Milletomics: a metabolomics centered integrated omics approach toward genetic progression.

Producing alternative staple foods like millet will be essential to feeding ten billion people by 2050. The increased demand for millet is driving researchers to improve its genetic variation. Millets include protein, dietary fiber, phenolic substances, and flavonoid components. Its climate resilience makes millet an appealing crop for agronomic sustainability. Integrative omics technologies could potentially identify and develop millets with desirable phenotypes that may have high agronomic value. Millets' salinity and drought tolerance have been enhanced using transcriptomics. In foxtail, finger, and pearl millet, proteomics has discovered salt-tolerant protein, phytohormone-focused protein, and drought tolerance. Metabolomics studies have revealed that certain metabolic pathways including those involving lignin, flavonoids, phenylpropanoid, and lysophospholipids are critical for many processes, including seed germination, photosynthesis, energy metabolism, and the synthesis of bioactive chemicals necessary for drought tolerance. Metabolomics integration with other omics revealed metabolome engineering and trait-specific metabolite creation. Integrated metabolomics and ionomics are still in the development stage, but they could potentially assist in comprehending the pathway of ionomers to control nutrient levels and biofortify millet. Epigenomic analysis has shown alterations in DNA methylation patterns and chromatin structure in foxtail and pearl millets in response to abiotic stress. Whole-genome sequencing utilizing next-generation sequencing is the most proficient method for finding stress-induced phytoconstituent genes. New genome sequencing enables novel biotechnological interventions including genome-wide association, mutation-based research, and other omics approaches. Millets can breed more effectively by employing next-generation sequencing and genotyping by sequencing, which may mitigate climate change. Millet marker-assisted breeding has advanced with high-throughput markers and combined genotyping technologies.

Unlocking the potential of Kodo millet: reviving an indigenous super grain for tomorrow's nutrition.

Kodo millet (Paspalum scrobiculatum L.) is an underutilized crop that encompasses nutritional benefits and climate resilience, making it a viable option for future crop development with nutraceutical properties. The cultivation of this crop has ancient roots, where it was revered for its ability to thrive in times of famine and was a vital companion crop to rice. Dishes made with Kodo millet are highly palatable and can be easily integrated into mainstream rice-based dishes. Among all cereals, Kodo millet is distinguished by its gluten-free composition, high phosphorus content, and significant antioxidant potential, which contributes to a diet that may reduce cardiovascular disease risk. Often grown in rainfed zones by marginal farmers, Kodo millet is valued for its grain and fodder. This less demanding crop can tolerate both biotic and abiotic stress, allowing it to thrive in soils with low organic matter and with minimal inputs, making it an ideal dual-purpose crop for rainfed areas. Despite its nutritional and agricultural benefits, Kodo millet's popularity is hindered by challenges such as low yield, market demand, lodging at harvest, and poor dehulling recovery, which necessitate the development of high-yielding varieties through the latest breeding advancements. Systematic investment and concerted breeding efforts are essential to harness the full potential of this nutrient-dense crop. The absence of whole genome sequence for Kodo millet poses a barrier to uncovering novel genetic traits. Consequently, there is an imperative to establish a millet-based value chain that elevates these underutilized crops, shaping smart cropping patterns and enhancing nutritional profiles for sustainable diets. Accordingly, this review highlights the significance of Kodo millet and the impact of breeding to establish it as a smart food choice for the future.

Brown-top millet: an overview of breeding, genetic, and genomic resources development for crop improvement.

MAIN CONCLUSION: Brown-top millet is a lesser-known millet with a high grain nutrient value, early maturation, and drought tolerance that needs basic research to understand and conserve food security. Brown-top millet [Urochloa ramosa (L.)] is currently cultivated in some developing countries (especially in India) for food and fodder, although it is less known among the small millets. Like other millets, it contains macro- and micronutrients, vitamins, minerals, proteins, and fiber, all of which have rich health benefits. The nutritional importance and health benefits of brown-top millet are still unknown to many people due to a lack of awareness, wide cultivation, and research. Hence, this millet is currently overshadowed by other major cereals. This review article aims to present the nutritional, breeding, genetic, and genomic resources of brown-top millet to inform millet and other plant researchers. It is important to note that genetic and genomic resources have not yet been created for this millet. To date, there are no genomic and transcriptomic resources for brown-top millet to develop single nucleotide polymorphisms (SNP) and insertion/Deletions (InDels) for breeding studies. Furthermore, studies regarding nutritional significance and health benefits are required to investigate the exact nutritional contents and health benefits of the brown-top millet. The present review delves into the nutritional value and health advantages of brown-top millet, as supported by the available literature. The limitations of producing brown-top millet have been enumerated. We also cover the status of marker-assisted breeding and functional genomics research on closely related species. Lastly, we draw insights for further research such as developing omics resources and applying genome editing to study and improve brown-top millet. This review will help to start breeding and other molecular studies to increase the growth and development of this cereal.

