Satendra K. Mangrauthia

Genome-wide changes in microRNA expression during short and prolonged heat stress and recovery in contrasting rice cultivars

Highlight Heat susceptible and tolerant rice genotypes have different landscapes of miRNAs and the tolerant genotypes show efficient recovery mediated by miRNAs. Roots regulate miRNAs more sensitively than shoots during heat stress.

Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas

RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of chemically synthesized 5′ FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12–15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach.

Phenotyping Rice for Molecular Plant Breeding

Rice is an important food crop, has the plasticity in growing in different ecologies in many countries around the world, which makes this crop to expose to many diseases and pests. The recent development in the genomics has led to the intensive efforts in molecular breeding for improvements of some of the qualitative traits. To make the successful molecular breeding programme, accurate phenotyping techniques need to be coupled with high-throughput genotyping. The chapter discusses the various phenotypic methods available for different diseases, pests and abiotic stress like drought.

Transcriptome Analysis of Oryza sativa (Rice) Seed Germination at High Temperature Shows Dynamics of Genome Expression Associated with Hormones Signalling and Abiotic Stress Pathways

Seed germination directly affects growth and yield of rice. Germination process is highly dependent on temperature which regulates expression of several genes and proteins in germinating tissue. Whole genome mRNA sequencing of rice germinated at optimum (30 °C) and high temperature (42 °C) was performed using Ion Torrent Sequencing. Bioinformatics analysis of sequence data was done to survey the expressed genes, gene ontology, and pathway analysis. High temperature altered the expression of genes involved in fatty acid, amino acid, carbohydrate, and energy metabolism. Genes involved in reserve degradation, protein folding and stress responses were also significantly affected. Induced expression of genes like dehydration responsive element binding (DREB), RAB (responsive to ABA), and late embryogenesis abundant (LEA) proteins suggested important role of these proteins in high temperature germinated (HTG) rice. The pathway analysis of differentially expressed genes at high temperature suggested the altered expression of transcripts associated with ABA and jasmonate signalling, antioxidants (peroxidase and ascorbate peroxidase), heat shock proteins (HSP 20 and HSP70 family), heat shock protein binding protein1 (HSPBP1, HSP70-interacting protein) and other stress associated pathways. In present scenario where very limited information is available on genome wide transcripts expression during germination process, the transcriptome study done in rice provides an opportunity to better understand the genes expression and regulation of seed germination, which is highly influenced by temperature.

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica

AbstractKey messagePollen-specific expression.AbstractPromoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 5′ deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 5′ promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3-∆1 (1504xa0bp), OSIPP3-∆2 (968xa0bp), OSIPP3-∆3 (388xa0bp) and OSIPP3-∆4 (286xa0bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated thatnthey were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504- and 968-bp fragments directed GUS expression in roots and anthers, while the 388- and 286-bp fragments restricted the GUS expression to only pollen, of which 388xa0bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1–P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-Δ1 followed by OSIPP3-Δ2 (eightfold) and OSIPP3-Δ3 (threefold) when compared to OSIPP3-Δ4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.

Nagina 22 mutants tolerant or sensitive to low P in field show contrasting response to double P in hydroponics and pots

ABSTRACT A simple screening method is needed to identify rice genotypes tolerant to low phosphorus (P) in soil. Nagina 22 (N22) mutants were classified as gain of function (gof) and loss of function (lof) for tolerance to very low P (soil Olsen P 2.01 mg kg−1). Two gof and two lof mutants were grown in hydroponics and in pot experiment at three P levels (-P, +P and +2P). Comparing response at +P and +2P in hydroponics we found that shoot and root dry weight were significantly less in gof and significantly more in lof in +2P. In pots with soil, tiller number and yield/plant was 3 fold less in gof but 2.5 to 3 fold more in lof and N22 in +2P compared to +P. That 2P can be used to identify low-P tolerant genotypes was validated using 3 low-P tolerant and 3 sensitive varieties whose response to low P soil is well documented. Both shoot and root dry weight was significantly less in +2P than in +P in tolerant and significantly more in sensitive genotypes. Thus screening in hydroponics in +2P can help identify low-P- tolerant genotypes easily and rapidly avoiding field screening.

