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Foxtail millet ( Setaria italica ) is an important crop species and an emerging model plant for C 4 grasses. However, functional genomics research on foxtail millet is challenging because of its long generation time, relatively large stature and recalcitrance to genetic transformation. Here we report the development of xiaomi , a rapid-cycling mini foxtail millet mutant as a C 4 model system. Five to six generations of xiaomi can be grown in a year in growth chambers due to its short life cycle and small plant size, similar to Arabidopsis . A point mutation in the Phytochrome C ( PHYC ) gene was found to be causal for these characteristics. PHYC encodes a light receptor essential for photoperiodic flowering. A reference-grade xiaomi genome comprising 429.94 Mb of sequence was assembled and a gene-expression atlas from 11 different tissues was developed. These resources, together with an established highly efficient transformation system and a multi-omics database, make xiaomi an ideal model system for functional studies of C 4 plants. This study developed xiaomi , a mini foxtail millet mutant, as a C 4 model plant that has a short life cycle and small stature. To further enhance its model plant function, xiaomi ’s genome was sequenced and an efficient transformation system was established.

Background Foxtail millet ( Setaria italica ) harbors the small diploid genome (~ 450 Mb) and shows the high inbreeding rate and close relationship to several major foods, feed, fuel and bioenergy grasses. Previously, we created a mini foxtail millet, xiaomi , with an Arabidopsis-like life cycle. The de novo assembled genome data with high-quality and an efficient Agrobacterium -mediated genetic transformation system made xiaomi an ideal C 4 model system. The mini foxtail millet has been widely shared in the research community and as a result there is a growing need for a user-friendly portal and intuitive interface to perform exploratory analysis of the data. Results Here, we built a Multi-omics Database for Setaria italica (MDSi, http://sky.sxau.edu.cn/MDSi.htm ), that contains xiaomi genome of 161,844 annotations, 34,436 protein-coding genes and their expression information in 29 different tissues of xiaomi (6) and JG21 (23) samples that can be showed as an Electronic Fluorescent Pictograph (xEFP) in-situ. Moreover, the whole-genome resequencing (WGS) data of 398 germplasms, including 360 foxtail millets and 38 green foxtails and the corresponding metabolic data were available in MDSi. The SNPs and Indels of these germplasms were called in advance and can be searched and compared in an interactive manner. Common tools including BLAST, GBrowse, JBrowse, map viewer, and data downloads were implemented in MDSi. Conclusion The MDSi constructed in this study integrated and visualized data from three levels of genomics, transcriptomics and metabolomics, and also provides information on the variation of hundreds of germplasm resources that can satisfies the mainstream requirements and supports the corresponding research community.

Foxtail millet (Setaria italica), a very important grain crop in China, has become a new model plant for cereal crops and biofuel grasses. Although its reference genome sequence was released recently, quantitative trait loci (QTLs) controlling complex agronomic traits remains limited. The development of massively parallel genotyping methods and next-generation sequencing technologies provides an excellent opportunity for developing single-nucleotide polymorphisms (SNPs) for linkage map construction and QTL analysis of complex quantitative traits. In this study, a high-throughput and cost-effective RAD-seq approach was employed to generate a high-density genetic map for foxtail millet. A total of 2,668,587 SNP loci were detected according to the reference genome sequence; meanwhile, 9,968 SNP markers were used to genotype 124 F2 progenies derived from the cross between Hongmiaozhangu and Changnong35; a high-density genetic map spanning 1648.8 cM, with an average distance of 0.17 cM between adjacent markers was constructed; 11 major QTLs for eight agronomic traits were identified; five co-dominant DNA markers were developed. These findings will be of value for the identification of candidate genes and marker-assisted selection in foxtail millet.

