Empowering rice breeding with NextGen genomics tools for rapid enhancement nitrogen use efficiency.

As rice has no physiological capacity of fixing nitrogen in the soil, its production had always been reliant on the external application of nitrogen (N) to ensure enhanced productivity. In the light of improving nitrogen use efficiency (NUE) in rice, several advanced agronomic strategies have been proposed. However, the soared increase of the prices of N fertilizers and subsequent environmental downfalls caused by the excessive use of N fertilizers, reinforces the prerequisite adaptation of other sustainable, affordable, and globally acceptable strategies. An appropriate alternative approach would be to develop rice cultivars with better NUE. Conventional breeding techniques, however, have had only sporadic success in improving NUE, and hence, this paper proposes a new schema that employs the wholesome benefits of the recent advancements in omics technologies. The suggested approach promotes multidisciplinary research, since such cooperation enables the synthesis of many viewpoints, approaches, and data that result in a comprehensive understanding of NUE in rice. Such collaboration also encourages innovation that leads to developing rice varieties that use nitrogen more effectively, facilitate smart technology transfer, and promotes the adoption of NUE practices by farmers and stakeholders to minimize ecological impact and contribute to a sustainable agricultural future.

Pilot-scale genome-wide association mapping in diverse sorghum germplasms identified novel genetic loci linked to major agronomic, root and stomatal traits.

This genome-wide association studies (GWAS) used a subset of 96 diverse sorghum accessions, constructed from a large collection of 219 accessions for mining novel genetic loci linked to major agronomic, root morphological and physiological traits. The subset yielded 43,452 high quality single nucleotide polymorphic (SNP) markers exhibiting high allelic diversity. Population stratification showed distinct separation between caudatum and durra races. Linkage disequilibrium (LD) decay was rapidly declining with increasing physical distance across all chromosomes. The initial 50% LD decay was ~ 5 Kb and background level was within ~ 80 Kb. This study detected 42 significant quantitative trait nucleotide (QTNs) for different traits evaluated using FarmCPU, SUPER and 3VmrMLM which were in proximity with candidate genes related and were co-localized in already reported quantitative trait loci (QTL) and phenotypic variance (R2) of these QTNs ranged from 3 to 20%. Haplotype validation of the candidate genes from this study resulted nine genes showing significant phenotypic difference between different haplotypes. Three novel candidate genes associated with agronomic traits were validated including Sobic.001G499000, a potassium channel tetramerization domain protein for plant height, Sobic.010G186600, a nucleoporin-related gene for dry biomass, and Sobic.002G022600 encoding AP2-like ethylene-responsive transcription factor for plant yield. Several other candidate genes were validated and associated with different root and physiological traits including Sobic.005G104100, peroxidase 13-related gene with root length, Sobic.010G043300, homologous to Traes_5BL_8D494D60C, encoding inhibitor of apoptosis with iWUE, and Sobic.010G125500, encoding zinc finger, C3HC4 type domain with Abaxial stomatal density. In this study, 3VmrMLM was more powerful than FarmCPU and SUPER for detecting QTNs and having more breeding value indicating its reliable output for validation. This study justified that the constructed subset of diverse sorghums can be used as a panel for mapping other key traits to accelerate molecular breeding in sorghum.

Plant Metabolomics: Current Initiatives and Future Prospects.

Plant metabolomics is a rapidly advancing field of plant sciences and systems biology. It involves comprehensive analyses of small molecules (metabolites) in plant tissues and cells. These metabolites include a wide range of compounds, such as sugars, amino acids, organic acids, secondary metabolites (e.g., alkaloids and flavonoids), lipids, and more. Metabolomics allows an understanding of the functional roles of specific metabolites in plants' physiology, development, and responses to biotic and abiotic stresses. It can lead to the identification of metabolites linked with specific traits or functions. Plant metabolic networks and pathways can be better understood with the help of metabolomics. Researchers can determine how plants react to environmental cues or genetic modifications by examining how metabolite profiles change under various crop stages. Metabolomics plays a major role in crop improvement and biotechnology. Integrating metabolomics data with other omics data (genomics, transcriptomics, and proteomics) provides a more comprehensive perspective of plant biology. This systems biology approach enables researchers to understand the complex interactions within organisms.

Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis.

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

Comparative Metabolomics Profiling Reveals Key Metabolites and Associated Pathways Regulating Tuber Dormancy in White Yam (Dioscorea rotundata Poir.).

Yams are economic and medicinal crops with a long growth cycle, spanning between 9-11 months due to their prolonged tuber dormancy. Tuber dormancy has constituted a major constraint in yam production and genetic improvement. In this study, we performed non-targeted comparative metabolomic profiling of tubers of two white yam genotypes, (Obiaoturugo and TDr1100873), to identify metabolites and associated pathways that regulate yam tuber dormancy using gas chromatography-mass spectrometry (GC-MS). Yam tubers were sampled between 42 days after physiological maturity (DAPM) till tuber sprouting. The sampling points include 42-DAPM, 56-DAPM, 87DAPM, 101-DAPM, 115-DAPM, and 143-DAPM. A total of 949 metabolites were annotated, 559 in TDr1100873 and 390 in Obiaoturugo. A total of 39 differentially accumulated metabolites (DAMs) were identified across the studied tuber dormancy stages in the two genotypes. A total of 27 DAMs were conserved between the two genotypes, whereas 5 DAMs were unique in the tubers of TDr1100873 and 7 DAMs were in the tubers of Obiaoturugo. The differentially accumulated metabolites (DAMs) spread across 14 major functional chemical groups. Amines and biogenic polyamines, amino acids and derivatives, alcohols, flavonoids, alkaloids, phenols, esters, coumarins, and phytohormone positively regulated yam tuber dormancy induction and maintenance, whereas fatty acids, lipids, nucleotides, carboxylic acids, sugars, terpenoids, benzoquinones, and benzene derivatives positively regulated dormancy breaking and sprouting in tubers of both yam genotypes. Metabolite set enrichment analysis (MSEA) revealed that 12 metabolisms were significantly enriched during yam tuber dormancy stages. Metabolic pathway topology analysis further revealed that six metabolic pathways (linoleic acid metabolic pathway, phenylalanine metabolic pathway, galactose metabolic pathway, starch and sucrose metabolic pathway, alanine-aspartate-glutamine metabolic pathways, and purine metabolic pathway) exerted significant impact on yam tuber dormancy regulation. This result provides vital insights into molecular mechanisms regulating yam tuber dormancy.

Marker Assisted Introgression of Resistance Genes and Phenotypic Evaluation Enabled Identification of Durable and Broad-Spectrum Blast Resistance in Elite Rice Cultivar, CO 51.

Across the globe, rice cultivation is seriously affected by blast disease, caused by Magnaporthe oryzae. This disease has caused heavy yield loss to farmers over the past few years. In this background, the most affordable and eco-friendly strategy is to introgress blast-resistant genes from donors into elite rice cultivars. However, it is not only challenging to evolve such resistance lines using conventional breeding approaches, but also a time-consuming process. Therefore, the marker-assisted introduction of resistance genes has been proposed as a rapid strategy to develop durable and broad-spectrum resistance in rice cultivars. The current study highlights the successful introgression of a blast resistance gene, i.e., Pi9, into CO 51, an elite rice cultivar which already has another resistance gene named Pi54. The presence of two blast resistance genes in the advanced backcross breeding materials (BC2F2:3) was confirmed in this study through a foreground selection method using functional markers such as NBS4 and Pi54MAS. The selected positive introgressed lines were further genotyped for background selection with 55 SSR markers that are specific to CO 51. Consequently, both Pi9 as well as Pi54 pyramided lines, with 82.7% to 88.1% of the recurrent parent genome recovery, were identified and the selected lines were evaluated under hotspot. The analysis outcomes found that both the lines possessed a high level of resistance against blast disease during the seedling stage itself. In addition to this, it was also noticed that the advanced breeding rice lines that carry Pi9 + Pi54 were effective in nature and exhibited a higher degree of resistance against blast disease compared to the lines that were introgressed with a single blast resistance gene. Thus, the current study demonstrates a rapid and a successful introgression and pyramiding of two blast resistance genes, with the help of markers, into a susceptible yet high-yielding elite rice cultivar within a short period of time. Those gene pyramided rice lines can be employed as donors to introgress the blast-resistant genes in other popular susceptible cultivars.