Protein research in millets: current status and way forward.

MAIN CONCLUSION: Millets' protein studies are lagging behind those of major cereals. Current status and future insights into the investigation of millet proteins are discussed. Millets are important small-seeded cereals majorly grown and consumed by people in Asia and Africa and are considered crops of future food security. Although millets possess excellent climate resilience and nutrient supplementation properties, their research advancements have been lagging behind major cereals. Although considerable genomic resources have been developed in recent years, research on millet proteins and proteomes is currently limited, highlighting a need for further investigation in this area. This review provides the current status of protein research in millets and provides insights to understand protein responses for climate resilience and nutrient supplementation in millets. The reference proteome data is available for sorghum, foxtail millet, and proso millet to date; other millets, such as pearl millet, finger millet, barnyard millet, kodo millet, tef, and browntop millet, do not have any reference proteome data. Many studies were reported on stress-responsive protein identification in foxtail millet, with most studies on the identification of proteins under drought-stress conditions. Pearl millet has a few reports on protein identification under drought and saline stress. Finger millet is the only other millet to have a report on stress-responsive (drought) protein identification in the leaf. For protein localization studies, foxtail millet has a few reports. Sorghum has the highest number of 40 experimentally proven crystal structures, and other millets have fewer or no experimentally proven structures. Further proteomics studies will help dissect the specific proteins involved in climate resilience and nutrient supplementation and aid in breeding better crops to conserve food security.

The role of phenomics and genomics in delineating the genetic basis of complex traits in millets.

Millets, comprising a diverse group of small-seeded grains, have emerged as vital crops with immense nutritional, environmental, and economic significance. The comprehension of complex traits in millets, influenced by multifaceted genetic determinants, presents a compelling challenge and opportunity in agricultural research. This review delves into the transformative roles of phenomics and genomics in deciphering these intricate genetic architectures. On the phenomics front, high-throughput platforms generate rich datasets on plant morphology, physiology, and performance in diverse environments. This data, coupled with field trials and controlled conditions, helps to interpret how the environment interacts with genetics. Genomics provides the underlying blueprint for these complex traits. Genome sequencing and genotyping technologies have illuminated the millet genome landscape, revealing diverse gene pools and evolutionary relationships. Additionally, different omics approaches unveil the intricate information of gene expression, protein function, and metabolite accumulation driving phenotypic expression. This multi-omics approach is crucial for identifying candidate genes and unfolding the intricate pathways governing complex traits. The review highlights the synergy between phenomics and genomics. Genomically informed phenotyping targets specific traits, reducing the breeding size and cost. Conversely, phenomics identifies promising germplasm for genomic analysis, prioritizing variants with superior performance. This dynamic interplay accelerates breeding programs and facilitates the development of climate-smart, nutrient-rich millet varieties and hybrids. In conclusion, this review emphasizes the crucial roles of phenomics and genomics in unlocking the genetic enigma of millets.

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus.

Supernumerary mini-chromosomes-a unique type of genomic structural variation-have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.

The potentialities of omics resources for millet improvement.

