Gene expression profiles in early leaf of rice(Oryza sativa)and foxtail millet(Setaria italica)

Plant anatomy is patterned early during leaf development which suggests studying the spatial–temporal transcriptomes of primordia will help identify critical regulative and functional genes.We successfully isolated the leaf primordia tissues from the C3grass rice and the C4grass foxtail millet by laser capture microdissection(LCM)and studied the gene expression throughout leaf developmental stages.Our data analysis uncovered the conserved expression patterns of certain gene clusters both in rice and foxtail millet during leaf development.We revealed genes and transcription factors involved in vein formation,stomatal development,and suberin accumulation.We identified 79 candidate genes associated with functional regulation of C4anatomy formation.Screening phenotype of the candidate genes revealed that knock-out of a putative polar auxin transport related gene NAL1 resulted significantly reduced veinal space in rice leaf.Our present work provides a foundation for future analyses of genes with novel functions in grasses and their role in leaf development,in particular the role in leaves with a contrasting C3vs.C4biosynthetic pathway.

SiRAP2-12,a Positive Regulatory Factor,Effectively Improves the Waterlogging Tolerance of Foxtail Millet(Setaria italica)

Foxtail millet(Setaria italica)growth was inhibited because of waterlogging stress,which has caused yield reduc-tion.ERF family plays an important role to plant adversity tolerance.In our study,we obtained 19,819 differential expressed genes(DEGs)between the two treatments based on the RNA-seq sequencing of foxtail millet of water-logging stress.Furthermore,a total of 28 ERF family members were obtained,which have a complete open read-ing frame.We studied the evolution and function of SiERF family and how they affected the waterlogging tolerance.It was found that SiERF1A/B/C(GenBank ID:OR775217,OR775219,OR775218)and SiRAP2-12(GenBank ID:OR775216)have similar functions to the known waterlogging tolerance genes of other plants.Among them,the SiRAP2-12 expression was obviously significantly up-regulated in foxtail millet after 5d water-logging stress.After SiRAP2-12 was silenced,the activity of defense enzymes in millet decreased significantly.In details,superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD),the osmotic regulator proline(Pro),and the activity of the anaerobic respiratory enzyme alcohol dehydrogenase(ADH)content were decreased by 78.61%,29.52%,79.95%,19.41%and 54.77%,respectively.In contrast,the relative electrical conductivity contents(REC),malondialdehyde(MDA),and hydrogen peroxide(H_(2)O_(2))of the foxtail millet subjected to virus-induced gene silencing clearly increased by 1.03-fold,36.09%,and 15.21%,respectively.The content of sodium(Na^(+))in the SiRAP2-12-silenced foxtail millet also increased,but that of potassium(K^(+))decreased.Interestingly,we found that ethylene content was significantly reduced.Further,the SiAOC1 expression,an essential gene for ethylene synthesis,was inhibited in SiRAP2-12-silenced foxtail millet after waterlogging stress.Taken together,we hypothesized that SiRAP2-12 might be a positive regulator of millet tolerance to waterlogging stress.

Salt stress responses in foxtail millet:Physiological and molecular regulation

Foxtail millet(Setaria italica L.),a member of the Paniceae family,is a temperate and tropical grass species that is widely cultivated on the Eurasian continent.It is Chinese in origin and possesses a small genome,short growth cycle,and strong natural abiotic stress resistance.Elucidating the mechanism of millet tolerance to salt stress is becoming increasingly important with increasing soil salinization limiting crop productivity.The responses and mechanisms of tolerance to salt stress from other model plants such as Arabidopsis and rice,were compared with those from foxtail millet to summarize current research on responses to salt stress.Numerous processes are involved in these processes,including physiological reactions,sensing,signaling,and control at the transcriptional,post-transcriptional,and epigenetic levels.To increase crop productivity and agricultural sustainability,a variety of technologies can be used to investigate how salt tolerance is mediated by physiological and molecular processes in foxtail millet.

