A comprehensive review on genomic resources in medicinally and industrially important major spices for future breeding programs: Status, utility and challenges.

In the global market, spices possess a high-value but low-volume commodities of commerce. The food industry depends largely on spices for taste, flavor, and therapeutic properties in replacement of cheap synthetic ones. The estimated growth rate for spices demand in the world is ∼3.19%. Since spices grow in limited geographical regions, India is one of the leading producer of spices, contributing 25-30 percent of total world trade. Hitherto, there has been no comprehensive review of the genomic resources of industrially important major medicinal spices to overcome major impediments in varietal improvement and management. This review focuses on currently available genomic resources of 24 commercially significant spices, namely, Ajwain, Allspice, Asafoetida, Black pepper, Cardamom large, Cardamom small, Celery, Chillies, Cinnamon, Clove, Coriander, Cumin, Curry leaf, Dill seed, Fennel, Fenugreek, Garlic, Ginger, Mint, Nutmeg, Saffron, Tamarind, Turmeric and Vanilla. The advent of low-cost sequencing machines has contributed immensely to the voluminous data generation of these spices, cracking the complex genomic architecture, marker discovery, and understanding comparative and functional genomics. This review of spice genomics resources concludes the perspective and way forward to provide footprints by uncovering genome assemblies, sequencing and re-sequencing projects, transcriptome-based studies, non-coding RNA-mediated regulation, organelles-based resources, developed molecular markers, web resources, databases and AI-directed resources in candidate spices for enhanced breeding potential in them. Further, their integration with molecular breeding could be of immense use in formulating a strategy to protect and expand the production of the spices due to increased global demand.

Revitalizing miRNAs mediated agronomical advantageous traits improvement in rice.

One of the key enigmas in conventional and modern crop improvement programmes is how to introduce beneficial traits without any penalty impairment. Rice (Oryza sativa L.), among the essential staple food crops grown and utilized worldwide, needs to improve genotypes in multifaceted ways. With the global view to feed ten billion under the climatic perturbation, only a potent functional master regulator can withstand with hope for the next green revolution and food security. miRNAs are such, miniature, fine tuners for crop improvement and provide a value addition in emerging technologies, namely large-scale genotyping, phenotyping, genome editing, marker-assisted selection, and genomic selection, to make rice production feasible. There has been surplus research output generated since the last decade on miRNAs in rice, however, recent functional knowledge is limited to reaping the benefits for conventional and modern improvements in rice to avoid ambiguity and redundancy in the generated data. Here, we present the latest functional understanding of miRNAs in rice. In addition, their biogenesis, intra- and inter-kingdom signaling and communication, implication of amiRNAs, and consequences upon integration with CRISPR-Cas9. Further, highlights refer to the application of miRNAs for rice agronomical trait improvements, broadly classified into three functional domains. The majority of functionally established miRNAs are responsible for growth and development, followed by biotic and abiotic stresses. Tabular cataloguing reveals and highlights two multifaceted modules that were extensively studied. These belong to miRNA families 156 and 396, orchestrate multifarious aspects of advantageous agronomical traits. Moreover, updated and exhaustive functional aspects of different supplemental miRNA modules that would strengthen rice improvement are also being discussed.

Recent advances on genome-wide association studies (GWAS) and genomic selection (GS); prospects for Fusarium head blight research in Durum wheat.

PURPOSE: Wheat is an important cereal crop that is cultivated in different parts of the world. The biotic stresses are the major concerns in wheat-growing nations and are responsible for production loss globally. The change in climate dynamics makes the pathogen more virulent in foothills and tropical regions. There is growing concern about FHB in major wheat-growing nations, and until now, there has been no known potential source of resistance identified in wheat germplasm. The plant pathogen interaction activates the cascade of pathways, genes, TFs, and resistance genes. Pathogenesis-related genes' role in disease resistance is functionally validated in different plant systems. Similarly, Genomewide association Studies (GWAS) and Genomic selection (GS) are promising tools and have led to the discovery of resistance genes, genomic regions, and novel markers. Fusarium graminearum produces deoxynivalenol (DON) mycotoxins in wheat kernels, affecting wheat productivity globally. Modern technology now allows for detecting and managing DON toxin to reduce the risk to humans and animals. This review offers a comprehensive overview of the roles played by GWAS and Genomic selection (GS) in the identification of new genes, genetic variants, molecular markers and DON toxin management strategies. METHODS: The review offers a comprehensive and in-depth analysis of the function of Fusarium graminearum virulence factors in Durum wheat. The role of GWAS and GS for Fusarium Head Blight (FHB) resistance has been well described. This paper provides a comprehensive description of the various statistical models that are used in GWAS and GS. In this review, we look at how different detection methods have been used to analyze and manage DON toxin exposure. RESULTS: This review highlights the role of virulent genes in Fusarium disease establishment. The role of genome-based selection offers the identification of novel QTLs in resistant wheat germplasm. The role of GWAS and GS selection has minimized the use of population development through breeding technology. Here, we also emphasized the function of recent technological developments in minimizing the impact of DON toxins and their implications for food safety.

