QTL mapping for foxtail millet plant height in multi-environment using an ultra-high density bin map.

KEY MESSAGE: Using a fixed RIL population derived from a widely used foxtail millet backbone breeding line and an elite cultivar, we constructed a high-density bin map and identified six novel multi-environment effect QTLs and seven candidate genes for dwarf phenotype. Plant height is an important trait that determines tradeoffs between competition and resource allocation, which is crucial for yield potential. To improve the C4 model plant foxtail millet (Setaria italica) productivity, it is necessary to isolate plant height-related genes that contribute to ideal plant architecture in breeding. In the present study, we generated a foxtail millet population of 333 recombinant inbred lines (RILs) derived from a cross between a backbone line Ai 88 and an elite cultivar Liaogu 1. We evaluated plant height in 13 environmental conditions across 4 years, the mean plant height of the RIL population ranged from 89.5 to 149.9 cm. Using deep re-sequencing data, we constructed a high-density bin map with 3744 marker bins. Quantitative trait locus (QTL) mapping identified 26 QTLs significantly associated with plant height. Of these, 13 QTLs were repeatedly detected under multiple environments, including six novel QTLs that have not been reported before. Seita.1G242300, a gene encodes gibberellin 2-oxidase-8, which was detected in nine environments in a 1.54-Mb interval of qPH1.3, was considered as an important candidate gene. Moreover, other six genes involved in GA biosynthesis or signaling pathways, and fifteen genes encode F-box domain proteins which might function as E3 ligases, were also considered as candidate genes in different QTLs. These QTLs and candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height, and the linked markers will be useful for marker-assistant selection of varieties with ideal plant architecture and high yield potential.

Genetic control and phenotypic characterization of panicle architecture and grain yield-related traits in foxtail millet (Setaria italica).

KEY MESSAGE: Multi-environment QTL mapping identified 23 stable loci and 34 co-located QTL clusters for panicle architecture and grain yield-related traits, which provide a genetic basis for foxtail millet yield improvement. Panicle architecture and grain weight, both of which are influenced by genetic and environmental factors, have significant effects on grain yield potential. Here, we used a recombinant inbred line (RIL) population of 333 lines of foxtail millet, which were grown in 13 trials with varying environmental conditions, to identify quantitative trait loci (QTL) controlling nine agronomic traits related to panicle architecture and grain yield. We found that panicle weight, grain weight per panicle, panicle length, panicle diameter, and panicle exsertion length varied across different geographical locations. QTL mapping revealed 159 QTL for nine traits. Of the 159 QTL, 34 were identified in 2 to 12 environments, suggesting that the genetic control of panicle architecture in foxtail millet is sensitive to photoperiod and/or other environmental factors. Eighty-eight QTL controlling different traits formed 34 co-located QTL clusters, including the triple QTL cluster qPD9.2/qPL9.5/qPEL9.3, which was detected 23 times in 13 environments. Several candidate genes, including Seita.2G388700, Seita.3G136000, Seita.4G185300, Seita.5G241500, Seita.5G243100, Seita.9G281300, and Seita.9G342700, were identified in the genomic intervals of multi-environmental QTL or co-located QTL clusters. Using available phenotypic and genotype data, we conducted haplotype analysis for Seita.2G002300 and Seita.9G064000,which showed high correlations with panicle weight and panicle exsertion length, respectively. These results not only provided a basis for further fine mapping, functional studies and marker-assisted selection of traits related to panicle architecture in foxtail millet, but also provide information for comparative genomics analyses of cereal crops.

Fine mapping of qPH9, a major quantitative trait locus, responsible for plant height in foxtail millet [Setaria italica (L.) P. Beauv.].

Plant height is vital for crop yield by influencing plant architecture and resistance to lodging. Although lots of quantitative trait loci (QTLs) controlling plant height had been mapped in foxtail millet, their contributions to phenotypic variation were generally small and mapping regions were relatively large, indicating the difficult application in molecular breeding using marker-assisted selection (MAS). In the present paper, a total of 23 QTLs involving in 15 traits were identified via a high-density Bin map containing 3024 Bin markers with an average distance of 0.48 cM through an F2 population. Among them, qPH9, with a large phenotypic variation explained (51.6%) related to plant height, was one of the major QTLs. Furthermore, qPH9 was repeatedly detected in multi-environments under field conditions using two new developed F2 populations from the same F1 plant, and was narrowed down to a smaller interval of 281 kb using 1024 recessive F2 individuals from the same F1 plant. Finally, we found that there was an extremely significant correlation between marker MRI1016 and plant height, and further speculated that Seita.9G088900 and Seita.9G089700 could be key candidates of qPH9. This study laid an important foundation for the cloning of qPH9 and molecular breeding of dwarf varieties via MAS. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01261-w.

