Senthil Natesan

Transcriptome profiling and comparative analysis of Panax ginseng adventitious roots

Background Panax ginseng Meyer is a traditional medicinal plant famous for its strong therapeutic effects and serves as an important herbal medicine. To understand and manipulate genes involved in secondary metabolic pathways including ginsenosides, transcriptome profiling of P. ginseng is essential. Methods RNA-seq analysis of adventitious roots of two P. ginseng cultivars, Chunpoong (CP) and Cheongsun (CS), was performed using the Illumina HiSeq platform. After transcripts were assembled, expression profiling was performed. Results Assemblies were generated from ∼85 million and ∼77 million high-quality reads from CP and CS cultivars, respectively. A total of 35,527 and 27,716 transcripts were obtained from the CP and CS assemblies, respectively. Annotation of the transcriptomes showed that approximately 90% of the transcripts had significant matches in public databases. We identified several candidate genes involved in ginsenoside biosynthesis. In addition, a large number of transcripts (17%) with different gene ontology designations were uniquely detected in adventitious roots compared to normal ginseng roots. Conclusion This study will provide a comprehensive insight into the transcriptome of ginseng adventitious roots, and a way for successful transcriptome analysis and profiling of resource plants with less genomic information. The transcriptome profiling data generated in this study are available in our newly created adventitious root transcriptome database (http://im-crop.snu.ac.kr/transdb/index.php) for public use.

Comparative Root Protein Profiles of Korean Ginseng (Panax ginseng) and Indian Ginseng (Withania somnifera)

Ginsenosides and withanolides are the secondary metabolites from Panax ginseng and Withania somnifera, respectively. These compounds have similar biological properties. Two-dimensional electrophoresis (2-DE) analysis was utilized to reveal the protein profile in the roots of both plants, with the aim of clarifying similarly- and differentially-expressed proteins. Total proteins of Korea ginseng (P. ginseng) and Indian ginseng (W. somnifera) roots were separated by 2-DE using a pH 4-7 immobilized pH gradient strip in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The protein spots were visualized by silver staining. Twenty-one P. ginseng proteins and 35 W. somnifera proteins were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; of these, functions were ascribed to 14 and 22 of the P. ginseng and W. somnifera proteins, respectively. Functions mainly included general cell metabolism, defense and secondary metabolism. ATPase and alcohol dehydrogenase proteins were expressed in both plants. The results of this study, to our knowledge, are the first to provide a reference 2-DE map for the W. somnifera root proteome, and will aid in the understanding of the expression and functions of proteins in the roots of Korean ginseng and Indian ginseng.

Fingerprinting in cucumber and melon ( Cucumis spp.) Genotypes using morphological and ISSR markers

In the present investigation, 13 Cucumis genotypes from different geographical areas of India were screened for genetic diversity using 19 morphological traits and 15 ISSR primers. The analysis of morphological traits grouped the accessions into six clusters. Cluster V contained the maximum number of genotypes namely Kanivellari, Long Green, Andaman Local, Perundurai Local, and Sempatti Local. Clusters I and VI contained the minimum number of genotypes. Among all the characters, the highest mean value was observed in fruit length (23.38) and the lowest mean value was observed in stripes on the blossom end (1.31). The 15 ISSR primers generated 109 polymorphic alleles. The average number of ISSR alleles generated was 8.3 per primer and the level of polymorphism was 87.20%. The ISSR primer UBC 825 was highly informative with a PIC value of 0.8934. The 13 genotypes were grouped into six clusters based on ISSR markers. Cluster III contained the maximum number of genotypes, namely Kanivellari, Sankagiri Local, Perundurai Local, Long Melon, and Sempatti Local, while Clusters I, II, IV, and V (Karur Local, Andaman Local, Edapaddi Local, and N 78, respectively) contained the minimum number of genotypes. The ISSR profile generated genotypes specific allele namely, UBC 812700bp and UBC 8121000bp for Cluster VI which contained Cucumis genotypes collected from the northern part of India. Similarly, UBC 808 produced specific allele UBC 808650bp formed in Cluster III which contained genotypes collected from Tamil Nadu and Kerala.

