Vandna Rai

Combining QTL mapping and transcriptome profiling of bulked RILs for identification of functional polymorphism for salt tolerance genes in rice (Oryza sativa L.).

Identification of genes for quantitative traits is difficult using any single approach due to complex inheritance of the traits and limited resolving power of the individual techniques. Here a combination of genetic mapping and bulked transcriptome profiling was used to narrow down the number of differentially expressed salt-responsive genes in rice in order to identify functional polymorphism of genes underlying the quantitative trait loci (QTL). A population of recombinant inbred lines (RILs) derived from cross between salt-tolerant variety CSR 27 and salt-sensitive variety MI 48 was used to map QTL for salt ion concentrations in different tissues and salt stress susceptibility index (SSI) for spikelet fertility, grain weight, and grain yield. Eight significant QTL intervals were mapped on chromosomes 1, 8, and 12 for the salt ion concentrations and a QTL controlling SSI for spikelet fertility was co-located in one of these intervals on chromosome 8. However, there were total 2,681 genes in these QTL intervals, making it difficult to pinpoint the genes responsible for the functional differences for the traits. Similarly, transcriptome profiling of the seedlings of tolerant and sensitive parents grown under control and salt-stress conditions showed 798 and 2,407 differentially expressed gene probes, respectively. By analyzing pools of RNA extracted from ten each of extremely tolerant and extremely sensitive RILs to normalize the background noise, the number of differentially expressed genes under salt stress was drastically reduced to 30 only. Two of these genes, an integral transmembrane protein DUF6 and a cation chloride cotransporter, were not only co-located in the QTL intervals but also showed the expected distortion of allele frequencies in the extreme tolerant and sensitive RILs, and therefore are suitable for future validation studies and development of functional markers for salt tolerance in rice to facilitate marker-assisted breeding.

Single-copy gene based 50 K SNP chip for genetic studies and molecular breeding in rice

Single nucleotide polymorphism (SNP) is the most abundant DNA sequence variation present in plant genomes. Here, we report the design and validation of a unique genic-SNP genotyping chip for genetic and evolutionary studies as well as molecular breeding applications in rice. The chip incorporates 50,051 SNPs from 18,980 different genes spanning 12 rice chromosomes, including 3,710 single-copy (SC) genes conserved between wheat and rice, 14,959 SC genes unique to rice, 194 agronomically important cloned rice genes and 117 multi-copy rice genes. Assays with this chip showed high success rate and reproducibility because of the SC gene based array with no sequence redundancy and cross-hybridisation problems. The usefulness of the chip in genetic diversity and phylogenetic studies of cultivated and wild rice germplasm was demonstrated. Furthermore, its efficacy was validated for analysing background recovery in improved mega rice varieties with submergence tolerance developed through marker-assisted backcross breeding.

Differences in the arbuscular mycorrhizal fungi-improved rice resistance to low temperature at two N levels: aspects of N and C metabolism on the plant side.

We performed an experiment to determine how N and C metabolism is involved in the low-temperature tolerance of mycorrhizal rice (Oryza sativa) at different N levels and examined the possible signaling molecules involved in the stress response of mycorrhizal rice. Pot cultures were performed, and mycorrhizal rice growth was evaluated based on treatments at two temperatures (15 °C and 25 °C) and two N levels (20 mg pot(-1) and 50 mg pot(-1)). The arbuscular mycorrhizal fungi (AMF) colonization of rice resulted in different responses of the plants to low and high N levels. The mycorrhizal rice with the low N supplementation had more positive feedback from the symbiotic AMF, as indicated by accelerated N and C metabolism of rice possibly involving jasmonic acid (JA) and the up-regulation of enzyme activities for N and C metabolism. Furthermore, the response of the mycorrhizal rice plants to low temperature was associated with P uptake and nitric oxide (NO).