Integrated genomic and transcriptomic analysis reveals genes associated with plant height of foxtail millet

Foxtail millet(Setaria italica)is an important C4 model crop;however,due to its high-density planting and high stature,lodging at the filling stage resulted in a serious reduction in yield and quality.Therefore,it is imperative to identify and deploy the genes controlling foxtail millet plant height.In this study,we used a semi-dwarf line 263A and an elite high-stalk breeding variety,Chuang 29 to construct an F2 population to identify dwarf genes.We performed transcriptome analysis(RNA-seq)using internode tissues sampled at three jointing stages of 263A and Chuang 29,as well as bulk segregant analysis(BSA)on their F2 population.A total of 8918 differentially expressed genes(DEGs)were obtained from RNA-seq analysis,and GO analysis showed that DEGs were enriched in functions such as‘‘gibberellin metabolic process”and‘‘oxidoreductase activity”,which have previously been shown to be associated with plant height.A total 593 mutated genes were screened by BSA-seq method.One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines,and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions,which indicated they play a crucial role in plant height determination.Among them,we found a gibberellin biosynthesis related GA20 oxidase gene(Seita.5G404900),which had a single-base at the third exon,leading to the frameshift mutation at 263A.Cleaved amplified polymorphic sequence assay and association analysis proved the single-base in Seita.5G404900 co-segregated with dwarf phenotype in two independent F2 populations planted in entirely different environments.Taken together,the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height,and the molecular marker will be useful for marker-assisted dwarf breeding.

Identification and Analysis of Mature Plant Type Mutants of M_1 Generation Foxtail Millet Changnong35 Treated with EMS Mutagenesis

[Objective] The aim was to identify the mutants of millet Changnong35 induced by different concentrations of EMS, so as to construct a millet mutant library. [Method] Foxtail millet cultivar Changnong35 which is widely used in agricultural production, was treated with 0.8% and 1.0% EMS; and then seven traits of mutants were investigated analyzed, to classify the mutants into different groups. [Result] 282 mutants in the M1 generation related to plant type were obtained, of which, 100 mutant plants treated with 0.8% EMS can be divided into 10 groups; 182 mutant plants obtained by using 1.0% EMS can be divided into 17 groups. The analysis results of the mature plant type traits of the M1 Generation showed that, plant height, diameter of stem under spike, diameter of the first internode under spike and internode number of the mutants treated with 1.0% EMS were significantly different from those of control, while those of mutants treated with 0.8% EMS did not show significant difference from those of control. [Conclusion] The inducing with 1.0% EMS was more conducive to obtain a large number and different types of mutants from Changnong35.

Technical Regulation of Foxtail Millet Production by Combining Machinery and Agronomy

In order to solve the problems of low production efficiency,great loss and low yield,Millet Research Institute of Hebei Academy of Agriculture and Forestry Sciences integrated the plastic film mulching technique and mechanized production technique,forming a foxtail millet production technique combining machinery and agronomy.The foxtail millet production technique combining machinery and agronomy regulates millet production from the links of soil preparation,fertilization,variety selection,seeding,intertillage and fertilization and harvest,so as to achieve the effects of promoting the matching between agro-machinery and agronomy,improving the level of millet production mechanization,realizing light simplified production and saving labor cost.This technical regulation has a broad application prospect.

Effects of Water-permeability Plastic Film Mulching plus Bunch Planting on Root and Yield of Foxtail Millet

Effects of water-permeability plastic film plus bunch planting on root growth and development and yield of foxtail millet were studied by randomized block design. The results showed that water-permeability plastic film mulching plus bunch planting had a significant promoting effect on root growth and development and yield of foxtail millet. Compared with the CK, the total root length, total surface area, total root volume and number of. root tips increased by 51.30%, 47.89%, 48.39% and 41.63%, respectively. The yield increased by 48.57%, and there was significant positive correlation between root length, total surface area, total volume, number of root tips and dry matter weight of roots with yield. Developed roots are the main reason for the yield increasing effect of water-permeability plastic film mulching plus bunch planting.

