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.

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.

Identification of no pollen 1 provides a candidate gene for heterosis utilization in foxtail millet(Setaria italica L.)

Male sterility is a common biological phenomenon in plant kingdom and has been used to generate male-sterile lines, which are important genetic resources for commercial hybrid seed production. Although increasing numbers of male-sterility genes have been identified in rice(Oryza sativa) and Arabidopsis(Arabidopsis thaliana), few male-sterility-related genes have been characterized in foxtail millet(Setaria italica). In this study, we isolated a male-sterile ethyl methanesulfonate-generated mutant in foxtail millet, no pollen 1(sinp1), which displayed abnormal Ubisch bodies, defective pollen exine and complete male sterility. Using bulk segregation analysis, we cloned SiNP1 and confirmed its function with CRISPR/Cas9 genome editing. SiNP1 encoded a putative glucose-methanol-choline oxidoreductase.Subcellular localization showed that the SiNP1 protein was preferentially localized to the endoplasmic reticulum and was predominantly expressed in panicle. Transcriptome analysis revealed that many genes were differentially expressed in the sinp1 mutant, some of which encoded proteins putatively involved in carbohydrate metabolism, fatty acid biosynthesis, and lipid transport and metabolism, which were closely associated with pollen wall development. Metabolome analysis revealed the disturbance of flavonoids metabolism and fatty acid biosynthesis in the mutant. In conclusion, identification of SiNP1 provides a candidate male-sterility gene for heterosis utilization in foxtail millet and gives further insight into the mechanism of pollen reproduction in plants.

Effects of Fertility and Density on Biomass Production,Translocation and Lodging Resistance of Millet(Setaria italica L.)in North China

In this study, the plant biomass production, biomass translocation rates across tissues and the lodging resistant-associated traits of millet （ Setaria italica L.） in North China were investigated. Among the four summer millet cultivars, Baogu 19 exhibited improved plant biomass （PB） production at flowering and maturity stages, biomass translocation amount （BTA） from vegetative tissues to seeds during filling period, and lodging resistant-associated （LRA） traits compared with other cultivars, including enhanced stem lignin contents, increased anti-broken resistance （ABR）, anti-puncturing resistance （APR）, and stem diameter （SD） of plants. Compared with treatment regular cultivation （RC）, high fertility treatment （HF） increased the plant BP, BTA from vegetative tissue to seed at filling stage, and the plant LRA traits; whereas high density treatment （HD） decreased the plant BP at plant level, plant BTA from vegetative tissues to seeds at filling stage, and the plant LRA traits. Correlation analysis revealed that stem ABR is significantly correlated with the plant lodging resistant-associated traits including APR and SD in the summer millet cultivars examined under various cultivation treatments. Our investigation indicates that cultivar Baogu 19 together with suitable fertilization and density can promote the plant biomass production, enhance vegetative tissue biomass translocation to seeds, and improve the lodging resistance of summer millet plants in North China.

Genetic Diversity and Classification of Chinese Elite Foxtail Millet [Setaria italica (L.) P. Beauv.] Revealed by Acid-PAGE Prolamin

Arid and semi-arid regions of China account for more than half of the country. Because of drought resistance and high nutritive value, elite foxtail millet (Setaria Italica (L.) P. Beauv.) is one of the most important cereal crops in China. Evaluation of germplasm and genetic diversity of foxtail millet is still in its infancy, but prolamin could play an important role as a protein marker. To investigate the genetic diversity and population structure of foxtail millet from different ecological zones of China, 90 accessions of foxtail millet were collected from three major ecological areas: North, Northwest, and Northeast China. The prolamin contents were examined by acid polyacrylamide gel electrophoresis (acid-PAGE). Five to twenty-two prolamin bands appeared in tested varieties, of which were polymorphic, so prolamin patterns of foxtail millet varieties can be used in variety identification and evaluation. Structure analysis identified six groups, which matches their pedigree information but not their geographic origins. This indicated a high degree (87.78%) of consistency with a phylogenetic classification based on SSR. The results showed prolamin banding patterns were an effective method for analyzing foxtail millet genetic variability.