Transcriptome and metabolite analysis reveal the drought tolerance of foxtail millet significantly correlated with phenylpropanoids-related pathways during germination process under PEG stress.

BACKGROUND: Foxtail millet [Setaria italica (L.) P. Beauv.] is an excellent crop known for its superior level of drought tolerance across the world. Especially, less water is needed during its germination period than the other cereal crops. However, the knowledge of the mechanisms underlying the abiotic stress effects on seed germination of foxtail millet is largely unknown. RESULTS: The water uptake pattern of foxtail millet seeds was ploted during germination period, according to which the germination time course of millet was separated into three phases. We sequenced the transcriptome of foxtail millet seeds, which were treated by PEG during different germination phases after sowing. The transcriptional studies revealed that more DEGs were identified during the further increase in water uptake period (phase III) than during the rapid initial uptake period (phase I) and the plateau period (phase II) under PEG stress. The pathway analysis of DEGs showed that the highly enriched categories were related to phenylpropanoid biosynthesis, plant hormone signal transduction and phenylalanine metabolism during phase III. The 20 phenylpropanoids-related genes of germinating foxtail millet were found to be down-regulated during the further increase in water uptake period under PEG stress. Further expression analysis identified 4 genes of phenylalanine ammonia-lyase, 4-coumarate-CoA ligase 3, cinnamoyl-CoA reductase 1, cationic peroxidase SPC4 in phenylpropanoids-related pathway, which played important roles in foxtail millet in response to PEG stress during different germination periods. The studies of metabolites in phenylpropanoid biosynthesis pathway revealed that higher amount of cinnamic acid was accumulated in germinating seeds under PEG stress, while the contents of p-coumaric acid, caffeic acid, ferulic acid and sinapic acid were decreased. And the effects of five phenolic compounds on germination and growth of foxtail millet showed that 1 mM concentration of cinnamic acid inhibited shoot and root growth, especially root development. Ferulic acid, caffeic acid, sinapic acid and p-coumaric acid could increase the root length and root/sprout in lower concentration. CONCLUSIONS: These findings suggest that key genes and metabolites of foxtail millet related with phenylpropanoids pathway may play prominent roles in the regulation of resistance to drought during germination. Foxtail millet can probably avoid drought by regulating the levels of endogenous allelochemicals.

Manure and mineral fertilization change enzyme activity and bacterial community in millet rhizosphere soils.

Fertilization is a key agricultural practice for increasing millet yields and influencing soil properties, enzyme activities and rhizosphere bacterial communities. High throughput Illumina sequencing of the 16S rDNA was applied to compare the bacterial community structures and diversities among six different soil samples. The experiments involved the following: no fertilizer (CK), phosphate (P) and potassium (K) plus organic manure (M) (PKM), nitrogen (N) and K plus M (NKM), NPM, NPK and NPKM fertilization. The results showed that the NPKM fertilization of the millet field had a maximal yield of 3617 kg ha-1 among the six different treatments. The abundances of the Actinobacteria and Bacteroidetes phyla, especially the Devosia, Mycobacterium, Opitutus and Chitinophaga genera, were higher in NPKM than those in the other samples. Redundancy analysis showed that the soil organic matter (SOM), available phosphorus (AP), and urease (UR) activity were significantly correlated with bacterial communities, while SOM and AP were strongly correlated with soil enzyme activities. Pearson's correlation showed that the available nitrogen was strongly correlated with Devosia and Mycobacterium, and SOM was strongly correlated with Opitutus and Chitinophaga. Besides, catalase was significantly related to Iamia, the UR was significantly related to Devosia, phosphatase was significantly related to Luteimonas and Chitinophaga. Based on the soil quality and millet yield, NPKM treatment was a better choice for the millet field fertilization practices. These findings provide a better understanding of the importance of fertilization in influencing millet yield, soil fertility and microbial diversity, and they lead to a choice of scientific fertilization practices for sustainable development of the agroecosystem.

