Epigenome editing for targeted DNA (de)methylation: a new perspective in modulating gene expression.

Traditionally, it has been believed that inheritance is driven as phenotypic variations resulting from changes in DNA sequence. However, this paradigm has been challenged and redefined in the contemporary era of epigenetics. The changes in DNA methylation, histone modification, non-coding RNA biogenesis, and chromatin remodeling play crucial roles in genomic functions and regulation of gene expression. More importantly, some of these changes are inherited to the next generations as a part of epigenetic memory and play significant roles in gene expression. The sum total of all changes in DNA bases, histone proteins, and ncRNA biogenesis constitutes the epigenome. Continuous progress in deciphering epigenetic regulations and the existence of heritable epigenetic/epiallelic variations associated with trait of interest enables to deploy epigenome editing tools to modulate gene expression. DNA methylation marks can be utilized in epigenome editing for the manipulation of gene expression. Initially, genome/epigenome editing technologies relied on zinc-finger protein or transcriptional activator-like effector protein. However, the discovery of clustered regulatory interspaced short palindromic repeats CRISPR)/deadCRISPR-associated protein 9 (dCas9) enabled epigenome editing to be more specific/efficient for targeted DNA (de)methylation. One of the major concerns has been the off-target effects, wherein epigenome editing may unintentionally modify gene/regulatory element which may cause unintended change/harmful effects. Moreover, epigenome editing of germline cell raises several ethical/safety issues. This review focuses on the recent developments in epigenome editing tools/techniques, technological limitations, and future perspectives of this emerging technology in therapeutics for human diseases as well as plant improvement to achieve sustainable developmental goals.

Comparative miRNome and transcriptome analyses reveal the expression of novel miRNAs in the panicle of rice implicated in sustained agronomic performance under terminal drought stress.

MAIN CONCLUSION: Differential expression of 128 known and 111 novel miRNAs in the panicle of Nagina 22 under terminal drought stress targeting transcription factors, stress-associated genes, etc., enhances drought tolerance and helps sustain agronomic performance under terminal drought stress. Drought tolerance is a complex multigenic trait, wherein the genes are fine-tuned by coding and non-coding components in mitigating deleterious effects. MicroRNA (miRNA) controls gene expression at post-transcriptional level either by cleaving mRNA (transcript) or by suppressing its translation. miRNAs are known to control developmental processes and abiotic stress tolerance in plants. To identify terminal drought-responsive novel miRNA in contrasting rice cultivars, we constructed small RNA (sRNA) libraries from immature panicles of drought-tolerant rice [Nagina 22 (N 22)] and drought-sensitive (IR 64) cultivars grown under control and terminal drought stress. Our analysis of sRNA-seq data resulted in the identification of 169 known and 148 novel miRNAs in the rice cultivars. Among the novel miRNAs, 68 were up-regulated while 43 were down-regulated in the panicle of N 22 under stress. Interestingly, 31 novel miRNAs up-regulated in N 22 were down-regulated in IR 64, whereas 4 miRNAs down-regulated in N 22 were up-regulated in IR 64 under stress. To detect the effects of miRNA on mRNA expression level, transcriptome analysis was performed, while differential expression of miRNAs and their target genes was validated by RT-qPCR. Targets of the differentially expressed miRNAs include transcription factors and stress-associated genes involved in cellular/metabolic/developmental processes, response to abiotic stress, programmed cell death, photosynthesis, panicle/seed development, and grain yield. Differential expression of the miRNAs could be validated in an independent set of the samples. The findings might be useful in genetic improvement of drought-tolerant rice.

DNA methylome analysis provides insights into gene regulatory mechanism for better performance of rice under fluctuating environmental conditions: epigenomics of adaptive plasticity.