Light Simplified Foxtail Millet Cultivation Technique Adopting Furrow Sowing beside Plastic Film Covering Micro-ridges

In order to solve the problem that dry-land foxtail millet production completely relies on rainwater with the characteristics of low instable yield, manual thinning and weeding, high labor intensity, and labor and time saving, Millet Research Institute of Hebei Academy of Agriculture and Forestry Sciences integrated furrow sowing beside plastic film covering micro-ridges, simplified cultivation and mechanized production, forming the simplified foxtail millet cultivation technique adopting furrow sowing beside plastic film covering micro-ridges. This study introduced the technique points of the simplified foxtail millet cultivation technique adopting furrow sowing beside plastic film covering micro-ridges, including preparation before sowing,sowing, attached agricultural machines, field management, harvest and residual film recovery.

Light Simplified Cultivation Techniques of Wide Row and Double Ridge with Filming, Fertilizing and Sowing on One for Foxtail Millet

In view of the problems of completely depending on rain, low and unstable yield and complicated planting of dry land foxtail millet, the light simplified cultivation techniques of wide row and double ridge with filming, fertilizing and sowing on one for foxtail millet was formed through the integration of plastic film mulching technology and mechanized production technology by Institute of Millet crops of Hebei Academy of Agriculture and Forestry Sciences, and the techniques were introduced from the key technologies of pre-sowing preparation, sowing, supporting equipment, field management, harvesting, plastic film recycling.

Genetic variation of yellow pigment and its components in foxtail millet(Setaria italica(L.) P.Beauv.) from different eco-regions in China

Kernel color is an important trait for assessing the commercial and nutritional quality of foxtail millet. Yellow pigment content （YPC） and carotenoid components （lutein and zeaxanthin） of 270 foxtail millet accessions, including 50 landraces and 220 improved cultivars, from four different eco-regions in China were surveyed using spectrophotometry and high performance liquid chromatography methods. Results indicated that YPC had rich variance, ranging from 1.91 to 28.54 mg kg-1, with an average value of 17.80 mg kg-1. The average YPC of improved cultivars （18.31 mg kg-1） was significantly higher than that of landraces （15.51 mg kg-l）. The YPC in cultivars from the Loess Plateau spring sowing region （LPSSR） was the highest （20.59 mg kg-~）, followed by the North China summer sowing region （NCSSR, 18.25 mg kg-1）, the northeast spring sowing region （NSSR, 17.25 mg kg-1）, and the Inner Mongolia Plateau spring sowing region （IMPSSR, 13.92 mg kg-1）. The variation coefficients of YPC in cultivars from NSSR, LPSSR, and IMPSSR were higher than that from NCSSR. A similar carotenoid profile was also obtained for 270 foxtail millet cultivars. Lutein and zeaxanthin accounted for approximately 55-65% of YPC in accessions. The lutein content was higher than zeaxanthin content in all cultivars. The ratio of lutein to zeaxanthin ranged from 1.51 to 6.06 with an average of 3.34. YPC was positively correlated with lutein （r=0.935, P〈0.01）, zeaxanthin （r=0.808, P〈0.01 ）, and growth duration （t=0.488, P〈0.01 ）, whereas it was negatively correlated with grain protein （t=-0.332, P〈0.01） and 1 000-kernel weight （t=-0.153, P〈0.05）. Our study is useful for screening and selecting cultivars with high levels of yellow pigment and for enhancing phytochemical concentrations in breeding programs.

Genetic Analysis and Preliminary Mapping of a Highly Male-Sterile Gene in Foxtail Millet(Setaria italica L.Beauv.) Using SSR Markers

Breeding of male-sterile lines has become the mainstream for the heterosis utilization in foxtail millet,but the genetic basis of most male-sterile lines used for the hybrid is still an area to be elucidated.In this study,a highly male-sterile line Gao146A was investigated.Genetic analysis indicated that the highly male-sterile phenotype was controlled by a single recessive gene a single recessive gene.Using F 2 population derived from cross Gao146A/K103,one gene controlling the highly male- sterility,tentatively named as ms1,which linked to SSR marker b234 with genetic distance of 16.7 cM,was mapped on the chromosome VI.These results not only laid the foundation for fine mapping of this highly male-sterile gene,but also helped to accelerate the improvement of highly male-sterile lines by using molecular marker assisted breeding method.

