Elucidation of dominant energy metabolic pathways of methane, sulphur and nitrogen in respect to mangrove-degradation for climate change mitigation.

Mangroves play a key role in ecosystem balancing and climate change mitigation. It acts as a source and sink of methane (CH4), a major greenhouse gas responsible for climate change. Energy metabolic pathways of methane production (methanogenesis) and oxidation (methanotrophy) are directly driven by sulphur (S) and nitrogen (N) metabolism and salinity in coastal wetlands. To investigate, how mangrove-degradations, affect the source-sink behaviour of CH4; the pathways of CH4, S and N were studied through whole-genome metagenomic approach. Soil samples were collected from degraded and undisturbed mangrove systems in Sundarban, India. Structural and functional microbial diversities (KEGG pathways) of CH4, S and N metabolism were analysed and correlated with labile carbon pools and physico-chemical properties of soil. Overall, the acetoclastic pathway of methanogenesis was dominant. However, the relative proportion of conversion of CO2 to CH4 was more in degraded mangroves. Methane oxidation was higher in undisturbed mangroves and the serine pathway was dominant. After serine, the ribulose monophosphate pathway of CH4 oxidation was dominant in degraded mangrove, while the xylulose monophosphate pathway was dominant in undisturbed site as it is more tolerant to salinity and higher pH. The assimilatory pathway (AMP) of S-metabolism was dominant in both systems. But in AMP pathway, adenosine triphosphate sulfurylase enzyme reads were higher in degraded mangrove, while NADPH-sulfite reductase abundance was higher in undisturbed mangrove due to higher salinity, and pH. In N-metabolism, the denitrification pathway was predominant in degraded sites, whereas the dissimilatory nitrate reduction pathway was dominant in undisturbed mangroves. The relative ratios of sulphur reducing bacteria (SRB): methanogens were higher in degraded mangrove; however, methanotrophs:methanogens was higher in undisturbed mangrove indicated lower source and greater sink capacity of CH4 in the system. Microbial manipulation in mangrove-rhizosphere for regulating major energy metabolic pathways of methane could open-up a new window of climate change mitigation in coastal wetlands.

Distinct morpho-physiological and biochemical features of arid and hyper-arid ecotypes of Ziziphus nummularia under drought suggest its higher tolerance compared with semi-arid ecotype.

Tree species in the arid and semi-arid regions use various strategies to combat drought stress. Ziziphus nummularia (Burm. f.) Wight et Arn., native to the Thar Desert in India, is highly drought-tolerant. To identify the most drought-tolerant ecotype of Z. nummularia, one ecotype each from semi-arid (Godhra, annual rainfall >750 mm), arid (Bikaner, 250-350 mm) and hyper-arid (Jaisalmer, <150 mm) regions was selected along with two other Ziziphus species, Ziziphus mauritiana Lamk. and Ziziphus rotundifolia Lamk., and screened for parameters contributing to drought tolerance. Among these, Z. nummularia (Jaisalmer) (CIAHZN-J) was the most drought - tolerant. The tolerance nature of CIAHZN-J was associated with increased membrane stability, root length and number, length of hairs and thorns, root dry/fresh weight ratio, seed germination (at -0.5 MPa), proline content (31-fold), catalase and sugar content (two- to three-fold). Apart from these characteristics, it also exhibited the longest duration to reach highest cumulative drought stress rating, maintained higher relative water content for a longer period of time with reduced leaf size, leaf rolling and falling of older leaves, and displayed sustained shoot growth during drought stress. To determine drought tolerance in Ziziphus, we developed a morphological symptom-based screening technique in this study. Additionally, transcriptome profiling of CIAHZN-J in response to drought revealed the up-regulation of genes involved in sugar metabolism and transport, abscisic acid biosynthesis, osmoregulation, reactive oxygen species homeostasis and maintaining water potential. Expression profiles and semi-quantitative reverse transcription PCR results further correlated with the physiological and biochemical mechanisms. In conclusion, CIAHZN-J is an excellent genetic stock for the identification of drought-responsive genes and can also be deployed in crop improvement programs for drought tolerance.