MAIN CONCLUSION: Roots play an important role in adaptive plasticity of rice under dry/direct-sown conditions. However, hypomethylation of genes in leaves (resulting in up-regulated expression) complements the adaptive plasticity of Nagina-22 under DSR conditions. Rice is generally cultivated by transplanting which requires plenty of water for irrigation. Such a practice makes rice cultivation a challenging task under global climate change and reducing water availability. However, dry-seeded/direct-sown rice (DSR) has emerged as a resource-saving alternative to transplanted rice (TPR). Though some of the well-adapted local cultivars are used for DSR, only limited success has been achieved in developing DSR varieties mainly because of a limited knowledge of adaptability of rice under fluctuating environmental conditions. Based on better morpho-physiological and agronomic performance of Nagina-22 (N-22) under DSR conditions, N-22 and IR-64 were grown by transplanting and direct-sowing and used for whole genome methylome analysis to unravel the epigenetic basis of adaptive plasticity of rice. Comparative methylome and transcriptome analyses indicated a large number (4078) of genes regulated through DNA methylation/demethylation in N-22 under DSR conditions. Gene × environment interactions play important roles in adaptive plasticity of rice under direct-sown conditions. While genes for pectinesterase, LRK10, C2H2 zinc-finger protein, splicing factor, transposable elements, and some of the unannotated proteins were hypermethylated, the genes for regulation of transcription, protein phosphorylation, etc. were hypomethylated in CG context in the root of N-22, which played important roles in providing adaptive plasticity to N-22 under DSR conditions. Hypomethylation leading to up-regulation of gene expression in the leaf complements the adaptive plasticity of N-22 under DSR conditions. Moreover, differential post-translational modification of proteins and chromatin assembly/disassembly through DNA methylation in CHG context modulate adaptive plasticity of N-22. These findings would help developing DSR cultivars for increased water-productivity and ecological efficiency.

Transcriptome and Physio-Biochemical Profiling Reveals Differential Responses of Rice Cultivars at Reproductive-Stage Drought Stress.

Drought stress severely affects the growth and development of rice, especially at the reproductive stage, which results in disturbed metabolic processes, reduced seed-set/grain filling, deteriorated grain quality, declined productivity, and lower yield. Despite the recent advances in understanding the responses of rice to drought stress, there is a need to comprehensively integrate the morpho-physio-biochemical studies with the molecular responses/differential expression of genes and decipher the underlying pathways that regulate the adaptability of rice at various drought-sensitive growth stages. Our comparative analysis of immature panicle from a drought-tolerant (Nagina 22) and a drought-sensitive (IR 64) rice cultivar grown under control (well-watered) and water-deficit/drought stress (treatment, imposed at the reproductive stage) conditions unraveled some novel stress-responsive genes/pathways responsible for reproductive-stage drought stress tolerance. The results revealed a more important role of upregulated (6706) genes in the panicle of N 22 at reproductive-stage drought stress compared to that (5590) in IR 64. Functional enrichment and MapMan analyses revealed that majority of the DEGs were associated with the phytohormone, redox signalling/homeostasis, secondary metabolite, and transcription factor-mediated mitigation of the adverse effects of drought stress in N 22. The upregulated expression of the genes associated with starch/sucrose metabolism, secondary metabolites synthesis, transcription factors, glutathione, linoleic acid, and phenylalanine metabolism in N 22 was significantly more than that in the panicle of IR 64. Compared to IR 64, 2743 genes were upregulated in N 22 under control conditions, which further increased (4666) under drought stress in panicle of the tolerant cultivar. Interestingly, we observed 6706 genes to be upregulated in the panicle of N 22 over IR 64 under drought and 5814 genes get downregulated in the panicle of N 22 over IR 64 under the stress. In addition, RT-qPCR analysis confirmed differential expression patterns of the DEGs. These genes/pathways associated with the reproductive-stage drought tolerance might provide an important source of molecular markers for genetic manipulation of rice for enhanced drought tolerance.

Transcriptome analysis of a near-isogenic line and its recurrent parent reveals the role of Pup1 QTL in phosphorus deficiency tolerance of rice at tillering stage.

