Molecular Plant ll

Research Article

Multi-omics analyses of 398 foxtail millet

accessions reveal genomic regions associated
with domestication, metabolite traits, and anti-
inflammatory effects
Xukai Li1,14, Jianhua Gao1,14, Jingyi Song2,14, Kai Guo3,14, Siyu Hou4, Xingchun Wang1,
Qiang He5, Yanyan Zhang5, Yakun Zhang4, Yulu Yang4, Jiaoyan Tang4, Hailang Wang6,
Staffan Persson7,8, Mingquan Huang2, Lishuai Xu9, Linlin Zhong10, Dongqin Li11,
Yongming Liu12, Hua Wu2,*, Xianmin Diao5,*, Peng Chen6,*, Xiaowen Wang13,*
and Yuanhuai Han4,*
1
Shanxi Key Laboratory of Minor Crop Germplasm Innovation and Molecular Breeding, College of Life Sciences, Shanxi Agricultural University, Taigu, China
2
Key Laboratory of Brewing Molecular Engineering of China Light Industry, Beijing Technology and Business University, Beijing, China
3
Department of Neurology, University of Michigan, Ann Arbor, MI, USA
4
Shanxi Key Laboratory of Minor Crop Germplasm Innovation and Molecular Breeding, College of Agriculture, Shanxi Agricultural University, Taigu, China
5
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
6
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
7
Copenhagen Plant Science Centre, Department of Plant & Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark
8
Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University,
Minhang, Shanghai 200240, China
9
College of Resources and Environment, Shanxi Agricultural University, Taigu, China
10
College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, China
11
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China
12
Grandomics Biosciences Company Limited, Beijing, China
13
College of Food Science and Engineering, Shanxi Agricultural University, Taigu, China
14These authors contributed equally to this article.
*Correspondence: Hua Wu (wuhua@btbu.edu.cn), Xianmin Diao (diaoxianmin@caas.cn), Peng Chen (chenpeng@mail.hzau.edu.cn), Xiaowen Wang
(wangx@sxau.edu.cn), Yuanhuai Han (hanyuanhuai@sxau.edu.cn)
https://doi.org/10.1016/j.molp.2022.07.003

ABSTRACT
Foxtail millet (Setaria italica), which was domesticated from the wild species green foxtail (Setaria viridis), is a
rich source of phytonutrients for humans. To evaluate how breeding changed the metabolome of foxtail millet
grains, we generated and analyzed the datasets encompassing the genomes, transcriptomes, metabolomes,
and anti-inflammatory indices from 398 foxtail millet accessions. We identified hundreds of common variants
that influence numerous secondary metabolites. We observed tremendous differences in natural variations
of the metabolites and their underlying genetic architectures between distinct sub-groups of foxtail millet.
Furthermore, we found that the selection of the gene alleles associated with yellow grains led to altered pro-
files of metabolites such as carotenoids and endogenous phytohormones. Using CRISPR-mediated genome
editing we validated the function of PHYTOENE SYNTHASE 1 (PSY1) gene in affecting millet grain color and
quality. Interestingly, our in vitro cell inflammation assays showed that 83 metabolites in millet grains have
anti-inflammatory effects. Taken together, our multi-omics study illustrates how the breeding history of fox-
tail millet has shaped its metabolite profile. The datasets we generated in this study also provide important
resources for further understanding how millet grain quality is affected by different metabolites, laying the
foundations for future millet genetic research and metabolome-assisted improvement.
Key words: foxtail millet, multi-omics, genetic association, metabolome, transcriptome, anti-inflammatory effects
Published by the Molecular Plant Shanghai Editorial Office in association with
Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author. 1367
Molecular Plant Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
Li X., Gao J., Song J., Guo K., Hou S., Wang X., He Q., Zhang Y., Zhang Y., Yang Y., Tang J., Wang H., Persson
S., Huang M., Xu L., Zhong L., Li D., Liu Y., Wu H., Diao X., Chen P., Wang X., and Han Y. (2022). Multi-omics
analyses of 398 foxtail millet accessions reveal genomic regions associated with domestication, metabolite traits,
and anti-inflammatory effects. Mol. Plant. 15, 1367–1383.
INTRODUCTION
Foxtail millet (Setaria italica) is an ancient cereal crop with a history
of cultivation of 11 000 years in northern China (Mamidi et al., 2020;
Yang et al., 2012). Due to its excellent drought tolerance and high
water-use efficiency, foxtail millet is widely cultivated as a dietary
staple in arid and semi-arid regions, particularly in China and India
(Bettinger et al., 2010). It also contributed to the origin and early
dispersal of Transeurasian languages (Robbeets et al., 2021).
During its domestication from green foxtail (Setaria viridis), many
traits were selected for cultivation, such as reduced or no
tillering, higher yield with larger panicles and grains, shatterproof
seeds, and loss of dormancy (Hu et al., 2018).
A striking aspect of foxtail millet domestication, compared with that
of other cereal crops such as wheat (Triticum aestivum) and rice
(Oryza sativa), is the selection of yellow millets (dehusked grain).
Most landraces and varieties of foxtail millet produce yellow millets,
whose color is mainly due to the accumulation of carotenoids and
flavonoids in the pericarp, aleurone, and endosperm. The ‘‘yellow-
ness’’ of the millets has been a major selection index for consumers.
This selection criterion may reflect the contribution of color to flavor
and nutritional content, although it is not clear whether selection
arose from a preference for color or taste. As stated in the Great
Pharmacopoeia ‘‘Ben Cao Gang Mu’’ (1578), ancient Chinese
had long realized that foxtail millets have beneficial effects on the
digestive system, and millet porridge is still popular not only as
food but also as a ‘‘stomach care’’ meal in northern China. A num-
ber of cases show that chronic digestive disorders can be allevi-
ated, or even cured, by ingesting foxtail millet (Wu and Liang, 2018).
Cytokines are regulators of host responses to infection, immune
responses, inflammation, and trauma. Pro-inflammatory cytokines
may worsen disease and induce fever, inflammation, tissue
destruction, and, in some cases, shock and death (Dinarello,
2000). For example, inflammatory bowel disease (IBD) is a
chronic inflammatory disorder of the gastrointestinal tract,
including ulcerative colitis and Crohn’s disease (Strober et al.,
2007). Although the pathogenesis of IBD remains unknown, IBD
patients frequently exhibit elevated levels of pro-inflammatory cyto-
kines such as tumor necrosis factor a (TNF-a), interleukin 1b (IL-1b),
and IL-6 (Fonseca-Camarillo and Yamamoto-Furusho, 2015).
Metabolite-based genome-wide association studies (mGWASs)
enable the simultaneous screening of many accessions for any as-
sociation between their genomes and different metabolites and can
reveal the genetic contribution to metabolic diversity and complex
traits (Riedelsheimer et al., 2012; Chen et al., 2014, 2016; Peng
et al., 2017; Tieman et al., 2017; Zhu et al., 2018; Fang and Luo,
2019). mGWASs have been successfully applied on various
model plants and agriculturally important species to establish
new links between genes and metabolites (Chan et al., 2010a,
2011; Chen et al., 2014; Zeng et al., 2020; Zhu et al., 2018),
although the biological significance of these associations remains
to be validated. Foxtail millet is an ideal candidate system for
GWAS because of its self-fertilization and high-quality reference
1368 Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author.
genome (Bennetzen et al., 2012; Zhang et al., 2012; Yang et al.,
2020).
To gain a better understanding of the genetic and metabolomic
variations across foxtail millet accessions, we performed a
high-throughput and comprehensive study on a diverse collec-
tion of foxtail millet germplasms. Through the mGWAS, we iden-
tified key genetic changes underlying the variations in metabolite
contents and profiles, and established associations between me-
tabolites and anti-inflammatory effects. Our study provides
important resources for future molecular breeding and selection
of elite foxtail millet varieties that would benefit human health.
RESULTS
Generation and analyses of multi-omics datasets
We conducted whole-genome resequencing of 398 geographically
diverse foxtail millet accessions (Supplemental Note), including 162
cultivars, 198 landraces, and 38 wild S. viridis, mostly from China
(Figure 1A and Supplemental Table 1). To characterize the
distribution of sequence variation across the accessions, we
identified a set of 4 158 075 filtered single-nucleotide polymor-
phisms (SNPs) and insertion-deletion polymorphisms (InDels),
from an average resequencing depth of 11.953 and 94.4%
coverage of the assembled xiaomi reference genome (Yang et al.,
2020) for analysis (minor allele frequency > 0.05 and missing rate
< 10%) (Supplemental Table 1). Of these SNPs, 30.5% occurred
in gene-coding regions (Supplemental Table 2). To assess
the genomic regions in foxtail millet that were targets of
domestication from S. viridis, we performed a genetic diversity
analysis. A neighbor-joining tree and a principal-component anal-
ysis (PCA) (Supplemental Figure 1A) revealed a clear separation
between Northern and Southern foxtail millet and between S.
italica and wild S. viridis. However, the landraces and cultivars
were not separated within the Northern and Southern branches
(Figure 1B). The linkage disequilibrium (LD) decay was about 35
kb (Supplemental Figure 1B).
To explore the extent of metabolic variation between different
foxtail millet accessions, we quantified metabolite levels using
ultra-performance liquid chromatography-tandem mass spec-
trometry (UPLC–MS/MS) from foxtail millet grains harvested
in 2018 and 2019 (Supplemental Tables 3 and 4). The
2018 metabolome data consisted of measurements from three
locations and detected 785 distinct metabolites (Supplemental
Tables 3, 5, 6, and 7), and the 2019 data identified 2557
metabolites from one location sampled as three biological
replicates (Supplemental Tables 4 and 8). In total, we identified
518 and 1050 metabolites in the 2018 and 2019 datasets,
respectively. Among the metabolites quantified in 2018, 21%
displayed broad-sense heritability (H2) greater than 0.50, with
5% of metabolites even reaching H2 values over 0.70
(Supplemental Figure 2A). The observed coefficients of
variation were above 50% for 56% of all metabolites
(Supplemental Figure 2B).
Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet Molecular Plant
A B
Temperature: <0
0 5
5 10
10 28