A unique bacterial and archaeal diversity make mangrove a green production system compared to rice in wetland ecology: A metagenomic approach.

Mangrove provides significant ecosystem services, however, 40% of tropical mangrove was lost in last century due to climate change induced sea-level rise and anthropogenic activities. Sundarban-India, the largest contiguous mangrove of the world lost 10.5% of its green during 1930-2013 which primarily converted to rice-based systems. Presently degraded mangrove and adjacent rice ecology in Sundarban-India placed side by side and create typical ecology which is distinct in nature in respect to soil physicochemical properties, carbon dynamics, and microbial diversities. We investigated the structural and functional diversities of bacteria and archaea through Illumina MiSeq metagenomic analysis using V3-V4 region of 16S rRNA gene approach that drives greenhouse gases emission and carbon-pools. Remote sensing-data base were used to select the sites for collecting the soil and gas samples. The methane and nitrous oxide emissions were lower in mangrove (-0.04 mg m-2 h-1 and -52.8 µg m-2 h-1) than rice (0.26 mg m-2 h-1 and 44.7 µg m-2 h-1) due to less availability of carbon-substrates and higher sulphate availability (85.8% more than rice). The soil labile carbon-pools were more in mangrove, but lower microbial activities were noticed due to stress conditions. A unique microbial feature indicated by higher methanotrophs: methanogens (11.2), sulphur reducing bacteria (SRB): methanogens (93.2) ratios and lower functional diversity (7.5%) in mangrove than rice. These could be the key drivers of lower global warming potential (GWP) in mangrove that make it a green production system. Therefore, labile carbon build-up potential (38%) with less GWP (63%) even in degraded-mangrove makes it a clean production system than wetland-rice that has high potential to climate change mitigation. The whole genome metagenomic analysis would be the future research priority to identify the predominant enzymatic pathways which govern the methanogenesis and methanotrophy in this system.

Draft genome assembly of Tenualosa ilisha, Hilsa shad, provides resource for osmoregulation studies.

This study provides the first high-quality draft genome assembly (762.5 Mb) of Tenualosa ilisha that is highly contiguous and nearly complete. We observed a total of 2,864 contigs, with 96.4% completeness with N50 of 2.65 Mbp and the largest contig length of 17.4 Mbp, along with a complete mitochondrial genome of 16,745 bases. A total number of 33,042 protein coding genes were predicted, among these, 512 genes were classified under 61 Gene Ontology (GO) terms, associated with various homeostasis processes. Highest number of genes belongs to cellular calcium ion homeostasis, followed by tissue homeostasis. A total of 97 genes were identified, with 16 GO terms related to water homeostasis. Claudins, Aquaporins, Connexins/Gap junctions, Adenylate cyclase, Solute carriers and Voltage gated potassium channel genes were observed to be higher in number in T. ilisha, as compared to that in other teleost species. Seven novel gene variants, in addition to claudin gene (CLDZ), were found in T. ilisha. The present study also identified two putative novel genes, NKAIN3 and L4AM1, for the first time in fish, for which further studies are required for pinpointing their functions in fish. In addition, 1.6 million simple sequence repeats were mined from draft genome assembly. The study provides a valuable genomic resource for the anadromous Hilsa. It will form a basis for future studies, pertaining to its adaptation mechanisms to different salinity levels during migration, which in turn would facilitate in its domestication.

Mechanism of plant mediated methane emission in tropical lowland rice.