Phosphorus (P) is essential for cellular processes like respiration, photosynthesis, biosynthesis of membrane phospholipids, etc. To cope with P deficiency stress, plants adopt reprograming of the expression of genes involved in different metabolic/signaling pathways for survival, growth, and development. Plants use transcriptional, post-transcriptional, and/or post-translational machinery to achieve P homeostasis. Several transcription factors (TFs), miRNAs, and P transporters play important roles in P deficiency tolerance; however, the underlying mechanisms responsible for P deficiency tolerance remain poorly understood. Studies on P starvation/deficiency responses in plants at early (seedling) stage of growth have been reported but only a few of them focused on molecular responses of the plant at advanced (tillering or reproductive) stage of growth. To decipher the strategies adopted by rice at tillering stage under P deficiency stress, a pair of contrasting genotypes [Pusa-44 (a high-yielding, P deficiency sensitive cultivar) and its near-isogenic line (NIL-23, P deficiency tolerant) for Pup1 QTL] was used for morphophysiological, biochemical, and molecular analyses. Comparative analyses of shoot and root tissues from 45-day-old plants grown hydroponically under P sufficient (16 ppm) or P deficient (4 ppm) medium confirmed some of the known morphophysiological responses. Moreover, RNA-seq analysis revealed the important roles of phosphate transporters, TFs, auxin-responsive proteins, modulation in the cell wall, fatty acid metabolism, and chromatin architecture/epigenetic modifications in providing P deficiency tolerance to NIL-23, which were brought in due to the introgression of the Pup1 QTL in Pusa-44. This study provides insights into the molecular functions of Pup1 for P deficiency tolerance, which might be utilized to improve P-use efficiency of rice for better productivity in P deficient soils. KEY MESSAGE: Introgression of Pup1 QTL in high-yielding rice cultivar modulates mainly phosphate transporters, TFs, auxin-responsive proteins, cell wall structure, fatty acid metabolism, and chromatin architecture/epigenetic modifications at tillering stage of growth under phosphorus deficiency stress.

MicroRNA: noncoding but still coding, another example of self-catalysis.

MicroRNAs (miRNAs) are known to interact with specific mRNAs to regulate gene expression at the post-transcriptional level by cleaving/repressing the translation process. MiRNA-mediated regulation of gene expression has become an interesting area of research on biological processes like growth, development, and stress responses. Studies suggest that some of the noncoding RNAs possess short open reading frames (ORFs) that code for micropeptides (miPEPs) having a regulatory function. Dual functions of some MIR genes are being deciphered, wherein the gene is transcribed into a longer transcript having a stem-loop structure and a shorter alternatively spliced transcript with no stem-loop. While the longer transcript is processed into miRNA, the shorter one is translated into miPEP. The miPEP enhances the transcription/production of the pri-miRNA from which it originates. Regulatory action of miPEP being species-specific, synthetic miPEP being is tested for exogenous application on crop plant to improve stress tolerance/agronomic performance. Deployment of the miPEP-mediated regulatory function might be a promising strategy to modulated miRNA-facilitated regulation of gene/trait of interest towards developing climate-resilient crops. In this review, we describe the newly identified and verified function of the MIR gene in the coding of miPEPs along with the comparison of the features of miRNA and miPEP in plant. We also discuss about their potential role in crop improvement and some of the yet unanswered question about miPEP.

A comprehensive transcriptome analysis of contrasting rice cultivars highlights the role of auxin and ABA responsive genes in heat stress response.

Sensing a change in ambient temperature is key to survival among all living organisms. Temperature fluctuations due to climate change are a matter of grave concern since it adversely affects growth and eventually the yield of crop plants, including two of the major cereals, i.e., rice and wheat. Thus, to understand the response of rice seedlings to elevated temperatures, we performed microarray-based transcriptome analysis of two contrasting rice cultivars, Annapurna (heat tolerant) and IR64 (heat susceptible), by subjecting their seedlings to 37 °C and 42 °C, sequentially. The transcriptome analyses revealed a set of uniquely regulated genes and related pathways in red rice cultivar Annapurna, particularly associated with auxin and ABA as a part of heat stress response in rice. The changes in expression of few auxin and ABA associated genes, such as OsIAA13, OsIAA20, ILL8, OsbZIP12, OsPP2C51, OsDi19-1 and OsHOX24, among others, were validated under high-temperature conditions using RT-qPCR. In particular, the expression of auxin-inducible SAUR genes was enhanced considerably at both elevated temperatures. Further, using genes that expressed inversely under heat vs. cold temperature conditions, we built a regulatory network between transcription factors (TF) such as HSFs, NAC, WRKYs, bHLHs or bZIPs and their target gene pairs and determined regulatory coordination in their expression under varying temperature conditions. Our work thus provides useful insights into temperature-responsive genes, particularly under elevated temperature conditions, and could serve as a resource of candidate genes associated with thermotolerance or downstream components of temperature sensors in rice.