MicroRNAs: potential target for genome editing in plants for traits improvement

The success of plant breeding for crop improvement is primarily dependent on effective utilization of coding and non-coding genetic elements. MicroRNAs are short non-coding RNAs transcribed from specific class of genes. These tiny RNAs regulate key biological processes in plants by targeting messenger RNAs for cleavage or translational inhibition. We herein describe the diverse functions of some of the well-studied miRNAs in different plant species and the possibility of using these miRNAs for crop improvement through genome editing. Some of the successful applications of genome editing of miRNAs are also described. This review presents key information on strategies and considerations to utilize miRNAs for genome editing with an example of rice. The major challenges of genome editing of miRNAs and the necessity of further studies to develop comprehensive knowledge of miRNA mediated gene regulatory networks and trait improvement, have been described in detail.

Trait and Marker Associations in Oryza nivara and O. rufipogon Derived Rice Lines under Two Different Heat Stress Conditions

Wild species and derived introgression lines (ILs) are a good source of genes for improving complex traits such as heat tolerance. The effect of heat stress on 18 yield traits was studied in four treatments in two seasons, under field conditions by subjecting 37 ILs and recurrent parents Swarna and KMR3, N22 mutants, and wild type and 2 improved rice cultivars to heat stress using polycover house method in wet season and late sowing method in dry season. Normal grown unstressed plants were controls. Both correlation and path coefficient analysis showed that the major contributing traits for high yield per plant (YPP) under heat stress conditions were tiller number, secondary branches in panicle, filled grain number, and percent spikelet fertility. Three ILs, K-377-24, K-16-3, and S-148 which gave the highest YPP of 12.30–32.52 g under heat stress in both the seasons were considered the most heat tolerant. In contrast, K-363-12, S-75, and Vandana which gave the least YPP of 5.36–10.84 g were considered heat susceptible. These lines are a good genetic resource for basic and applied studies on heat tolerance in rice. Genotyping using 49 SSR markers and single marker analysis (SMA) revealed 613 significant marker- trait associations in all four treatments. Significantly, nine markers (RM243, RM517, RM225, RM518, RM525, RM195, RM282, RM489, and RM570) on chromosomes 1, 2, 3, 4, 6, and 8 showed association with six traits (flag leaf spad, flag leaf thickness, vegetative leaf temperature, plant height, panicle number, and tiller number) under heat stress conditions in both wet and dry seasons. Genes such as heat shock protein binding DnaJ, Hsp70, and temperature-induced lipocalin-2 OsTIL-2 close to these markers are candidates for expression studies and evaluation for use in marker assisted selection for heat tolerance.

Proteomic and transcriptomic approaches to identify resistance and susceptibility related proteins in contrasting rice genotypes infected with fungal pathogen Rhizoctonia solani

The devastating sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) causes major yield loss in most rice growing regions of the world. In this study, two moderately tolerant and four susceptible genotypes of rice were selected for R. solani induced proteome analysis using two-dimensional polyacrylamide gel electrophoresis. Forty five differentially expressed proteins (DEPs) were identified and analyzed by Mass Spectrometry. Based on their functions, these proteins were classified into different groups, viz., photosynthesis, resistance and pathogenesis, stress, cell wall metabolism and cytoskeleton development associated proteins, and hypothetical or uncharacterized proteins. Expression of 14 genes encoding DEPs was analyzed by quantitative PCR which showed consistency in transcripts and genes expression pattern. Furthermore, the expression of 16 other genes involved in diverse biological functions was analyzed. Up-regulation of these genes in the tolerant genotype Pankaj during sheath blight disease suggested efficient genetic regulation of this cultivar under stress. Also, expression analysis of conserved microRNAs (miRNAs) and their target genes revealed important role of miRNAs in post-transcriptional gene regulation during development of rice sheath blight disease. Genome-wide discovery of miRNAs and further characterization of DEPs and genes will help in better understanding of the molecular events during sheath blight disease development in rice.

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 improvexa0the 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.