Millets are nutrient-rich (nutri-rich) cereals with climate resilience attributes. However, its full productive potential is not realized due to the lack of a focused yield improvement approach, as evidenced by the available literature. Also, the lack of well-characterized genomic resources significantly limits millet improvement. But the recent availability of genomic data and advancement in omics tools has shown its enormous potential to enhance the efficiency and precision faced by conventional breeding in millet improvement. The development of high throughput genotyping platforms based on next-generation sequencing (NGS) has provided a low-cost method for genomic information, specifically for neglected nutri-rich cereals with the availability of a limited number of reference genome sequences. NGS has created new avenues for millet biotechnological interventions such as mutation-based study, GWAS, GS, and other omics technologies. The simultaneous discovery of high-throughput markers and multiplexed genotyping platform has aggressively aided marker-assisted breeding for millet improvement. Therefore, omics technology offers excellent opportunities to explore and combine useful variations for targeted traits that could impart high nutritional value to high-yielding cultivars under changing climatic conditions. In millet improvement, an in-depth account of NGS, integrating genomics data with different biotechnology tools, is reviewed in this context.

Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets.

Millets are a class of nutrient-rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress-related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi-omics analysis for millets, which seriously hinders the mining of stress-related genes and the molecular breeding of millets. Here, a free, web-accessible, user-friendly millets multi-omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph-based pan-genomics of pearl millet, and stress-related multi-omics data, which enable Milletdb to be the most complete millets multi-omics database available. We stored GWAS (genome-wide association study) results of 20 yield-related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress-related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., 'Gene mapping', 'Co-expression', 'KEGG/GO Enrichment' analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through 'GWAS', which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress-related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.

Comprehensive physiological, transcriptomic, and metabolomic analysis of the key metabolic pathways in millet seedling adaptation to drought stress.

Drought is one of the leading environmental constraints that affect the growth and development of plants and, ultimately, their yield and quality. Foxtail millet (Setaria italica) is a natural stress-resistant plant and an ideal model for studying plant drought resistance. In this study, two varieties of foxtail millet with different levels of drought resistance were used as the experimental material. The soil weighing method was used to simulate drought stress, and the differences in growth, photosynthetic physiology, metabolite metabolism, and gene transcriptional expression under drought stress were compared and analyzed. We aimed to determine the physiological and key metabolic regulation pathways of the drought-tolerant millet in resistance to drought stress. The results showed that drought-tolerant millet exhibited relatively stable growth and photosynthetic parameters under drought stress while maintaining a relatively stable level of photosynthetic pigments. The metabolomic, transcriptomic, and gene co-expression network analysis confirmed that the key to adaptation to drought by millet was to enhance lignin metabolism, promote the metabolism of fatty acids to be transformed into cutin and wax, and improve ascorbic acid circulation. These findings provided new insights into the metabolic regulatory network of millet adaptation to drought stress.

5M approach to decipher starch-lipid interaction in minor millets.

KEY MESSAGE: The 5M approach can be applied to understand genetic complexity underlying nutritional traits of minor millets. It will help to systematically identify genomic regions/candidate genes imprinting metabolite profiles.

Maize ANT1 modulates vascular development, chloroplast development, photosynthesis, and plant growth.

Arabidopsis AINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of millet SCR1, GNC, and AN3, which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of the ANT1 ortholog in the C4 millet Setaria viridis by the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model for ANT1 regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles of ANT1 in several developmental processes beyond its known roles in plant growth and floral organogenesis.

Genome-editing in millets: current knowledge and future perspectives.

Millets are small seeded cereal crops predominantly cultivated and consumed by resource-poor farmers in the semi-arid tropics of Asia and Africa. Millets possess rich nutrients and a climate resilience property when compared to the other cereals such as rice and wheat. Millet improvement using modern genetic and genomic tools is falling behind other cereal crops due to their cultivation being restricted to less developed countries. Genome editing tools have been successfully applied to major cereal crops and, as a result, many key traits have been introduced into rice, wheat and maize. However, genome editing tools have not yet been used for most millets although they possess rich nutrients. The foxtail millet is the only millet utilised up to now for genome editing works. Limited genomic resources and lack of efficient transformation systems may slow down genome editing in millets. As millets possess many important traits of agricultural importance, high resolution studies with genome editing tools will help to understand the specific mechanism and transfer such traits to major cereals in the future. This review covers the current status of genome editing studies in millets and discusses the future prospects of genome editing in millets to understand key traits of nutrient fortification and develop climate resilient crops in the future.

Transcriptome Sequencing and Metabolome Analysis Reveals the Molecular Mechanism of Drought Stress in Millet.