增强UV-B辐射与不同水平氮素对谷子(Setaria italica(L．)Beauv．)叶片保护物质及保护酶的影响

研究了生长在1.875 mmol.L-1和15 mmol.L-1硝态氮素水平条件下的谷子(Setaria italica(L.)Beauv.)开花期间在进行强度为7.12 kJ.m-.2d-1增强UV-B辐射处理时叶片类黄酮含量、苯丙氨酸解氨酶(PAL)和保护酶活性变化差异。主要结果表明在开花期无论是否进行增强UV-B辐射处理,较低水平氮素均比较高水平氮素更有利于提高谷子叶片PAL活性;叶片类黄酮含量除在进行增强UV-B处理时较低氮素条件下生长的谷子在开花末期显著高于较高氮素条件下生长的谷子外,受氮素水平影响不甚明显。而在开花期不进行与进行增强UV-B辐射处理,氮素水平对叶片保护酶的影响有所差异:不进行增强UV-B辐射处理,整个开花期氮素水平对谷子叶片SOD活性有显著影响而对ASP活性无显著影响,对CAT和POD活性则在开花期部分阶段有显著影响。进行增强UV-B辐射处理,整个开花期氮素水平对谷子叶片SOD与CAT活性有显著影响而对ASP、POD活性影响不显著。

古粟(Setaria italica Beauv.)研究综述

粟于中国北方俗称"谷子",脱壳后称"小米",南方则通称"小米",在古代粮食作物中的地位举足轻重。近世学者对粟的史前考古发现、起源与传播、稷的粟黍之争、生产与文化等方面行了研究,并且已经取得了很多成果,但在某些方面的探索还非常不够,如史前人类生活中粟作的地位与演变;古粟的栽培、储藏、加工与利用技术;历史上粟的生产布局、价格与产量的变动;粟与古代经济、政治、文化的关系等。今后应加强在这些方面的投入,不断拓展粟研究的深度和广度。

The application of Fourier transform mid-infrared(FTIR) spectroscopy to identify variation in cell wall composition of Setaria italica ecotypes

Cell wall composition in monocotyledonous grasses has been identified as a key area of research for developing better feedstocks for forage and biofuel production.Setaria viridis and its close domesticated relative Setaria italica have been chosen as suitable monocotyledonous models for plants possessing the C4 pathway of photosynthesis including sorghum,maize,sugarcane,switchgrass and Miscanthus×giganteus.Accurate partial least squares regression（PLSR）models to predict S.italica stem composition have been generated,based upon Fourier transform mid-infrared（FTIR）spectra and calibrated with wet chemistry determinations of ground S.italica stem material measured using a modified version of the US National Renewable Energy Laboratory（NREL）acid hydrolysis protocol.The models facilitated a high-throughput screening analysis for glucan,xylan,Klason lignin and acid soluble lignin（ASL）in a collection of 183 natural S.italica variants and clustered them into classes,some possessing unique chemotypes.The predictive models provide a highly efficient screening tool for large scale breeding programs aimed at identifying lines or mutants possessing unique cell wall chemotypes.Genes encoding key catalytic enzymes of the lignin biosynthesis pathway exhibit a high level of conservation with matching expression profiles,measured by RT-q PCR,among accessions of S.italica,which closely mirror profiles observed in the different developmental regions of an elongating internode of S.viridis by RNASeq.

