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IntroductionRice, one of the major staple food crops is frequently affected by various biotic/abiotic stresses including drought, salinity, submergence, heat, Bacterial leaf blight, Brown plant hopper, Gall midge, Stem borer, Leaf folder etc. Sustained increase of yield growth is highly necessary to meet the projected demand in rice production during the year 2050. Hence, development of high yielding and multiple stress tolerant rice varieties adapted to wider environments will serve the need. MethodsA systematic MAB approach was followed to pyramid eight major QTLs/genes controlling tolerance to major abiotic/biotic stresses viz., drought (qDTY1.1 and qDTY2.1), salinity (Saltol), submergence (Sub1), bacterial leaf blight (xa13 and Xa21), blast (Pi9) and gall midge (Gm4) in the genetic background of an elite rice culture CBMAS 14065 possessing high yield and desirable grain quality traits. Two advanced backcross derivatives of CBMAS 14065 possessing different combinations of target QTLs namely #27-1-39 (qDTY1.1+qDTY2.1+Sub1+xa13+Xa21+Gm4+Pi9) and #29-2-2 (qDTY1.1+qDTY2.1+Saltol+Xa21+Gm4+Pi9) were inter-mated.ResultsInter-mated F1 progenies harboring all the eight target QTLs/genes were identified through foreground selection. Genotyping of the inter-mated F4 population identified 14 progenies possessing all eight target QTLs/genes under homozygous conditions. All the fourteen progenies were forwarded up to F8 generation and evaluated for their yield and tolerance to dehydration, salinity, submergence, blast and bacterial leaf blight. All the 14 progenies exhibited enhanced tolerance to dehydration and salinity stresses by registering lesser reduction in their chlorophyll content, relative water content, root length, root biomass etc., against their recurrent parent Improved White Ponni/CBMAS 14065. All the 14 progenies harboring Sub1 loci from FR13A exhibited enhanced survival (90 - 95%) under 2 weeks of submergence /flooding when compared to their recurrent parent CBMAS 14065 which showed 100% susceptibility The inter-mated population showed a enhanced level of resistance to bacterial leaf blight (Score = 0 to 2) against blast (Score – 0) whereas the susceptible check CO 39 and the recurrent parent CBMAS 14065 recorded high level of susceptibility (Score = 7 to 9). Conclusion or discussionOur study demonstrated the accelerated development of multiple stress tolerant rice genotypes through marker assisted pyramiding of target QTLs/genes using tightly linked markers. These multiple stress tolerant rice lines will serve as excellent genetic stocks for field testing/variety release and also as parental lines in future breeding programs for developing climate resilient super rice varieties.

Carotenoids are potential antioxidants offering extensive human health benefits including protection against chronic diseases. Augmenting the supply of health-benefiting compounds/metabolites through dietary supplements is the most sustainable way for a healthy life. Our study compares the traditional rice cultivar Kavuni and the white rice variety ASD 16. RNA-Seq analysis was carried out in the maturing panicles of Kavuni, which are enriched with antioxidants such as the therapeutic carotenoid lutein, polyphenols, and anthocyanins, along with “ASD 16”, a popularly eaten white rice variety, to elucidate the molecular networks regulating accumulation of health benefiting compounds. Systematic analysis of transcriptome data identified preferential up-regulation of carotenoid precursors (OsDXS, OsGGPS) and key carotenoid biosynthetic genes (OsPSY1, OsZ-ISO) in the maturing grains of Kavuni. Our study also identified enhanced expression of OsLYC-E, OsCYP97A, and OsCYP97C transcripts involved in the alpha-carotenoid biosynthetic pathway and thereby leading to elevated lutein content in the grains of Kavuni. Kavuni grains showed preferential down-regulation of negative regulators of carotenoid metabolism viz., AP2 and HY5 and preferential up-regulation of positive modulators of carotenoid metabolism viz., Orange, OsDjB7, and OsSET29, thus creating a favorable molecular framework for carotenoid accumulation. Our study has unearthed valuable gene control points for precise manipulation of carotenoid profiles through CRISPR-based gene editing in rice grains. Perturbation of carotenoid biosynthesis holds unprecedented potential for the rapid development of the next generation of ‘Golden rice’.

Abstract 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 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.

The increased use of chemicals in rice farming poses significant issues regarding the emergence of pesticide/fungicide resistance and environmental sustainability concerns. This study was aimed at the genetic improvement of blast, bacterial leaf blight (BB) and gall midge resistance in a popular rice variety CO 51 which already harbours a blast resistance gene Pi54. Efforts were made to pyramid an additional blast resistance gene Pi9 along with two BB resistance genes (xa13 and Xa21) and two gall midge resistance genes (Gm1 and Gm4) into an elite rice variety CO 51 to enhance the resistance level to biotic stresses. The superior lines were selected using functional markers conferring resistance to blast (NBS4 and Pi54MAS linked to Pi9 and Pi54 genes, respectively) and BB [(xa13Prom (xa13) and pTA248 (Xa21)] and SSR markers linked to Gm1 (RM1328) and Gm4 (RM22550) for phenotypic screening and agronomic evaluation. The genotyping and phenotyping of F6 and BC2F6 progenies of CO 51 X 562-4, for agronomic traits and resistance to BB and blast, identified ten superior progenies in F6 and five superior progenies in BC2F6. The breeding lines harbouring both xa13+Xa21 exhibited high levels of resistance to BB (score ≤ 1 cm) and Pi9+Pi54 exhibited strong resistance to blast (score ≤ 2). Identified lines can be evaluated further for varietal improvement or utilised as genetic stocks in breeding programs.