Characterization of contrasting rice (Oryza sativa L.) genotypes reveals the Pi-efficient schema for phosphate starvation tolerance.

BACKGROUND: Phosphorus (P), being one of the essential components of nucleic acids, cell membranes and enzymes, indispensable for diverse cellular processes like photosynthesis/carbohydrate metabolism, energy production, redox homeostasis and signaling. Crop yield is severely affected due to Phosphate (Pi) deficiency; and to cope with Pi-deficiency, plants have evolved several strategies. Some rice genotypes are compatible with low Pi availability, whereas others are sensitive to Pi deficiency. However, the underlying molecular mechanism for low Pi tolerance remains largely unexplored. RESULT: Several studies were carried out to understand Pi-deficiency responses in rice at seedling stage, but few of them targeted molecular aspects/responses of Pi-starvation at the advanced stage of growth. To delineate the molecular mechanisms for low Pi tolerance, a pair of contrasting rice (Oryza sativa L.) genotypes [viz. Pusa-44 (Pi-deficiency sensitive) and its near isogenic line (NIL-23, Pi-deficiency tolerant) harboring Phosphorus uptake 1 (Pup1) QTL from an aus landrace Kasalath] were used. Comparative morphological, physiological, and biochemical analyses confirmed some of the well-known findings. Transcriptome analysis of shoot and root tissues from 45-day-old rice plants grown hydroponically under P-sufficient (16 ppm Pi) or P-starved (0 ppm Pi) medium revealed that Pi-starvation stress causes global transcriptional reprogramming affecting several transcription factors, signaling pathways and other regulatory genes. We could identify several significantly up-regulated genes in roots of NIL-23 under Pi-starvation which might be responsible for the Pi starvation tolerance. Pathway enrichment analysis indicated significant role of certain phosphatases, transporters, transcription factors, carbohydrate metabolism, hormone-signaling, and epigenetic processes in improving P-starvation stress tolerance in NIL-23. CONCLUSION: We report the important candidate mechanisms for Pi acquisition/solubilization, recycling, remobilization/transport, sensing/signalling, genetic/epigenetic regulation, and cell wall structural changes to be responsible for P-starvation tolerance in NIL-23. The study provides some of the novel information useful for improving phosphorus-use efficiency in rice cultivars.

Characterization of haplotypes and single nucleotide polymorphisms associated with Gn1a for high grain number formation in rice plant.

Rice serves as one of the essential staple food for half of the global human population. However, due to rapid human population growth, there is an increase in food demand across the globe. Thus, to lessen the gap between food demand and supply, there is an urgent requirement for grain yield enhancement in various important cereals crops, including rice. In the present study, the authors attempted to characterize haplotypes and single nucleotide polymorphisms associated with Gn1a for high grain number formation in rice plants. Result obtained reveals that high grain number gene sequences are under balancing selection and four high grain number specific missense SNPs decreases the stability of Gn1a. Earlier studies have also suggested that decreases Gn1a expression causes cytokinin accretion in inflorescence meristems, which in turn led to increase in grain yield. Hence, these four SNPs may be utilized for increasing grain yield in rice plants.

Tolerance mechanisms in maize identified through phenotyping and transcriptome analysis in response to water deficit stress.

Water deficit is a key limiting factor for maize (Zea mays L.) productivity. Elucidating the molecular regulatory networks of stress tolerance is crucial for genetic enhancement of drought tolerance. Two genotypes of maize contrasting in their yield response to water deficit were evaluated for tolerance traits of water relations, net CO2 assimilation rate, antioxidative metabolism and grain yield in relation to the expression levels, based on transcription profiling of genes involved in stress signaling, protein processing and energy metabolism to identify functional tolerance mechanisms. In the genotype SNJ201126 upregulation of calcium mediated signaling, plasma membrane and tonoplast intrinsic proteins and the membrane associated transporters contributed to better maintenance of water relations as evident from the higher relative water content and stomatal conductance at seedling and anthesis stages coupled with robust photosynthetic capacity and antioxidative metabolism. Further the protein folding machinery consisting of calnexin/calreticulin (CNX/CRT) cycle was significantly upregulated only in SNJ201126. While the down regulation of genes involved in photosystems and the enzymes of carbon fixation led to the relative susceptibility of genotype HKI161 in terms of reduced net CO2 assimilation rate, biomass and grain yield. Our results provide new insight into intrinsic functional mechanisms related to tolerance in maize. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01003-4.