Calcineurin B-like protein 5(SiCBL5)in Setaria italica enhances salt tolerance by regulating Na^(+)homeostasis

Salinity,a major abiotic stress,reduces plant growth and severely limits agricultural productivity.Plants regulate salt uptake via calcineurin B-like proteins(CBLs).Although extensive studies of the functions of CBLs in response to salt stress have been conducted in Arabidopsis,their functions in Setaria italica are still poorly understood.The foxtail millet genome encodes seven CBLs,of which only SiCBL4 was shown to be involved in salt response.Overexpression of SiCBL5 in Arabidopsis thaliana sos3-1 mutant rescued its salt hypersensitivity phenotype,but that of other SiCBLs(SiCBL1,SiCBL2,SiCBL3,SiCBL6,and SiCBL7)did not rescue the salt hypersensitivity of the Atsos3-1 mutant.SiCBL5 harbors an N-myristoylation motif and is located in the plasma membrane.Overexpression of SiCBL5 in foxtail millet increased its salt tolerance,but its knockdown increased salt hypersensitivity.Yeast two-hybrid and firefly luciferase complementation imaging assays showed that SiCBL5 physically interacted with SiCIPK24 in vitro and in vivo.Cooverexpression of SiCBL5,SiCIPK24,and SiSOS1 in yeast conferred a high-salt-tolerance phenotype.Compared to wild-type plants under salt stress conditions,SiCBL5 overexpressors showed lower accumulations of Na^(+) and stronger Na^(+) efflux,whereas RNAi-SiCBL5 plants showed higher accumulations of Na^(+) and weaker Na^(+) efflux.These results indicate that SiCBL5 confers salt tolerance in foxtail millet by modulating Na^(+) homeostasis.

Chromosome Location of the Male-sterility and Yellow Seedling Gene in Line 1066A of Foxtail Millet

Using foxtail millet (Setaria italica (L.) Beauv.) male-sterile line 1066A as female parent and Yugu 1 primary trisomic series (1 - 7) and tetrasomics 8, 9 as male parents, chromosome location of gene for male-sterility and yellow seedling in line 1066A was studied by primary trisomic analysis. The plants of F-1 generation of trisomics 2 - 9 were obtained by crossing with a great many plants of 1066A. F-1 generation of trisomics was similar to their male parent in morphologic characters, the color of their seedling was green, and pollen was partially fertile. The segregation ratio of fertility to sterility is 3:1 in F-2 generation of trisomics 2, 3, 4, 5, 7, 8 and 9; and 14:1 only in F-2 generation of trisomic 6 (chi(0.05)(2) = 0.012). The segregation ratio of green seedling to yellow seedling is 12:1 only in F-2 generation of trisomic 7 (chi(0.05)(2) = 0.31), but in other cases, this ratio is 3:1. The results indicated that the male-sterility gene was located on chromosome 6, and the gene for yellow seedling was monogenic recessive and located on chromosome 7. The rate of trisomics transmission by pollen was tested, trisomics 8 and 9 were the highest in rates of trisomics transmission and followed by trisomics 6 and 4.

Retrotransposon-mediated DELLA transcriptional reprograming underlies semi-dominant dwarfism in foxtail millet

Retrotransposons account for a large proportion of the genome and genomic variation, and play key roles in creating novel genes and diversifying the genome in many eukaryotic species. Although retrotransposons are abundant in plants, their roles had been underestimated because of a lack of research. Here, we characterized a gibberellin Acid (GA)-insensitive dwarf mutant, 84133, in foxtail millet. Map-based cloning revealed a 5.5-kb Copia-like retrotransposon insertion in DWARF1 (D1), which encodes a DELLA protein. Transcriptional analysis showed that the Copia retrotransposon mediated the transcriptional reprogramming of D1 leading to a novel N-terminal-deleted truncated DELLA transcript that was putatively driven by Copia's LTR, namely D1-TT, and another chimeric transcript. The presence of D1-TT was confirmed by protein immunodetection analysis. Furthermore, D1-TT protein was resistant to GA3 treatment compared with the intact DELLA protein due to its inability to interact with the GA receptor, SiGID1. Overexpression of D1-TT in foxtail millet resulted in dwarf plants, confirming that it determines the dwarfism of 84133. Thus, our study documents a rare instance of long terminal repeat (LTR) retrotransposon-mediated transcriptional reprograming in the plant kingdom. These results shed light on the function of LTR retrotransposons in generating new gene functions and genetic diversity.