Background Modification of histone acetylation is a ubiquitous and reversible process in eukaryotes and prokaryotes and plays crucial roles in the regulation of gene expression during plant development and stress responses. Histone acetylation is co-regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC). HAT plays an essential regulatory role in various growth and development processes by modifying the chromatin structure through interactions with other histone modifications and transcription factors in eukaryotic cells, affecting the transcription of genes. Comprehensive analyses of HAT genes have been performed in Arabidopsis thaliana and Oryza sativa . However, little information is available on the HAT genes in foxtail millet ( Setaria italica [L.] P. Beauv ). Results In this study, 24 HAT genes ( SiHAT s) were identified and divided into four groups with conserved gene structures via motif composition analysis. Phylogenetic analysis of the genes was performed to predict functional similarities between Arabidopsis thaliana , Oryza sativa , and foxtail millet; 19 and 2 orthologous gene pairs were individually identified. Moreover, all identified HAT gene pairs likely underwent purified selection based on their non-synonymous/synonymous nucleotide substitutions. Using published transcriptome data, we found that SiHAT genes were preferentially expressed in some tissues and organs. Stress responses were also examined, and data showed that SiHAT gene transcription was influenced by drought, salt, low nitrogen, and low phosphorus stress, and that the expression of four SiHAT s was altered as a result of infection by Sclerospora graminicola . Conclusions Results indicated that histone acetylation may play an important role in plant growth and development and stress adaptations. These findings suggest that SiHATs play specific roles in the response to abiotic stress and viral infection. This study lays a foundation for further analysis of the biological functions of SiHATs in foxtail millet.

Background Broomcorn millet is highly tolerant to drought and barren soil. Changes in chlorophyll content directly affect leaf color, which subsequently leadsleading to poor photosynthetic performance and reduced crop yield. Herein, we isolated a yellow leaf mutant ( YX-yl ) using a forward genetics approach and evaluated its agronomic traits, photosynthetic pigment content, chloroplast ultrastructure, and chlorophyll precursors. Furthermore, the molecular mechanism of yellowing was explored using transcriptome sequencing. Results The YX-yl mutant showed significantly decreased plant height and low yield. The leaves exhibited a yellow-green phenotype and poor photosynthetic capacity during the entire growth period. The content of chlorophyll a, chlorophyll b, and carotenoids in YX-yl leaves was lower than that in wild-type leaves. Chlorophyll precursor analysis results showed that chlorophyll biosynthesis in YX-yl was hindered by the conversion of porphobilinogen to protoporphyrin IX. Examination of chloroplast ultrastructure in the leaves revealed that the chloroplasts of YX-yl accumulated on one side of the cell. Moreover, the chloroplast structure of YX-yl was degraded. The inner and outer membranes of the chloroplasts could not be distinguished well. The numbers of grana and grana thylakoids in the chloroplasts were low. The transcriptome of the yellowing mutant YX-yl was sequenced and compared with that of the wild type. Nine chlorophyll-related genes with significantly different expression profiles were identified: PmUROD , PmCPO , PmGSAM , PmPBDG , PmLHCP , PmCAO , PmVDE , PmGluTR , and PmPNPT . The proteins encoded by these genes were located in the chloroplast, chloroplast membrane, chloroplast thylakoid membrane, and chloroplast matrix and were mainly involved in chlorophyll biosynthesis and redox-related enzyme regulation. Conclusions YX-yl is an ideal material for studying pigment metabolism mechanisms. Changes in the expression patterns of some genes between YX-yl and the wild type led to differences in chloroplast structures and enzyme activities in the chlorophyll biosynthesis pathway, ultimately resulting in a yellowing phenotype in the YX-yl mutant. Our findings provide an insight to the molecular mechanisms of leaf color formation and chloroplast development in broomcorn millet.