Leaf mass area determines water use efficiency through its influence on carbon gain in rice mutants.

Saving water and enhancing rice productivity are consensually the most important research goals globally. While increasing canopy cover would enhance growth rates by higher photosynthetic carbon gain, an accompanied increase in transpiration would have a negative impact on saving water as well as for sustainability under water-limited conditions. Increased water use efficiency (WUE) by virtue of higher carbon assimilatory capacity can significantly circumvent this trade-off. Here, we report leaf mass area (LMA) has an important canopy architecture trait which when combined with superior carboxylation efficiency (CE) would achieve higher water productivity in rice. A set of 130 ethyl methanesulfonate induced mutants of an upland cultivar Nagina-22 (N22), was screened for leaf morphological traits leading to the identification of mutants differing in LMA. The wild-type, N22, along with a selected low-LMA (380-4-3) and two high-LMA mutants (392-9-1 and 457-1-3), all with comparable total leaf area, were raised under well-watered (100% Field Capacity (FC)) and water-limited (60% FC) conditions. Low Δ13 C and a higher RuBisCO content in high-LMA mutants indicated higher carboxylation efficiency, leading to increased carbon gain. Single parent backcross populations developed by crossing high and the low-LMA mutants with N22, separately, were screened for LMA, Δ13 C and growth traits. Comparison of dry matter accumulation per unit leaf area among the progenies differing in LMA and Δ13 C reiterated the association of LMA with CE. Results illustrated that high-LMA when combined with higher CE (low Δ13 C) lead to increased WUE and growth rates.

Differential Expression of Genes at Panicle Initiation and Grain Filling Stages Implied in Heterosis of Rice Hybrids.

RNA-Seq technology was used to analyze the transcriptome of two rice hybrids, Ajay (based on wild-abortive (WA)-cytoplasm) and Rajalaxmi (based on Kalinga-cytoplasm), and their respective parents at the panicle initiation (PI) and grain filling (GF) stages. Around 293 and 302 million high quality paired-end reads of Ajay and Rajalaxmi, respectively, were generated and aligned against the Nipponbare reference genome. Transcriptome profiling of Ajay revealed 2814 and 4819 differentially expressed genes (DEGs) at the PI and GF stages, respectively, as compared to its parents. In the case of Rajalaxmi, 660 and 5264 DEGs were identified at PI and GF stages, respectively. Functionally relevant DEGs were selected for validation through qRT-PCR, which were found to be co-related with the expression patterns to RNA-seq. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated significant DEGs enriched for energy metabolism pathways, such as photosynthesis, oxidative phosphorylation, and carbon fixation, at the PI stage, while carbohydrate metabolism-related pathways, such as glycolysis and starch and sucrose metabolism, were significantly involved at the GF stage. Many genes involved in energy metabolism exhibited upregulation at the PI stage, whereas the genes involved in carbohydrate biosynthesis had higher expression at the GF stage. The majority of the DEGs were successfully mapped to know yield related rice quantitative trait loci (QTLs). A set of important transcription factors (TFs) was found to be encoded by the identified DEGs. Our results indicated that a complex interplay of several genes in different pathways contributes to higher yield and vigor in rice hybrids.

Marker-assisted selection for grain number and yield-related traits of rice (Oryza sativa L.).