Methane (CH4) is predominantly produced in lowland rice soil, but its emission from soil to atmosphere primarily depends on passage/conduit or capillary pore spaces present in rice plants. The gas transport mechanism through aerenchyma pore spaces of rice cultivars was studied to explore the plant mediated CH4 emission. Seven rice cultivars, based on the life cycle duration (LCD), were tested in tropical eastern India. Three LCD groups were, (a) Kalinga 1 and CR Dhan 204 (LCD: 110-120 days); (b) Lalat, Pooja and CR 1014 (LCD: 130-150 days); and (c) Durga and Varshadhan (LCD: 160-170 days). Rate of CH4emission, root exudates, root oxidase activities and shoot aerenchyma pore spaces were analyzed to study the mechanism of plant mediated emission from rice. Aerenchyma pore space was quantified in the hypothesis that it regulates the CH4 transportation from soil to atmosphere. The ratio of pore space area to total space was lowest in Kalinga 1 cultivar (0.29) and highest was in Varshadhan (0.43). Significant variations in the methane emission were observed among the cultivars with an average emission rate ranged from 0.86 mg m-2 h-1 to 4.96 mg m-2 h-1. The CH4 emission rates were lowest in short duration cultivars followed by medium and long duration ones. The greenhouse gas intensity considering average CH4 emission rate per unit grain yield was also lowest (0.35) in Kalinga 1 and relatively less in short and medium duration cultivars. Root exudation was higher at panicle initiation (PI) than maximum tillering (MT) stage. Lowest exudation was noticed in (197.2 mg C plant-1 day-1) Kalinga 1 and highest in Varsadhan (231.7 mg C plant-1 day-1). So we can say, the rate of CH4 emission was controlled by aerenchyma orientation, root exudation and biomass production rate which are the key specific traits of a cultivar. Identified traits were closely associated with duration and adaptability to cultivars grown in specific ecology. Therefore, there is possibility to breed rice cultivars depending on ecology, duration and having less CH4 emission potential, which could be effectively used in greenhouse gas mitigation strategies.

Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.).

BACKGROUND: Increased water and labour scarcity in major rice growing areas warrants a shift towards direct seeded rice cultivation under which management of weeds is a major issue. Use of broad spectrum non-selective herbicides is an efficient means to manage weeds. Availability of rice genotypes with complete tolerance against broad-spectrum non-selective herbicides is a pre-requisite for advocating use of such herbicides. In the present study, we developed an EMS induced rice mutant, 'HTM-N22', exhibiting tolerance to a broad spectrum herbicide, 'Imazethapyr', and identified the mutations imparting tolerance to the herbicide. RESULTS: We identified a stable and true breeding rice mutant, HTM-N22 (HTM), tolerant to herbicide, Imazethapyr, from an EMS-mutagenized population of approximately 100,000 M2 plants of an upland rice variety, Nagina 22 (N22). Analysis of inheritance of herbicide tolerance in a cross between Pusa 1656-10-61/HTM showed that this trait is governed by a single dominant gene. To identify the causal gene for Imazethapyr tolerance, bulked segregant analysis (BSA) was followed using microsatellite markers flanking the three putative candidate genes viz., an Acetolactate Synthase (ALS) on chromosome 6 and two Acetohydroxy Acid Synthase (AHAS) genes, one on chromosomes 2 and another on chromosome 4. RM 6844 on chromosome 2 located 0.16 Mbp upstream of AHAS (LOC_Os02g30630) was found to co-segregate with herbicide tolerance. Cloning and sequencing of AHAS (LOC_Os02g30630) from the wild type, N22 and the mutant HTM and their comparison with reference Nipponbare sequence revealed several Single Nucleotide Polymorphisms (SNPs) in the mutant, of which eight resulted in non-synonymous mutations. Three of the eight amino acid substitutions were identical to Nipponbare and hence were not considered as causal changes. Of the five putative candidate SNPs, four were novel (at positions 30, 50, 81 and 152) while the remaining one, S627D was a previously reported mutant, known to result in Imidazolinone tolerance in rice. Of the novel ones, G152E was found to alter the hydrophobicty and abolish an N myristoylation site in the HTM compared to the WT, from reference based modeling and motif prediction studies. CONCLUSIONS: A novel mutant tolerant to the herbicide "Imazethapyr" was developed and characterized for genetic, sequence and protein level variations. This is a HTM in rice without any IPR (Intellectual Property Rights) infringements and hence can be used in rice breeding as a novel genetic stock by the public funded organizations in the country and elsewhere.

Metagenomic assessment of methane production-oxidation and nitrogen metabolism of long term manured systems in lowland rice paddy.

