Ram Kumar Sharma

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

BackgroundDespite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.ResultsConsidering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (HE) and observed (Ho) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.ConclusionUGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.

Next Generation Sequencing Technologies: The Doorway to the Unexplored Genomics of Non-Model Plants

Non-model plants i.e., the species which have one or all of the characters such as long life cycle, difficulty to grow in the laboratory or poor fecundity, have been schemed out of sequencing projects earlier, due to high running cost of Sanger sequencing. Consequently, the information about their genomics and key biological processes are inadequate. However, the advent of fast and cost effective next generation sequencing (NGS) platforms in the recent past has enabled the unearthing of certain characteristic gene structures unique to these species. It has also aided in gaining insight about mechanisms underlying processes of gene expression and secondary metabolism as well as facilitated development of genomic resources for diversity characterization, evolutionary analysis and marker assisted breeding even without prior availability of genomic sequence information. In this review we explore how different Next Gen Sequencing platforms, as well as recent advances in NGS based high throughput genotyping technologies are rewarding efforts on de-novo whole genome/transcriptome sequencing, development of genome wide sequence based markers resources for improvement of non-model crops that are less costly than phenotyping.

Structure and genetic diversity of natural populations of Morus alba in the trans-Himalayan Ladakh region.

Sequence-related amplified polymorphism markers were used to assess the genetic structure in three natural populations of Morus alba from trans-Himalaya. Multilocation sampling was conducted across 14 collection sites. The overall genetic diversity estimates were high: percentage polymorphic loci 89.66%, Nei’s gene diversity 0.2286, and Shannon’s information index 0.2175. At a regional level, partitioning of variability assessed using analysis of molecular variance (AMOVA), revealed 80% variation within and 20% among collection sites. Pattern appeared in STRUCTURE, BARRIER, and AMOVA, clearly demonstrating gene flow between the Indus and Suru populations and a geographic barrier between the Indus-Suru and Nubra populations, which effectively hinders gene flow. The results showed significant genetic differentiation, population structure, high to restricted gene flow, and high genetic diversity. The assumption that samples collected from the three valleys represent three different populations does not hold true. The fragmentation present in trans-Himalaya was more natural and less anthropogenic.

Identification and cross-species amplification of EST derived SSR markers in different bamboo species.

The availability of expressed sequence data derived from gene discovery programs became an alternative source of mining simple sequence repeat (SSR) and developing inexpensive genetic markers for the crop improvements. In present study, 10 express sequence tags (EST)-SSR markers were identified from Bambusa oldhamii public sequence data base. Transferability to 25 species of Bambusoideae ranged from 30% to 100%. The number of alleles detected per locus ranged from 2 to 10. All the newly identified SSR markers were found to be moderately to highly polymorphic with an average Polymorphic Information Content (PIC) value of 0.54. As these loci represents transcribed region and recorded high level of cross transferability and reliable amplification across the species, demonstrating the utility of these markers for functional and genetic analyses of bamboo species.

Identification and cross-species transferability of 112 novel unigene-derived microsatellite markers in tea (Camellia sinensis)

PREMISE OF THE STUDY Tea Unigene-derived MicroSatellite (TUGMS) markers were identified from the publicly available EST data in Camellia sinensis for characterization and future genome mapping studies in tea. METHODS AND RESULTS One hundred twelve novel TUGMS markers were identified from 4356 unigenes derived by clustering of 12788 random ESTs in C. sinensis. Amplification-based validation of the TUGMS loci proved them to be highly polymorphic [an average (av.) of 5.24 alleles], heterozygous (H(E), av. 0.746; H(o), av. 0.566) and informative (PIC, av. 0.392). TUGMS loci were 100% transferable in cultivated C. assamica and C. assamica subsp. lasiocalyx and highly cross-transferrable to the related species C. japonica, C. rosiflora, and C. sasanqua. CONCLUSIONS These 112 novel highly polymorphic TUGMS markers with proven cross-species transferability will facilitate the future genetic diversity and genome mapping studies in tea.

Transcriptome Analysis Reveals Candidate Genes involved in Blister Blight defense in Tea ( Camellia sinensis (L) Kuntze)

To unravel the molecular mechanism of defense against blister blight (BB) disease caused by an obligate biotrophic fungus, Exobasidium vexans, transcriptome of BB interaction with resistance and susceptible tea genotypes was analysed through RNA-seq using Illumina GAIIx at four different stages during ~20-day disease cycle. Approximately 69 million high quality reads were assembled de novo, yielding 37,790 unique transcripts with more than 55% being functionally annotated. Differentially expressed, 149 defense related transcripts/genes, namely defense related enzymes, resistance genes, multidrug resistant transporters, transcription factors, retrotransposons, metacaspases and chaperons were observed in RG, suggesting their role in defending against BB. Being present in the major hub, putative master regulators among these candidates were identified from predetermined protein-protein interaction network of Arabidopsis thaliana. Further, confirmation of abundant expression of well-known RPM1, RPS2 and RPP13 in quantitative Real Time PCR indicates salicylic acid and jasmonic acid, possibly induce synthesis of antimicrobial compounds, required to overcome the virulence of E. vexans. Compendiously, the current study provides a comprehensive gene expression and insights into the molecular mechanism of tea defense against BB to serve as a resource for unravelling the possible regulatory mechanism of immunity against various biotic stresses in tea and other crops.