Indian Wheat Genomics Initiative for Harnessing the Potential of Wheat Germplasm Resources for Breeding Disease-Resistant, Nutrient-Dense, and Climate-Resilient Cultivars.

Wheat is one of the major staple cereal food crops in India. However, most of the wheat-growing areas experience several biotic and abiotic stresses, resulting in poor quality grains and reduced yield. To ensure food security for the growing population in India, there is a compelling need to explore the untapped genetic diversity available in gene banks for the development of stress-resistant/tolerant cultivars. The improvement of any crop lies in exploring and harnessing the genetic diversity available in its genetic resources in the form of cultivated varieties, landraces, wild relatives, and related genera. A huge collection of wheat genetic resources is conserved in various gene banks across the globe. Molecular and phenotypic characterization followed by documentation of conserved genetic resources is a prerequisite for germplasm utilization in crop improvement. The National Genebank of India has an extensive and diverse collection of wheat germplasm, comprising Indian wheat landraces, primitive cultivars, breeding lines, and collection from other countries. The conserved germplasm can contribute immensely to the development of wheat cultivars with high levels of biotic and abiotic stress tolerance. Breeding wheat varieties that can give high yields under different stress environments has not made much headway due to high genotypes and environmental interaction, non-availability of truly resistant/tolerant germplasm, and non-availability of reliable markers linked with the QTL having a significant impact on resistance/tolerance. The development of new breeding technologies like genomic selection (GS), which takes into account the G × E interaction, will facilitate crop improvement through enhanced climate resilience, by combining biotic and abiotic stress resistance/tolerance and maximizing yield potential. In this review article, we have summarized different constraints being faced by Indian wheat-breeding programs, challenges in addressing biotic and abiotic stresses, and improving quality and nutrition. Efforts have been made to highlight the wealth of Indian wheat genetic resources available in our National Genebank and their evaluation for the identification of trait-specific germplasm. Promising genotypes to develop varieties of important targeted traits and the development of different genomics resources have also been highlighted.

Genes determining panicle morphology and grain quality in rice (Oryza sativa).

The world's increase in rice (Oryza sativa L.) production is not keeping up with the increase in its population. To boost the introduction of new high-yielding cultivars, knowledge is being gained on the genes and quantitative trait loci (QTLs) determining the panicle phenotype. The important are those determining yield of the crop, such as grain numbers per panicle and size and weight of the grains. Biochemical and molecular functions of many of them are understood in some details. Among these, OsCKX2 and OsSPL14 have been shown to increase panicle branching and grain numbers when overexpressed. Furthermore, miRNAs appear to play an important role in determining the panicle morphology by regulating the expressions of the genes like OsSPL14 and GRF4 involved in panicle branching and grain numbers and length. Mutations also greatly influence the grain shape and size. However, the information gained so far on the genetic regulation of grain filling and panicle morphology has not been successfully put into commercial application. Furthermore, the identification of the gene(s)/QTLs regulating panicle compactness is still lacking, which may enable the researchers to convert a compact-panicle cultivar into a lax/open one, and thereby increasing the chances of enhancing the yield of a desired compact-panicle cultivar obtained by the breeding effort.

Multi-locus genome-wide association studies (ML-GWAS) reveal novel genomic regions associated with seedling and adult plant stage leaf rust resistance in bread wheat (Triticum aestivum L.).