Untargeted Metabolite Profiling of Specialty Rice Grains Using Gas Chromatography Mass Spectrometry.

With-ever increasing demand food grains for the increasing population, it has also increased the importance of quality rice with nutritional and therapeutic properties. The quality of rice includes nutritional value, therapeutic properties, and further generation of aroma. Initial studies on sensory analysis using potassium hydroxide (1.7% KOH) identified the presence of a distinct aroma of the traditional rice cultivar Chakhao Amubi in comparison with other aromatic rice varieties were conducted. The metabolomic profiling of aromatic rice grains Chakhao Amubi, Pusa Basmati 1, and nonaromatic rice, Improved White Ponni was attempted to use gas chromatography-mass spectrometry (GC-MS). A total of fifty volatile aromatic compounds, including aromatic hydrocarbons, alkanes, alkenes, ketones, and aromatic aldehydes, have been identified. Detected compounds include six crucial volatile i.e., pentanal, hexanal, 2-pentylfuran, pyridine, (Z)-7-Decenal, and Mesitylene for distinct flavor and presence of aroma in Chakhao Amubi. The findings showed a distinct difference in the metabolic profile of Chakhao Amubi compared to Pusa Basmati 1 and Improved White Ponni. Thus, this study paved the way for a new understanding of the aromatic aspects of traditional rice germplasm and its utilization in rice breeding programs to improve the aroma, therapeutic, and nutritional characteristics of rice.

Comparative RNA-Seq analysis unravels molecular mechanisms regulating therapeutic properties in the grains of traditional rice Kavuni.

Developing rice varieties with enhanced levels of functional bioactives is an important intervention for achieving food and nutritional security in Asia where rice is the staple food and Type II diabetes incidences are higher. The present study was aimed at dissecting out the molecular events underlying the accumulation of bio active compounds in pigmented traditional rice Kavuni. Comparative transcriptome profiling in the developing grains of Kavuni and a white rice variety ASD 16 generated 37.7 and 29.8 million reads respectively. Statistical analysis identified a total of 9177 exhibiting significant differential expression (DEGs) between the grains of Kavuni and ASD 16. Pathway mapping of DEGs revealed the preferential up-regulation of genes involved in the biosynthesis of amylose and dietary fibres in Kavuni accounting for its low glycemic index (GI). Transcripts involved in the biosynthesis of carotenoids, flavonoids, anthocyanins, phenolic acids and phenylpropanoids were also found to be up-regulated in the grains of Kavuni. This study identified up-regulation of key transcripts involved in the accumulation of phenolic acids having potential for inhibiting major hydrolytic enzymes α-amylase and α-glucosidase and thus accounting for the slow digestibility leading to low GI. Overall, this study has identified molecular targets for the genetic manipulation of anti-diabetic and anti-oxidant traits in rice.

Comparative transcriptome and metabolome profiling in the maturing seeds of contrasting cluster bean (Cyamopsis tetragonoloba L. Taub) cultivars identified key molecular variations leading to increased gum accumulation.