Broomcorn millet, renowned for its strong stress tolerance and rich nutritional value, serves as a crucial germplasm resource in the arid and semi-arid regions of northern China. It exhibits advantages such as a short growth period, high water use efficiency, salt tolerance, and pest resistance, which guarantee the stability of grain supply in local areas. Accurately identifying its growth saturation threshold is one of the core elements of precision agriculture technology and has become a research hotspot in the field of agricultural science both domestically and internationally in recent years.In this study, 8 representative broomcorn millet varieties were selected from typical dryland farming areas in Shanxi Province. Based on functional limits and growth models, temporal identification and comparative analysis of the phenotypic saturation thresholds of these 8 varieties were conducted, providing a scientific basis for variety selection and precision cultivation in arid regions.The Logistic model was used to fit the plant height growth dynamics, yielding a growth limit of 134.86–171.74 cm and a threshold achievement time of 59.60–73.80 days, with a model fitting degree R 2 > 97%. The Richards model was applied to fit the stem diameter growth, resulting in a growth limit of 8.47–10.28 cm and a threshold achievement time of 70.50–182.20 days, with an R 2 also > 97%. A quadratic polynomial regression model was employed to simulate the dynamic changes in chlorophyll content (R 2 > 70%), clarifying the chlorophyll content characteristics of different plant parts.The results indicated significant differences in plant height, stem diameter, and chlorophyll content thresholds among different varieties. Pinshu 4 ranked first due to its dual advantages in plant height and stem diameter; Xiaohongruan Proso Millet 6 followed closely by virtue of its high photosynthetic efficiency; White Proso Millet 8 showed balanced performance in stem diameter and chlorophyll content; Jinshu 7 had stable plant height and relatively high chlorophyll content; the remaining varieties ranked lower due to weak performance in one or more traits.

Foxtail millet is an important minor cereal crop rich in nutrients. Due to the small size of its seeds, there is little information on the diversity of its seed structure among germplasms, limiting the identification of genes controlling seed development and germination. This paper utilized X-ray computed tomography (CT) scanning technology and deep learning models to reveal the microstructure of foxtail millet seeds, gaining insights into their internal features, distribution, and composition. A total of 100 foxtail millet varieties were scanned with X-ray computed tomography to obtain 3D reconstruction images and slices. Pre-processing steps were adopted to improve image segmentation accuracy, including noise reduction, rotation, contrast enhancement, and brightness enhancement. The experiment revealed that traditional OpenCV image processing methods failed to achieve precise segmentation, whereas deep learning models exhibited outstanding performance in segmenting seed CT slice images. We compared UNet, PSPNet, and DeepLabV3 models, selected different backbones and optimizers based on the dataset, and continuously adjusted learning rates and maximum training epochs to train the models. Results demonstrated that VGG16-UNet achieved an accuracy of 99.19% on the foxtail millet seed CT slice image dataset, outperforming PSPNet and DeepLabV3 models. Compared to ResNet-UNet, VGG16-UNet shows an improvement of approximately 3.18% in accuracy, demonstrating superior performance in accurately segmenting the inner glume, outer glume, embryo, and endosperm under various adhesion conditions. Accurate segmentation of foxtail millet CT images enables analysis of embryo size, endosperm size, and glume thickness, which impact germination, growth, and nutrition. This study fills a gap in small grain structure research, offering insights to optimize agriculture and molecular breeding for improved yield and quality.

Background Kernel color is an important characteristic of foxtail millet ( Setaria italica ) associated with its market ability, quality, and nutritional value, which is mainly due to the accumulation of carotenoids. Despite its importance, the genetic basis of carotenoid variation in foxtail millet remains largely unexplored. Herein, the molecular mechanisms governing carotenoid accumulation in the kernel of foxtail millet were investigated by an exhaustive methodology encompassing Genome-Wide Association Study (GWAS), Bulk Segregant Analysis sequencing (BSA-seq), and integrated transcriptomic and metabolomic analyses. Results The total carotenoid content in kernels across 201 foxtail millet germplasms showed a spectrum of variations, which indicated that the kernel color is a quantitative genetic trait controlled by multiple genes. Using GWAS on these germplasms and BSA-seq on an F 6 generation Recombinant Inbred Line (RIL) population derived from the GBS (yellow kernel) and NMB (white kernel) cross, we identified genome regions linked with total carotenoid content in foxtail millet kernels. Integrated transcriptomic and metabolomic profiling during grain filling in both yellow and white varieties pinpointed SiPSY1 and SiCCD1 as key genes controlling carotenoid accumulation. Notably, the SNP (G/A) at 364 bp and the Indel (29 bp insertion) at 856 bp within the SiPSY1 promoter predominantly contributed to the variance in promoter activity. These variations markedly affected SiPSY1 expression levels, ultimately determining the phenotypic difference between yellow and white kernels. Conclusions These findings provide crucial genetic insights for the molecular mechanisms involved in carotenoid metabolism and lay a solid foundation for millet color breeding in foxtail millet.