Biochemical pathways of methanogenesis and methanotrophy coupled with carbon (C)-nitrogen (N) metabolism were studied in long term (13years) manured systems in lowland rice paddy through metagenomics approach. Manured systems included in this study were, control (exclusion of application of any manure), farm yard manure (FYM, @5Mgha-1yr-1) and green manuring (GM with Sesbania aculeata). Metagenomic sequence data revealed the dominance of C decomposing bacterial communities' like Proteobacteria, Planctomycetes, Actinobacteria, Firmicutes, Acidobacteria, in manure amended soils as compared to control. Diversities for assimilatory and dissimilatory N-fixing microorganisms at phylum level were found higher under GM as compared to rest. Two genera responsible for methanogenesis, viz. Methanolobus and Methanotorris were absent in manured systems as compared to control. The acetoclastic and serine pathway was found as the predominant pathway for methanogenesis and methanotrophy, respectively, in tropical lowland rice paddy. Abundance reads of enzymes were in the range of 254-445 in the acetoclastic methanogenesis pathway. On the other hand, these were varied from 165 to 216 in serine pathway of methanotrophy. Lowland paddy soil exhibited higher functional and structural diversities in manured systems as compared to unamended control in respect to labile C pools and CH4 production. Methane (CH4) emission was 31% higher in FYM system than GM. However, nitrous oxide (N2O) emission was found 25% higher in GM as compared to FYM. As a whole, bacterial diversities were higher under FYM system in tropical lowland rice paddy as compared to GM and unamended systems.

Unraveling the molecular basis of oxidative stress management in a drought tolerant rice genotype Nagina 22.

BACKGROUND: Drought stress tolerance for crop improvement is an important goal worldwide. Drought is a complex trait, and it is vital to understand the complex physiological, biochemical, and molecular mechanisms of drought tolerance to tackle it effectively. Osmotic adjustment, oxidative stress management (OSM), and cell membrane stability (CMS) are major components of cellular tolerance under drought stress. In the current study, we explored the molecular basis of OSM in the drought tolerant rice variety, Nagina 22 and compared it with the popular drought sensitive rice variety, IR 64, under drought imposed at the reproductive stage, to understand how the parental polymorphisms correlate with the superiority of Nagina 22 and tolerant bulk populations under drought. RESULTS: We generated recombinant inbred lines (RIL) from contrasting parents Nagina 22 and IR 64 and focussed on spikelet fertility (SF), in terms of its correlation with OSM, which is an important component of drought tolerance in Nagina 22. Based on SF under drought stress and its correlations with other yield related traits, we used superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) activity assays to establish the relationship between SF and OSM genes in the tolerant and sensitive lines. Among the OSM enzymes studied, GR had a significant and positive correlation with single plant yield (SPY) under drought stress. GR was also positively correlated with APX but negatively so with SOD. Interestingly, none of the enzyme-morphology correlations were significant under irrigated control (IC). Through genome-wide SNP analysis of the 21 genes encoding for OSM enzymes, we identified the functional polymorphisms between the parents and identified superior alleles. By using network analysis of OSM genes in rice, we identified the genes that are central to the OSM network. CONCLUSIONS: From the biochemical and morphological data and the SNP analysis, the superiority of Nagina 22 in spikelet fertility under drought stress is because of its superior alleles for SOD (SOD2, SODCC1, SODA) and GR (GRCP2) rather than for APX, for which IR 64 had the superior allele (APX8). Nagina 22 can bypass APX8 by directly interacting with SODA. For nine of the 11 genes present in the central network, Nagina 22 had the superior alleles. We propose that Nagina 22 tolerance could mainly be because of SODA which is a reactive oxygen scavenger in mitochondria which is directly associated with spikelet fertility.

Elucidation of rice rhizosphere metagenome in relation to methane and nitrogen metabolism under elevated carbon dioxide and temperature using whole genome metagenomic approach.