The NAC-like transcription factor Si NAC110 in foxtail millet(Setaria italica L.) confers tolerance to drought and high salt stress through an ABA independent signaling pathway

Foxtail millet（Setaria italica（L.）P.Beauv）is a naturally stress tolerant crop.Compared to other gramineous crops,it has relatively stronger drought and lower nutrition stress tolerance traits.To date,the scope of functional genomics research in foxtail millet（S.italic L.）has been quite limited.NAC（NAM,ATAF1/2 and CUC2）-like transcription factors are known to be involved in various biological processes,including abiotic stress responses.In our previous foxtail millet（S.italic L.）RNA seq analysis,we found that the expression of a NAC-like transcription factor,SiNAC110,could be induced by drought stress;additionally,other references have reported that SiNAC110 expression could be induced by abiotic stress.So,we here selected SiNAC110 for further characterization and functional analysis.First,the predicted SiNAC110 protein encoded indicated SiNAC110 has a conserved NAM（no apical meristem）domain between the 11–139 amino acid positions.Phylogenetic analysis then indicated that SiNAC110 belongs to subfamily III of the NAC gene family.Subcellular localization analysis revealed that the SiNAC110-GFP fusion protein was localized to the nucleus in Arabidopsis protoplasts.Gene expression profiling analysis indicated that expression of SiNAC110 was induced by dehydration,high salinity and other abiotic stresses.Gene functional analysis using SiNAC110 overexpressed Arabidopsis plants indicated that,under drought and high salt stress conditions,the seed germination rate,root length,root surface area,fresh weight,and dry weight of the SiNAC110 overexpressed lines were significantly higher than the wild type（WT）,suggesting that the SiNAC110 overexpressed lines had enhanced tolerance to drought and high salt stresses.However,overexpression of SiN AC110 did not affect the sensitivity of SiNAC110 overexpressed lines to abscisic acid（ABA）treatment.Expression analysis of genes involved in proline synthesis,Na＋/K＋transport,drought responses,and aqueous transport proteins were higher in the SiNAC110overexpressed lines than in the WT,whereas expression of ABA-dependent pathway genes did not change.These results indicated that overexpression of SiNAC110 conferred tolerance to drought and high salt stresses,likely through influencing the regulation of proline biosynthesis,ion homeostasis and osmotic balance.Therefore,SiNAC110 appears to function in the ABA-independent abiotic stress response pathway in plants.

Assessment of Genetic Relationship of Foxtail Millet with Its Wild Ancestor and Close Relatives by ISSR Markers

Inter simple sequence repeat（ISSR） analysis was applied to samples of foxtail millet and its wild ancestor and other close relatives of the genus Setaria in order to detect domestication-related geographical structure and phylogenetic relationship of those species.Eighty-one accessions of nine Setaria species that originated from different regions were used in this study.Fourteen out of the 27 ISSR primers screened amplified successfully and a total of 156 markers were scored for all the accessions,with a high level of polymorphism being detected.The dendrogram based on UPGMA cluster analysis showed clear geographic structure among foxtail millet and its wild ancestor green foxtail,which implies that northern China is the domestication center for both the Chinese and European foxtail millet landraces used in this study.This result did not support the hypothesis that China and Europe are independent domestication centers for foxtail millet proposed by several previous studies based on morphological and isozyme data.The dendrogram also clearly classified the Setaria sample used into two groups,a viridis and a pumila groups.The viridis group was composed of S.viridis,S.italica,S.faberii,S.verticillata,S.leucopila,and S.queenslandica,and the pumila group consisted of S.parviflora and S.pumila,which is consistent with the recently proposed hypothesis of multiple origin of Setaria species.The phylogenetic relationships among different species are discussed.