Setaria viridis
1000 Km

Content
1.0

0.8

0.6

0.4

0.2

0.0
Sub-groups: North_cultivar North_landrace Setaria_viridis South_cultivar South_landrace

D
40
30
20
-log10(P Value)

10
0 Color AA and NA der Flavonoids Others / Unkonwn
10
20
30
40

Figure 1. Phylogenetic relationships and metabolic accumulation in 398 foxtail millet accessions.
(A) Geographic distributions of foxtail millet accessions are indicated on the map of China under different temperature conditions. The annual average
temperature was obtained from the Chinese meteorological dataset in resource and environmental science and data center (https://www.resdc.cn).
(B) Neighbor-joining tree calculated from whole-genome SNPs. The genepools are colored: orange, North cultivar lines; yellow, North landrace lines;
green, S. viridis; navy blue, South cultivar lines; sky blue, South landrace lines.
(C) Heatmap visualization of relative differences in grains metabolites in foxtail millet accessions. The contents of each metabolite were normalized before
performing linkage hierarchical clustering. Red indicates high abundance, and blue indicates low relative metabolic content.
(D) The strength of association for known (top) and unknown (bottom) metabolites is indicated as the negative logarithm of the p value for the mixed linear
model (MLM). All metabolite-SNP associations with p values below 8.81 3 10
7 are plotted against genome location in intervals of 1 Mb. AA, amino acid;
NA ders, nucleic acid derivatives.

Based on the metabolite profiles of the grains, all varieties formed metabolites showed greater variation and lower overall contents
three main clusters in a PCA (Figure 2A), which was consistent than in foxtail millet samples (Figure 1C and Supplemental
with the evolutionary relationships observed between genetic Table 9), including serotonin (m0051, wh0485, wh0483),
markers (Supplemental Figure 1C). In S. viridis, 1104 N-acetylserotonin (mt1088, wh0770), N-(p-coumaroyl) serotonin
Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author. 1369
Molecular Plant Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet

A B
0.10 P

0.05
PC2 (6.54%)

0.00

-0.05

-0.10
Setaria viridis

-0.15
-0.06 -0.05 -0.04
PC1 (43.30%)
C ROF1
AFC2 FLK
GAE6
IDH1 CBL3
SHN1 FaTA
WR3 MIPS1
CAD2AGG2 IAA13 MUR4 CLC-A TRFL5 SPX1 GI
AGG3 APX1ACHT2 IBR5 galactosidase
TED4 GCS1 DMC1 NOL PCB2 XYP1
LAZ5 G4 XERICO D27

D E F G
40 25 25
-log10 (P value)

-log10 (P value)

-log10 (P value)
-log10 (P value)

20 12 20
30
15 8 15
20
10 10
10 4
5 5
0 0 0 0
10 20 30 40 Mb 10 20 30 40 Mb 5 10 20 30 Mb 10 20 30 40 50 Mb

H I J K
20
-log10 (P value)

15
-log10 (P value)

-log10 (P value)
-log10 (P value)

15 15 15
10 10 10 10
5 5 5 5
0 0 0 0
10 20 30 40 Mb 10 20 30 40 Mb 5 10 20 30 Mb 10 20 30 40 50 Mb

Figure 2. Genome-wide screening of domestication sweeps and GWAS on metabolite traits.

(A) PCA plots of the first two components of 355 foxtail millet accessions with 1977 metabolites.
(B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis for the top 5% of XP-CLR (cross-population composite likelihood ratio).
Abbreviations: Nc-Nl, North_cultivar vs. North_landrace; Nc-Sv, North_cultivar vs. Setaria_viridis; Nl-Sv, North_landrace vs. Setaria_viridis; Sc-Sl,
South_cultivar vs. South_landrace; Sc-Sv, South_cultivar vs. Setaria_viridis; Sl-Sv, South_landrace vs. Setaria_viridis.
(C) Genome-wide distribution of selective sweeps between all sub-groups (XP-CLR).
(D–K) Genome-wide p values for the MLM.

(wh1431), trimethoprim (mt1254, wh1214, wh1215), androgra- explore the relationship between gene expression and
pholide (mt762, wh1495, wh1497), (E)-3,4,5-trimethoxycinnamic metabolite levels and contents (Supplemental Figure 3). We
acid (wh0892), gibberellin A24 (wh1581), 9,10-dihydroxystearic detected 24 800 expressed genes in the RNA-seq data, ac-
acid (wh1398), ciprofloxacin (wh1486), L-(–)-methionine (mt82, counting for 72.0% of the annotated genes. We performed a
wh0286), and succinic anhydride (wh0002, wh0003). In addition, weighted correlation network analysis and identified 23 highly
betaine (mt7, wh0084) and homocitric acid (wh0691) levels were correlated co-expression modules (Supplemental Figure 3D).
higher in foxtail millet cultivars than in landraces. Several metab- We imported these modules into the Kyoto Encyclopedia
olites also showed a geographical difference in their levels; of Genes and Genomes database to determine their potential
D-glutamine (wh0261, wh0262) and D-mannitol (wh0528) levels biological functions. While multiple metabolic pathways
were higher in Northern foxtail millet accessions than in Southern co-existed within a single module, we identified several
accessions (Supplemental Table 9). metabolic patterns (Supplemental Table 10). For example,
flavonoid and carotenoid biosyntheses were highly enriched
We also analyzed grain transcriptomes at the late grain in the ‘‘blue’’ and ‘‘cyan’’ modules; fatty acid metabolism
filling stage via transcriptome deep sequencing (RNA-seq) to was enriched in the ‘‘green’’ modules; and diterpenoid
1370 Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author.
Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet Molecular Plant

