Generation of High-Value Genomic Resource in Rice: A "Subgenomic Library" of Low-Light Tolerant Rice Cultivar Swarnaprabha.

Rice is the major staple food crop for more than 50% of the world's total population, and its production is of immense importance for global food security. As a photophilic plant, its yield is governed by the quality and duration of light. Like all photosynthesizing plants, rice perceives the changes in the intensity of environmental light using phytochromes as photoreceptors, and it initiates a morphological response that is termed as the shade-avoidance response (SAR). Phytochromes (PHYs) are the most important photoreceptor family, and they are primarily responsible for the absorption of the red (R) and far-red (FR) spectra of light. In our endeavor, we identified the morphological differences between two contrasting cultivars of rice: IR-64 (low-light susceptible) and Swarnaprabha (low-light tolerant), and we observed the phenological differences in their growth in response to the reduced light conditions. In order to create genomic resources for low-light tolerant rice, we constructed a subgenomic library of Swarnaprabha that expedited our efforts to isolate light-responsive photoreceptors. The titer of the library was found to be 3.22 × 105 cfu/mL, and the constructed library comprised clones of 4-9 kb in length. The library was found to be highly efficient as per the number of recombinant clones. The subgenomic library will serve as a genomic resource for the Gramineae community to isolate photoreceptors and other genes from rice.

Selection of suitable internal control gene for assaying gene expression in rice through qRT-PCR during sheath blight infection.

qRT-PCR is a globally accepted technique for assaying gene expression in relative terms which compares the difference between critical threshold (CT) values of a gene calculated form two independently isolated RNA samples. Independent RNA isolations, however, include error due to batch effect which must be normalized for error-free calculation of relative gene expression. Hence, CT values of internal control (IC) genes are used for normalization during the calculation of expression fold-change in gene expression analysis. The expression of ICs genes expected to be stable in all the experimental conditions. However, it is almost impossible to find such a gene which do not depict expression fluctuation in response to the changes in experimental conditions. Hence, it is necessary to identify suitable IC gene(s) for any given experimental condition before conducting any particular gene expression study. Here, we examined the suitability of eight candidate IC genes, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic elongation factor-1 (eEF-1α), 25 S rRNA (25 S), 18 S rRNA (18 S), ubiquitin C E2 ligase (UBC), Actin (Act), ubiquitin 5 (UBQ5) and ubiquitin 10 (UBQ10), for assaying gene expression in rice during sheath blight infection. Our analysis suggest that GAPDH might be the IC of choice when expression studies include contrasting genotypes differing in their tolerance to sheath blight pathogen as well as progressive infection time. While if expression analysis have to be performed only in one genotype but under progressive sheath blight infection, UBQ5 might be chosen as IC because of its high expression stability under the proposed experimental setup.

Development of early maturing salt-tolerant rice variety KKL(R) 3 using a combination of conventional and molecular breeding approaches.

Introduction: Soil salinity poses a severe threat to rice production, resulting in stunted growth, leaf damage, and substantial yield losses. This study focuses on developing an early maturing seedling stage salinity tolerant rice variety by integrating conventional breeding methods with marker assisted breeding (MAB) approaches. Methods: Seedling-stage salinity tolerance Quantitative Trait Locus (QTL) "Saltol" from the salt-tolerant parent FL478 was introduced into the high-yielding but salt-sensitive rice variety ADT 45. This was achieved through a combination of conventional breeding and MAB. The breeding process involved rigorous selection, screening, and physiological parameter assessments. Results: KKL(R) 3 (KR 15066) identified as the top performing Recombinant Inbred Line (RIL), consistently demonstrating maximum mean grain yields under both salinity (3435.6 kg/ha) and normal (6421.8 kg/ha) conditions. In comparison to the early maturing, salt-tolerant national check variety CSR 10, KKL(R) 3 exhibited a substantial yield increase over 50%. Discussion: The notable improvement observed in KKL(R) 3 positions it as a promising variety for release, offering a reliable solution to maximize yields, ensure food security, and promote agricultural sustainability in both saline and non-saline environments. The study highlights the effectiveness of MAB in developing salt-tolerant rice varieties and emphasizes the significance of the Saltol QTL in enhancing seedling stage salinity tolerance. The potential release of KKL(R) 3 has the capacity to revolutionize rice production in salt affected regions, providing farmers with a reliable solution to maximize yields and contribute to food security while ensuring agricultural sustainability.

Physio-biochemical and molecular stress regulators and their crosstalk for low-temperature stress responses in fruit crops: A review.