Influences of four processing methods on main nutritional components of foxtail millet: A review

Foxtail millet, originated from China and now cultivated worldwide, is a kind of high dietary fiber whole grain food, and has a high level of vitamins and proteins. Furthermore, foxtail millet has many positive effects on the adjuvant treatment of diabetes, cancer, and cardiovascular diseases because of the abundance in polyphenols. Nonetheless, foxtail millet has poor processing characteristics due to the absence of gluten, restricting the development of foxtail millet products. Studies have demonstrated that heat-moisture treatment, extrusion, superfine grinding, and microbial fermentation are promising methods to improve the processing qualities of foxtail millet. Heat-moisture treatment is helpful to increase the content of resistant starch but has less influence on other components, further reduce the GI value of foxtail millet. The extrusion has positive effects on improving the solubility of foxtail millet starch and increasing the contents of polyunsaturated fatty acid, linoleic and linolenic acids, and adverse effects on reducing the solubility of foxtail millet proteins and causing losses of nutrients due to Maillard reaction. Superfine grinding can reduce the particle size of foxtail millet to obtain a better mouthfeel of foxtail millet products. The superfine foxtail millet flour has better solubility, higher freeze-thaw stability, and lower gelatinization temperature. Microbial fermentation contributes positively to reducing the molecular weight and retrogradation value of foxtail millet starch, degrading rapidly digested starch, and improving the digestibility of foxtail millet protein. This paper briefly introduced the effects of different processing methods on foxtail millet nutrients, aiming to provide references for increasing the variety and improving the quality of foxtail millet products.

Identification of blast-resistance loci through genome-wide association analysis in foxtail millet(Setaria italica (L.)Beauv.)

Blast disease caused by the fungus Magnaporthe grisea results in significant yield losses of cereal crops across the world.To date,very few regulatory genes contributing to blast resistance in grass species have been identified and the genetic basis of blast resistance in cereals remains elusive.Here,a core collection of foxtail millet(Setaria italica)containing 888 accessions was evaluated through inoculation with the blast strain HN-1 and a genome-wide association study(GWAS)was performed to detect regulators responsible for blast disease resistance in foxtail millet.The phenotypic variation of foxtail millet accessions inoculated with the blast strain HN-1 indicated that less than 1.60% of the samples were highly resistant,35.25% were moderately resistant,57.09% were moderately susceptible,and 6.08% were highly susceptible.The geographical pattern of blast-resistant samples revealed that a high proportion of resistant accessions were located in lower latitude regions where the foxtail millet growing season has higher rain precipitation.Using 720000 SNP markers covering the Setaria genome,GWAS showed that two genomic loci from chromosomes 2 and 9 were significantly associated with blast disease resistance in foxtail millet.Finally,eight putative genes were identified using rice blast-related transcriptomic data.The results of this work lay a foundation for the foxtail millet blast resistance biology and provide guidance for breeding practices in this promising crop species and other cereals.

Foxtail millet supplementation improves glucose metabolism and gut microbiota in rats with high-fat diet/streptozotocin-induced diabetes

Foxtail millet(FM)whole grain has received special attention in recent years.To confirm the hypoglycemic effects of FM,we investigated the effects of FM supplementation on glucose metabolism and gut microbiota in rats with high-fat diet/streptozotocin(HFD/STZ)-induced diabetes.Specifically,we fully assessed the blood biochemical profiles,pancreatic histopathology,insulin-glucagon immunofluorescence,short-chain fatty acids,and gut microbiota composition of rats with HFD/STZ-induced diabetes before and after FM supplementation.Results showed that both 30% and 48% FM supplementation significantly decreased concentrations of fasting blood glucose,60-min blood glucose,and blood triglycerides(P<0.05);additionally,48% FM supplementation significantly improved blood glucose tolerance and insulin resistance(P<0.05).However,FM supplementation could not effectively repair damage to β-cells over a short period of time.In addition,4 weeks of 48% FM supplementation siginificantly increased the relative abundance of Bifidobacterium and concentration of butyrate,suggesting that the hypoglycemic effects of FM supplementation might be partially mediated by gut microbiota.Collectively,we found a dose-dependent relationship between FM supplementation and improvement of blood glucose metabolism,but did not find a synergistic effect between FM supplementation and metformin(Met)treatment.Our findings provide further support that consuming more whole-grain FM might be beneficial to individuals suffering from type 2 diabetes.