Genome-Wide Association Mapping for Leaf Tip Necrosis and Pseudo-black Chaff in Relation to Durable Rust Resistance in Wheat

The partial rust resistance genes Lr34 and Sr2 have been used extensively in wheat (Triticum aestivum L.) improvement, as they confer exceptional durability. Interestingly, the resistance of Lr34 is associated with the expression of leaf tip necrosis (LTN) and Sr2 with pseudo‐black chaff (PBC). Genome‐wide association mapping using CIMMYTs stem rust resistance screening nursery (SRRSN) wheat lines was done to identify genotyping‐by‐sequencing (GBS) markers linked to LTN and PBC. Phenotyping for these traits was done in Ithaca, New York (fall 2011); Njoro, Kenya (main and off‐seasons, 2012), and Wellington, India (winter, 2013). Using the mixed linear model (MLM), 18 GBS markers were significantly associated with LTN. While some markers were linked to loci where the durable leaf rust resistance genes Lr34 (7DS), Lr46 (1BL), and Lr68 (7BL) were mapped, significant associations were also detected with other loci on 2BL, 5B, 3BS, 4BS, and 7BS. Twelve GBS markers linked to the Sr2 locus (3BS) and loci on 2DS, 4AL, and 7DS were significantly associated with PBC. This study provides insight into the complex genetic control of LTN and PBC. Further efforts to validate and study these loci might aid in determining the nature of their association with durable resistance.

Novel Genetic Resources in the Genus Vigna Unveiled from Gene Bank Accessions.

The genus Vigna (Fabaceae) consists of five subgenera, and includes more than 100 wild species. In Vigna, 10 crops have been domesticated from three subgenera, Vigna, Plectrotropis, and Ceratotropis. The habitats of wild Vigna species are so diverse that their genomes could harbor various genes responsible for environmental stress adaptation, which could lead to innovations in agriculture. Since some of the gene bank Vigna accessions were unidentified and they seemed to be novel genetic resources, these accessions were identified based on morphological traits. The phylogenetic positions were estimated based on the DNA sequences of nuclear rDNA-ITS and chloroplast atpB-rbcL spacer regions. Based on the results, the potential usefulness of the recently described species V. indica and V. sahyadriana, and some wild Vigna species, i.e., V. aconitifolia, V. dalzelliana, V. khandalensis, V. marina var. oblonga, and V. vexillata, was discussed.

Prevalence, Patterns, and Genetic Association Analysis of Modic Vertebral Endplate Changes

Study Design A prospective genetic association study. Purpose The etiology of Modic changes (MCs) is unclear. Recently, the role of genetic factors in the etiology of MCs has been evaluated. However, studies with a larger patient subset are lacking, and candidate genes involved in other disc degeneration phenotypes have not been evaluated. We studied the prevalence of MCs and genetic association of 41 candidate genes in a large Indian cohort. Overview of Literature MCs are vertebral endplate signal changes predominantly observed in the lumbar spine. A significant association between MCs and lumbar disc degeneration and nonspecific low back pain has been described, with the etiopathogenesis implicating various mechanical, infective, and biochemical factors. Methods We studied 809 patients using 1.5-T magnetic resonance imaging to determine the prevalence, patterns, distribution, and type of lumbar MCs. Genetic association analysis of 71 single nucleotide polymorphisms (SNPs) of 41 candidate genes was performed based on the presence or absence of MCs. SNPs were genotyped using the Sequenome platform, and an association test was performed using PLINK software. Results The mean age of the study population (n=809) was 36.7±10.8 years. Based on the presence of MCs, the cohort was divided into 702 controls and 107 cases (prevalence, 13%). MCs were more commonly present in the lower (149/251, 59.4%) than in the upper (102/251, 40.6%) endplates. L4–5 endplates were the most commonly affected levels (30.7%). Type 2 MCs were the most commonly observed pattern (n=206, 82%). The rs2228570 SNP of VDR (p=0.02) and rs17099008 SNP of MMP20 (p=0.03) were significantly associated with MCs. Conclusions Genetic polymorphisms of SNPs of VDR and MMP20 were significantly associated with MCs. Understanding the etiopathogenetic mechanisms of MCs is important for planning preventive and therapeutic strategies.

The complete chloroplast genome sequence of Indian barnyard millet, Echinochloa frumentacea (Poaceae)

Abstract We report the complete chloroplast genome sequence of the important nutritious millet crop Indian barnyard millet, Echinochloa frumentacea Link (Poaceae). The size of the circular chloroplast genome is 139 593 bp in length with a typical quadripartite structure, containing pair of inverted repeats of 22 618 bp, flanked by large single copy and small single copy regions of 81 839 bp, 12 518 bp, respectively. Overall GC content of the genome was 38.6% and consists of 112 individual genes, including 77 protein coding genes, 30 tRNA genes, four rRNA genes and one conserved open reading frame. In addition, phylogenetic analysis with grass species has revealed that E. frumentacea is diverged around 1.9–2.7 million years with its close relatives, E. oryzicola and E. crus-galli.