Minor Gene-
Traits or Causative allele GWAS based TWAS Candidate
compounds SNPs frequency p value p value p value gene Description Variant
b* of millet 4:37 904 472 0.073 2.20E
21 1.97E
12 NaN Si4g27520 PSY1 intronic:T>C
color
4:37 904 866 0.057 1.08E
21 9.71E
13 intronic:A>G
4:37 905 652 0.061 4.15E
22 7.42E
13 UTR5: c.-187G>C
b* of powder 4:37 904 472 0.073 9.78E
11 1.66E
10 NaN Si4g27520 PSY1 intronic:T>C
color
4:37 904 866 0.057 5.48E
11 8.15E
11 intronic:A>G
4:37 905 652 0.061 4.96E
11 7.35E
11 UTR5: c.-187G>C
Propylparaben 1:5 754 594 0.111 1.59E
38 7.30E
25 NaN Si1g06500 GT2 UTR5:c.-62G>C
1:5 756 024 0.111 9.56E
38 2.68E
24 exon1:c.G1369A:p.A457T
1:5 770 226 0.106 1.63E
39 7.31E
25 3.37E
26 Si1g06520 CAD exon3:c.C303T:p.R101R
9:54 605 172 0.216 2.25E
15 3.77E
15 3.76E
15 Si9g49990 TDC stopgain:exon1:c.
C37T:p.Q13X
9:54 644 260 0.133 1.98E
24 3.77E
15 NaN Si9g50050 FLDH UTR5:c.-41G>A
Tyramine 9:54 604 517 0.221 3.17E
17 1.23E
19 6.17E
20 Si9g49990 TDC upstream:G>T
9:54 605 172 0.216 1.06E
18 1.75E
17 stopgain:exon1:c.
C37T:p.Q13X
C-Hex-C- 7:17 121 970 0.500 1.98E
08 5.70E
09 NaN Si7g08740 F-box UTR3:c.*960T>C
Pen-Api
Schaftoside 7:17 121 970 0.500 1.52E
12 1.71E
18 NaN Si7g08740 F-box UTR3:c.*960T>C
Stearic acid 5:45 631 957 0.148 1.43E
06 2.64E
17 3.89E
06 Si5g42810 SAD frameshift
insertion:exon1:c.
99dupG:p.Q34fs
Chr O-Rut 3:14 240 202 0.234 1.58E
10 2.10E
17 NaN Si3g18760 UGT73E1 stopgain:: exon1:
c.C8G:p.S3X
Neodiosmin 3:14 240 202 0.234 9.90E
07 2.81E
12 NaN Si3g18760 UGT73E1 stopgain:: exon1:
c.C8G:p.S3X

Table 1. Summary of the key 15 candidate genes assigned from GWAS results.
More information is listed in Supplemental Table 13. Abbreviations: 30 GT, anthocyanin 3-O-beta-glucosyltransferase; CAD, cinnamyl-alcohol dehydro-
genase; FLDH, farnesol dehydrogenase; GT2, gallate 1-beta-glucosyltransferase; PSY1, phytoene synthase 1; SAD, stearoyl-ACP desaturase; TDC,
tyrosine decarboxylase; UGT73E1, UDP-glycosyltransferase 73E1; UGT89B1, UDP-glycosyltransferase 89B2; UROIIIS, uroporphyrinogen-III synthase.

biosynthesis was enriched in the ‘‘pink’’ module (Supplemental
Table 10).
Genetic basis of natural variations of the metabolites
To uncover the genetic architecture underlying the variation in
metabolic traits detected across millets, we performed a mGWAS
on all 398 foxtail millet accessions using the mixed linear model
(MLM) algorithm with TASSEL (Bradbury et al., 2007) (Figure 1D
and methods). We employed a Bonferroni correction to
determine the significance threshold for association, resulting in a
cutoff value of 8.81 3 10
7 by genetic type I error calculator (Li
et al., 2012) and 15 248 significant SNPs for 636 metabolites
(Supplemental Table 11). Each detected metabolite had at least 1
significant association, with an average of 24 associations per
metabolite.
Given the false-positive and false-negative issues associated with
GWAS (Atwell et al., 2010; Chan et al., 2010a, 2010b), we
combined biological and bioinformatic approaches to mine
candidate genes. The filtering procedure was set as follows: (1)
haplotype analysis for all genes in the LD regions of a significant
SNP using CandiHap (Li et al., 2020) (Supplemental Figure 4); (2)
Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author. 1371
search for proteins that are biochemically related to the
associated metabolic traits encoded at the candidate loci; (3)
cluster analysis of candidate genes and homologous genes with
known function; (4) cross-reference with statistical significance re-
sults of causal genes from gene-based GWAS using CandiHap;
and (5) prediction of functional/molecular phenotypes from
GWAS results by performing transcriptome-wide association
studies (TWASs) (Supplemental Table 12). After the filtering steps
above, we identified 511 candidate genes associated with 690
lead SNPs that might be responsible for the variation in
metabolic traits (Table 1 and Supplemental Table 13). These
analyses provide insights into the genetic basis of metabolome
variation and functional insights into the underlying pathways.
Selection signals during foxtail millet domestication and
GWAS identification of genes associated with millet gain
color and quality traits
To reveal the genetic basis for foxtail millet domestication, we
identified 57 genomic hotspots that are associated with several
major traits and overlap with selection sweeps (Figures 2C
and 3A and Supplemental Table 14). Based on gene annotation
and studies of homologous genes in Arabidopsis thaliana or
Molecular Plant Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet

A KT1
CLPC1 CPK5 PIL5
PAP19 PSY1 PDF1B
OZS1 PHB SDS
DGL1 WRKY64 FAH1 C/VIF2 SAY1 Rap2.6L ABCG25 CYP71B SUS3
COP1 PYD4 CSLD5 ISI1 Les1 BR6OX2
FST

B C D
r2
-log10 (P value)

12 Kernel color intronic UTR5: c.-187G>C

20

-log10 (P value)
Powder color gRNA spacer

0.2
0
0.1
8
15 WT In/Del
4 4:37906380 psy1-1 -39 bp
10 psy1-2 +1 bp
0 5 SQUALEN_PHYTOEN_SYN_1 and 2
5 10 15 20 25 30 35 Mb (415 aa) 242-257 278-303
0
20 r2 5'-UTR psy1-1: -39 bp, -13 aa, 402 aa
-log10 (P value)

CDS psy1-2: +1 bp, Stopgain, 105 aa

0.1
0

0.35

15
3'-UTR
10
5
0
5 10 15 20 25 30 35 Mb
WT psy1-1 psy1-2
20
-log10 (P value)

Carotenoids content ( g/g)

Carotenoids content ( g/g)

15 8

4.4E-05
6.2E-06
10 0.9

1.3E-04
1.4E-03
6
5 0.6
4
0
2 0.3
Kernel Si4g27520 (PSY1)
Powder 0 0.0
37.86 37.88 37.9 37.92 Mb Allele code 0/0 0/1 or ./. 1/1 Lutein Zeaxanthin

E p = 7.25E-15
F G
1 2
40

97.7% 1
2.3%
b* of powder color

10 100 1
1 2
35

26.3% 4 2
1 2
9.4% 0.9% 2 H6
10.5% H7
30

6 4 2 H5
1 2
25

21.3% 32.5% H3 H1
Setaria viridis 1 1 6
20

5
1 1
H4 H2
2
A/A G/G 2.7%

Figure 3. Genome-wide screening of domestication sweeps and functional characterization of PSY1.

(A) Genome-wide distribution of selective sweeps between all sub-groups.
(B) Genome-wide p values for the MLM on kernel and powder color.
(C) Zoomed Manhattan plot, gene model and haplotype analyses of the domestication gene PSY1. The red vertical lines mark the proposed functional
site. Rows correspond to samples and columns correspond to SNPs. Left color bar represents the five subpopulations.
(D) Validation of PSY1 by genetic transformation in WT. The top panel shows two independent transgenic lines, where 1-bp insertion causing frameshift
variants and 39-bp deletion causing 13 amino acid deletions in exon 1. The middle panel shows the segregation of the In/Del on T2 grains. Scale bar
corresponds to 2 mm. The lower panel is the statistical analysis of carotenoids content between WT and mutant grains. Student’s t-test, three biological
replicates.
(E) Distribution of b* of powder color by genotype at SNP 4:37 906 380. The significance was calculated by the Wilcoxon rank-sum exact test.
(F) Genotype frequency distribution of SNP 4:37 906 380.
(G) Haplotype network of PSY1 alleles. The diameter of the colored circles is proportional to the log2 number of accessions with the haplotype. Number on
the branches indicate the number of mutations. The genepools are colored: orange, North cultivar; yellow, North landrace; green, S. viridis; navy blue,
South cultivar; sky blue, South landrace.

