Genetic mapping of regions associated with root system architecture in rice using MutMap QTL-seq.

The root system architecture is an important complex trait in rice. With changing climatic conditions and soil nutrient deficiencies, there is an immediate need to breed nutrient-use-efficient rice varieties with robust root system architectural (RSA) traits. To map the genomic regions associated with crucial component traits of RSA viz. root length and root volume, a biparental F2 mapping population was developed using TI-128, an Ethyl Methane Sulphonate (EMS) mutant of a mega variety BPT-5204 having high root length (RL) and root volume (RV) with wild type BPT-5204. Extreme bulks having high RL and RV and low RL and RV were the whole genome re-sequenced along with parents. Genetic mapping using the MutMap QTL-Seq approach elucidated two genomic intervals on Chr.12 (3.14-3.74 Mb, 18.11-20.85 Mb), and on Chr.2 (23.18-23.68 Mb) as potential regions associated with both RL and RV. The Kompetitive Allele Specific PCR (KASP) assays for SNPs with delta SNP index near 1 were associated with higher RL and RV in the panel of sixty-two genotypes varying in root length and volume. The KASP_SNPs viz. Chr12_S4 (C→T; Chr12:3243938), located in the 3' UTR region of LOC_Os12g06670 encoding a protein kinase domain-containing protein and Chr2_S6 (C→T; Chr2:23181622) present upstream in the regulator of chromosomal condensation protein LOC_Os2g38350. Validation of these genes using qRT-PCR and in-silico studies using various online tools and databases revealed higher expression in TI-128 as compared to BPT- 5204 at the seedling and panicle initiation stages implying the functional role in enhancing RL and RV.

Multiomics-assisted characterization of rice-Yellow Stem Borer interaction provides genomic and mechanistic insights into stem borer resistance in rice.

KEY MESSAGE: By deploying a multi-omics approach, we unraveled the mechanisms that might help rice to combat Yellow Stem Borer infestation, thus providing insights and scope for developing YSB resistant rice varieties. Yellow Stem Borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), is a major pest of rice, that can lead to 20-60% loss in rice production. Effective management of YSB infestation is challenged by the non-availability of adequate sources of resistance and poor understanding of resistance mechanisms, thus necessitating studies for generating resources to breed YSB resistant rice and to understand rice-YSB interaction. In this study, by using bulk-segregant analysis in combination with next-generation sequencing, Quantitative Trait Loci (QTL) intervals in five rice chromosomes were mapped that could be associated with YSB resistance at the vegetative phase in a resistant rice line named SM92. Further, multiple SNP markers that showed significant association with YSB resistance in rice chromosomes 1, 5, 10, and 12 were developed. RNA-sequencing of the susceptible and resistant lines revealed several genes present in the candidate QTL intervals to be differentially regulated upon YSB infestation. Comparative transcriptome analysis revealed a putative candidate gene that was predicted to encode an alpha-amylase inhibitor. Analysis of the transcriptome and metabolite profiles further revealed a possible link between phenylpropanoid metabolism and YSB resistance. Taken together, our study provides deeper insights into rice-YSB interaction and enhances the understanding of YSB resistance mechanism. Importantly, a promising breeding line and markers for YSB resistance have been developed that can potentially aid in marker-assisted breeding of YSB resistance among elite rice cultivars.

Strong culm: a crucial trait for developing next-generation climate-resilient rice lines.

Lodging, a phenomenon characterized by the bending or breaking of rice plants, poses substantial constraints on productivity, particularly during the harvesting phase in regions susceptible to strong winds. The rice strong culm trait is influenced by the intricate interplay of genetic, physiological, epigenetic, and environmental factors. Stem architecture, encompassing morphological and anatomical attributes, alongside the composition of both structural and non-structural carbohydrates, emerges as a critical determinant of lodging resistance. The adaptive response of the rice culm to various biotic and abiotic environmental factors further modulates the propensity for lodging. Advancements in next-generation sequencing technologies have expedited the genetic dissection of lodging resistance, enabling the identification of pertinent genes, quantitative trait loci, and novel alleles. Concurrently, contemporary breeding strategies, ranging from biparental approaches to more sophisticated methods such as multi-parent-based breeding, gene pyramiding, genomic selection, genome-wide association studies, and haplotype-based breeding, offer perspectives on the genetic underpinnings of culm strength. This review comprehensively delves into physiological attributes, culm histology, epigenetic determinants, and gene expression profiles associated with lodging resistance, with a specialized focus on leveraging next-generation sequencing for candidate gene discovery.