A graph-based genome and pan-genome variation of the model plant Setaria.

Setaria italica (foxtail millet), a founder crop of East Asian agriculture, is a model plant for C4 photosynthesis and developing approaches to adaptive breeding across multiple climates. Here we established the Setaria pan-genome by assembling 110 representative genomes from a worldwide collection. The pan-genome is composed of 73,528 gene families, of which 23.8%, 42.9%, 29.4% and 3.9% are core, soft core, dispensable and private genes, respectively; 202,884 nonredundant structural variants were also detected. The characterization of pan-genomic variants suggests their importance during foxtail millet domestication and improvement, as exemplified by the identification of the yield gene SiGW3, where a 366-bp presence/absence promoter variant accompanies gene expression variation. We developed a graph-based genome and performed large-scale genetic studies for 68 traits across 13 environments, identifying potential genes for millet improvement at different geographic sites. These can be used in marker-assisted breeding, genomic selection and genome editing to accelerate crop improvement under different climatic conditions.

Protein Isolates from Raw and Cooked Foxtail Millet Attenuate Development of Type 2 Diabetes in Streptozotocin-Induced Diabetic Mice.

SCOPE: Millet protein has received much attention due to its beneficial role in alleviating metabolic disease symptoms. This study aims to investigate the role and molecular mechanism of foxtail millet protein isolates, including protein isolates from raw and cooked foxtail millet in alleviating diabetes, including gut microbiota and intracellular signal pathways. METHODS AND RESULTS: Protein isolates from raw and cooked foxtail millet are orally administered to streptozotocin (STZ)-induced diabetic mice for 5 weeks before hypoglycemic effect evaluation. The results show that foxtail millet protein isolates improve glucose intolerance and insulin resistance in diabetic mice. However, only the protein isolate from cooked foxtail millet reverse the weight loss trend and alleviate lipid disorders in diabetic mice. Besides, 16S rRNA sequencing show that both raw and cooked foxtail millet protein isolates altered diabetes-induced gut dysbiosis. In addition, western blotting analysis indicated that the protein isolate from cooked foxtail millet increases the expression levels of glucagon-like peptide-1 receptor (GLP-1R), phosphoinositide 3-kinase (PI3K), and phosphoinositide-protein kinase B (p-AKT)/AKT while the protein isolate from raw foxtail millet downregulates stearoyl-coenzyme A desaturase 1 (SCD1) level. CONCLUSION: Both raw and cooked foxtail millet protein isolates can exert hypoglycemic effects in diabetic mice through rewiring glucose homeostasis, mitigating diabetes-induced gut dysbiosis, and affecting the GLP-1R/PI3K/AKT pathway.

Response of Multiple Tissues to Drought Revealed by a Weighted Gene Co-Expression Network Analysis in Foxtail Millet [Setaria italica (L.) P. Beauv.].

Characterization of drought-tolerance mechanisms during the jointing stage in foxtail millet under water-limited conditions is essential for improving the grain yield of this C4 crop species. In this trial, two drought-tolerant and two drought-sensitive cultivars were examined using transcriptomic dissections of three tissues (root, stem, and leaf) under naturally occurring water-limited conditions. We detected a total of 32,170 expressed genes and characterized 13,552 differentially expressed genes (DEGs) correlated with drought treatment. The majority of DEGs were identified in the root tissue, followed by leaf and stem tissues, and the number of DEGs identified in the stems of drought-sensitive cultivars was about two times higher than the drought-tolerant ones. A total of 127 differentially expressed transcription factors (DETFs) with different drought-responsive patterns were identified between drought-tolerant and drought-sensitive genotypes (including MYB, b-ZIP, ERF, and WRKY). Furthermore, a total of 34 modules were constructed for all expressed genes using a weighted gene co-expression network analysis (WGCNA), and seven modules were closely related to the drought treatment. A total of 1,343 hub genes (including RAB18, LEA14, and RD22) were detected in the drought-related module, and cell cycle and DNA replication-related transcriptional pathways were identified as vital regulators of drought tolerance in foxtail millet. The results of this study provide a comprehensive overview of how Setaria italica copes with drought-inflicted environments during the jointing stage through transcriptional regulating strategies in different organs and lays a foundation for the improvement of drought-tolerant cereal cultivars through genomic editing approaches in the future.