Continuous rise in the human population has resulted in an upsurge in food demand, which in turn demand grain yield enhancement of cereal crops, including rice. Rice yield is estimated via the number of tillers, grain number per panicles, and the number of spikes present per panicle. Marker-assisted selection (MAS) serve as one of the best ways to introduce QTLs/gene associated with yield in the rice plant. MAS has also been employed effectively in dissecting several other complex agricultural traits, for instance, drought, cold tolerance, salinity, etc. in rice plants. Thus, in this review, authors attempted to collect information about various genes/QTLs associated with high yield, including grain number, in rice and how different scheme of MAS can be employed to introduce them in rice (Oryza sativa L.) plant, which in turn will enhance rice yield. Information obtained to date suggest that, numerous QTLs, e.g., Gn1a, Dep1, associated with grain number and yield-related traits, have been identified either via mapping or cloning approaches. These QTLs have been successfully introduced into rice plants using various schemes of MAS for grain yield enhancement in rice. However, sometimes, MAS does not perform well in breeding, which might be due to lack of resources, skilled labors, reliable markers, and high costs associated with MAS. Thus, by overcoming these problems, we can enhance the application of MAS in plant breeding, which, in turn, may help us in increasing yield, which subsequently may help in bridging the gap between demand and supply of food for the continuously growing population.

Genes, pathways and transcription factors involved in seedling stage chilling stress tolerance in indica rice through RNA-Seq analysis.

BACKGROUND: Rice plants show yellowing, stunting, withering, reduced tillering and utimately low productivity in susceptible varieties under low temperature stress. Comparative transcriptome analysis was performed to identify novel transcripts, gain new insights into different gene expression and pathways involved in cold tolerance in rice. RESULTS: Comparative transcriptome analyses of 5 treatments based on chilling stress exposure revealed more down regulated genes in susceptible and higher up regulated genes in tolerant genotypes. A total of 13930 and 10599 differentially expressed genes (DEGs) were detected in cold susceptible variety (CSV) and cold tolerant variety (CTV), respectively. A continuous increase in DEGs at 6, 12, 24 and 48 h exposure of cold stress was detected in both the genotypes. Gene ontology (GO) analysis revealed 18 CSV and 28 CTV term significantly involved in molecular function, cellular component and biological process. GO classification showed a significant role of transcription regulation, oxygen, lipid binding, catalytic and hydrolase activity for tolerance response. Absence of photosynthesis related genes, storage products like starch and synthesis of other classes of molecules like fatty acids and terpenes during the stress were noticed in susceptible genotype. However, biological regulations, generation of precursor metabolites, signal transduction, photosynthesis, regulation of cellular process, energy and carbohydrate metabolism were seen in tolerant genotype during the stress. KEGG pathway annotation revealed more number of genes regulating different pathways resulting in more tolerant. During early response phase, 24 and 11 DEGs were enriched in CTV and CSV, respectively in energy metabolism pathways. Among the 1583 DEG transcription factors (TF) genes, 69 WRKY, 46 bZIP, 41 NAC, 40 ERF, 31/14 MYB/MYB-related, 22 bHLH, 17 Nin-like 7 HSF and 4C3H were involved during early response phase. Late response phase showed 30 bHLH, 65 NAC, 30 ERF, 26/20 MYB/MYB-related, 11 C3H, 12 HSF, 86 Nin-like, 41 AP2/ERF, 55 bZIP and 98 WRKY members TF genes. The recovery phase included 18 bHLH, 50 NAC, 31 ERF, 24/13 MYB/MYB-related, 4 C3H, 4 HSF, 14 Nin-like, 31 bZIP and 114 WRKY TF genes. CONCLUSIONS: Transcriptome analysis of contrasting genotypes for cold tolerance detected the genes, pathways and transcription factors involved in the stress tolerance.

Allele-specific analysis of single parent backcross population identifies HOX10 transcription factor as a candidate gene regulating rice root growth.