Carbon (C) and nitrogen (N) mineralization is one of the key processes of biogeochemical cycling in terrestrial ecosystem in general and rice ecology in particular. Rice rhizosphere is a rich niche of microbial diversity influenced by change in atmospheric temperature and concentration of carbon dioxide (CO2). Structural changes in microbial communities in rhizosphere influence the nutrient cycling. In the present study, the bacterial diversity and population dynamics were studied under ambient CO2 (a-CO2) and elevated CO2+temperature (e-CO2T) in lowland rice rhizosphere using whole genome metagenomic approach. The whole genome metagenomic sequence data of lowland rice exhibited the dominance of bacterial communities including Proteobacteria, Firmicutes, Acidobacteria, Actinobacteria and Planctomycetes. Interestingly, four genera related to methane production namely, Methanobacterium, Methanosphaera, Methanothermus and Methanothermococcus were absent in a-CO2 but noticed under e-CO2T. The acetoclastic pathway was found as the predominant pathway for methanogenesis, whereas, the serine pathway was found as the principal metabolic pathway for CH4 oxidation in lowland rice. The abundances of reads of enzymes in the acetoclastic methanogenesis pathway and serine pathways of methanotrophy were much higher in e-CO2T (328 and 182, respectively) as compared with a-CO2 (118 and 98, respectively). Rice rhizosphere showed higher structural diversities and functional activities in relation to N metabolism involving nitrogen fixation, assimilatory and dissimilatory nitrate reduction and denitrification under e-CO2T than that of a-CO2. Among the three pathways of N metabolism, dissimilarity pathways were predominant in lowland rice rhizosphere and more so under e-CO2T. Consequently, under e-CO2T, CH4 emission, microbial biomass nitrogen (MBN) and dehydrogenase activities were 45%, 20% and 35% higher than a-CO2, respectively. Holistically, a high bacterial diversity and abundances of C and N decomposing bacteria in lowland rice rhizosphere were found under e-CO2T, which could be explored further for their specific role in nutrient cycling, sustainable agriculture and environment management.

From QTL to variety-harnessing the benefits of QTLs for drought, flood and salt tolerance in mega rice varieties of India through a multi-institutional network.

Rice is a staple cereal of India cultivated in about 43.5Mha area but with relatively low average productivity. Abiotic factors like drought, flood and salinity affect rice production adversely in more than 50% of this area. Breeding rice varieties with inbuilt tolerance to these stresses offers an economically viable and sustainable option to improve rice productivity. Availability of high quality reference genome sequence of rice, knowledge of exact position of genes/QTLs governing tolerance to abiotic stresses and availability of DNA markers linked to these traits has opened up opportunities for breeders to transfer the favorable alleles into widely grown rice varieties through marker-assisted backcross breeding (MABB). A large multi-institutional project, "From QTL to variety: marker-assisted breeding of abiotic stress tolerant rice varieties with major QTLs for drought, submergence and salt tolerance" was initiated in 2010 with funding support from Department of Biotechnology, Government of India, in collaboration with International Rice Research Institute, Philippines. The main focus of this project is to improve rice productivity in the fragile ecosystems of eastern, northeastern and southern part of the country, which bear the brunt of one or the other abiotic stresses frequently. Seven consistent QTLs for grain yield under drought, namely, qDTY1.1, qDTY2.1, qDTY2.2, qDTY3.1, qDTY3.2, qDTY9.1 and qDTY12.1 are being transferred into submergence tolerant versions of three high yielding mega rice varieties, Swarna-Sub1, Samba Mahsuri-Sub1 and IR 64-Sub1. To address the problem of complete submergence due to flash floods in the major river basins, the Sub1 gene is being transferred into ten highly popular locally adapted rice varieties namely, ADT 39, ADT 46, Bahadur, HUR 105, MTU 1075, Pooja, Pratikshya, Rajendra Mahsuri, Ranjit, and Sarjoo 52. Further, to address the problem of soil salinity, Saltol, a major QTL for salt tolerance is being transferred into seven popular locally adapted rice varieties, namely, ADT 45, CR 1009, Gayatri, MTU 1010, PR 114, Pusa 44 and Sarjoo 52. Genotypic background selection is being done after BC2F2 stage using an in-house designed 50K SNP chip on a set of twenty lines for each combination, identified with phenotypic similarity in the field to the recipient parent. Near-isogenic lines with more than 90% similarity to the recipient parent are now in advanced generation field trials. These climate smart varieties are expected to improve rice productivity in the adverse ecologies and contribute to the farmer's livelihood.

Evaluation of random forest regression for prediction of breeding value from genomewide SNPs.