As one of the oldest agricultural crops in China, millet (Panicum miliaceum) has powerful drought tolerance. In this study, transcriptome and metabolome analyses of 'Hequ Red millet' (HQ) and 'Yanshu No.10' (YS10) millet after 6 h of drought stress were performed. Transcriptome characteristics of drought stress in HQ and YS10 were characterized by Pacbio full-length transcriptome sequencing. The pathway analysis of the differentially expressed genes (DEGs) showed that the highly enriched categories were related to starch and sucrose metabolism, pyruvate metabolism, metabolic pathways, and the biosynthesis of secondary metabolites when the two millet varieties were subjected to drought stress. Under drought stress, 245 genes related to energy metabolism were found to show significant changes between the two strains. Further analysis showed that 219 genes related to plant hormone signal transduction also participated in the drought response. In addition, numerous genes involved in anthocyanin metabolism and photosynthesis were confirmed to be related to drought stress, and these genes showed significant differential expression and played an important role in anthocyanin metabolism and photosynthesis. Moreover, we identified 496 transcription factors related to drought stress, which came from 10 different transcription factor families, such as bHLH, C3H, MYB, and WRKY. Further analysis showed that many key genes related to energy metabolism, such as citrate synthase, isocitrate dehydrogenase, and ATP synthase, showed significant upregulation, and most of the structural genes involved in anthocyanin biosynthesis also showed significant upregulation in both strains. Most genes related to plant hormone signal transduction showed upregulated expression, while many JA and SA signaling pathway-related genes were downregulated. Metabolome analysis was performed on 'Hequ red millet' (HQ) and 'Yanshu 10' (YS10), a total of 2082 differential metabolites (DEMs) were identified. These findings indicate that energy metabolism, anthocyanins, photosynthesis, and plant hormones are closely related to the drought resistance of millet and adapt to adversity by precisely regulating the levels of various molecular pathways.

Phosphate supply influenced the growth, yield and expression of PHT1 family phosphate transporters in seven millets.

MAIN CONCLUSION: Phosphate starvation altered the root morphology and phosphate uptake with the induction of PHT1 family transporter genes in root and shoot tissues of seven millets. Millets are nutrient-rich cereals majorly cultivated in Asia and Africa. Foxtail millet (FoxM), pearl millet (PeaM), finger millet (FinM), kodo millet (KodM), little millet (LitM), proso millet (ProM), and barnyard millet (BarM) were examined for the influence of external phosphorous (P) supply on phenotypic traits, P uptake, yield, and PHosphate Transporter1 (PHT1) family gene expression. Millet seedlings grown under low Pi condition (LPC) produced significantly lower mean values for all traits except for lateral root length (LRL) and lateral root number (LRN) which were increased under LPC. Under LPC, seed weight (SW) also reduced by > 75% and had significantly lower levels of total P (TP) and Pi contents in leaf and root tissues. Expression dynamics of 12 PHT1 family (PHT1;1-1;12) transporters genes were analyzed in 7 millets. PHT1;2 has been found to be a constitutive transporter gene in all millets. Under LPC, root tissues showed the overexpression of PHT1;2, 1;3, 1;4 and 1;9 in FoxM, PHT1;1, 1;2, 1;3, 1;4, 1;8 and 1;10 in PeaM, PHT1;2 and 1;3 in FinM and ProM and PHT1;3, 1;6 and 1;11 in BarM. In leaf, LPC induced the expression of PHT1;3, 1;4 and 1;6 in FoxM, PHT1;2, 1;3, 1;4 and 1;8 in PeaM, PHT1;2, 1;3 and 1;4 in FinM and KodM, PHT1;2 in LitM and PHT1;4 in ProM and BarnM. This comprehensive study on the influence of P in phenotype, physiology, and molecular responses may help to improve the P uptake and its use efficiency of millets in future.

Genetic Variation of Physicochemical Properties and Digestibility of Foxtail Millet (Setaria italica) Landraces of Taiwan.

