxtail Millet Markers for Millets and Bioenergy Grasses Research

The prominent attributes of foxtail millet (Setaria italica L.) including its small genome size, short life cycle, inbreeding nature, and phylogenetic proximity to various biofuel crops have made this crop an excellent model system to investigate various aspects of architectural, evolutionary and physiological significances in Panicoid bioenergy grasses. After release of its whole genome sequence, large-scale genomic resources in terms of molecular markers were generated for the improvement of both foxtail millet and its related species. Hence it is now essential to congregate, curate and make available these genomic resources for the benefit of researchers and breeders working towards crop improvement. In view of this, we have constructed the Foxtail millet Marker Database (FmMDb; http://www.nipgr.res.in/foxtail.html), a comprehensive online database for information retrieval, visualization and management of large-scale marker datasets with unrestricted public access. FmMDb is the first database which provides complete marker information to the plant science community attempting to produce elite cultivars of millet and bioenergy grass species, thus addressing global food insecurity.

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

Tomato genomic resources database: an integrated repository of useful tomato genomic information for basic and applied research.

Tomato Genomic Resources Database (TGRD) allows interactive browsing of tomato genes, micro RNAs, simple sequence repeats (SSRs), important quantitative trait loci and Tomato-EXPEN 2000 genetic map altogether or separately along twelve chromosomes of tomato in a single window. The database is created using sequence of the cultivar Heinz 1706. High quality single nucleotide polymorphic (SNP) sites between the genes of Heinz 1706 and the wild tomato S. pimpinellifolium LA1589 are also included. Genes are classified into different families. 5′-upstream sequences (5′-US) of all the genes and their tissue-specific expression profiles are provided. Sequences of the microRNA loci and their putative target genes are catalogued. Genes and 5′-US show presence of SSRs and SNPs. SSRs located in the genomic, genic and 5′-US can be analysed separately for the presence of any particular motif. Primer sequences for all the SSRs and flanking sequences for all the genic SNPs have been provided. TGRD is a user-friendly web-accessible relational database and uses CMAP viewer for graphical scanning of all the features. Integration and graphical presentation of important genomic information will facilitate better and easier use of tomato genome. TGRD can be accessed as an open source repository at http://59.163.192.91/tomato2/.

Structural and biochemical perspectives in plant isoprenoid biosynthesis

The isoprenoid family represents one of the most ancient and widespread classes of structurally and functionally rich biomolecules known to man. Although these natural products are synthesized in all organisms, the plant kingdom exhibits tremendous variation in their chemistry and roles, ranging from primary metabolism to secondary metabolism and specialized ecological interactions with the environment. Despite enormous diversity in structure and function, all isoprenoids are derived from the universal C5 precursor isoprene. The isoprenoid biosynthetic pathway has three major stages, viz., (1) synthesis of the isoprene building blocks, followed by their (2) assembly into flexible linear and branched hydrocarbon substrates, which then undergo (3) multistep reaction cascades to generate the vast assortment of isoprenoid end products. One of the most interesting aspects of isoprenoid biosynthesis is its being finely tuned by a multilayered and complex regulatory network, which excellently controls the machinery producing one of the most heterogeneous groups of molecules in plants. Terpene synthases, enzymes of the final stage, are key players in the generation of isoprenoid diversity, catalyzing one of the most complex reactions known to chemistry and biology. Unraveling the mechanism by which a minimal pool of substrates is thus converted into tens of thousands of regiospecific and stereospecific products, is a promising research avenue: This knowledge may be practically used for rational design of novel compounds by metabolic engineering, in order to yield plants with improved nutritional efficacy, stress resistance, bio-pharmaceutical properties etc. This review is an attempt to summarize the biochemical, molecular, physiological, structural, genomic and evolutionary aspects of isoprenoid biosynthesis, providing new insights into how these enzymes utilize various innovative strategies for creation of the so-called final terpenome.

Whole genome sequencing-based association study to unravel genetic architecture of cooked grain width and length traits in rice

In this study, we used 2.9 million single nucleotide polymorphisms (SNP) and 393,429 indels derived from whole genome sequences of 591 rice landraces to determine the genetic basis of cooked and raw grain length, width and shape using genome-wide association study (GWAS). We identified a unique fine-mapped genetic region GWi7.1 significantly associated with cooked and raw grain width. Additionally, GWi7.2 that harbors GL7/GW7 a cloned gene for grain dimension was found. Novel regions in chromosomes 10 and 11 were also found to be associated with cooked grain shape and raw grain width, respectively. The indel-based GWAS identified fine-mapped genetic regions GL3.1 and GWi5.1 that matched synteny breakpoints between indica and japonica. GL3.1 was positioned a few kilobases away from GS3, a cloned gene for cooked and raw grain lengths in indica. GWi5.1 found to be significantly associated with cooked and raw grain width. It anchors upstream of cloned gene GW5, which varied between indica and japonica accessions. GWi11.1 is present inside the 3′-UTR of a functional gene in indica that corresponds to a syntenic break in chromosome 11 of japonica. Our results identified novel allelic structural variants and haplotypes confirmed using single locus and multilocus SNP and indel-based GWAS.