Hypoglycemic Effect of Prolamin from Cooked Foxtail Millet (Setaria italic) on Streptozotocin-Induced Diabetic Mice.

Millet proteins have been demonstrated to possess glucose-lowering and lipid metabolic disorder modulation functions against diabetes; however, the molecular mechanisms underlying their anti-diabetic effects remain unclear. The present study aimed to investigate the hypoglycemic effect of prolamin from cooked foxtail millet (PCFM) on type 2 diabetic mice, and explore the gut microbiota and serum metabolic profile changes that are associated with diabetes attenuation by PCFM. Our diabetes model was established using a high-fat diet combined with streptozotocin before PCFM or saline was daily administrated by gavage for 5 weeks. The results showed that PCFM ameliorated glucose metabolism disorders associated with type 2 diabetes. Furthermore, the effects of PCFM administration on gut microbiota and serum metabolome were investigated. 16S rRNA gene sequencing analysis indicated that PCFM alleviated diabetes-related gut microbiota dysbiosis in mice. Additionally, the serum metabolomics analysis revealed that the metabolite levels disturbed by diabetes were partly altered by PCFM. Notably, the decreased D-Glucose level caused by PCFM suggested that its anti-diabetic potential can be associated with the activation of glycolysis and the inhibition of gluconeogenesis, starch and sucrose metabolism and galactose metabolism. In addition, the increased serotonin level caused by PCFM may stimulate insulin secretion by pancreatic ß-cells, which contributed to its hypoglycemic effect. Taken together, our research demonstrated that the modulation of gut microbiota composition and the serum metabolomics profile was associated with the anti-diabetic effect of PCFM.

A high-density genetic map and QTL analysis of agronomic traits in foxtail millet [Setaria italica (L.) P. Beauv.] using RAD-seq.

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.

Microsatellite Variations of Elite Setaria Varieties Released during Last Six Decades in China.

Crop improvement is a multifaceted micro-evolutionary process, involving changes in breeding approaches, planting configurations and consumption preferences of human beings. Recent research has started to identify the specific genes or genomic regions correlate to improved agronomic traits, however, an apparent blank between the genetic structure of crop elite varieties and their improving histories in diverse modern breeding programs is still in existence. Foxtail millet (Setaria italica) was one of the earliest cereal crops to be domesticated and served as a staple crop for early civilizations in China, where it is still widely grown today. In the present trial, a panel of foxtail millet elite varieties, which were released in the last sixty years in different geographical regions of China, was characterized using microsatellite markers (SSRs). A clear separation of two subpopulations corresponding to the two eco-geographical regions of foxtail millet production in China was identified by the dataset, which also indicated that in more recently released elite varieties, large quantities of accessions have been transferred from spring-sowing to summer-sowing ecotypes, likely as a result of breeding response to planting configurations. An association mapping study was conducted to identify loci controlling traits of major agronomic interest. Furthermore, selective sweeps involved in improvement of foxtail millet were identified as multi-diverse minor effect loci controlling different agronomic traits during the long-term improvement of elite varieties. Our results highlight the effect of transition of planting configuration and breeding preference on genetic evolvement of crop species.

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setaria italica).

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5'-RLM-RACE) method.

A haplotype map of genomic variations and genome-wide association studies of agronomic traits in foxtail millet (Setaria italica).

Foxtail millet (Setaria italica) is an important grain crop that is grown in arid regions. Here we sequenced 916 diverse foxtail millet varieties, identified 2.58 million SNPs and used 0.8 million common SNPs to construct a haplotype map of the foxtail millet genome. We classified the foxtail millet varieties into two divergent groups that are strongly correlated with early and late flowering times. We phenotyped the 916 varieties under five different environments and identified 512 loci associated with 47 agronomic traits by genome-wide association studies. We performed a de novo assembly of deeply sequenced genomes of a Setaria viridis accession (the wild progenitor of S. italica) and an S. italica variety and identified complex interspecies and intraspecies variants. We also identified 36 selective sweeps that seem to have occurred during modern breeding. This study provides fundamental resources for genetics research and genetic improvement in foxtail millet.