Genomic prediction is meant for estimating the breeding value using molecular marker data which has turned out to be a powerful tool for efficient utilization of germplasm resources and rapid improvement of cultivars. Model-based techniques have been widely used for prediction of breeding values of genotypes from genomewide association studies. However, application of the random forest (RF), a model-free ensemble learning method, is not widely used for prediction. In this study, the optimum values of tuning parameters of RF have been identified and applied to predict the breeding value of genotypes based on genomewide single-nucleotide polymorphisms (SNPs), where the number of SNPs (P variables) is much higher than the number of genotypes (n observations) (P ¼ n). Further, a comparison was made with the model-based genomic prediction methods, namely, least absolute shrinkage and selection operator (LASSO), ridge regression (RR) and elastic net (EN) under P ¼ n. It was found that the correlations between the predicted and observed trait response were 0.591, 0.539, 0.431 and 0.587 for RF, LASSO, RR and EN, respectively, which implies superiority of the RF over the model-based techniques in genomic prediction. Hence, we suggest that the RF methodology can be used as an alternative to the model-based techniques for the prediction of breeding value at genome level with higher accuracy.

Physiological, anatomical and transcriptional alterations in a rice mutant leading to enhanced water stress tolerance.

Water stress is one of the most severe constraints to crop productivity. Plants display a variety of physiological and biochemical responses both at the cellular and whole organism level upon sensing water stress. Leaf rolling, stomatal closure, deeper root penetration, higher relative water content (RWC) and better osmotic adjustment are some of the mechanisms that plants employ to overcome water stress. In the current study, we report a mutant, enhanced water stress tolerant1 (ewst1) with enhanced water stress tolerance, identified from the ethyl methanesulfonate-induced mutant population of rice variety Nagina22 by field screening followed by withdrawal of irrigation in pots and hydroponics (PEG 6000). Though ewst1 was morphologically similar to the wild type (WT) for 35 of the 38 morphological descriptors (except chalky endosperm/expression of white core, decorticated grain colour and grain weight), it showed enhanced germination in polyethylene glycol-infused medium. It exhibited increase in maximum root length without any significant changes in its root weight, root volume and total root number on crown when compared with the WT under stress in PVC tube experiment. It also showed better performance for various physiological parameters such as RWC, cell membrane stability and chlorophyll concentration upon water stress in a pot experiment. Root anatomy and stomatal microscopic studies revealed changes in the number of xylem and phloem cells, size of central meta-xylem and number of closed stomata in ewst1. Comparative genome-wide transcriptome analysis identified genes related to exocytosis, secondary metabolites, tryptophan biosynthesis, protein phosphorylation and other signalling pathways to be playing a role in enhanced response to water stress in ewst1. The possible involvement of a candidate gene with respect to the observed morpho-physiological and transcriptional changes and its role in stress tolerance are discussed. The mutant identified and characterized in this study will be useful for further dissection of water stress tolerance in rice.

Identification of heat-related ESTs in moth bean through suppression subtraction hybridization.

Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.

Neuroprotective potentials of neurotrophin rich olfactory ensheathing cell's conditioned media against 6OHDA-induced oxidative damage.

On the basis of recent reports, we propose that impaired neurotrophin signaling (PI3k/Akt), low antioxidant levels, and generation of reactive oxygen species (ROS) conjointly participate in the progressive events responsible for the dopaminergic cell loss in Parkinson's disease (PD). In the present study we tried to target these deficits collectively through multiple neurotrophic factors (NTFs) support in the form of Olfactory Ensheathing Cell's Conditioned Media (OEC CM) using human SH-SY5Y neuroblastoma cell line exposed to 6 hydroxydopamine (6OHDA). 6OHDA exposure induced, oxidative stress-mediated apoptotic cell death viz. enhanced ROS generation, diffused cytosolic cytochrome c (cyt c), impaired Bcl-2: Bax levels along with decrease in GSH content. These changes were accompanied by loss in Akt phosphorylation and TH levels in SH-SY5Y cells. OEC CM significantly checked apoptotic cell death by preserving pAkt levels which coincided with enhanced GSH and suppressed oxidative injury. Functional integrity of OEC CM supported cells was evident by maintained tyrosine hydroxylase (TH) expression. Intercepting Akt signaling by specific inhibitor LY294002 blocked the protective effect. Taken together our findings provide important evidence that the key to protective effect of multiple NTF support via OEC CM is enhanced Akt survival signaling which promotes antioxidant defense leading to suppression of oxidative damage.