rice, we linked the functions of these genes to major agronomic (Supplemental Table 14). The PHYTOENE SYNTHASE1 (PSY1)
traits, including grain yield, grain size, plant height, tiller gene (Si4g27520) was contained within a selective sweep
number, heading date, drought tolerance, cold resistance, region (the top 1% of the length of the entire genome
heat resistance, carotenoid contents, sugar contents, sequence), indicating that it is most likely a domestication-
chlorophyll contents, photosynthetic capacity, and millet color related key gene (Figure 3A and Supplemental Table 14).
1372 Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author.
Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
The yellow color of grain and millet quality were important
breeding traits during foxtail millet domestication. We collected
color data for foxtail millet color in 2018 and 2019 at the Jinz-
hong and Jincheng field locations using a non-contact spectro-
photometer. Other color-related traits such as powder in color
index and red-green-blue values are shown in Supplemental
Table 1. We then performed a GWAS using color information
of foxtail millet grains to identify candidate genes for color-
related traits. We obtained a total of 70 988 SNPs and 2113
lead SNPs for millet color-related traits with a suggestive p
value threshold of 8.8 3 10
7 with the MLM (Supplemental
Table 13). Most candidate genes in the LD regions of GWAS
signals were highly expressed during the grain filling stage
(Supplemental Table 15). We also detected four GWAS signals
for color-related traits that were under selection during domes-
tication (Figures 2C and 3A). We observed a GWAS signal
associated with the b* component and powder color of millet
on chromosomes 5 and 6, respectively (Figures 3B and 3C
and Supplemental Figure 5). The GWAS signal mapping to
chromosome six coincided with genes encoding cinnamoyl-
CoA reductase 1, shikimate O-hydroxy cinnamoyl transferase,
UDP-glycosyltransferase 89B1, and anthocyanin 3-O-beta-glu-
cosyltransferase. These genes participate in the biosynthesis
of flavonoids and lignin and may in turn affect millet color
(Supplemental Figure 6L). In addition, the GWAS signal
detected on chromosome 5 mapped to a genomic region
containing genes encoding a CYP94C1 (cytochrome P450, fam-
ily 94, subfamily C, polypeptide 1), which has omega-
hydroxylase activity and can metabolize lauric acid (C12:0)
and C18 unsaturated fatty acids in Arabidopsis (Kandel et al.,
2007) (Supplemental Figure 6K). Whether these genes are
indeed causal for color traits need further validation.
In addition to chromosomes 5 and 6, we also detected a GWAS
signal associated with the b* value of millet and powder color
on chromosome 4. This signal was possibly linked to PSY1, en-
coding a major rate-limiting enzyme in carotenoid biosynthesis
(Cunningham and Gantt, 1998; Cazzonelli et al., 2010).
Haplotype analysis showed that 68.75% of examined S. viridis
harbored the A haplotype at the lead SNP (4:37 906 380, for
chromosome 4, position 3 790 565 bp), whereas 98.4% of
foxtail millet accessions carried the G haplotype in the 50
untranslated region at the PSY1 locus (Figure 3C). The G
haplotype was associated with a higher b* value for millet and
powder color compared with the A haplotype (Figure 3E). The
frequency of the A haplotype decreased from 68.75% in S.
viridis to less than 2.6% in landraces and less than 0.7% in
cultivars, indicative of positive selection for the G haplotype
during domestication (Figure 3F). Most elite cultivars have
yellow grains, whereas the wild species S. viridis typically has
uniformly gray grains, and landraces exhibit variable grain
colors (Supplemental Table 1). The haplotype network indicated
that the PSY1 haplotypes have a geographically structured
distribution (Figure 3G): seven haplotypes were shared by
different geographical populations (H1–H7), whereas the other
haplotypes were only present in S. viridis. We also noticed that
the haplotype diversity and nucleotide diversity were higher in
the S. viridis populations than in other populations.
Furthermore, haplotypes H1, H3, and H4 were the most
common in Northern accessions, whereas H2 was the most
common haplotype in the Southern group (Figure 3G).
Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author. 1373
Molecular Plant
To establish a link between SiPSY1 and millet color, we gener-
ated mutant materials for SiPSY1 using clustered regularly inter-
spaced short palindromic repeats (CRISPR)/CRISPR-associated
nuclease 9 (Cas9)-mediated genome editing (Figure 3D). We
identified two gene editing events in the SiPSY1 gene. The T2
plant was derived from a positive heterozygous T1 plant that
carried a 1-bp insertion (c.116insT) and a 39-bp (c.85_123del)
deletion in exon 1 of SiPSY1, introducing a premature stop codon
leading to a truncated protein (p.W39Cfs, loss-of-function mu-
tants) and 13 amino acid deletions (p.29_41del). We determined
that the two editing mutations co-segregated with the millet light
yellow color phenotype: all T2 grains with a mutant phenotype
were homozygous for the mutations, whereas phenotypically
normal T2 grains were either heterozygous or homozygous for
the wild-type non-edited allele (Supplemental Table 16).
Carotenoid contents were much lower in the grains of
homozygous gene-edited plants than in wild-type grains. The
contents for lutein and zeaxanthin were reduced by 25.5% and
34.4%, respectively, in homozygous grains than in wild-type
grains (Figure 3D); however, their contents were unaffected or
slightly elevated in leaves (Supplemental Figure 7B). The foxtail
millet genome harbors three PSY-like genes, and SiPSY2 is
highly expressed in leaves, which could explain the lack of a
strong phenotype in the leaves of the Sipsy1-1 mutant
(Supplemental Figure 8). Taken together, these results establish
a link between SiPSY1 and millet color variation and
demonstrate the power of mGWAS to identify key candidate
genes associated with metabolite-related traits.
Anti-inflammatory response to foxtail millet powder
extracts
Cytokines are well known for their function during inflammation.
To investigate the anti-inflammatory properties of different foxtail
millet extracts, we determined the levels of IL-1b, IL-6, and TNF-a
in the monocyte/macrophage-like cells RAW264.7 co-cultured
with 1 mg/ml lipopolysaccharide (LPS) to simulation inflammation
and 200 mg/ml foxtail millet extract (Figure 4A). LPS treatment
significantly increased the levels of IL-1b, IL-6, and TNF-a,
which decreased upon the addition of millet extracts to the
culture medium (showing a ratio below 1; Supplemental
Table 17). More than 96% of the treatments with grain extracts
showed a significant (p < 0.05) capacity to prevent the
production of these three cytokines compared with the LPS
treatment alone, with some grain extracts from foxtail millet
accessions even yielding cytokine levels as low as those in
untreated controls (Figure 4B and Supplemental Table 17). The
millet extracts from B167, B263, and B056 accessions
displayed best anti-inflammatory effects, as reflected by the
levels of IL-1b, IL-6, and TNF-a, respectively (Supplemental
Table 17). These results indicated that grain extracts can
suppress the production of TNF-a to a greater extent than that
of IL-1b or IL-6. We calculated the anti-inflammatory index for
each millet extract and used the values as a trait for a GWAS.
This analysis identified loci associated with IL-1b, IL-6, and
TNF-a indices on chromosomes 2, 6, and 1, respectively. We de-
tected candidate genes within each genomic region: genes asso-
ciated with folic acid transport (Si2g05580) for the TNF-a index on
chromosome 2; lipid metabolism (Si6g04690) for IL-6 on chromo-
some 6; and aldehyde decarbonylase (Si1g36380) for IL-1b
on chromosome 1. These genes might play a role in the
Molecular Plant Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet

A
IL-1 Extract IL-1
IL-1 Extract IL-1
LPS

IL-6 IL-6 LPS

IL-6
IL-6
TNF- TNF- TNF-
TNF-
LPS

LPS IL-1 IL-6 TNF- Extract of foxtail millet

B
Protein expression (pg/mL) / LPS

1.00
p p p

0.75 Setaria viridis

0.50

0.25

0.00

IL-1 / LPS CK / LPS IL-6 / LPS CK / LPS TNF- / LPS CK / LPS

C 0.3
10 0.31
8 r2 0.15
-log10 (P value)

-log10 (P value)
-log10 (P value)

0.4
8 r2 0.15 6
r2 0.2 6 0
6 0
0 4
4 4
2 2 2
0 0 0
10 20 30 40 Mb 10 20 30 Mb 10 20 30 40 Mb

8
-log10 (P value)

-log10 (P value)
-log10 (P value)

8 6 6
4 4
4 2
2
0 0 0
Si2g05580 (Folic acid transport) Si6g04690 (Lipid metabolism) Si1g36380 (Aldehyde decarbonylase)
4.3 4.4 4.5 Mb 3210 3220 3230 3240 Kb 41.36 41.40 41.44 41.48 Mb

Figure 4. Anti-inflammatory model and GWAS on anti-inflammatory traits.