Understanding the molecular and physiological mechanisms of trait diversity is crucial for crop improvement to achieve drought adaptation. Root traits such as high biomass and/or deep rootedness are undoubtedly important drought adaptive traits. The major aim of this investigation was to functionally characterize a set of ethyl methane sulfonate-induced rice mutants for root traits. We report the identification of a high-root biomass mutant through a novel screening strategy for yield and Δ13 C measurements. The high-root mutant (392-9-1) thus identified, had a 66% higher root biomass compared to wild-type (Nagina-22). Better maintenance of leaf turgor and carbon assimilation rates resulted in lower drought susceptibility index in 392-9-1. Targeted resequencing revealed three non-synonymous single nucleotide variations in 392-9-1 for the genes HOX10, CITRATE SYNTHASE and ZEAXANTHIN EPOXIDASE. Segregation pattern of phenotype and mutant alleles in a single parent backcross F2 population revealed a typical 3:1 segregation for each of the mutant alleles. The number of F2 progeny with root biomass equal to or greater than that of 392-9-1 represented approximately one-third of the population indicating a major role played by HOX10 gene in regulating root growth in rice. Allele-specific Sanger sequencing in contrasting F2 progenies confirmed the co-segregation of HOX10 allele with the root biomass. The non-synonymous mutations in the other two genes did not reveal any specific pattern of co-segregation with root phenotype, indicating a strong role of HOX10, an upstream transcription factor, in regulating root biomass in rice.

Nutritional security through crop biofortification in India: Status & future prospects.

Malnutrition has emerged as one of the most serious health issues worldwide. The consumption of unbalanced diet poor in nutritional quality causes malnutrition which is more prevalent in the underdeveloped and developing countries. Deficiency of proteins, essential amino acids, vitamins and minerals leads to poor health and increased susceptibility to various diseases, which in turn lead to significant loss in Gross Domestic Product and affect the socio-economic structure of the country. Although various avenues such as dietary-diversification, food-fortification and medical-supplementation are available, biofortification of crop varieties is considered as the most sustainable and cost-effective approach where the nutrients reach the target people in natural form. Here, we have discussed the present status on the development of biofortified crop varieties for various nutritional and antinutritional factors. Ongoing programmes of the Indian Council of Agricultural Research on the improvement of nutritional traits in different crops have been presented. Challenges and future prospects of crop biofortification in India have also been discussed. The newly developed biofortified crop varieties besides serving as an important source for livelihood to poor people assume great significance in nutritional security.

RiceMetaSys for salt and drought stress responsive genes in rice: a web interface for crop improvement.

BACKGROUND: Genome-wide microarray has enabled development of robust databases for functional genomics studies in rice. However, such databases do not directly cater to the needs of breeders. Here, we have attempted to develop a web interface which combines the information from functional genomic studies across different genetic backgrounds with DNA markers so that they can be readily deployed in crop improvement. In the current version of the database, we have included drought and salinity stress studies since these two are the major abiotic stresses in rice. RESULTS: RiceMetaSys, a user-friendly and freely available web interface provides comprehensive information on salt responsive genes (SRGs) and drought responsive genes (DRGs) across genotypes, crop development stages and tissues, identified from multiple microarray datasets. 'Physical position search' is an attractive tool for those using QTL based approach for dissecting tolerance to salt and drought stress since it can provide the list of SRGs and DRGs in any physical interval. To identify robust candidate genes for use in crop improvement, the 'common genes across varieties' search tool is useful. Graphical visualization of expression profiles across genes and rice genotypes has been enabled to facilitate the user and to make the comparisons more impactful. Simple Sequence Repeat (SSR) search in the SRGs and DRGs is a valuable tool for fine mapping and marker assisted selection since it provides primers for survey of polymorphism. An external link to intron specific markers is also provided for this purpose. Bulk retrieval of data without any limit has been enabled in case of locus and SSR search. CONCLUSIONS: The aim of this database is to facilitate users with a simple and straight-forward search options for identification of robust candidate genes from among thousands of SRGs and DRGs so as to facilitate linking variation in expression profiles to variation in phenotype. Database URL: http://14.139.229.201.

Development and validation of cross-transferable and polymorphic DNA markers for detecting alien genome introgression in Oryza sativa from Oryza brachyantha.

African wild rice Oryza brachyantha (FF), a distant relative of cultivated rice Oryza sativa (AA), carries genes for pests and disease resistance. Molecular marker assisted alien gene introgression from this wild species to its domesticated counterpart is largely impeded due to the scarce availability of cross-transferable and polymorphic molecular markers that can clearly distinguish these two species. Availability of the whole genome sequence (WGS) of both the species provides a unique opportunity to develop markers, which are cross-transferable. We observed poor cross-transferability (~0.75 %) of O. sativa specific sequence tagged microsatellite (STMS) markers to O. brachyantha. By utilizing the genome sequence information, we developed a set of 45 low cost PCR based co-dominant polymorphic markers (STS and CAPS). These markers were found cross-transferrable (84.78 %) between the two species and could distinguish them from each other and thus allowed tracing alien genome introgression. Finally, we validated a Monosomic Alien Addition Line (MAAL) carrying chromosome 1 of O. brachyantha in O. sativa background using these markers, as a proof of concept. Hence, in this study, we have identified a set molecular marker (comprising of STMS, STS and CAPS) that are capable of detecting alien genome introgression from O. brachyantha to O. sativa.