Assessment of genetic diversity in Indian rice germplasm (Oryza sativa L.): use of random versus trait-linked microsatellite markers.

Assessment of genetic diversity in a crop germplasm is a vital part of plant breeding. DNA markers such as microsatellite or simple sequence repeat markers have been widely used to estimate the genetic diversity in rice. The present study was carried out to decipher the pattern of genetic diversity in terms of both phenotypic and genotypic variability, and to assess the efficiency of random vis-á-vis QTL linked/gene based simple sequence repeat markers in diversity estimation. A set of 88 rice accessions that included landraces, farmer's varieties and popular Basmati lines were evaluated for agronomic traits and molecular diversity. The random set of SSR markers included 50 diversity panel markers developed under IRRI's Generation Challenge Programme (GCP) and the trait-linked/gene based markers comprised of 50 SSR markers reportedly linked to yield and related components. For agronomic traits, significant variability was observed, ranging between the maximum for grains/panicle and the minimum for panicle length. The molecular diversity based grouping indicated that varieties from a common centre were genetically similar, with few exceptions. The trait-linked markers gave an average genetic dissimilarity of 0.45 as against that of 0.37 by random markers, along with an average polymorphic information constant value of 0.48 and 0.41 respectively. The correlation between the kinship matrix generated by trait-linked markers and the phenotype based distance matrix (0.29) was higher than that of random markers (0.19). This establishes the robustness of trait-linked markers over random markers in estimating genetic diversity of rice germplasm.

Salsolinol induced apoptotic changes in neural stem cells: amelioration by neurotrophin support.

Salsolinol (SAL), a catechol isoquinoline has invited considerable attention due to its structural similarity with dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Its high endogenous presence in Parkinsonian brain implicated its possible association with the disease process. SAL is also present in alcohol beverages and certain food materials and can get access to brain especially in conditions of immature or impaired BBB. Besides this, the effect of SAL on neural stem cells (NSCs) which are potential candidates for adult neurogenesis and transplantation mediated rejuvenating attempts for Parkinson's disease (PD) brain has not been known so far. NSCs in both the cases have to overcome suppressive cues of diseased brain for their survival and function. In this study we explored the toxicity of SAL toward NSCs focusing on apoptosis and status of PI3K survival signaling. NSCs cultured from embryonic day 11 rat fetal brain including those differentiated to TH(+ve) colonies, when challenged with SAL (1-100µM), elicited a concentration and time dependent cell death/loss of mitochondrial viability. 10µM SAL on which significant mitochondrial impairment initiated was further used to study mechanism of toxicity. Morphological impairment, enhanced TUNEL positivity, cleaved caspase-3 and decreased Bcl-2:Bax suggested apoptosis. Sal toxicity coincided with reduced pAkt level and its downstream effectors: pCREB, pGSK-3ß, Bcl-2 and neurotrophins GDNF, BDNF suggesting repressed PI3K/Akt signaling. Multiple neurotrophic factor support in the form of Olfactory Ensheathing Cell's Conditioned Media (OEC CM) potentially protected NSCs against SAL through activating PI3K/Akt pathway. This was confirmed on adding LY294002 the PI3K inhibitor which abolished the protection. We inferred that SAL exerts substantial toxicity toward NSCs. These findings will lead to better understanding of endogenous threats that might affect the fate of transplanted NSCs and their probable antidotes.

Construction and analysis of an SSH cDNA library of early heat-induced genes of Vigna aconitifolia variety RMO-40.