Background The grains of foxtail millet are enriched in carotenoids, which endow this plant with a yellow color and extremely high nutritional value. However, the underlying molecular regulation mechanism and gene coexpression network remain unclear. Methods The carotenoid species and content were detected by HPLC for two foxtail millet varieties at three panicle development stages. Based on a homologous sequence BLAST analysis, these genes related to carotenoid metabolism were identified from the foxtail millet genome database. The conserved protein domains, chromosome locations, gene structures and phylogenetic trees were analyzed using bioinformatics tools. RNA-seq was performed for these samples to identify differentially expressed genes (DEGs). A Pearson correlation analysis was performed between the expression of genes related to carotenoid metabolism and the content of carotenoid metabolites. Furthermore, the expression levels of the key DEGs were verified by qRT-PCR. The gene coexpression network was constructed by a weighted gene coexpression network analysis (WGCNA). Result The major carotenoid metabolites in the panicles of DHD and JG21 were lutein and β-carotene. These carotenoid metabolite contents sharply decreased during the panicle development stage. The lutein and β-carotene contents were highest at the S1 stage of DHD, with values of 11.474 μg /100 mg and 12.524 μg /100 mg, respectively. Fifty-four genes related to carotenoid metabolism were identified in the foxtail millet genome. Cis-acting element analysis showed that these gene promoters mainly contain ‘plant hormone’, ‘drought stress resistance’, ‘MYB binding site’, ‘endosperm specific’ and ‘seed specific’ cis-acting elements and especially the ‘light-responsive’ and ‘ABA-responsive’ elements. In the carotenoid metabolic pathways, SiHDS , SiHMGS3 , SiPDS and SiNCED1 were more highly expressed in the panicle of foxtail millet. The expression of SiCMT , SiAACT3 , SiPSY1 , SiZEP1/2 , and SiCCD8c/8d was significantly correlated with the lutein content. The expression of SiCMT , SiHDR , SiIDI2 , SiAACT3 , SiPSY1 , and SiZEP1/2 was significantly correlated with the content of β-carotene. WGCNA showed that the coral module was highly correlated with lutein and β-carotene, and 13 structural genes from the carotenoid biosynthetic pathway were identified. Network visualization revealed 25 intramodular hub genes that putatively control carotenoid metabolism. Conclusion Based on the integrative analysis of the transcriptomics and carotenoid metabonomics, we found that DEGs related to carotenoid metabolism had a stronger correlation with the key carotenoid metabolite content. The correlation analysis and WGCNA identified and predicted the gene regulation network related to carotenoid metabolism. These results lay the foundation for exploring the key target genes regulating carotenoid metabolism flux in the panicle of foxtail millet. We hope that these target genes could be used to genetically modify millet to enhance the carotenoid content in the future.

Efficient and fast water uptake by seeds, facilitated by optimal soil moisture, plays a critical role in timely germination and early seedling vigor for foxtail millet production in arid and semi-arid regions. The husk, as a unique structure through which the seed contacts the soil, plays an important role in water uptake and germination. Many foxtail millet germplasm accessions have papillae on the epidermis of their husks, yet the role of this trait in water uptake and germination, as well as the genetic basis and regulatory mechanism related to this trait, remain unknown. In this study, we demonstrated that the water uptake by the seeds from accessions with papillae was significantly higher than that of accessions without papillae two hours and four hours after sowing during a 10 h experiment, resulting in faster germination. Analysis of segregating ratios from two F2 populations derived from crossing between accessions with and without papillae indicated that husk papilla density was of monogenic dominance. Bulked Segregant Analysis Sequencing (BSA-Seq) showed that candidate regions on chromosome 5 were significantly associated with husk papilla density. The mapped region overlapped by the two BSA populations for papilla density included 72 genes. In combination with the expression profiles of these genes, five candidate genes were identified, encoding aquaporins, fructose transporter, and glycoside hydrolase. This study elucidated the role of husk papillae in enhancing water uptake and germination in foxtail millet, provided genetic insights into the trait, and laid the foundation for further study on the mechanism of husk papilla differentiation.