(A) Anti-inflammatory model of foxtail millet extract. Inflammatory factors are very few in normal cells (control). Their content increased significantly after
LPS treatment. Foxtail millet extract could inhibit the inflammatory factors increasing by co-cultured with LPS. Green circle, IL-1b; yellow diamond, IL-6;
red triangle, TNF-a; orange circle, foxtail millet extract.
(B) Bee swarm plot presenting the distribution of the difference of ratio between protein expression/LPS and CK/LPS. Student’s t-test.
(C) Genome-wide p values for the MLM on three anti-inflammatory indices.

anti-inflammatory effects of millet extracts (Figure 4C and We selected the top 2000 highly variable genes and 1629
Supplemental Figure 9). metabolites using two-way orthogonal partial least squares
(O2PLS) discriminant analysis to identify joint co-variation be-
tween the two datasets. The total joint variance between the da-
Network of transcriptome, metabolome, and anti- tasets reached 31.5% for the transcript-predictive structure and
inflammatory indices 17.8% for the metabolite-predictive structure. The extent of
In this multi-omics study, we attempted to connect transcrip- unique variance reached 34.0% for the transcript data and
tome, metabolome and anti-inflammatory properties by calcu- 26.8% for the metabolite data. A substantial joint variance sug-
lating correlation coefficients between the abundance of each gested that a considerable proportion of changes in metabolite
transcript, metabolite and anti-inflammatory index (Figure 5). levels are accompanied by changes in gene expression. Finally,
1374 Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author.
Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet Molecular Plant
A

Figure 5. Network built on correlation among metabolites, genes, and anti-inflammatory indices.
(A) Network of clusters harboring the top 200 of the metabolites and genes. The Spearman correlation coefficients were calculated between genes and
metabolites. The gene-metabolite pair showed in the network was selected by using the adjusted p value < 1E–5. The circle nodes indicate metabolites,
while the triangles are genes with a distinct color per co-expression module. The metabolites are colored based on the main classes. The edges are
colored based on the positive (red) or negative (blue) correlation coefficients between genes and metabolites.
(B) Network of anti-inflammatory and metabolites. The metabolites were selected from the lasso regression results. The diamond nodes are indicated
three anti-inflammatory indexes while the circles are metabolites. The metabolites are colored based on the main classes. The edges are colored based
on the positive (red) or negative (blue) correlation coefficients between genes and metabolites.

we included a total of 9415 gene-metabolite pairs in the lasso (least absolute shrinkage and selection operator) regres-
metabolite-transcript network, with most genes coming from sion model, we identified 25, 5, and 53 metabolites (beta s 0)
the ‘‘brown’’ co-expression module associated with vitamin B6 that were highly correlated with the levels of IL-1b, IL-6,
and thiamine metabolism (Figure 5A). Using the region-adjusted and TNF-a cytokines, respectively (81 metabolites are
Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author. 1375
Molecular Plant Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
non-duplicate items). We also identified metabolites, such as
flavonoids, amides, and organic acids that exhibited anti-
inflammatory properties. The metabolite-cytokine network
showed that most of the metabolites were unknown, or not
included in the current class, except for "wh1094" (naringenin
chalcone, flavanone) and "wh1526" (5-O-p-coumaroylquinic
acid, amino acids) (Figure 5B and Supplemental Table 17). The
two metabolites were used in earlier anti-inflammatory tests,
and showed good anti-inflammatory effects (Liang and Kitts,
2015; Escribano-Ferrer et al., 2019).
Multiple regression analysis (best subset regression) to estimate
anti-inflammatory index yielded the metabolite levels predictor
variables. Three-variable models were identified as optimal for
anti-inflammatory index, corresponding to IL-1b, IL-6, and TNF-
a, respectively. According to R2 (goodness-of-fit measure), the
best performer is the model with 9 variables from the 81 metab-
olites. The wh0155, wh1500, and wh2196 were present in IL-1b
and IL-6 models. The wh1502, wh2268, and wh2533 were shared
in IL-1b and TNF-a models (Supplemental Figure 10). These
results indicated that the levels of the 81 metabolites could
provide a valid sub-maximum assessment for anti-inflammatory
index. The three best equations were:
IL-1b = 3.636E
01 + (
8.263E
03 3 wh0155) + (
3.652E

03 3 wh0277**) + (
5.839E
03 3 wh0991****) + (2.470E
03 3
wh1500) + (
5.583E
03 3 wh1502**) + (3.264E
04 3 wh2012) +
(1.190E
03 3 wh2196) + (2.767E
03 3 wh2268) + (1.857E

03 3 wh2336); R2 = 0.40; p value: 6.762E
04
IL-6 = 1.723E
01 + (
5.192E
03 3 wh0136**) + (
2.988E

03 3 wh0155*) + (
3.900E
05 3 wh0766) + (4.867E
04 3
wh1500) + (
5.225E
04 3 wh1563) + (
1.777E
03 3 wh1831) +
(
7.263E
05 3 wh2196) + (9.213E
04 3 wh2484) + (
4.677E

05 3 wh2533); R2 = 0.34; p value: 1.223E
02
TNF-a = 4.260E
01 + (7.890E
03 3 wh0333****) + (
2.818E

02 3 wh0424) + (4.463E
03 3 wh0855) + (6.486E
03 3
wh0900**) + (
1.667E
02 3 wh1502) + (2.212E
02 3 wh2268) +
(3.498E
03 3 wh2341) + (
2.237E
03 3 wh2474) + (2.488E