Moth bean ( Vigna aconitifolia (Jacq.) Marechal) is an important grain legume crop grown in rain fed areas of hot desert regions of Thar, India, under scorching sun rays with very little supplementation of water. An SSH cDNA library was generated from leaf tissues of V. aconitifolia var. RMO-40 exposed to an elevated temperature of 42 °C for 5 min to identify early-induced genes. A total of 488 unigenes (114 contigs and 374 singletons) were derived by cluster assembly and sequence alignment of 738 ESTs; out of 206 ESTs (28%) of unknown proteins, 160 ESTs (14%) were found to be novel to moth bean. Only 578 ESTs (78%) showed significant BLASTX similarity (<1 × 10(-6)) in the NCBI non-redundant database. Gene ontology functional classification terms were retrieved for 479 (65%) sequences, and 339 sequences were annotated with 165 EC codes and mapped to 68 different KEGG pathways. Four hundred and fifty-two ESTs were further annotated with InterProScan (IPS), and no IPS was assigned to 153 ESTs. In addition, the expression level of 27 ESTs in response to heat stress was evaluated through semiquantitative RT-PCR assay. Approximately 20 different signaling genes and 16 different transcription factors have been shown to be associated with heat stress in moth bean for the first time.

Molecular breeding for the development of multiple disease resistance in Basmati rice.

BACKGROUND AND AIMS: Basmati rice grown in the Indian subcontinent is highly valued for its unique culinary qualities. Production is, however, often constrained by diseases such as bacterial blight (BB), blast and sheath blight (ShB). The present study developed Basmati rice with inbuilt resistance to BB, blast and ShB using molecular marker-assisted selection. METHODOLOGY: The rice cultivar 'Improved Pusa Basmati 1' (carrying the BB resistance genes xa13 and Xa21) was used as the recurrent parent and cultivar 'Tetep' (carrying the blast resistance gene Pi54 and ShB resistance quality trait loci (QTL), qSBR11-1) was the donor. Marker-assisted foreground selection was employed to identify plants possessing resistance alleles in the segregating generations along with stringent phenotypic selection for faster recovery of the recurrent parent genome (RPG) and phenome (RPP). Background analysis with molecular markers was used to estimate the recovery of RPG in improved lines. PRINCIPAL RESULTS: Foreground selection coupled with stringent phenotypic selection identified plants homozygous for xa13, Xa21 and Pi54, which were advanced to BC(2)F(5) through pedigree selection. Marker-assisted selection for qSBR11-1 in BC(2)F(5) using flanking markers identified seven homozygous families. Background analysis revealed that RPG recovery was up to 89.5%. Screening with highly virulent isolates of BB, blast and ShB showed that the improved lines were resistant to all three diseases and were on a par with 'Improved Pusa Basmati 1' for yield, duration and Basmati grain quality. CONCLUSIONS: This is the first report of marker-assisted transfer of genes conferring resistance to three different diseases in rice wherein genes xa13 and Xa21 for BB resistance, Pi54 for blast resistance, and a major QTL qSBR11-1 have been combined through marker-assisted backcross breeding. In addition to offering the potential for release as cultivars, the pyramided lines will serve as useful donors of gene(s) for BB, blast and ShB in future Basmati rice breeding programmes.

Glycoprotein C gene based molecular subtyping of a bovine herpesvirus -1 isolate from uttar pradesh, India.

Bovine herpesvirus -1 (BHV-1) is the etiological agent of many clinical syndromes in cattle which causes huge economic losses to the animal husbandry sector annually. Since the first report of its presence in India in 1976, the disease is considered to be endemic in the country. In the present study, a case of keratoconjunctivitis in a cow was investigated to find out the underlying cause of the condition. The clinical material (ocular swab) was tested by BHV-1 glycoprotein D gene specific PCR using in house designed primers and found to be positive by the presence of a 212 bp DNA product in agarose gel electrophoresis. The virus was isolated in MDBK cell line in the third passage and the serum from the animal, was positive for antibodies against BHV-1 by ELISA. A 575 bp segment of the glycoprotein C gene of the isolate was amplified by PCR, cloned and sequenced. On phylogenetic analysis, it was seen that the sequence matched with published BHV-1.1 sequences from USA and Uruguay whereas it was divergent from Brazilian BHV-1.1 isolates. This study highlights the isolation, rapid and sensitive detection of BHV-1 virus from clinical cases and its subtyping by nucleotide sequencing and subsequent phylogenetic analysis which gives invaluable information about the molecular epidemiology of BHV-1 subtypes prevalent in the country.