Nutrient Composition of Different Foxtail Millet Seeds

Ten millet cultivars from China and abroad were analyzed for their crude fat, amylose and amylopectin, crude protein and amino acid content. A wide variation was observed in amylopectin contain, ranging from 54.47% to 69.26%, while amylose content ranged from 11.26% to 23.22%. The average crude fat in all cultivates was 3.46%, and most cultivates ranged from 3.1% to 3.7%. Protein contents ranged from 8.61% to 15.54% with a mean value of 11.94%. For amino acid composition, the ten cultivates were particularly rich in leucine, and the mean was 34.57 mg/g. The millet cultivars were also high in glycine, glutamic acid and cysteine. Millet was known to be limiting in the essential amino lysine, and the mean was 5.18 mg/g. Other amino acids, tyrosine, histidine and arginine were also very low in the ten cultivars. In general, significant nutrient composition differences were observed between the different millet cultivars, and A2 （a kind of mother line of the hybrid millet） and hybrid millet registered a higher level of protein and amino acid composition which would be useful in millet breeding.

Comprehensive analysis of YABBY gene family in foxtail millet(Setaria italica)and functional characterization of SiDL

YABBY genes are plant-specific transcription factors(TFs)that function in plant growth and development.We investigated the functions of the YABBY genes in plants’stress tolerance by analyzing the YABBY genes in foxtail millet(Setaria italica)and studying their functions on plant growth and responses to different stresses.Eight YABBY genes were identified on five chromosomes,which showed strong relationships with YABBY genes in other monocot species.Phylogenetical Si YABs were classified into four clades:FIL/YAB3,YAB2,INO,and CRC.No monocot YABBY member was classified into the YAB5 clade.Four conserved motifs were identified,and motif 1 constituted the YABBY domain,whereas motifs 2 and 3 formed the C2-C2 region.Si YAB genes were highly expressed in reproductive tissues.Si DL,one of the Si YABs,was selected to be overexpressed in Arabidopsis thaliana to check the functions of the YABBY genes.Overexpression of Si DL in A.thaliana caused delayed flowering,leaf curling,and reduced seed size.In addition,Si DL acted as a negative regulator in plant response to salt stress.Our study provides information to assist in studying the YABBY gene functions in S.italica.

Control Effect of Different Concentrations of Herbicide Atrazine on Weed in Foxtail Millet Field

The control effect of different concentrations of herbicide atrazine （before seedling after sowing） on weed in foxtail millet field was compared, which provide theoretical basis for screening suitable herbicides concentration for foxtail millet production. As a result, after spraying 25 and 40 d, plant control effect and fresh weight control effect were best by 4 500 ml/hm^2 processing, and fresh weight control effect reached 83.16% and 86.43%. Compared with spraying water （CK）, yieldincreasing effect was the best in the treatment group with atrazine at 2 250 ml/hm^2, where yield growth rate was 54.31%, followed by the treatment group with atrazine at 3 000 ml/hm^2. Therefore, 2 250 ml/hm^2 was the optiaml concentration of herbicide atrazine for millet production.

Genetics, Development, and Application of Cytoplasmic Herbicide Resistance in Foxtail Millet

The effect of cytoplasmic herbicide resistant gene in millet plants was studied. The heterozygous populations and isogenic lines with homocaryotic alloplasmic genes were obtained by crossing and reciprocal crossing of cytoplasmic herbicide resistant plants with susceptive plants of foxtail millet. The characters of F1, F2, backcross and composite cross groups, and the growth and development of isogenic lines were compared. The cytoplasmic herbicide resistant gene slowed the development of seedling, delayed heading, and shortened the milking stage in the foxtail millet plant. Yield capacity and main agronomic characters were all affected by the cytoplasmic herbicide resistant gene in most of the backcross, composite cross, and F2 populations. However, there was stronger hybrid vigor in F1. The backcrosses, composite crosses, and F2 populations were widely separated and some of them had good characters similar to those of susceptive groups. The plant characters and development of foxtail millet were negatively affected by the cytoplasmic herbicide resistant gene. The authors proposed a method of using hybrid vigor to obtain high yield and avoid the negative effects of herbicide resistance cytoplasm in plant growth. The expected results could be obtained by selecting individuals in separate populations of fast developed seedlings, well-developed roots, and with capacities of early heading and fast milking. Guided by the principal mentioned above, many high yield lines and hybrid crosses of foxtail millet with herbicide resistant cytoplasm were obtained.