03 3 wh2533); R2 = 0.38; p value: 2.306E
03
The asterisks behind the metabolite ID indicate the significance of
a regression coefficient. Significance codes: 0 ‘****’, 0.001 ‘***’,
0.01 ‘**’, 0.05 ‘*’.
DISCUSSION
Foxtail millet has been cultivated for over 10 000 years, but it has
not been intensively bred for grain quality and high yield, unlike
other major cereal crops, such as wheat, rice, and maize (Zea
mays). The development of foxtail millet as a model system for
C4 photosynthesis has brought greater focus and interest to the
exploration of the genetic resources of this crop (Mamidi et al.,
2020). The demand from consumers for healthy millets is also
placing pressure on breeders to utilize molecular marker-
assisted breeding methods to select varieties with improved
nutritional traits that have not been achieved by traditional
breeding. As a result, multi-dimensional and multi-staged ana-
lyses are increasingly used to provide insights into biological
mechanisms (Holzinger and Ritchie, 2012). The data generated
1376 Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author.
in this study will offer a valuable resource for future research on
plant metabolic pathways and for improvement of nutritional
quality.
In GWASs, it is important to pay attention to both the quality of the
phenotypic data and the data analysis pipeline. We examined
traits separately for individual gene pools, sampling locations,
and years, as well as for aggregated results. We ensure that the
metabolomic and genomic data were suitable for multi-year
and multi-location standards. The broad-sense heritability and
coefficient variation indicate that our data, derived from three
populations, capture the diversity and inherent features of all ac-
cessions. Using our extensive dataset, we identified loci associ-
ated with thousands of metabolic traits. These datasets, coming
from multiple environments, also helped us identify loci with sig-
natures of recent selection. For instance, SiPSY1 was identified
as a candidate gene for intra-population millet color variation
and can now be utilized for breeding, as can other candidate
genes drawn from these GWASs and mGWASs, after they are
functionally verified and explored. These data will provide targets
for future genetic improvements of foxtail millet via CRISPR or
genomic selection. Genome resequencing and mGWASs of
several crops have been exploited to reveal the extent of standing
natural variation in chemical composition and have demonstrated
their power for trait association and candidate gene mining. For
example, mGWAS of rice accessions allowed the identification
of candidate genes associated with flavone and trigonelline
biosynthesis in rice, which regulate stress-tolerance and grain
width (Chen et al., 2014, 2016). Similarly, in a comprehensive
multi-omics analysis performed on a population of hundreds of
different tomato (Solanum lycopersicum) accessions, SlMYB12
was identified as a key transcription factor gene responsible for
flavonoid biosynthesis and the red fruit phenotype (Zhu et al.,
2018). Several homologous genes that catalyze the
C-glycosylation reaction and N-feruloyl tyramine biosynthesis
have been identified in Tibetan barley (qingke) using 196 diverse
qingke and barley (Hordeum vulgare) accessions (Zeng et al.,
2020). The development of high-throughput multi-omics tech-
niques and platforms can help decipher unknowns of domestica-
tion from a metabolic point of view (Zhan et al., 2022). Likewise,
using mGWAS and genetic and biochemical evidence, we
demonstrated here that SiPSY1 encodes a key limiting enzyme
in the carotenoid biosynthesis pathway in foxtail millet, which
contributes to desirable nutritional traits, such as the yellow
millet color and carotenoid production. Beyond SiPSY1, genes
and potential causative SNPs identified in this study could be
used as potential targets for marker-assisted breeding and
crop engineering to improve nutritional quality.
Understanding the genetics of domestication is critically impor-
tant because the gathered knowledge has implications for plant
breeding and crop improvement. In contrast to its wild ancestor,
foxtail millet cultivars and landraces have yellow millets rather
than gray grains (Supplemental Table 1), reflecting metabolome
differences that have accumulated over the course of
domestication. We detected many genes that were under
human-imposed selection, particularly those involved in the
metabolism of flavonoids and derivatives of phenylalanine. This
selection signature might be an adaptation to the biotic environ-
ment (Figure 2B and Supplemental Figure 11) and is consistent
with the expansion of gene families related to specialized
Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
metabolism and defense responses in S. viridis accession
ME034v (Thielen et al., 2020). Moreover, metabolism of starch,
sucrose, carotenoids, folate terpenoids, and fatty acids
indicates additional selection to improve energy, nutrition, and
taste (Figure 2B and Supplemental Figure 11). From the PCA
performed on Northern versus Southern cultivars/landraces
with S. viridis as an outgroup, we observed a clear separation
based on the resequencing data (Supplemental Figure 1A),
indicating that the two groups evolved in different directions.
The separation between Northern and Southern millet
accession was also reflected in the metabolome profiling
(Figure 2A), as sub-categories of metabolites showed distinct
accumulation in accessions collected from particular regions.
These data indicate that different climates and soils may affect
the accumulation of metabolites.
Visual and flavor preference may have imposed selective pres-
sure, further separating foxtail millet cultivars from wild green mil-
let. Selective breeding for yellow pigmentation, due mainly to the
accumulation of carotenoids and flavonoids in foxtail millet
grains, may have had other indirect effects on the grain metabo-
lome. Resequencing data can complement SNP and InDel infor-
mation available for Genebank entries in foxtail millet, just as
GWASs and mGWASs can complement quantitative trait
locus discovery methods in this self-fertilizing crop. The metabo-
lome data collected in our study provide a solid and comprehen-
sive metabolomic platform for foxtail millet accessions. Loci iden-
tified for specific traits, such as millet/powder color, revealed
genomic hotspots associated with important traits (Figure 3).
Inflammation is a localized protective response that involves
numerous cells and molecules (cytokines, transcription factors,
catabolic mediators). Excessive secretion of inflammatory fac-
tors, such as IL-1b, IL-6, or TNF-a, may damage the immune sys-
tem and hinder organ function and may induce or exacerbate
several diseases (Navarro-González and Mora-Fernández,
2008; Rivero et al., 2009; Favalli, 2020; Guo et al., 2021).
Traditional Chinese medicine deemed that the risk of chronic
diseases can be alleviated or cured by appropriate intake of
foods with pharmaceutical effects, such as foxtail millet, which
is well known in China for its beneficial effects on the human
digestive system. Accordingly, we performed anti-inflammatory
tests with grain extracts from all our analyzed foxtail millet acces-
sions. Most foxtail millet grains extracts appeared to significantly
suppress the production of pro-inflammatory mediators of
macrophage response induced by LPS. The anti-inflammatory
activities varied across accessions, which may be due to differ-
ences in both the metabolite categories and their contents in
different foxtail millet accessions, and need to be studied further
(Supplemental Table 17). Previous studies carried out on
naringenin chalcone (wh1094) (Escribano-Ferrer et al., 2019)
and 5-O-p-coumaroylquinic acid (wh1526) (Liang and Kitts,
2015) have shown that these compounds had promising anti-
inflammatory effects, supporting their contribution to the anti-
inflammatory trait in this study. However, foxtail millet extract is
a mixture, and so other anti-inflammatory metabolites and the
levels of these in the extracts need further research. Based on
the correlation analysis between the millet metabolome and
anti-inflammation activities, we identified lines or varieties whose
grain extracts exhibited high anti-inflammatory activities, which
could lay the foundation for a scientific exploration of the relation-
Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author. 1377
Molecular Plant
ship between nutritional value and specific metabolites. Further
investigation to determine the specific metabolite(s) that
contribute to anti-inflammation, and the underlying genes
involved in their metabolism, would be valuable for targeted mo-
lecular breeding for special metabolites and therapeutic foxtail
millet varieties.
METHODS
Plant materials
A total of 398 accessions, including 360 foxtail millets and 38 S. viridis,
were used in this study. The 360 foxtail millet accessions were kindly pro-
vided by Dr. Xianmin Diao in the Institute of Crop Sciences, Chinese Acad-
emy of Agriculture Sciences. Among them, 6 were from abroad, and 38
wild S. viridis were collected in China by our team (Supplemental
Table 1). This collection included 198 landraces, 162 cultivars lines, and
38 wild S. viridis, representing 49.75%, 40.70%, and 9.55% of the total,
respectively. All accessions were sown in the field of Shanxi Agricultural
University in China (37 250 N, 112 350 E) at the end of April in 2018.
Plant growth conditions
For 2018, 360 foxtail millet accessions were grown in a randomized design
field in three experiment stations at Datong (40 220 N, 113 120 E), Jinz-
hong (37 250 N, 112 350 E, Shanxi Agricultural University), and Jincheng
(35 320 N, 113 070 E), respectively. The planting density was 15 cm be-
tween plants in a row, with 25 cm between rows. The mature grains
were harvested from three different plants per accession for metabolite
extraction, and three plants were then combined to make one biological