Genetic diversity of high-elevation populations of an endangered medicinal plant

Despite its wide distribution across the entire Himalayan range, the current status of Podophyllum hexandrum, a highly important anti-cancerous herb, remains endangered. Genetic diversity characterization of 24 populations comprising of 209 individuals representing the whole of the Indian Himalayas revealed that regardless of geographic location, all of the populations are intermixed and are composed broadly of two types of genetic populations. Our findings also suggested that these populations have evolved well in response to the environment. This study will help in the formulation of conservation programs for P. hexandrum populations in this region.

Spatial transcriptome analysis provides insights of key gene(s) involved in steroidal saponin biosynthesis in medicinally important herb Trillium govanianum

Trillium govanianum, an endangered medicinal herb native to the Himalaya, is less studied at the molecular level due to the non-availability of genomic resources. To facilitate the basic understanding of the key genes and regulatory mechanism of pharmaceutically important biosynthesis pathways, first spatial transcriptome sequencing of T. govanianum was performed. 151,622,376 (~11.5 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 69,174 transcripts. Functional annotation with multiple public databases identified array of genes involved in steroidal saponin biosynthesis and other secondary metabolite pathways including brassinosteroid, carotenoid, diterpenoid, flavonoid, phenylpropanoid, steroid and terpenoid backbone biosynthesis, and important TF families (bHLH, MYB related, NAC, FAR1, bZIP, B3 and WRKY). Differentially expressed large number of transcripts, together with CYPs and UGTs suggests involvement of these candidates in tissue specific expression. Combined transcriptome and expression analysis revealed that leaf and fruit tissues are the main site of steroidal saponin biosynthesis. In conclusion, comprehensive genomic dataset created in the current study will serve as a resource for identification of potential candidates for genetic manipulation of targeted bioactive metabolites and also contribute for development of functionally relevant molecular marker resource to expedite molecular breeding and conservation efforts in T. govanianum.

Genome-Wide Transcriptional Profiling to Elucidate Key Candidates Involved in Bud Burst and Rattling Growth in a Subtropical Bamboo (Dendrocalamus hamiltonii)

Bamboo, one of the fastest growing plants, can be a promising model system to understand growth. The study provides an insight into the complex interplay between environmental signaling and cellular machineries governing initiation and persistence of growth in a subtropical bamboo (Dendrocalamus hamiltonii). Phenological and spatio-temporal transcriptome analysis of rhizome and shoot during the major vegetative developmental transitions of D. hamiltonii was performed to dissect factors governing growth. Our work signifies the role of environmental cues, predominantly rainfall, decreasing day length, and high humidity for activating dormant bud to produce new shoot, possibly through complex molecular interactions among phosphatidylinositol, calcium signaling pathways, phytohormones, circadian rhythm, and humidity responses. We found the coordinated regulation of auxin, cytokinin, brassinosteroid signaling and cell cycle modulators; facilitating cell proliferation, cell expansion, and cell wall biogenesis supporting persistent growth of emerging shoot. Putative master regulators among these candidates were identified using predetermined Arabidopsis thaliana protein-protein interaction network. We got clues that the growth signaling begins far back in rhizome even before it emerges out as new shoot. Putative growth candidates identified in our study can serve in devising strategies to engineer bamboos and timber trees with enhanced growth and biomass potentials.

Amplified fragment length polymorphism of clinical and environmental Vibrio cholerae from a freshwater environment in a cholera-endemic area, India

BackgroundThe region around Chandigarh in India has witnessed a resurgence of cholera. However, isolation of V. cholerae O1 from the environment is infrequent. Therefore, to study whether environmental nonO1-nonO139 isolates, which are native to the aquatic ecosystem, act as precursors for pathogenic O1 strains, their virulence potential and evolutionary relatedness was checked.MethodsV. cholerae was isolated from clinical cases of cholera and from water and plankton samples collected from freshwater bodies and cholera-affected areas. PCR analysis for the ctxA, ctxB, tcpA, toxT and toxR genes and AFLP with six primer combinations was performed on 52 isolates (13 clinical, 34 environmental and 5 reference strains).ResultsAll clinical and 3 environmental isolates belonged to serogroup O1 and remaining 31 environmental V. cholerae were nonO1-nonO139. Serogroup O1 isolates were ctxA, tcpA (ElTor), ctxB (Classical), toxR and toxT positive. NonO1-nonO139 isolates possessed toxR, but lacked ctxA and ctxB; only one isolate was positive for toxT and tcpA. Using AFLP, 2.08% of the V. cholerae genome was interrogated. Dendrogram analysis showed one large heterogeneous clade (n = 41), with two compact and distinct subclades (1a and 1b), and six small mono-phyletic groups. Although V. cholerae O1 isolates formed a distinct compact subclade, they were not clonal. A clinical O1 strain clustered with the nonO1-nonO139 isolates; one strain exhibited 70% similarity to the Classical control strain, and all O1 strains possessed an ElTor variant-specific fragment identified with primer ECMT. Few nonO1-nonO139 isolates from widely separated geographical locations intermingled together. Three environmental O1 isolates exhibited similar profiles to clinical O1 isolates.ConclusionIn a unique study from freshwater environs of a cholera-endemic area in India over a narrow time frame, environmental V. cholerae population was found to be highly heterogeneous, diverse and devoid of major virulence genes. O1 and nonO1-nonO139 isolates showed distinct lineages. Clinical isolates were not clonal but were closely related, indicating accumulation of genetic differences over a short time span. Though, environment plays an important role in the spread of cholera, the possibility of an origin of pathogenic O1 strains from environmental nonO1-nonO139 strains seems to be remote in our region.