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.

In the ever-changing climatic conditions, it has become important to enhance rice productivity to ensure global food security. Drought is one of the major limiting factors in rice pro- duction. Drought during the reproductive stage results in maximum or complete yield loss. Efforts have been taken to develop drought-tolerant rice lines by introgressing three major drought-effect QTLs, viz. qDTY1.1, qDTY2.1, and qDTY3.1, from Apo into a susceptible popular rice variety, Improved White Ponni (IWP). Backcross inbred lines of IWP × Apo were developed through the marker-assisted backcross breeding approach. Foreground analysis using linked markers resulted in the identification of 17 progenies carrying two or more QTLs, and the recurrent parent genome recoveries of these lines were >95.6% using 72 genome-wide SSR markers distributed throughout all chromosomes. Upon phenotypic evaluation of 17 IWP BILs, the water limited condition resulted in the identification of improved lines by recording the yield and the yield-related parameters. The promising performance of IWP BILs in terms of spikelet fertility (63.3%) and grain yield per plant (>10 g) under drought stress indicated the positive effects of introgressed qDTYs, while IWP recorded complete yield loss (94.2%). Out of the cultivars considered, the best-performing lines which truly exhibited drought tolerance, with more increased yield than the recurrent parent under water-limited conditions, and the effects of these QTLs and their interactions were examined in this research work.

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.

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.

By 2050, the rice production needs to be increased by at least 50% in order to meet the growing food demands of the global population. Among various yield limiting factors, high temperature is fast becoming a major threat to sustain rice yields due to its increased frequency of occurrence and severity of stress events. The development of heat-resilient rice cultivars has been slow due to the lack of relevant donors for heat tolerance traits and limited information regarding the genetic basis of these component traits. The early morning flowering (EMF) trait, contributing to heat escape by promoting flowering/anthesis during cooler hours in the morning is demonstrated to offer protection against high-temperature-induced failure of pollination and fertilization. In this study, evaluation of CO 51, IR64 and IR64-qEMF3 (NIL of IR64 harboring QTL promoting EMF revealed that qEMF3 promoted early morning flowering in IR64-qEMF3 (1½ to 2 h earlier than IR64) and thereby reduced the sterility by about 8.15%. Attempts through marker-assisted backcross breeding led to development of advanced backcross progenies (NILs) of CO 51, harboring qEMF3. Evaluation of 88 BC3F2 progenies identified 19 progenies harboring qEMF3 under homozygous conditions. Evaluation of NILs of CO 51 harboring qEMF3 during summer 2019 revealed that the NILs exhibited early (7.30 a.m.) onset of anthesis by 1½ h and completed its peak anthesis well around cooler hours (9.30 a.m.) of the day and thereby recorded reduced spikelet sterility (7.8–9.0%) than their recurrent parent CO 51 (19.2%). The current study clearly demonstrated the efficacy of early morning flowering in the mitigation of yield losses under high-temperature conditions in a farmer preferred rice variety.

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.

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.

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.

Engineering transcription factors (TF) hold promise in enhancing abiotic stress tolerance in plants. In this study, one of the popular rice varieties of South India, namely ADT 43, was engineered with a TF AtDREB1A driven by a stress-inducible rd29A promoter. PCR and Southern hybridization were employed to confirm the integration and copy number of the transgene. Transgenic lines (T1) of ADT 43 showed enhanced tolerance to drought and salinity compared to the non-transgenic ADT 43. Transgenic lines were found to maintain higher RWC %, lower leaf temperature, and partially closed stomata, enabling better survival under stress conditions. qRT-PCR analysis revealed the strong induction of AtDREB1A transcripts during drought. Transgenic lines of ADT 43 exhibited increased germination and retention of chlorophyll in their leaves under salinity. Evaluation of transgenic lines under transgenic screen house conditions revealed that line # A16 exhibited on par agronomic performance against its non-transgenic counterpart under normal conditions. Under drought, non-transgenic ADT 43 showed >20% reduction in the total number of spikelets per panicle, whereas transgenic line # A16 registered only a 2% reduction. Non-transgenic ADT 43 recorded 80% yield reduction under drought, whereas line # A16 recorded only 54% yield loss. The above results demonstrated the effectiveness of controlled expression of DREB1A in regulating dehydration responses in rice.

Rice production system is greatly affected by blast disease caused by Magnaporthe grisea Barr which causes significant yield reduction throughout Asia and different parts of the globe. Developing resistant varieties has been most efficient and economical method for controlling blast disease in rice. Identification of broad spectrum resistance genes against prevailing isolates of the pathogen is a key determining success of developing resistant varieties. However, the blast resistance characterization and evaluation has not been well studied. In the current study, 3 parental lines namely CO 51 (Pi54), 562-4 (a NIL of CO 43 harboring Pi 9) and a susceptible genotype Improved White Ponni (IWP) were evaluated against local isolates of blast pathogen. Presence of Pi9 and Pi54 blast resistance genes in the parental lines was confirmed by using functional markers such as NBS4 and Pi54MAS respectively. All the three genotypes were evaluated against blast disease in a hot spot environment. Both CO51 and 562-4 carrying the Pi54 and Pi9 genes respectively showed moderate resistance against blast disease whereas IWP showed a susceptibility reaction against blast disease. Results indicated that pyramiding of these genes would improve resistance against blast disease. The present study has favored the selection of blast resistant parental lines which can be utilized as donor in breeding programs.