Impairment in a member of AP2/ERF and F-box family protein enhances complete panicle exsertion, an economically important trait in rice.

Complete panicle exsertion (CPE) is an economically important quantitative trait that contributes to grain yield in rice. We deployed an integrated approach for understanding the molecular mechanism of CPE using a stable EMS mutant line, CPE-109 of Samba Mahsuri (SM) exhibiting CPE. Two consistent genomic regions have been identified for CPE through QTL mapping [qCPE-4 (28.24-31.22 Mb) and qCPE-12 (2.30-3.18 Mb)] and QTL-sequencing [Chr-4 (31.21-33.69 Mb) and Chr-12 (0.12-3.15 Mb)]. Two non-synonymous SNPs, viz; KASP 12-12 (T→C; Chr12:1269983) in Os12g0126300; AP2/ERF transcription factor and KASP 12-16 (G→A; Chr12:1515198) in Os12g0131400; F-box domain-containing protein explained 81.05 and 59.61% phenotypic variance respectively and exhibited strong co-segregation with CPE in F2 mapping populations, advanced generation lines and CPE exhibiting SM mutants through KASP assays. The downregulation of these genes in CPE-109 compared to SM was observed in transcriptome sequencing of flag leaves which was validated through qRT-PCR. We propose that the abrogation of Os12g0126300 and Os12g0131400 in CPE-109 combinatorially influences the downregulation of ethylene biosynthetic genes viz. ACC synthase, ethylene-responsive factor-2, and up-regulation of gibberellic acid synthetic genes viz. ent-kaurene synthase and two cytokinin biosynthesis genes viz. cytokinin-O-glucosyltransferase 2, carboxy-lyase which result in complete panicle exsertion.

Long non-coding RNA-mediated epigenetic response for abiotic stress tolerance in plants.

Plants perceive environmental fluctuations as stress and confront several stresses throughout their life cycle individually or in combination. Plants have evolved their sensing and signaling mechanisms to perceive and respond to a variety of stresses. Epigenetic regulation plays a critical role in the regulation of genes, spatiotemporal expression of genes under stress conditions and imparts a stress memory to encounter future stress responses. It is quintessential to integrate our understanding of genetics and epigenetics to maintain plant fitness, achieve desired genetic gains with no trade-offs, and durable long-term stress tolerance. The long non-coding RNA >200 nts having no coding potential (or very low) play several roles in epigenetic memory, contributing to the regulation of gene expression and the maintenance of cellular identity which include chromatin remodeling, imprinting (dosage compensation), stable silencing, facilitating nuclear organization, regulation of enhancer-promoter interactions, response to environmental signals and epigenetic switching. The lncRNAs are involved in a myriad of stress responses by activation or repression of target genes and hence are potential candidates for deploying in climate-resilient breeding programs. This review puts forward the significant roles of long non-coding RNA as an epigenetic response during abiotic stresses in plants and the prospects of deploying lncRNAs for designing climate-resilient plants.

Combined strategy employing MutMap and RNA-seq reveals genomic regions and genes associated with complete panicle exsertion in rice.