Cluster bean (Guar) is the major source of industrial gum. Knowledge on the molecular events regulating galactomannan gum accumulation in guar will pave way for accelerated development of gummy guar genotypes. RNA Seq analysis in the immature seeds of contrasting cluster bean genotypes HGS 563 (gum type) and Pusa Navbahar (vegetable type) resulted in the generation of 19,855,490 and 21,488,472 quality reads. Data analysis identified 4938 differentially expressed genes between the gummy vs vegetable genotypes. A set of 2241 genes were up-regulated and 2587 genes were down-regulated in gummy guar. Significant up-regulation of genes involved in the biosynthesis of galactomannan and cell wall storage polysaccharides was observed in the gummy HGS 563. Genes involved in carotenoids, flavonoids, non mevalonic acid, terpenoids, and wax metabolism were also up-regulated in HGS 563. Mannose and galactose were the major nucleotide sugars in Pusa Navbahar and HGS 563 immature seeds. Immature seeds of HGS 563 showed high concentration of mannose and galactose accumulation compared to Pusa Navbahar. qRT-PCR analysis of selected genes confirmed the findings of transcriptome data.

Creation of novel alleles of fragrance gene OsBADH2 in rice through CRISPR/Cas9 mediated gene editing.

Fragrance in rice grains is a key quality trait determining its acceptability and marketability. Intensive research on rice aroma identified mutations in betaine aldehyde dehydrogenase (OsBADH2) leading to production of aroma in rice. Gene editing technologies like CRISPR/Cas9 system has opened new avenues for accelerated improvement of rice grain quality through targeted mutagenesis. In this study, we have employed CRISPR/Cas9 tool to create novel alleles of OsBADH2 leading to introduction of aroma into an elite non-aromatic rice variety ASD16. PCR analysis of putative transformants using primers targeting the flanking regions of sgRNA in the 7th exon of OsBADH2 identified 37.5% potential multi-allelic mutations in T0 generation. Sensory evaluation test in the leaves of T0 lines identified thirteen lines belonging to five independent events producing aroma. Sequence analysis of these aromatic T0 lines identified 22 different types of mutations located within -17 bp to +15bp of sgRNA region. The -1/-2 bp deletion in the line # 8-19 and -8/-5 bp deletion in the line # 2-16 produced strong aroma and the phenotype was stably inherited in the T1 generation. Comparative volatile profiling detected novel aromatic compounds viz., pyrrolidine, pyridine, pyrazine, pyradazine and pyrozole in the grains of T1 progenies of line # 8-19. This study has demonstrated the use of CRISPR/Cas9 in creating novel alleles of OsBADH2 to introduce aroma into any non-aromatic rice varieties.

Pyramiding QTLs controlling tolerance against drought, salinity, and submergence in rice through marker assisted breeding.

Increases in rice productivity are significantly hampered because of the increase in the occurrence of abiotic stresses, including drought, salinity, and submergence. Developing a rice variety with inherent tolerance against these major abiotic stresses will help achieve a sustained increase in rice production under unfavorable conditions. The present study was conducted to develop abiotic stress-tolerant rice genotypes in the genetic background of the popular rice variety Improved White Ponni (IWP) by introgressing major effect quantitative trait loci (QTLs) conferring tolerance against drought (qDTY1.1, qDTY2.1), salinity (Saltol), and submergence (Sub1) through a marker assisted backcross breeding approach. Genotyping of early generation backcrossed inbred lines (BILs) resulted in the identification of three progenies, 3-11-9-2, 3-11-11-1, and 3-11-11-2, possessing all four target QTLs and maximum recovery of the recurrent parent genome (88.46%). BILs exhibited consistent agronomic and grain quality characters compared to those of IWP and enhanced performance against dehydration, salinity, and submergence stress compared with the recurrent parent IWP. BILs exhibited enhanced tolerance against salinity during germination and increased shoot length, root length, and vigor index compared to those of IWP. All three BILs exhibited reduced symptoms of injury because of salinity (NaCl) and dehydration (PEG) than did IWP. At 12 days of submergence stress, BILs exhibited enhanced survival and greater recovery, whereas IWP failed completely. BILs were found to exhibit on par grain and cooking quality characteristics with their parents. Results of this study clearly demonstrated the effects of the target QTLs in reducing damage caused by drought, salinity, and submergence and lead to the development of a triple stress tolerant version of IWP.