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.

Variations in chlorophyll content, stomatal conductance, and photosynthesis in Setaria EMS mutants

Chlorophyll (Chl) content,especially Chl b content,and stomatal conductance (G_s) are the key factors affecting the net photosynthetic rate (P_n).Setaria italica,a diploid C_4 panicoid species with a simple genome and high transformation efficiency,has been widely accepted as a model in photosynthesis and drought-tolerance research.The current study characterized Chl content,G_s,and P_n of 48 Setaria mutants induced by ethyl methanesulfonate.A total of 24,34,and 35 mutants had significant variations in Chl content,G_s,and P_n,respectively.Correlation analysis showed a positive correlation between increased G_s and increased P_n,and a weak correlation between decreased Chl b content and decreased P_n was also found.Remarkably,two mutants behaved with significantly decreased Chl b content but increased P_n compared to Yugu 1.Seven mutants behaved with significantly decreased G_s but did not decrease P_(n )compared to Yugu 1.The current study thus identified various genetic lines,further exploration of which would be beneficial to elucidate the relationship between Chl content,G_s,and P_n and the mechanism underlying why C_4 species are efficient at photosynthesis and water saving.

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.

The boron transporter SiBOR1 functions in cell wall integrity, cellular homeostasis, and panicle development in foxtail millet

Boron(B) is an essential micronutrient for vascular plant growth. Both B deficiency and toxicity can impair tissue development in diverse plant species, but little is known about the effect of B on reproductive panicle development and grain yield. In this study, a mutant of Setaria italica exhibiting necrotic panicle apices was identified and designated as sibor1. Sequencing revealed a candidate gene, Si BOR1, with a G-to-A alteration at the seventh exon. Knockout transgenic lines generated by clustered regularly interspaced short palindromic repeats and their associated protein-9 also had necrotic panicles, verifying the function of Si BOR1. Si BOR1 encoded a membrane-localized B efflux transporter, co-orthologous to the rice BOR1 protein. Si BOR1 was dominantly expressed in panicles and displayed a distinct expression pattern from those of its orthologs in other species. The induced mutation in Si BOR1 caused a reduction in the B content of panicle primary branches, and B deficiency-associated phenotypes such as thicker cell walls and higher cell porosity compared with Yugu 1. Transcriptome analysis indicated that differentially expressed genes involved in cell wall biogenesis, jasmonic acid synthesis, and programmed cell death response pathways were enriched in sibor1. q PCR analysis identified several key genes, including phenylalanine ammonia-lyase(Si PAL) and jasmonate-ZIM-domain(Si JAZ) genes, responsive to B-deficient conditions. These results indicate that Si BOR1 helps to regulate panicle primary branch development to maintain grain yield in S. italica. Our findings shed light on molecular mechanisms underlying the relationship between B transport and plant development in S. italica.

Plants regeneration from protoplast and their genetic variation in foxtail millet

Compact calli derived from immature spikelet of a foxtail millet variety—Jigu 11cann’t be directly used for protoplast isolation because of its firm physical structure,and must beloosened with subculturing in M1,M2 and M3 media successively and altering these media compo-sitions.The loosened calli can be selected from the regulation and used for protoplast isolationsuccessfully.Rate of protoplast division in KM8P medium was 12.3—33.5%.Calli derivedthrough protoplast division are loose and cann’t be used directly for plan regeneration because ofits soft physical structure.When they were subcultured in N6—1,N6—2,N6—3 and N6—4 media,in which the media compositions were changed,the compact calli were obtained and 129 plantletswere regenerated from them.101 plants,which grew to maturity after transplanting the plantletsinto field,exhibited sterility in some degree.Most of the subsequent lines derived from the regen-erated plants were sterile and only two lines could get normal reproduction.