replicate, with a total of three biological replicates for each accession.
For 2019, the 398 accessions were grown at Shanxi Agricultural University
experiment station (Shanxi, China, 37 250 N, 112 340 E). The experi-
mental design and replicates were the same as described above, except
that two different plants per accession were collected for metabolite
extraction. Data collected from the two biological sample sets at different
locations in 2018 were used to calculate H2.
DNA extraction and genome re-sequencing
For each accession, young leaves from a single healthy plant were
collected, immediately frozen in liquid nitrogen, and stored at
80 C until
used. Total genomic DNAs were extracted using Plant Genomic DNA Kits
(Tiangen, Beijing) according to the manufacturer’s instructions. About
10 mg genomic DNA of each sample was used to construct a 350-bp
paired-end library. The libraries were sequenced to generate 150-bp
paired-end reads on an Illumina NovaSeq 6000 system using the whole-
genome resequencing approach by Biomarker Technologies (Beijing,
China). Forty-eight accessions, including Changgu1, Changnong35,
Changsheng08, Fenxuan3, GBS, Huangsu, Jigu38, Jingu21, Jingu29,
Jingu35, Jingu40, Jingu42, Jingu54, Yugu1, and 34 wild S. viridis acces-
sions, were deep sequenced with more than 303 coverage. The other
350 accessions were sequenced about 103 coverage. Detailed informa-
tion about the geographic origin and sequencing coverage of the 398 fox-
tail millet accessions is listed in Supplemental Table 1.
Alignment, variant discovery, and annotation
The raw reads were qualified and filtered using Trimmomatic (Bolger et al.,
2014) (ver. 0.39) to generate clean reads with the parameters phred score
R20 and read length R30 bp. The obtained clean reads were then
mapped onto the xiaomi reference genome (Yang et al., 2020) using
Burrows–Wheeler aligner software (Li and Durbin, 2010) (BWA mem,
ver. 0.7.17) with default parameters. Mapped reads were converted into
BAM files using SAMtools (ver. 1.7), and duplicate reads were removed
with the MarkDuplicates program (ver. 2.18.9) in Picard (https://
broadinstitute.github.io/picard/) to avoid any influence on variant detec-
tion. After removing duplicate reads, SNPs and InDels were detected us-
ing GATK (McKenna et al., 2010) (ver. 3.8.0). Genome coverage of
Molecular Plant Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
mapped reads on the reference genome was estimated using bamdst
(ver. 1.0.9) (https://github.com/shiquan/bamdst). A strict filtering
procedure was applied to the raw variant set using GATK with the
following parameters ‘‘QD < 2.0 || MQ < 40.0 || FS > 60.0 || SOR >3.0 ||
MQRankSum < -12.5 || ReadPosRankSum < -8.0’’ applied to SNPs, and
‘‘QD < 2.0 || FS > 200.0 || SOR >10.0 || MQRankSum < -12.5 ||
ReadPosRankSum < -8.0’’ applied to indels. The SNP data output from
GATK, using SelectVariants, was further filtered using the VCFtools
(Danecek et al., 2011) (ver. 0.1.15). The SNPs and InDels were
considered valid if they met the following requirements: (1) two alleles
only; (2) excluding sites on the basis of the proportion of missing data
>0.9 (defined to be between 0 and 1, where 0 allows sites that are
completely missing and 1 indicates no missing data allowed); (3) minor
allele frequency R0.05; and (4) mean depth values R5. SNPs that did
not meet these four criteria were excluded from the study. All identified
SNPs that passed quality screening were further annotated with
ANNOVAR (ver. 2015 Dec 14) (Wang et al., 2010) based on the gene
annotation of the xiaomi reference genome (Yang et al., 2020). The
mutation analysis was processed and visualized using R package
maftools (Mayakonda et al., 2018).
Phylogenetic and population analysis
Bi-allelic SNPs with no missing data and a minor allele frequency greater
than 0.05 were kept for population genomic analysis. The neighbor-
joining phylogenetic tree was built using SNPhylo (Lee et al., 2014) (ver.
20180901) with standard settings and 500 bootstrap replicates. The
phylogenetic tree was drawn using an online tool iTOL (Letunic and Bork,
2019) (ver. 4) (https://itol.embl.de). PCA was performed using Plink
(Purcell et al., 2007) (ver. 1.90p) and EIGENSOFT (Price et al., 2006) (ver.
6.1.4) with the entire set of SNPs (3 826 381 SNPs, minor allele frequency
>0.05 and missing data <10%). Population structure analysis was
performed using fastStructure (Raj et al., 2014) (ver. 2.3.4). fastStructure
was run for each possible number of clusters (K = 2–20). Next, the most
suitable K (K = 3) was selected for the full set of 3 826 381 SNPs using a
python script chooseK.py in fastStructure. LD decay was calculated for
all SNP pairs within 300 kb, and plotted in PopLDdecay (Zhang et al.,
2019) (ver. 3.40) with parameters ‘‘-MaxDist 300 and -break 50 -bin1 5
-bin2 50’’. The p, Tajima’s D and FST were calculated using VCFtools
(Danecek et al., 2011) (ver. 0.1.15) based on filtered SNPs with high
confidence (3 826 381 SNPs). The p and FST value for each SNP
was calculated using a 20-kb window with a step size of 5 kb for each
sub-population. We calculated Tajima’s D value in a 20-kb non-
overlapping window. The following formula was used to calculate ROD:
ROD = 1
pcultivar lines/plandrace or ROD = 1
pcultivar lines/pwild.
Detection of selective sweeps
A cross-population composite likelihood ratio test (ver. 1.0) (Chen et al.,
2010) was used to scan the xiaomi genome for selective sweeps. In
brief, selective sweeps were identified in the following five comparisons
representing different foxtail millet speciation and breeding processes:
North cultivar, North landrace, South cultivar, South landrace, and S.
viridis. For each chromosome, the cross-population composite
likelihood ratio score was calculated with parameter ‘‘-w1 0.005 200
2000 chrN -p0 0.95’’. The top 1% outliers of chromosome-level test statis-
tics from each population were considered as the genomic segments
under selective sweep. The integrated haplotype score and the cross-
population extended haplotype homozygosity were performed using sels-
can (Szpiech and Hernandez, 2014) (ver. 1.2.0) with default setting
parameters. The integrated haplotype score and cross-population
extended haplotype homozygosity scores were normalized by norm
(ver. 1.2.1) in selscan. The five comparisons representing different
foxtail millet accessions from other geographical quadrants were
modeled in TreeMix (Pickrell and Pritchard, 2012) (ver.1.12).
Segregating SNPs across the nine chromosomes were filtered using
Plink (Purcell et al., 2007) (ver. 1.90p) to include sites with genotyping
rate >90% and exclude sites with minor allele frequency <5%.
1378 Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author.
Admixture trees were built using the S. viridis accessions as the
outgroup while allowing m = 0–10 migration events. The model fit for
each migration event was examined by estimating the proportion of
variance in relatedness between populations explained by each
migration model.
PSMC’ analysis
Evolutionary demographic changes in foxtail millet and S. viridis were in-
ferred using PSMC’ (Schiffels and Durbin, 2014) with default parameters.
Nine samples with >203 genome coverage was used: three foxtail millet
(B067, B165, and B309) and six S. viridis (B509, B514, B515, B517, B534,
and B538). Mutation scaled time and effective population sizes estimated
by MSMC were converted assuming a mutation rate of 7.0 3 10
9 base
substitutions per site per year (Ossowski et al., 2010) and a generation
time of one generation per year.
Color measurement
Color reading of each sample was performed using a non-contact spec-
trophotometer X-Rite VS540 (X-Rite, Grand Rapids, MI, USA). The color
parameters corresponded to the uniform CIELAB space (L*, a*, b*, and
CCI) and were directly obtained from the apparatus software Color iQC
ver. 9.0.94 (X-Rite). A standard white background (Ceram, Staffordshire,
UK) and an optical coupling using 400 polyethyleneglycols were used.
The device was set to make three consecutive readings, automatically
calculating the mean values of L*, a*, and b*. The color is expressed in
CIELAB, L* defines lightness, a* denotes the red/green value, b* the yel-
low/blue value, and CCI the orange index (CCI = 1000 3 a*/(L* 3 b*))
(Gandı́a-Herrero et al., 2013).
Sample preparation and extraction
We carried out metabolic profiling using mature grains. For each sample,
5 g well-grown grains was randomly harvested from at least three individ-
ual plants. The de-hulled grains were ground using a mixer mill (MM 400,
Retsch) with zirconia beads for 2 min at 30 Hz. After grinding, the powder
was partitioned into two sample sets and stored at
80 C until use. For
each set, 100 mg powder was extracted overnight at 4 C with 1.0 ml ab-
solute methanol (for lipid-soluble metabolites) or 70% methanol (for
water-soluble metabolites) containing 0.1 mg/l lidocaine. The extract
was centrifuged at 10 000 g for 10 min, and the supernatant was purified
using a CNWBOND Carbon-GCB SPE Cartridge (250 mg, 3 ml; ANPEL,
Shanghai, China) and filtered (SCAA-104, 0.22 mm pore size; ANPEL)
before injection for UPLC–MS/MS analysis (Chen et al., 2013).
Metabolite profiling
Two sets of data were collected in 2018 and 2019, respectively, for the
collection of foxtail millet accessions used in this study. Metabolite iden-
tification and quantification were carried out using an MS/MS spectral tag
(MS2T) library-based method (Matsuda et al., 2009). For 2018 data, a
MS2T library previously established for plant secondary metabolites
from rice was used (Chen et al., 2013, 2014, 2016). A C18 reverse-
phase column (VP-ODS, 5 mm particle size, 2 mm, 150 mm) was used
for metabolite separation on a UPLC–ESI–MS/MS system (UPLC, Nexera
X2 LC-30AD; MS, Applied Biosystems 4000 Q-TRAP). The gradient con-
ditions were as follows: two solvents system, solvent A-water with 0.10%
(v/v) acetic acid, solvent B-acetonitrile with 0.10% acetic acid; solvent