Foxtail millet is considered a 'smart food' because of nutrient richness and resilience to environments. A diversity panel of 92 foxtail millet landraces preserved by Taiwan indigenous peoples containing amylose content (AC) in the range of 0.7% to 16.9% exhibited diverse physiochemical properties revealed by a rapid viscosity analyzer (RVA). AC was significantly correlated with 5 RVA parameters, and some RVA parameters were also highly correlated with one another. In comparison to rice, foxtail millet contained less starch (65.9-73.1%) and no significant difference in totals of resistant starch (RS), slowly digestible starch (SDS), hydrolysis index (HI), and expected glycemic index (eGI) according to in vitro digestibility assays of raw flour with similar AC. RS was significantly positively correlated with AC and four RVA parameters, cold paste viscosity (CPV), setback viscosity (SBV), peak time (PeT), and pasting temperature (PaT), implying that suitable food processing to alter physicochemical properties of foxtail millet might mitigate hyperglycemia. This investigation of pasting properties and digestibility of diverse foxtail millet germplasm revealed much variation and showed potential for multi-dimensional utilizations in daily staple food and food industries.

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration.

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

An expression profiling analysis of hybrid millet and its parents at grain filling stage.

Heterosis has been widely used in crop breeding and production. However, a shortage of genes known to function in heterosis significantly limits our understanding of the molecular basis underlying heterosis. Here, we report 740 differentially expressed genes (DEGs) in the leaves of the hybrid millet Zhang No.5 and its parents at the grain filling stage determined using Solexa Illumina digital gene expression. Of the 740 DEGs, 546 were from the hybrid and its parents and most were up-regulated in the hybrid. Particularly, a large number of DEGs related to starch and carbohydrate metabolism and 2 DEGs encoding chlorophyll a/b binding proteins were up-regulated in hybrid millet. Moreover, all DEGs were enriched in the biological process and molecular function, and no DEGs were found to be enriched in the cellular component of GO terms. Pathway enrichment using KEGG showed that several DEGs were enriched in the circadian rhythm pathway. Further analysis revealed that the altered circadian rhythm, which mediates photosynthesis and carbohydrate accumulation, may play an important role in heterosis of the hybrid millet.

Overview of the Metabolite Composition and Antioxidant Capacity of Seven Major and Minor Cereal Crops and Their Milling Fractions.

Cereal grains play an important role in human health as a source of macro- and micronutrients, besides phytochemicals. The metabolite diversity was investigated in cereal crops and their milling fractions by untargeted metabolomics ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) of 69 samples: 7 species (barley, oat, pearl millet, rye, sorghum, triticale, and wheat), 23 genotypes, and 4 milling fractions (husk, bran, flour, and wholegrain). Samples were also analyzed by in vitro antioxidant activity. UHPLC-MS/MS signals were processed using XCMS, and metabolite annotation was based on SIRIUS and GNPS libraries. Bran and husk showed the highest antioxidant capacity and phenolic content/diversity. The major metabolite classes were phenolic acids, flavonoids, fatty acyls, and organic acids. Sorghum, millet, barley, and oats showed distinct metabolite profiles, especially related to the bran fraction. Molecular networking and chemometrics provided a comprehensive insight into the metabolic profiling of cereal crops, unveiling the potential of coproducts and super cereals such as sorghum and millet as sources of polyphenols.

Ancient genomes reveal millet farming-related demic diffusion from the Yellow River into southwest China.

The study of southwest China is vital for understanding the dispersal and development of farming because of the coexistence of millet and rice in this region since the Neolithic period.1,2 However, the process of the Neolithic transition in southwest China is largely unknown, mainly due to the lack of ancient DNA from the Neolithic period. Here, we report genome-wide data from 11 human samples from the Gaoshan and Haimenkou sites with mixed farming of millet and rice dating to between 4,500 and 3,000 years before present in southwest China. The two ancient groups derived approximately 90% of their ancestry from the Neolithic Yellow River farmers, suggesting a demic diffusion of millet farming to southwest China. We inferred their remaining ancestry to be derived from a Hòabìnhian-related hunter-gatherer lineage. We did not detect rice farmer-related ancestry in the two ancient groups, which indicates that they likely adopted rice farming without genetic assimilation. We, however, observed rice farmer-related ancestry in the formation of some present-day Tibeto-Burman populations. Our results suggested the occurrence of both demic and cultural diffusion in the development of Neolithic mixed farming in some parts of southwest China.