Haplotype distribution and association of candidate genes with salt tolerance in Indian wild rice germplasm

Key messageThe association of natural genetic variations of salt-responsive candidate genes belonging to different gene families with salt-tolerance phenotype and their haplotype variation in different geographic regions.AbstractSoil salinity covers a large part of the arable land of the world and is a major factor for yield losses in salt-sensitive crops, such as rice. Different gene families that respond to salinity have been identified in rice, but limited success has been achieved in developing salt-tolerant cultivars. Therefore, 21 salt stress-responsive candidate genes belonging to different gene families were re-sequenced to analyse their genetic variation and association with salt tolerance. The average single nucleotide polymorphism (SNP) density was 16 SNPs per kbp amongst these genes. The identified nucleotide and haplotype diversity showed comparatively higher genetic variation in the transporter family genes. Linkage disequilibrium (LD) analysis showed significant associations of SNPs in BADH2, HsfC1B, MIPS1, MIPS2, MYB2, NHX1, NHX2, NHX3, P5CS1, P5CS2, PIP1, SIK1, SOS1, and SOS2 genes with the salt-tolerant phenotype. A combined analysis of SNPs in the 21 candidate genes and eight other HKT transporter genes produced two separate clusters of tolerant genotypes, carrying unique SNPs in the ion transporter and osmoticum-related genes. Haplotype network analysis showed all the major and few minor alleles distributed over distant geographic regions. Minor haplotypes may be recently evolved alleles which migrated to distant geographic regions and may represent recent expansion of Indian wild rice. The analysis of genetic variation in different gene families identified the relationship between adaptive variations and functional significance of the genes. Introgression of the identified alleles from wild relatives may enhance the salt tolerance and consequently rice production in the salinity-affected areas.

Deciphering Phosphate Deficiency-Mediated Temporal Effects on Different Root Traits in Rice Grown in a Modified Hydroponic System

Phosphate (Pi), an essential macronutrient for growth and development of plant, is often limiting in soils. Plants have evolved an array of adaptive strategies including modulation of root system architecture (RSA) for optimal acquisition of Pi. In rice, a major staple food, RSA is complex and comprises embryonically developed primary and seminal roots and post-embryonically developed adventitious and lateral roots. Earlier studies have used variant hydroponic systems for documenting the effects of Pi deficiency largely on primary root growth. Here, we report the temporal effects of Pi deficiency in rice genotype MI48 on 15 ontogenetically distinct root traits by using easy-to-assemble and economically viable modified hydroponic system. Effects of Pi deprivation became evident after 4 days- and 7 days-treatments on two and eight different root traits, respectively. The effects of Pi deprivation for 7 days were also evident on different root traits of rice genotype Nagina 22 (N22). There were genotypic differences in the responses of primary root growth along with lateral roots on it and the number and length of seminal and adventitious roots. Notably though, there were attenuating effects of Pi deficiency on the lateral roots on seminal and adventitious roots and total root length in both these genotypes. The study thus revealed both differential and comparable effects of Pi deficiency on different root traits in these genotypes. Pi deficiency also triggered reduction in Pi content and induction of several Pi starvation-responsive (PSR) genes in roots of MI48. Together, the analyses validated the fidelity of this modified hydroponic system for documenting Pi deficiency-mediated effects not only on different traits of RSA but also on physiological and molecular responses.

A comparative method for protein extraction and 2-D gel electrophoresis from different tissues of Cajanus cajan.

Pigeonpea is an important legume crop with high protein content. However, it is often subjected to various abiotic and biotic stresses. Proteomics is a state-of-the-art technique used to analyze the protein profiling of a tissue for deciphering the molecular entities that could be manipulated for developing crops resistant to these stresses. In this context, developing a comprehensive proteome profile from different vegetative and reproductive tissues has become mandatory. Although several protein extraction protocols from different tissues of diverse plant species have been reported, there is no report for pigeonpea. Here, we report tissue-specific protein extraction protocols representing vegetative (young leaves), and reproductive (flowers and seeds) organs and their subsequent analysis on 2-dimensional gel electrophoresis. The study explicitly demonstrated that the efficacy of a particular protein extraction protocol is dependent on the different tissues, such as leaves, flowers and seeds that differ in their structure and metabolic constituents. For instance, phenol-based protocol showed an efficacy toward higher protein yield, better spot resolution and a minimal streaking on 2-DE gel for both leaves and flowers. Protein extraction from seeds was best achieved by employing phosphate-TCA-acetone protocol.