Leaf rust is one of the important diseases limiting global wheat production and productivity. To identify quantitative trait nucleotides (QTNs) or genomic regions associated with seedling and adult plant leaf rust resistance, multilocus genome-wide association studies (ML-GWAS) were performed on a panel of 400 diverse wheat genotypes using 35 K single-nucleotide polymorphism (SNP) genotyping assays and trait data of leaf rust resistance. Association analyses using six multi-locus GWAS models revealed a set of 201 significantly associated QTNs for seedling and 65 QTNs for adult plant resistance (APR), explaining 1.98-31.72% of the phenotypic variation for leaf rust. Among these QTNs, 51 reliable QTNs for seedling and 15 QTNs for APR were consistently detected in at least two GWAS models and were considered reliable QTNs. Three genomic regions were pleiotropic, each controlling two to three pathotype-specific seedling resistances to leaf rust. We also identified candidate genes, such as leucine-rich repeat receptor-like (LRR) protein kinases, P-loop containing nucleoside triphosphate hydrolase and serine-threonine/tyrosine-protein kinases (STPK), which have a role in pathogen recognition and disease resistance linked to the significantly associated genomic regions. The QTNs identified in this study can prove useful in wheat molecular breeding programs aimed at enhancing resistance to leaf rust and developing next-generation leaf rust-resistant varieties.

Biochemical and molecular processes contributing to grain filling and yield in rice.

The increase in much required rice production through breeding programmes is on decline. The primary reason being poor filling of grains in the basal spikelets of the heavy and compact panicle rice developed. These spikelets are genetically competent to develop into well filled grains, but fail to do so because the carbohydrate assimilates available to them remain unutilized, reportedly due to poor activities of the starch biosynthesizing enzymes, high production of ethylene leading to enhanced synthesis of the downstream signaling component RSR1 protein that inhibits GBSS1 activity, poor endosperm cell division and endoreduplication of the endosperm nuclei, altered expression of the transcription factors influencing grain filling, enhanced expression and phosphorylation of 14-3-3 proteins, poor expression of the seed storage proteins, reduced synthesis of the hormones like cytokinins and IAA that promote grain filling, and altered expression of miRNAs preventing their normal role in grain filling. Since the basal spikelets are genetically competent to develop into well filled mature grains, biotechnological interventions in terms of spikelet-specific overexpression of the genes encoding enzymes involved in grain filling and/or knockdown/overexpression of the genes influencing the activities of the starch biosynthesizing enzymes, various cell cycle events and hormone biosynthesis could increase rice production by as much as 30%, much more than the set production target of 800 mmt. Application of these biotechnological interventions in the heavy and compact panicle cultivars producing grains of desired quality would also maintain the quality of the grains having demand in market besides increasing the rice production per se.

Study of expressions of miRNAs in the spikelets based on their spatial location on panicle in rice cultivars provided insight into their influence on grain development.

Development of rice cultivars bearing numerous spikelets by breeding approach to increase the yearly production of rice to approximately 800 million metric tons to feed the ever increasing population of the world accompanies poor grain filling in the inferior spikelets preventing achievement of the yield potential. As the initial stages of caryopses development are of much importance for grain filling, spatio-temporal expressions of the miRNAs were studied during these periods in the spikelets of a compact-panicle rice cultivar, Oryza sativa cv. Mahalaxmi, bearing numerous spikelets per panicle to understand the reason of poor grain filling at the level of the initial biochemical events. Differential expression of several known miRNAs between the superior and inferior spikelets suggested great difference in metabolism related to grain filling in the spikelets based on their spatial location on compact panicle. Expressions of five known and four novel miRNAs were validated by Northern. Their targets included the enzymes directly involved in starch biosynthesis like sucrose synthase, starch synthase and pullulanase, besides others. Spatio-temporal expression studies of these miRNAs in the spikelets of Mahalaxmi revealed a pattern of mostly a greater expression in the inferior spikelets compared with the superior ones concomitant with an inverse expression of the target genes, which was not observed in the lax-panicle cultivar Upahar. The study thus revealed that the grain filling in rice is greatly regulated by miRNAs, and these miRNAs or their target genes could be considered for biotechnological interventions for improving grain filling in the rice cultivars of interest.

Cell cycle events and expression of cell cycle regulators are determining factors in differential grain filling in rice spikelets based on their spatial location on compact panicles.

Rice being a staple crop for human, its production is required to be increased significantly, particularly keeping in view the expected world's population of 9.6 billion by the year 2050. In this context, although the rice breeding programs have been successful in increasing the number of spikelets per panicle, the basal spikelets remain poorly filled, undermining the yield potential. The present study also found the grain filling to bear negative correlation with the panicle grain density. The poorly filled basal spikelets of the compact-panicle cultivars showed a lower endosperm cell division rate and ploidy status of the endosperm nuclei coupled with no significant greater expression of CYCB;1 and CYCH;1 compared with the apical spikelets, unlike that observed in the lax-panicle cultivars, which might have prevented them from overcoming apical dominance. Significantly greater expression of CYCB2;2 in the basal spikelets than in the apical spikelets might also have prevented the former to enter into endoreduplication. Furthermore, expression studies of KRPs in the caryopses revealed that a higher expression of KRP;1 and KRP;4 in the basal spikelets than in the apical spikelets of the compact-panicle cultivars could also be detrimental to grain filling in the former, as KRPs form complex primarily with CDKA-CYCD that promotes S-phase activity and G1/S transition, and thus inhibits endosperm cell division. The study indicates that targeted manipulation of expression of CYCB1;1, CYCB2;2, CYCH1;1, KRP;1 and KRP4 in the basal spikelets of the compact-panicle cultivars may significantly improve their yield performance.