Single nucleotide polymorphism in sugar pathway and disease resistance genes in sugarcane.

KEY MESSAGE: Single nucleotide polymorphism in sugar pathway and disease resistance genes showing genetic association with sugar content and red rot resistance would be useful in marker-assisted genetic improvement of sugarcane. Validation and genotyping of potential sequence variants in candidate genes are necessary to understand their functional significance and trait association potential. We discovered, characterized, validated and genotyped SNPs and InDels in sugar pathway and disease resistance genes of Saccharum complex and sugarcane varieties using amplicon sequencing and CAPS assays. The SNPs were abundant in the non-coding 3'UTRs than 5'UTRs and coding sequences depicting a strong bias toward C to T transition substitutions than transversions. Sequencing of cloned amplicons validated 61.6 and 45.2 % SNPs detected in silico in 21 sugar pathway and 16 disease resistance genes, respectively. Sixteen SNPs in four sugar pathway genes and 10 SNPs in nine disease resistance genes were validated through cost-effective CAPS assay. Functional and adaptive significance of SNP and protein haplotypes identified in sugar pathway and disease resistance genes was assessed by correlating their allelic variation with missense amino acid substitutions in the functional domains, alteration in protein structure models and possible modulation of catalytic enzyme activity in contrasting high and low sugar and moderately red rot resistant and highly susceptible sugarcane genotypes. A strong genetic association of five SNPs in the sugar pathway and disease resistance genes, and an InDel marker in the promoter sequence of sucrose synthase-2 gene, with sugar content and red rot resistance, was evident. The functionally relevant SNPs and InDels, detected and validated in sugar pathway and disease resistance genes, and genic CAPS markers designed, would be of immense use in marker-assisted genetic improvement of sugarcane for sugar content and disease resistance.

Differentiation and description of aromatic short grain rice landraces of eastern Indian state of Odisha based on qualitative phenotypic descriptors.

BACKGROUND: Speciality rice, in general, and aromatic rice in particular, possess enormous market potential for enhancing farm profits. However, systematic characterization of the diversity present in this natural wealth is a major pre requisite for using it in the breeding programs. This study reports qualitative phenotypic trait based characterization of 126 short grain aromatic rice genotypes, collected from different areas of the state of Odisha, India. RESULTS: Out of the 24 descriptors employed, highest variability (8 different types) was observed for lemma-palea colour with a genetic diversity index (He) of 0.696. The principal component analysis reveals that the tip colour of lemma, colour of awn and colour of stigma, cumulatively explain 74 % of the total variation. The Population STRUCTURE analysis classified the population into two subpopulations which were subdivided further into four distinct groups. The western and southern districts of Odisha are endowed with maximum diversity in comparison to eastern and northern districts and at district level comparisons, Koraput and Puri districts are rich with a genetic diversity values of 0.324 and 0.303 respectively. With this set of morphological qualitative traits, based on 'phenoprinting', a newly proposed bar coding system, unique fingerprints of each genotype can be effectively generated that can help in easy identification of these genotypes. CONCLUSION: Though aromatic rices represent a tiny fraction of the total rice germplasm, a small collection of 126 land races did exhibit rich diversity for all the qualitative traits. For lemma-palea colour, eight different types were detected while for tip colour of lemma, six different types were recorded, suggesting the presence of rich variability in short grain aromatic rices that are conserved in this region. The proposed 'phenoprinting' can be an effective descriptor with the unique finger prints generated for each genotype and coupled with molecular (DNA) finger printing, we can discriminate and identify each and every aromatic short grain rice genotype. The proposed system not only help in conservation but also can confer IPR protection to these specialty rices.