Low-temperature stress (LTS) drastically affects vegetative and reproductive growth in fruit crops leading to a gross reduction in the yield and loss in product quality. Among the fruit crops, temperate fruits, during the period of evolution, have developed the mechanism of tolerance, i.e., adaptive capability to chilling and freezing when exposed to LTS. However, tropical and sub-tropical fruit crops are most vulnerable to LTS. As a result, fruit crops respond to LTS by inducing the expression of LTS related genes, which is for climatic acclimatization. The activation of the stress-responsive gene leads to changes in physiological and biochemical mechanisms such as photosynthesis, chlorophyll biosynthesis, respiration, membrane composition changes, alteration in protein synthesis, increased antioxidant activity, altered levels of metabolites, and signaling pathways that enhance their tolerance/resistance and alleviate the damage caused due to LTS and chilling injury. The gene induction mechanism has been investigated extensively in the model crop Arabidopsis and several winter kinds of cereal. The ICE1 (inducer of C-repeat binding factor expression 1) and the CBF (C-repeat binding factor) transcriptional cascade are involved in transcriptional control. The functions of various CBFs and aquaporin genes were well studied in crop plants and their role in multiple stresses including cold stresses is deciphered. In addition, tissue nutrients and plant growth regulators like ABA, ethylene, jasmonic acid etc., also play a significant role in alleviating the LTS and chilling injury in fruit crops. However, these physiological, biochemical and molecular understanding of LTS tolerance/resistance are restricted to few of the temperate and tropical fruit crops. Therefore, a better understanding of cold tolerance's underlying physio-biochemical and molecular components in fruit crops is required under open and simulated LTS. The understanding of LTS tolerance/resistance mechanism will lay the foundation for tailoring the novel fruit genotypes for successful crop production under erratic weather conditions.

Identification and functional prediction of long non-coding RNAs of rice (Oryza sativa L.) at reproductive stage under salinity stress.

Salinity adversely affects the yield and growth of rice (Oryza sativa L.) plants severely, particularly at reproductive stage. Long non-coding RNAs (lncRNAs) are key regulators of diverse molecular and cellular processes in plants. Till now, no systematic study has been reported for regulatory roles of lncRNAs in rice under salinity at reproductive stage. In this study, total 80 RNA-seq data of Horkuch (salt-tolerant) and IR-29 (salt-sensitive) genotypes of rice were used and found 1626 and 2208 transcripts as putative high confidence lncRNAs, among which 1529 and 2103 were found to be novel putative lncRNAs in root and leaf tissue respectively. In Horkuch and IR-29, 14 and 16 lncRNAs were differentially expressed in root tissue while 18 and 63 lncRNAs were differentially expressed in leaf tissue. Interaction analysis among the lncRNAs, miRNAs and corresponding mRNAs indicated that these modules are involved in different biochemical pathways e.g. phenyl propanoid pathway during salinity stress in rice. Interestingly, two differentially expressed lncRNAs such as TCONS_00008914 and TCONS_00008749 were found as putative target mimics of known rice miRNAs. This study indicates that lncRNAs are involved in salinity adaptation of rice at reproductive stage through certain biochemical pathways.

Current status of genomic resources on wild relatives of rice.

Rice is a food crop of global importance, cultivated in diverse agro-climatic zones of the world. However, in the process of domestication many beneficial alleles have been eroded from the gene pool of the rice cultivated globally and eventually has made it vulnerable to a plethora of stresses. In contrast, the wild relatives of rice, despite being agronomically inferior, have inherited a potential of surviving in a range of geographical habitats. These adaptations enrich them with novel traits that upon introgression to modern cultivated varieties offer tremendous potential of increasing yield and adaptability. But, due to the unavailability of their genetic as well as genomic resources, identification and characterisation of these novel beneficial alleles has been a challenging task. Nevertheless, with the unprecedented surge in the area of conservation genomics, researchers have now shifted their focus towards these natural repositories of beneficial traits. Presently, there are several generic and specialized databases harboring genome-wide information on wild species of rice, and are acting as a useful resource for identification of novel genes and alleles, designing of molecular markers, comparative analysis and evolutionary biology studies. In this review, we introduce the key features of these databases focusing on their utility in rice breeding programs.

Improving plant-resistance to insect-pests and pathogens: The new opportunities through targeted genome editing.