Complete panicle exsertion (CPE) in rice is an important determinant of yield and a desirable trait in breeding. However, the genetic basis of CPE in rice still remains to be completely characterized. An ethyl methane sulfonate (EMS) mutant line of an elite cultivar Samba Mahsuri (BPT 5204), displaying stable and consistent CPE, was identified and named as CPE-110. MutMap and RNA-seq were deployed for unraveling the genomic regions, genes, and markers associated with CPE. Two major genomic intervals, on chromosome 8 (25668481-25750456) and on chromosome 11 (20147154-20190400), were identified to be linked to CPE through MutMap. A non-synonymous SNP (G/A; Chr8:25683828) in the gene LOC_Os08g40570 encoding pyridoxamine 5'-phosphate oxidase with the SNP index 1 was converted to Kompetitive allele-specific PCR (KASP) marker. This SNP (KASP 8-1) exhibited significant association with CPE and further validated through assay in the F2 mapping population, released varieties and CPE exhibiting BPT 5204 mutant lines. RNA-seq of the flag leaves at the booting stage, 1100 genes were upregulated and 1305 downregulated differentially in CPE-110 and BPT 5204. Metabolic pathway analysis indicated an enrichment of genes involved in photosynthesis, glyoxylate, dicarboxylate, porphyrin, pyruvate, chlorophyll, carotenoid, and carbon metabolism. Further molecular and functional studies of the candidate genes could reveal the mechanistic aspects of CPE. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01412-1.

Genome-wide association studies in rice germplasm reveal significant genomic regions for root and yield-related traits under aerobic and irrigated conditions.

The development of nutrient-use efficient rice lines is a priority amidst the changing climate and depleting resources viz., water, land, and labor for achieving sustainability in rice cultivation. Along with the traditional transplanted irrigated system of cultivation, the dry direct-seeded aerobic system is gaining ground nationwide. The root-related traits play a crucial role in nutrient acquisition, adaptation and need to be concentrated along with the yield-attributing traits. We phenotyped an association panel of 118 rice lines for seedling vigour index (SVI) traits at 14 and 21 days after sowing (DAS), root-related traits at panicle initiation (PI) stage in polythene bags under controlled aerobic condition, yield and yield-related traits under the irrigated condition at ICAR-IIRR, Hyderabad, Telangana; irrigated and aerobic conditions at ARS, Dhadesugur, Raichur, Karnataka. The panel was genotyped using simple sequence repeats (SSR) markers and genome-wide association studies were conducted for identifying marker-trait associations (MTAs). Significant correlations were recorded for root length, root dry weight with SVI, root volume at the PI stage, number of productive tillers per plant, spikelet fertility, the total number of grains per panicle with grain yield per plant under irrigated conditions, and the total number of grains per panicle with grain yield per plant under aerobic condition. The panel was divided into three sub-groups (K = 3) and correlated with the principal component analysis. The maximum number of MTAs were found on chromosomes 2, 3, and 12 with considerable phenotypic variability. Consistent MTAs were recorded for SVI traits at 14 and 21 DAS (RM25310, RM80, RM22961, RM1385), yield traits under irrigated conditions (RM2584, RM5179, RM410, RM20698, RM14753) across years at ICAR-IIRR, grain yield per plant (RM22961, RM1146) under the aerobic condition, grain yield per plant at irrigated ICAR-IIRR and SVI (RM5501), root traits at PI stage (RM2584, RM80, RM410, RM1146, RM18472). Functionally relevant genes near the MTAs through in-silico expression analysis in root and panicle tissues viz., HBF2 bZIP transcription factor, WD40 repeat-like domain, OsPILS6a auxin efflux carrier, WRKY108, OsSCP42, OsMADS80, nodulin-like domain-containing protein, amino acid transporter using various rice expression databases were identified. The identified MTAs and rice lines having high SVI traits (Langphou, TI-128, Mouli, TI-124, JBB-631-1), high yield under aerobic (Phouren, NPK-43, JBB-684, Ratnamudi, TI-112), irrigated conditions (KR-209, KR-262, Phouren, Keibi-Phou, TI-17), robust root traits like root length (MoirangPhou-Angouba, Wangoo-Phou, JBB-661, Dissi, NPK-45), root volume (Ratnachudi, KJ-221, Mow, Heimang-Phou, PUP-229) can be further employed in breeding programs for the targeted environments aimed at improving seedling vigour, yield-related traits under irrigated condition, aerobic condition as adaptability to water-saving technology.

Draft Genome Sequence of Paenibacillus sonchi IIRRBNF1, a Nitrogen-Fixing and Plant Growth-Promoting Bacterium Isolated from Rice Rhizosphere.