Towards a common bean proteome atlas: looking at the current state of research and the need for a comprehensive proteome

1 School of Biotechnology, SK University of Agricultural Sciences and Technology of Jammu, Jammu, India, Department of Botany, Jamia Hamdard University, New Delhi, India, National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India, 4 Reproduction and Developmental Biology, Institute of Plant Genetics and Biotechnology, Slovak Academy of Science, Nitra, Slovakia, 5 Research Laboratory for Biotechnology and Biochemistry, Kathmandu, Nepal, Organization for Educational Initiatives, University of Tsukuba, Tsukuba, Japan

Nitrate and amino acid availability affects glycine betaine and mycosporine-2-glycine in response to changes of salinity in a halotolerant cyanobacterium Aphanothece halophytica

A halotolerant cyanobacterium Aphanothece halophytica thrives in extreme salinity with accumulation of a potent osmoprotectant glycine betaine. Recently, this cyanobacterium was shown to accumulate sunscreen molecule mycosporine-2-glycine significantly at high salinity. In this study, we investigated effects of nitrate and amino acid provision on the accumulation of glycine betaine and mycosporine-2-glycine. With elevated nitrate concentrations at high salinity, intracellular levels of both metabolites were enhanced. Six-fold high nitrate concentration increased the relative amounts of glycine betaine and mycosporine-2-glycine to be 1.5 and 2.0 folds compared with control condition : Increased levels were time- and dose-dependent manner. Exogenous supply of glycine/serine at high salinity resulted in the similar trends as observed in excess nitrate experiment. Intracellular level of glycine betaine increased ∼1.6 folds with glycine/serine supplementation. These supplementations also caused the increased level of mycosporine-2-glycine, namely 1.4 and 2 folds by glycine and serine, respectively. The transcription of glycine betaine and mycosporine-2-glycine biosynthetic genes was strongly induced under high-nitrate-salt condition. These results suggest the dependence of glycine betaine and mycosporine-2-glycine productions on substrate availability, and the effect of nitrate was possibly associated with stimulation of osmoprotectant increment in this extremophile.

Shift from Gel Based to Gel Free Proteomics to Unlock Unknown Regulatory Network in Plants: A Comprehensive Review

Sajad Majeed Zargar1*, Nancy Gupta2, Rakeeb A Mir3, Vandna Rai4 1Centre for Plant Biotechnology, Division of Biotechnology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, Shalimar, Srinagar, J&K, India 2School Biotechnology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu, Chatha, Jammu, J&K, India 3School of Bio resources & Biotechnology, BGSB University, Rajouri, J&K, India 4NRCPB, New Delhi, India Journal of Advanced Research in Biotechnology Open Access Review Article

Comparative biochemical analysis of wild introgression lines in response to short-term exposure to salinity.

A probiotic is a viable microbial dietary supplement that is beneficial for the host through its effects in the intestinal tract. Probiotics are widely used to prepare fermented dairy products such as yoghurt and it is an important source of probiotic Lactobacilli. In this study, the samples were collected from different superstores of Chittagong and Bogra city of Bangladesh. Pure culture of specific probiotic isolates from each sample was performed and identified on the basis of their colonies morphologies and some biochemical tests such as catalase, oxidase and IMViC (indole, methylred, voges proskavar, citrate utilization) test. Their identification as Lactobacillus spp., was confirmed through PCR reaction using genus specific primer. The isolated Lactobacillus spp., were resistant to inhibitory substances like NaCl (1-9%) and bile acid (0.05-0.3%). In addition, the satisfactory growth of isolated Lactobacillus spp., was observed in alkaline condition. The isolated Lactobacillus spp., showed positive result in different carbohydrate source such as glucose, xylose, galactose, etc and were able to produce organic acid in skim milk which was determined by titrimetic method. The Lactobacillus spp., was examined for their antimicrobial activities against some test pathogens by modified agar overlay method and the high inhibition zones showed their potential antibacterial effects. The yoghurt is suggested as a source of potential probiotic strains and there were variations in probiotic properties of the isolated Lactobacillus spp., obtained from selective regional yoghurt samples in this study.