Identification and expression analysis of miRNAs and elucidation of their role in salt tolerance in rice varieties susceptible and tolerant to salinity.

Soil salinization is a serious problem for cultivation of rice, as among cereals rice is the most salt sensitive crop, and more than 40% of the total agricultural land amounting to approximately 80 million ha the world over is salt affected. Salinity affects a plant in a varieties of ways, including ion toxicity, osmotic stress and oxidative damage. Since miRNAs occupy the top place in biochemical events determining a trait, understanding their role in salt tolerance is highly desirable, which may allow introduction of the trait in the rice cultivars of choice through biotechnological interventions. High throughput sequencing of sRNAs in the root and shoot tissues of the seedlings of the control and NaCl treated Pokkali, a salt-tolerant rice variety, identified 75 conserved miRNAs and mapped 200 sRNAs to the rice genome as novel miRNAs. Expression of nine novel miRNAs and two conserved miRNAs were confirmed by Northern blotting. Several of both conserved and novel miRNAs that expressed differentially in root and/or shoot tissues targeted transcription factors like AP2/EREBP domain protein, ARF, NAC, MYB, NF-YA, HD-Zip III, TCP and SBP reported to be involved in salt tolerance or in abiotic stress tolerance in general. Most of the novel miRNAs expressed in the salt tolerant wild rice Oryza coarctata, suggesting conservation of miRNAs in taxonomically related species. One of the novel miRNAs, osa-miR12477, also targeted L-ascorbate oxidase (LAO), indicating build-up of oxidative stress in the plant upon salt treatment, which was confirmed by DAB staining. Thus, salt tolerance might involve miRNA-mediated regulation of 1) cellular abundance of the hormone signaling components like EREBP and ARF, 2) synthesis of abiotic stress related transcription factors, and 3) antioxidative component like LAO for mitigation of oxidative damage. The study clearly indicated importance of osa-miR12477 regulated expression of LAO in salt tolerance in the plant.

Identification and characterization of drought responsive miRNAs in a drought tolerant upland rice cultivar KMJ 1-12-3.

Shortfall of rain that creates drought like situation in non-irrigated agriculture system often limits rice production, necessitating introduction of drought tolerance trait into the cultivar of interest. The mechanism governing drought tolerance is, however, largely unknown, particularly the involvement of miRNAs, the master regulators of biochemical events. In this regard, response study on a drought tolerant rice variety KMJ 1-12-3 to 20% PEG (osmolality- 315 mOsm/kg) as drought stress revealed significant changes in abundance of several conserved miRNAs targeting transcription factors like homeodomain-leucine zipper, MADS box family protein, C2H2 zinc finger protein and Myb, well known for their importance in drought tolerance in plants. The response study also revealed significant PEG-induced decrease in abundance of the miRNAs targeting cyclin A, cyclin-dependent kinase, guanine nucleotide exchange factor, GTPase-activating protein, 1-aminocyclopropane-1-carboxylic acid oxidase and indole-3-acetic beta-glucosyl transferase indicating miRNA-regulated role of the cell cycle regulators, G-protein signalling and the plant hormones ethylene and IAA in drought tolerance in plants. The study confirmed the existence of four novel miRNAs, including osa-miR12470, osa-miR12471, osa-miR12472 and osa-miR12473, and the targets of three of them could be successfully validated. The PEG-induced decrease in abundance of the novel miRNAs osa-miR12470 and osa-miR12473 targeting RNA dependent RNA polymerase and equilibrative nucleoside transporter, respectively suggested an overall increase in both degradation and synthesis of nucleic acids in plants challenged with drought stress. The drought-responsive miRNAs identified in the study may be proved useful in introducing the trait in the rice cultivars of choice by manipulation of their cellular abundance.

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo• "Free Radical" Is Never Free.