Paenibacillus sonchi IIRRBNF1 is a rice-rhizospheric, endospore-forming, Gram-positive, plant growth-promoting rhizobacterium. Here, we report the draft genome sequence of Paenibacillus sonchi IIRRBNF1, which consists of an∼7.3-Mb (7,323,556-bp) genome with 6,271 coding sequences (CDSs), 13 rRNAs, and 67 tRNAs. The genome reveals the presence of a nitrogen-fixing gene cluster and genes associated with multiple plant growth-promoting traits.

Performance of Novel Antimicrobial Protein Bg_9562 and In Silico Predictions on Its Properties with Reference to Its Antimicrobial Efficiency against Rhizoctonia solani.

Bg_9562 is a potential broad-spectrum antifungal effector protein derived from the bacteria Burkholderia gladioli strain NGJ1 and is effective against Rhizoctonia solani, the causal agent of sheath blight in rice. In the present study, in vitro antifungal assays showed that Bg_9562 was efficient at 35 °C and 45 °C and ineffective either at high acidic pH (3.0) or alkaline pH (9.5) conditions. Compatibility studies between the native bioagents Trichoderma asperellum TAIK1 and Bacillus subtilis BIK3 indicated that Bg_9562 was compatible with the bioagents. A field study using foliar spray of the Bg_9562 protein indicated the need of formulating the protein before its application. In silico analysis predicted that Bg_9562 possess 111 amino acid residues (46 hydrophobic residues, 12 positive and 8 negative residues) with the high aliphatic index of 89.92, attributing to its thermostability with a half-life of 30 h. Bg_9562 (C491H813N137O166S5) possessed a protein binding potential of 1.27 kcal/mol with a better possibility of interacting and perturbing the membrane, the main target for antimicrobial proteins. The secondary structure revealed the predominance of random coils in its structure, and the best 3D model of Bg_9562 was predicted using an ab initio method with Robetta and AlphaFold 2. The predicted binding ligands were nucleic acids and zinc with confidence scores of 0.07 and 0.05, respectively. The N-terminal region (1-14 residues) and C-terminal region (101 to 111) of Bg_9562 residues were predicted to be disordered regions. Stability and binding properties of the protein from the above studies would help to encapsulate Bg_9562 using a suitable carrier to maintain efficiency and improve delivery against Rhizoctonia solani in the most challenging rice ecosphere.

Whole genome sequencing data of native isolates of Bacillus and Trichoderma having potential biocontrol and plant growth promotion activities in rice.

Six native isolates of Trichoderma and Bacillus having potential for biocontrol and plant growth-promoting activities in rice were isolated from different rice growing regions of India. These isolates were screened for their efficiency in both in vitro and in vivo conditions for three years. The identity of the isolates was confirmed both by morphological and molecular characterization. Three Bacillus spp. viz., Bacillus velenzensis strain BIK2, Bacillus cabrialesii strain BIK3 and Bacillus paralicheniformis strain BIK4 and Trichoderma spp. viz., Trichoderma asperellum strain TAIK1, and T. asperellum strain TAIK5, native to the Telangana state, in Southern India except for strain TAIK4 (Rewa district in the state of Madhya Pradesh in Central India). These promising isolates were subjected for whole genome sequencing using the Illumina platform and data was presented. The data was emanated for Trichoderma asperellum (TAIK1), Trichoderma asperellum (TAIK4), Trichoderma asperellum (TAIK5), Bacillus velezensis (BIK2), Bacillus cabrialesii (BIK3) and Bacillus paralicheniformis (BIK4) isolates had an average 100X coverage of 109X, 150X and 116X; 1447X, 905X and 585X respectively. Further studies on the annotation of the data obtained in correlation with the lab and field performance of these microbes would enable them to be used in metagenomics studies to compare their performance under natural conditions with different microbiota and popular rice varieties. Bioformulation of these strains would be more appropriate with the availability of this genomic data.

Current technological interventions and applications of CRISPR/Cas for crop improvement.