Innovation of Super Early-Mature Millet Germplasm with Sweet-Stem and Multi-Spikes

A new millet (Setaria italica Beauv) variety, super early-mature millet No.1, was bred by means of gene bank breedingmethod of target characters. This variety has the following outstanding characters. (1) Super early-mature. This varietyonly needs 1550C effective accumulated temperature and can normally maturate in the Bashang Region in Hebei Provinceof Chi na, which can break through the limit zone of millet cultivation and move the cultivation zone northward greatly. (2)Multi-spikes, in addition to the effect tilling at the top, the nodes in the low-middle part also can produce spikes. (3) Sweetstem have high sugar content. The contents of whole-sugar, soluable sugar and deoxidized sugar are 74.8, 200.5, 237.2%higher than the regular varieties respectively. (4) High gross protein content. The content of gross protein is higher thanthe regular varieties by 3.9-30.4%. (5)Changeable grain color. The grain color of super early-mature millet No.1 is red inShijiazhuang, but yellow in the Bashang region. In addition, this variety is characterized by good quality, high yield, andgood synthetic traits

A nucleotide substitution at the 5′ splice site of intron 1 of rice HEADING DATE 1(HD1) gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

We investigated genetic variation of a rice HEADING DATE 1(HD1) homolog in foxtail millet.First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions(including Yugu 1, a Chinese cultivar used for genome sequencing) from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by d CAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

Effects of Nitrogen Amount on the Photosynthesis Parameters of Summer Millet in North China

In this study,effects of nitrogen（ N） amount applied on photosynthesis behaviors of the summer millet in North China was investigated. Photosynthetic rates（ Pn）,chlorophyll contents（ Chl）,photosynthetic active duration（ PAD）,and chlorophyll relative steady phase（ RSP） in flag and the upper third leaves were assessed in cultivars of Baogu 19,Jigu 19,9050,and 60 D under three N treatments [i. e.,0（ control）,75,and 150 kg/hm2]. Results indicated that the photosynthesis parameters were drastically regulated by external N levels,all of them showing elevation along with the increased N input in both assayed leaves.Among the cultivars examined,behaviors of the photosynthetic parameters were much better in Baogu 19 and worse in 60 D. The plant yields in the cultivars under various N treatments were shown to be in consistent with the behavior of the photosynthesis parameters. Correlation analysis revealed that plant yield is positively correlated with Pn and Chl and significantly positively correlated with PAD and RSP,suggesting that longer effective photosynthetic duration of leaves impacts largely on plant biomass production and the yield formation potential. Our investigation indicates that suitable external N applied can increase the yield of summer millet associating with the improvement of photosynthesis behaviors in upper leaves that contribute to plant biomass at the late growth stage. Baogu 19 exhibited higher plant yield together with improved photosynthetic parameters in upper leaves,suggesting its potential as an elite cultivar in planting in the summer season of North China.

Haplotype variation and KASP markers for SiPSY1-A key gene controlling yellow kernel pigmentation in foxtail millet

Carotenoid biosynthesis and accumulation are important in determining nutritional and commercial value of crop products.Yellow pigmentation of mature kernels caused by carotenoids is considered a vital quality trait in foxtail millet,an ancient and widely cultivated cereal crop across the world.Genomic regions associated with yellow pigment content(YPC),lutein and zeaxanthin in foxtail millet grains were identified by genome-wide association analysis(GWAS),and SiPSY1(Phytoene synthase 1 which regulates formation of the 40-carbon backbone of carotenoids)was confirmed as the main contributor to all three components by knockout and overexpression analysis.SiPSY1 was expressed in seedlings,leaves,panicles,and mature seeds,and was subcellularly localized to chloroplasts.Transcription of SiPSY1 in 15 DAP immature grains was responsible for YPC in mature seeds.Selection of SiPSY1 combined with increased YPC in mature grains during domestication of foxtail millet was confirmed.Haplotype analysis suggested that expression level of SiPSY1 could be a selection target for future breeding programs,and a KASP marker was developed for selection of favorable SiPSY1 alleles in breeding.The results of this work will benefit nutritional and commercial improvement of foxtail millet varieties,as well as other cereal crops.