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S-C5' bond, which creates the highly reactive 5'-deoxyadenosyl radical (5'-dAdo•), the same radical generated by homolytic Co-C bond cleavage in B12 radical enzymes. The SAM surrogate S-3',4'-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-ß-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of (13)C, (2)H, and (15)N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 "tames" the 5'-dAdo• radical, preventing it from carrying out harmful side reactions: this "free radical" in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S-C5' bond, thereby enabling the 5'-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ∼0.6 Å toward the target and ∼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5' radical, with "van der Waals control" of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.

Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase.

Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe-4S](+) cluster accounting for 3-4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe-4S](+) cluster is observed that accounts for 34-45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-L: -methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 +/- 0.6 nmol min(-1) mg(-1), whereas no repair was observed for the 5S diastereomer.

An efficient deprotection of N-trimethylsilylethoxymethyl (SEM) groups from dinucleosides and dinucleotides.

A convenient and efficient method for deprotection of N-(trimethyl)silylethoxymethyl (SEM) groups from thymidine dinucleoside and dinucleotide has been achieved. The SEM groups were easily removed in excellent yields from protected nucleosides, dinucleosides, and dinucleotides.

Spore photoproduct lyase catalyzes specific repair of the 5R but not the 5S spore photoproduct.

Bacterial spores are remarkable in their resistance to chemical and physical stresses, including exposure to UV radiation. The unusual UV resistance of bacterial spores is a result of the unique photochemistry of spore DNA, which results in accumulation of 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), coupled with the efficient repair of accumulated damage by the enzyme spore photoproduct lyase (SPL). SPL is a member of the radical AdoMet superfamily of enzymes, and utilizes an iron-sulfur cluster and S-adenosylmethionine to repair SP by a direct reversal mechanism initiated by H atom abstraction from C-6 of the thymine dimer. While two distinct diastereomers of SP (5R or 5S) could in principle be formed upon UV irradiation of bacterial spores, only the 5R configuration is possible for SP formed from adjacent thymines in double helical DNA, due to the constraints imposed by the DNA structure; the 5S configuration is possible in less well-defined DNA structures or as an interstrand cross-link. We report here results from HPLC and MS analysis of in vitro enzymatic assays on stereochemically defined SP substrates demonstrating that SPL specifically repairs only the 5R isomer of SP. The observation that 5R-SP, but not 5S-SP, is a substrate for SPL is consistent with the expectation that 5R is the SP isomer produced in vivo upon UV irradiation of bacterial spore DNA.

Chemoselective deprotection of triethylsilyl ethers.

An efficient and selective method was developed for the deprotection of triethylsilyl (TES) ethers using formic acid in methanol (5-10%) or in methylene chloride 2-5%) with excellent yields. TES ethers are selectively deprotected to the corresponding alcohols in high yields using formic acid in methanol under mild reaction conditions. Other hydroxyl protecting groups like t-butyldimethylsilyl (TBDMS) remain unaffected.

Determination of the anomeric configuration in carbohydrates by longitudinal cross-correlated relaxation studies: application to mono- and disaccharides.

The role of cross-correlated relaxation between the anomeric proton chemical shift anisotropy (CSA) and its dipolar relaxation with nearby proton on the longitudinal relaxation in mono- and disaccharides at two magnetic field strengths has been investigated and shown to directly report the identity of their anomeric configuration.

Chemoselective deprotection of alpha-indole and imidazole ribonucleosides.

A series of 2 ',3 '-isopropylidene and 5 '-trityl-protected alpha-indole and alpha/beta-benzimidazole and imidazole ribonucleosides were deprotected with different acids. Selectivity was achieved for 5 '-versus 2 ',3 '- deprotection by using formic acid in the alpha-indole ribonucleoside series. Treatment of alpha-indole ribonucleosides with a mixture of formic acid and ether at room temperature afforded 2 ',3 '-deprotected alpha-ribonucleosides, whereas treatment of the alpha-benzimidazole ribonucleosides with the same acid afforded the 5 '-deprotected ribonucleoside without any 2 ', 3 '-deprotected products. The structures of these ribonucleosides were elucidated with 2D (NOESY, COSY, and HMQC) NMR spectroscopy.

Regio- and stereoselective glycosylation: synthesis of 5-haloimidazole alpha-ribonucleosides.

We describe the synthesis of novel 5-haloimidazole ribonucleosides as precursors of modified cobalamins. A regio- and stereoselective glycosylation of protected ribose with silylated 4(5)-haloimidazoles produces 5-haloimidazole ribonucleosides predominantly in the alpha-configuration (60-75%) without any 4-substituted imidazole ribonucleoside. The structure of the 5-fluoroimidazole ribonucleoside was confirmed by X-ray crystallography and 2D NMR spectroscopy.