Efficient and innovative breeding strategies are immensely required to meet the global food demand, nutritional security and sustainable agriculture. Genome editing tools have emerged as an effective technology for site-directed genome modification causing the change in gene expression and protein function for the improvement of various important traits in particular the CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein). As the technology evolved with time, advances have been observed like prime editing, base editing, PAMless editing, Drosha based editing with multiple targets having the potential to fulfill the regulatory processes around the world. These recent interventions are highly proficient, cost-efficient, user-friendly, and holds promise for a major revolution in basic and applied plant biology research in the ever-evolving climatic conditions. In the review, we have discussed the most recent technologies and advances for CRISPR/Cas editing in plants.

The Draft Genome of Yellow Stem Borer, an Agriculturally Important Pest, Provides Molecular Insights into Its Biology, Development and Specificity Towards Rice for Infestation.

Yellow stem borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), a major monophagous insect pest of rice, causes significant yield losses. The rice-YSB interaction is very dynamic, making it difficult for management. The development of resistant lines has been unsuccessful as there are no effective resistant sources in the germplasm. Genome information is necessary for a better understanding of interaction with rice in terms of its recognition, response, and infestation mechanism. The draft genome of YSB is predicted to have 46,057 genes with an estimated size of 308 Mb, being correlated with the flow cytometry analysis. The existence of complex metabolic mechanisms and genes related to specific behavior was identified, being conditioned by a higher level of regulation. We deciphered the possible visual, olfactory, and gustatory mechanisms responsible for its evolution as a monophagous pest. Comparative genomic analysis revealed that YSB is unique in the way it has evolved. The obvious presence of high-immunity-related genes, well-developed RNAi machinery, and diverse effectors provides a means for developing genomic tools for its management. The identified 21,696 SSR markers can be utilized for diversity analysis of populations across the rice-growing regions. We present the first draft genome of YSB. The information emanated paves a way for biologists to design novel pest management strategies as well as for the industry to design new classes of safer and specific insecticide molecules.

Down regulation of transcripts involved in selective metabolic pathways as an acclimation strategy in nitrogen use efficient genotypes of rice under low nitrogen.

To understand the molecular mechanism of nitrogen use efficiency (NUE) in rice, two nitrogen (N) use efficient genotypes and two non-efficient genotypes were characterized using transcriptome analyses. The four genotypes were evaluated for 3 years under low and recommended N field conditions for 12 traits/parameters of yield, straw, nitrogen content along with NUE indices and 2 promising donors for rice NUE were identified. Using the transcriptome data generated from GS FLX 454 Roche and Illumina HiSeq 2000 of two efficient and two non-efficient genotypes grown under field conditions of low N and recommended N and their de novo assembly, differentially expressed transcripts and pathways during the panicle development were identified. Down regulation was observed in 30% of metabolic pathways in efficient genotypes and is being proposed as an acclimation strategy to low N. Ten sub metabolic pathways significantly enriched with additional transcripts either in the direction of the common expression or contra-regulated to the common expression were found to be critical for NUE in rice. Among the up-regulated transcripts in efficient genotypes, a hypothetical protein OsI_17904 with 2 alternative forms suggested the role of alternative splicing in NUE of rice and a potassium channel SKOR transcript (LOC_Os06g14030) has shown a positive correlation (0.62) with single plant yield under low N in a set of 16 rice genotypes. From the present study, we propose that the efficient genotypes appear to down regulate several not so critical metabolic pathways and divert the thus conserved energy to produce seed/yield under long-term N starvation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02631-5.

Mapping QTL hotspots associated with weed competitive traits in backcross population derived from Oryza sativa L. and O. glaberrima Steud.

To improve grain yield under direct seeded and aerobic conditions, weed competitive ability of a rice genotype is a key desirable trait. Hence, understanding and dissecting weed competitive associated traits at both morphological and molecular level is important in developing weed competitive varieties. In the present investigation, the QTLs associated with weed competitive traits were identified in BC1F2:3 population derived from weed competitive accession of O. glaberrima (IRGC105187) and O. sativa cultivar IR64. The mapping population consisting of 144 segregating lines were phenotyped for 33 weed competitive associated traits under direct seeded condition. Genetic analysis of weed competitive traits carried out in BC1F2:3 population showed significant variation for the weed competitive traits and predominance of additive gene action. The population was genotyped with 81 genome wide SSR markers and a linkage map covering 1423 cM was constructed. Composite interval mapping analysis identified 72 QTLs linked to 33 weed competitive traits which were spread on the 11 chromosomes. Among 72 QTLs, 59 were found to be major QTLs (> 10% PVE). Of the 59 major QTLs, 38 had favourable allele contributed from the O. glaberrima parent. We also observed nine QTL hotspots for weed competitive traits (qWCA2a, qWCA2b, qWCA2c, qWCA3, qWCA5, qWCA7, qWCA8, qWCA9, and qWCA10) wherein several QTLs co-localised. Our study demonstrates O. glaberrima species as potential source for improvement for weed competitive traits in rice and identified QTLs hotspots associated with weed competitive traits.