Starch content and cassava mosaic disease genetic diversity with relation to yield in south Indian cassava (Manihot esculenta Crantz) germplasm

Cassava, family Euphorbiaceae, is the fifth most important staple food crop. The study of variability and diversity present in diverse cassava accessions maintained at the field genebank of TCRS, TNAU is essential to design the breeding program of cassava in TNAU. Hence, a study was carried out to assess the genetic diversity based on morphological traits and Simple Sequence Repeat (SSR) markers, identifying the SSR markers linked to a cassava mosaic disease-resistant QTL region. Genetic diversity among cassava germplasm accessions was estimated using 28 morphological traits and three SSR markers. The polymorphic information content (PIC) result gives high information for all the SSR markers. Morphological traits revealed 10 clusters and the SSR markers revealed 13 clusters at a similarity coefficient of 2.514 and 0.75, respectively. The SSR marker diversity revealed that the most promising clusters (II and XII) possessing accessions with yield attributing traits and cluster IX were grouped together due to low yield and CMD susceptibility. Morphological descriptors revealed variance for yield contributing traits. The first three principle components accounted for 10.8, 9.1, and 8.4%, respectively. Correlation studies showed significant correlation among yield and yield-attributing traits, which in turn influences yield. The morphological traits diversity reveals that cluster VI was the most promising cluster in which genotypes ME209 and ME460 possess higher yield and starch content with cassava mosaic disease (CMD) resistance. Most of the accessions grouped under cluster X belong to selection from International Centre for Tropical Agriculture (CIAT) collections and the cultivars under clusters I and II were true cassava seed (TCS) selections of India. The CMD-linked SSR marker which is reported in the previous studies have generated eight alleles and each were regressed with CMD resistance and resulted in non-significant linkage to CMD resistance.

Phenotype Standardization is the Urgent Need in Genetic Association Studies of Disc Degeneration - Results of Analysis of Genetic Association of 71 SNPs with Highly Specific Phenotypes in 809 Subjects:

Introduction Although the influence of genetics on etio-pathogenesis of disc degeneration is well recognized, there is a wide variation in the different genes observed to have association with disc degeneration. The clinical and radiographic features of disc degeneration used as phenotypes are quite variable in previously published studies. It is unclear whether the variations in genetic association studies depict variations in ethnicity or selection of phenotypes. Methods The purpose of the study was to evaluate the allelic diversity of 71 SNPs related to six different MRI markers of lumbar degenerative disc disease (annular tear, Pfirmanns grading, Schmorls nodes, Modic changes, Total Endplate Damage score and disc bulge) in a single study population, and analyze how genetic associations can vary in the same study subjects with the choice of phenotype, based on age and sequence of selection of study subjects. Genotyping of cases and controls was performed on Genome wide SNP array to identify potentially associated disease loci. The results from the GWAS were then used to facilitate SNP selection and genotype validation was conducted using Sequenom based genotyping. Results The mean age of 809 subjects (M: 455, F:354) was 36.76 ± 10.8 years (range 10 – 80). Highly significant association (p < 0.01) was observed with three SNPs of CILP for disc bulge and rs2249350 of ADAMTS5 and rs11247361IGF1R for annular tears. Significant association (p < 0.05) was observed with polymorphisms of VDR and MMP20 for Modic changes, three SNPs of MMP20 for Schmorls node and SNPs of CALM1 and FN1 for Pfirmanns grading. None of the SNPs had significant association with TEPS. Subgroup analysis based on age (<30, 30–40 and > 40 years) showed new set of genetic associations for all the six radiographic parameters. Similarly the population was divided into two groups based on numerical order and the association patterns completely differed as compared with the total group. Conclusions In the same study population with DDD, SNP associations completely change when phenotypes changed. Variations in age, sequence of study subjects, number of population apart from radiographic description of DDD significantly change the genetic association. Based on current results, it is difficult to consider one set of genes as responsible for disc degeneration considering these variations. Our study results demand an urgent need for standardizing the description of DDD, phenotype selection, and study criteria for genetic association studies.

Phenomics: Technologies and Applications in Plant and Agriculture

To meet the challenges of global food security in the changing climatic scenario, it would be most imperative to enhance crop productivity under resource competence. It is estimated that approximately 70 % of reduction in crop yield is due to the direct impact of abiotic stresses such as drought, salinity, and extreme temperatures. In the present context, one of the major challenges is large-scale screening of crop performance as a consequence of its genetic makeup. The development of advanced biotechnological and next-generation sequencing tools has lead to the accumulation of enormous data on genomics; nevertheless, data on phenotype and functions is yet to be fully annotated. To exploit the wealth of large-scale genomic data sets, it is necessary to characterize the crop performance quantitatively and link it to the genomic data. High-throughput phenomics studies offer strategies to screen large-scale population (s) for a particular phenotype employing advanced robotics, high-tech sensors, imaging systems, and computing power. Advanced bioinformatics tools further facilitate the analysis of large-scale multi-dimensional, high-resolution data collected through phenotyping. With the integrated approach of genotyping and phenotyping, gene functions and environmental responses can be well understood. It will also help in finding more relevant solutions for the major problems that are currently limiting crop production.