The advantages of high input agriculture are fading away due to degenerating soil health and adverse effects of climate change. Safeguarding crop yields in the changing environment and dynamics of pest and pathogens, has posed new challenges to global agriculture. Thus, integration of new technologies in crop improvement has been imperative for achieving the breeding objectives in faster ways. Recently, enormous potential of genome editing through engineered nucleases has been demonstrated in plants. Continuous refinements of the genome editing tools have increased depth and breadth of their applications. So far, genome editing has been demonstrated in more than fifty plant species. These include model species like Arabidopsis, as well as important crops like rice, wheat, maize etc. Particularly, CRISPR/Cas9 based two component genome editing system has been facile with wider applicability. Potential of genome editing has unfurled enormous possibilities for engineering diverse agronomic traits including durable resistance against insect-pests and pathogens. Novel propositions of developing insect and pathogen resistant crops by genome editing include altering the effector-target interaction, knocking out of host-susceptibility genes, engineering synthetic immune receptor eliciting broad spectrum resistance, uncoupling of antagonistic action of defense hormones etc. Alternatively, modification of insect genomes has been used either to create gene drive or to counteract resistance to various insecticides. The distinct advantage of genome editing system is that it can knock out specific target region in the genome without leaving the unwanted vector backbone. In this article, we have reviewed the novel opportunities offered by the genome editing technologies for developing insect and pathogen resistant crop-types, their future prospects and anticipated challenges.

Status and Prospects of Next Generation Sequencing Technologies in Crop Plants.

The history of DNA sequencing dates back to 1970s. During this period the two first generation nucleotide sequencing techniques were developed. Subsequently the Sanger's dideoxy method of sequencing gained popularity over Maxam and Gilbert's chemical method of sequencing. However, in the last decade, we have observed revolutionary changes in DNA sequencing technologies leading to the emergence of next-generation sequencing (NGS) techniques. NGS technologies have enhanced the throughput and speed of sequencing combined with bringing down the overall cost of the process over a time. The major applications of NGS technologies being genome sequencing and resequencing, transcriptomics, metagenomics in relation to plant-microbe interactions, exon and genome capturing, development of molecular markers and evolutionary studies. In this review, we present a broader picture of evolution of NGS tools, its various applications in crop plants, and future prospects of the technology for crop improvement.

Identification of long non-coding RNA in rice lines resistant to Rice blast pathogen Maganaporthe oryzae.

Rice blast disease caused by a fungus Magnaporthae oryzae is one of the most important biotic factors that severely damage the rice crop. Several molecular approaches are now being applied to tackle this issue in rice. It is of interest to study long non-coding RNA (lncRNA) in rice to control the disease. lncRNA, a non-coding transcript that does not encode protein, is known to play an important role in gene regulation of various biological processes. Here we describe a computational pipeline to identify lncRNA from a resistant rice line. The number of lncRNA found in resistant line was 1429, 1927 and 1981 in mock and M. oryzae (ZB13 and Zhong) inoculated samples, respectively. Functional classification of these lncRNA reveals a higher number of long intergenic non-coding RNA compared to antisense lncRNA in both mock and M. oryzae inoculated resistant rice lines. Many intergenic lncRNA candidates were identified from resistant rice line and their role to regulate the resistance mechanism in rice during M. oryzae invasion is implied.

Cytoplasmic male sterility (CMS) in hybrid breeding in field crops.

KEY MESSAGE: A comprehensive understanding of CMS/Rf system enabled by modern omics tools and technologies considerably improves our ability to harness hybrid technology for enhancing the productivity of field crops. Harnessing hybrid vigor or heterosis is a promising approach to tackle the current challenge of sustaining enhanced yield gains of field crops. In the context, cytoplasmic male sterility (CMS) owing to its heritable nature to manifest non-functional male gametophyte remains a cost-effective system to promote efficient hybrid seed production. The phenomenon of CMS stems from a complex interplay between maternally-inherited (mitochondrion) and bi-parental (nucleus) genomic elements. In recent years, attempts aimed to comprehend the sterility-inducing factors (orfs) and corresponding fertility determinants (Rf) in plants have greatly increased our access to candidate genomic segments and the cloned genes. To this end, novel insights obtained by applying state-of-the-art omics platforms have substantially enriched our understanding of cytoplasmic-nuclear communication. Concomitantly, molecular tools including DNA markers have been implicated in crop hybrid breeding in order to greatly expedite the progress. Here, we review the status of diverse sterility-inducing cytoplasms and associated Rf factors reported across different field crops along with exploring opportunities for integrating modern omics tools with CMS-based hybrid breeding.