RNA-seq reveals the involvement of key genes for aerobic adaptation in rice.

Adaptation of rice to the aerobic condition is needed to cope with the water scarcity as well as to ensure sustainable yield in future. To understand the molecular mechanisms responsible for aerobic adaptation in rice, we performed RNA-seq analysis of root and shoot i.e. developing panicle tissues at panicle initiation stage in two cultivars adapted to aerobic (CR Dhan 202) and traditional transplanted anaerobic (BPT 5204) conditions. The RNA-seq data emanated from 1.65 billion clean reads with approximately 37 million reads per sample. The number of differentially expressed transcripts was higher in the root than that in the shoot under both aerobic and anaerobic conditions. The transcription factors viz. MADS4, MADS5, MADS6, MADS7, MADS15 and transporters involved in sugar (SWEET3A) and nutrient uptake (PHT1;6, MDR-like ABC and vacuolar iron transporter homolog 2) were highly and uniquely expressed in the aerobic adapted cultivar (AAC) CR Dhan 202 under aerobic condition indicating their role in adaptation. The hormones such as ethylene and abscisic acid might be significantly involved in imparting aerobic adaptation. The higher expression of root related genes in the AAC under aerobic conditions suggests the involvement and sensitivity of roots to the water limiting condition. The metabolic activities are also more pronounced in the roots which impart rigorous plant establishment under the aerobic condition. The presence of alternative splice variants in the transcripts viz. Tetratrico peptide repeat (TPR) domain containing protein and GOLDEN2-LIKE1 (GLK1) additionally confirms that post transcriptional regulation is also crucial for aerobic adaptation. The QTLs related to root traits and stress tolerance harboring the uniquely expressed genes, which were identified in the present study can be deployed in molecular breeding programs to develop elite, high yielding aerobic rice cultivars.

Studies on root anatomy, morphology and physiology of rice grown under aerobic and anaerobic conditions.

With the changing climate and rainfall abrasions, there is a gradual shift in the system of rice cultivation from traditional transplanted anaerobic to aerobic system. Studies on the root anatomical and morpho-physiological traits provide insights about the adaptation under aerobic conditions. We investigated the root anatomical and morpho-physiological traits in anaerobic (BPT 5204) and aerobic (CR Dhan 202) adapted rice genotypes grown under anaerobic and aerobic conditions. It was observed that the formation of fewer aerenchyma, thickened root and larger xylem area were critical anatomical traits associated with aerobic adaptation as compared to anaerobic conditions. The root length of CR Dhan 202 significantly increased under aerobic condition which may be attributed to its aerobic adaptation in terms of water acquisition. The photosynthetic rate was significantly higher in CR Dhan 202 as compared to that of BPT 5204 under the aerobic condition. The morpho-physiological results showed that the root length, total dry weight and photosynthetic rate are the key parameters for imparting aerobic adaptation. These root anatomical and morpho-physiological traits associated with the adaptation can be used as screening criteria for phenotyping and selection of genotypes suitable for aerobic system of cultivation. Such study is expected to expedite the development of rice aerobic varieties in aerobic breeding programmes.

New breeding technique "genome editing" for crop improvement: applications, potentials and challenges.

Crop improvement is a continuous process in agriculture which ensures ample supply of food, fodder and fiber to burgeoning world population. Despite tremendous success in plant breeding and transgenesis to improve the yield-related traits, there have been several limitations primarily with the specificity in genetic modifications and incompatibility of host species. Because of this, new breeding techniques (NBTs) are gaining worldwide attention for crop improvement programs. Among the NBTs, genome editing (GE) using site-directed nucleases (SDNs) is an important and potential technique that overcomes limitations associated with classical breeding and transgenesis. These SDNs specifically target a compatible region in the gene/genome. The meganucleases (MgN), zinc finger nucleases (ZFN), transcription activator-like effectors nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonuclease (Cas) are being successfully employed for GE. These can be used for desired or targeted modifications of the native endogenous gene(s) or targeted insertion of cis/trans elements in the genomes of recipient organisms. Applications of these techniques appear to be endless ever since their discovery and several modifications in original technologies have further brought precision and accuracy in these methods. In this review, we present an overview of GE using SDNs with an emphasis on CRISPR/Cas system, their advantages, limitations and also practical considerations while designing experiments have been discussed. The review also emphasizes on the possible applications of CRISPR for improving economic traits in crop plants.

Genes encoding membrane proteins showed stable expression in rice under aerobic condition: novel set of reference genes for expression studies.

A set of reference genes expressing stably under aerobic and anaerobic conditions in rice is essential to execute omics studies relating to aerobic adaptations. Stability of expression of ten rice reference genes, viz. Actin, eEF-1a, eIF-5C, Exp1, Exp2, Memp, SKP1A, TF-SUI1, TPH, and UBQ5 was validated across six experimental sets in shoot and root tissues at seedling, tillering, and panicle initiation stages. Comprehensively, Memp (Membrane protein), TPH (Tumor protein homolog), and Exp1 (Expressed protein) were revealed as the most stable set with acceptable M and V value according to the gold standards of qRT-PCR using various algorithms/tools. The identified set of reference genes was validated using root trait genes, which showed concurrence with the functional expression patterns in the aerobic and anaerobic adapted cultivars. The Memp (Membrane protein), TPH (Tumor protein homolog), and Exp1 (Expressed protein) genes are the most stable reference genes across the root and shoot at various developmental stages under aerobic and anaerobic conditions in rice. This is the first study for accurate and reliable relative gene expression analysis in rice grown in aerobic and anaerobic conditions.

RNA-seq of Rice Yellow Stem Borer Scirpophaga incertulas Reveals Molecular Insights During Four Larval Developmental Stages.

The yellow stem borer (YSB), Scirpophaga incertulas, is a prominent pest in rice cultivation causing serious yield losses. The larval stage is an important stage in YSB, responsible for maximum infestation. However, limited knowledge exists on the biology and mechanisms underlying the growth and differentiation of YSB. To understand and identify the genes involved in YSB development and infestation, so as to design pest control strategies, we performed de novo transcriptome analysis at the first, third, fifth, and seventh larval developmental stages employing Illumina Hi-seq. High-quality reads (HQR) of ∼229 Mb were assembled into 24,775 transcripts with an average size of 1485 bp. Genes associated with various metabolic processes, i.e., detoxification mechanism [CYP450, GSTs, and carboxylesterases (CarEs)], RNA interference (RNAi) machinery (Dcr-1, Dcr-2, Ago-1, Ago-2, Sid-1, Sid-2, Sid-3, and Sid-1-related gene), chemoreception (CSPs, GRs, OBPs, and ORs), and regulators [transcription factors (TFs) and hormones] were differentially regulated during the developmental stages. Identification of stage-specific transcripts made it possible to determine the essential processes of larval development. Comparative transcriptome analysis revealed that YSB has not evolved much with respect to the detoxification mechanism, but showed the presence of distinct RNAi machinery. The presence of strong specific visual recognition coupled with chemosensory mechanisms supports the monophagous nature of YSB. Designed expressed sequenced tags-simple-sequence repeats (EST-SSRs) will facilitate accurate estimation of the genetic diversity of YSB. This is the first report on characterization of the YSB transcriptome and the identification of genes involved in key processes, which will help researchers and industry to devise novel pest control strategies. This study also opens up a new avenue to develop next-generation resistant rice using RNAi or genome editing approaches.

CRISPR/Cas9: a promising way to exploit genetic variation in plants.

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.