gradients (A/B, v/v): 95/5 at 0 min, 5/95 at 13.0 min, 5/95 at 17.0 min,
95/5 at 17.1 min, 95/5 at 20.0 min; flow rate, 0.25 ml/min; temperature,
40 C; injection volume: 10 ml. The abundance of each metabolite was
calculated based on peak area, using lidocaine (0.1 mg/l) as internal stan-
dard during sample extraction and ESI–MS/MS analysis.
Since foxtail millet may contain metabolites not included in the rice or
maize MS2T library, a de novo MS2T library construction was applied
for the 2019 sample set. First, a library was constructed using non-
redundant metabolite signals collected from pooled samples of all mature
foxtail millet grains (Chen et al., 2014; Wen et al., 2014, 2016). A Dionex
Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
UltiMate 3000 UHPLC system (Thermos Scientific) coupled with a Thermo
qE Plus high-sensitivity MS detection system was used for metabolite
identification. Metabolite annotation was made based on the high-
accurate mass values, fragmentation pattern, and the retention time
with help from authentic standards including amino acids, flavonoids, ly-
sophosphatidylcholine, fatty acids, and phytohormones (Supplemental
Table 4). For metabolites that could not be identified by available
standards, peaks in the MS2T library were then queried against the MS/
MS spectral data taken from the literature or available databases
(MassBank, KNApSAcK, HMDB, MoTo DB, and METLIN). Best matches
were searched in the Dictionary of Natural Products and the Kyoto Ency-
clopedia of Genes and Genomes for possible structures. After library
setup, samples were analyzed using a Dionex UltiMate 3000 UHPLC sys-
tem coupled with a TSQ Altis Triple Quadrupole Mass Spectrometer
(Thermo Scientific). A C18 reverse-phase column (VP-ODS, 5 mm particle
size, 2 mm, 150 mm) was used, using a two-solvent system (solvent
A-water with 0.04% acetic acid, solvent B-acetonitrile with 0.04% acetic
acid) with the following gradients: (A/B, v/v) 98:2 at 0 min, 98:2 at 1.0 min,
80:20 at 4.0 min, 2:98 at 12.0 min, 2:98 at 15.0 min, 98:2 at 15.1 min, 98:2
at 18.0 min (Wen et al., 2014, 2016). Flow rate, 0.25 ml/min; temperature,
40 C; sample injection volume: 10 ml. Lidocaine (0.1 mg/l) was used as
internal standard. Metabolite (m-trait) raw data were assessed and
corrected for peak area by QC sample runs and internal standard
(Matsuda et al., 2015) using MetaboDrift (ver. 1.1) (Thonusin et al.,
2017), and then log2 transformed for statistical analysis (Chen et al.,
2013). The m-trait data was taken from three biological replicates for
each accession.
Fatty acid extraction and derivatization
Fatty acid extraction and derivatization were conducted according to
China National Standard GB 5009.168-2016. In brief, 4.00 g of finely
grounded millet powder was mixed with 10 ml 8.3 M hydrochloric acid,
2 ml 95% (v/v) ethanol, 4 ml water, 100 mg pyrogallic acid, and 2.00 ml
of 0.5 mg/ml methyl heptadecanoate as internal standard. The mixture
was vortexed and heated in a water bath at 80 C for 40 min. The mixture
was cooled to room temperature and then extracted with a solvent con-
sisting of 10 ml 95% (v/v) ethanol and 50 ml n-hexane. The extraction
was performed three times and the obtained upper layers were combined
in a flask. After drying in a vacuum rotavapor at 35 C (Buchi R215, Flawil,
Switzerland), 8 ml methanol containing 2% (w/v) of sodium hydroxide was
added, and heated in a water bath at 80 C until no oil drops were
observed. Then 7 ml 15% boron trifluoride-methanol was added into the
flask. The flask was kept in a water bath for another 2 min and then cooled
to room temperature. A 10-ml aliquot of n-hexane was added to the flask
followed by vigorously shaking for 2 min. A proper volume of saturated so-
dium chloride solution was added and the mixture was kept still for
another 10 min. Then a total of 5 ml of the upper layer was carefully taken
and 5 g of anhydrous sodium sulfate was added to remove the water
before the samples were subjected to GC–MS analysis.
GC–MS analysis of fatty acid methyl esters
The analysis of fatty acid methyl esters was performed using an Agilent
7890B gas chromatograph coupled to a 5977A mass spectrometer
(GC–MS, Agilent, USA), and equipped with an Agilent DB-WAX column
(30 m 3 0.25 mm id, 0.25 mm film thickness). The GC–MS analysis was
carried out in a split mode (split ratio 1:40), using helium as the carrier
gas. The flow rate in the column was 3.075 ml/min. The injector tempera-
ture was set at 220 C. The volume of the injected sample was 1 ml. The
oven temperature started at 135 C, then increased to 185 C at 2 C/min
and held for 2 min, then to 195 C at a speed of 1 C/min (holding time:
1 min), and finally to 230 C at a speed of 8 C/min. The detection temper-
ature was set at 250 C. The data were acquired with a mass spectrometer
operating in EI mode with an electron impact ionization energy 70 eV and
mass scanning range 40–400 m/z. For identification, the retention times
and the mass spectra of methylated fatty acids were compared with those
obtained from the standards (Nu-Chek, USA) analyzed under the same
Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author. 1379
Molecular Plant
chromatographic conditions. For quantification, calibration curves were
created using methylated fatty acid standards (Nu-Chek) under the
same chromatographic conditions, with methyl heptadecanoate as inter-
nal standard (Supplemental Table 5).
Inflammatory response in RAW264.7
A total of 129 millet accessions from different locations were randomly
chosen for extraction. For each set, 2000 mg powder was weighed and
extracted overnight at 4 C with 50.0 ml absolute methanol (for lipid-
soluble metabolites) or 70% methanol (for water-soluble metabolites).
The extractions were carried out at room temperature overnight with
gentle stirring. The supernatant and sediment were separated by centri-
fuging at 10 000 g for 10 min at 4 C. The supernatant solutions were com-
bined and concentrated using a rotary evaporator at 40 C. Finally, the ex-
tracts were freeze-dried and stored in a desiccator in the dark until use.
The dried extracts for cell culture were weighed and thoroughly dissolved
in sterilized PBS with 20% DMSO (v/v) to a final concentration of 100 mg/
ml. Murine RAW264.7 macrophages were purchased from Stem Cell
Bank, Institute of Zoology (China Academy of Sciences, Beijing). The cells
were cultured as an adherent cell in Dulbecco’s modified Eagle
medium containing 10% (v/v) heat-inactivated fetal bovine serum (brand
company), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (Gibco,
USA), 1% (w/v) glutamax, and 1% (w/v) sodium pyruvate in 5% CO2 atmo-
sphere at 37 C. RAW264.7 Cells were seeded onto 60-mm cell culture
plates at approximately 1.2 3 106 cells/dish in standard growth medium
and grown for 20 h until confluent. After confluence, medium was aspi-
rated and 1 mg/ml LPS and 200 mg/ml foxtail millet extract were added.
The dose of 200 mg/ml millet extract was decided through preliminary ex-
periments with a range (5–800 mg/ml) of three millet extracts. Plates were
incubated for 24 h and the supernatants were collected and centrifuged at
2000 g for 5 min at 4 C. The resultant supernatant was collected and
stored immediately at
80 C. Mouse TNF-a, IL-6, and IL-1b ELISA kits
(ABclonal, China) were used to determine the production of TNF-a, IL-6,
and IL-1b generated by the cells under challenge with LPS. All experi-
ments used the blank corrected mean of three independent experiments
with two replicates per experiment, unless otherwise stated. The t-test for
treatments and relative positive controls required p values of less than
0.05 and 0.01 and were considered as statistically significant and
extremely significant, respectively. Experimental data were presented us-
ing the following formula: mean (treatments)/related positive controls.
GWAS
SNP loci for which more than 10% of data missing across accessions
were excluded, and those with minor allele frequency less than 5%
were used for mGWAS. We performed GWAS using a MLM (Zhang
et al., 2010), accounting for the population structure (Q) and familial
relationship (K) to examine the association between SNPs and
metabolic traits, provided by TASSEL (Bradbury et al., 2007) (ver.
5.2.51). The most suitable Q was analyzed using fastStructure (Raj
et al., 2014) (ver. 2.3.4), and the K was performed using KinshipPlugin in
TASSEL. The modified Bonferroni correction was used to determine the
genome-wide significance thresholds of the GWAS using a genetic type
I error calculator (Li et al., 2012), based on a nominal level of suggestive,
significant, and highly significant, corresponding to raw p values of
8.81 3 10
7, 4.41 3 10
8, 8.81 3 10
10, and –log10(P) values of 6.06,
7.36, and 9.06, respectively.
CandiHap: An R platform for haplotype analysis of variation
study
To the best of our knowledge, haplotype analysis has usually been con-
ducted manually, which is laborious, time-consuming, and prone to errors
and omissions. To solve this problem, we developed a user-friendly soft-
ware, CandiHap (https://github.com/xukaili/CandiHap) (Li et al., 2020).
With CandiHap, users can complete the following analysis within a minute,
which usually costs hours or even days manually: (1) local GWAS for a
gene; (2) haplotype analysis for a gene (CandiHap); (3) haplotype analysis
Molecular Plant Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
for all genes in the LD regions of a significant SNP position one by one
(GWAS_LD2haplotypes); (4) haplotype analysis of Sanger sequencing
population variation data run on Unix-like systems (sanger_CandiHap.sh).
More importantly, in addition to command lines, we also developed a
graphical user interface version that can be run on personal computers
with Windows, Mac, or UNIX platforms. Therefore, researchers without
any bioinformatics knowledge could analyze the haplotype easily.
TWAS
The fusion program (http://gusevlab.org/projects/fusion/) (Gusev et al.,
2016) was used to conduct TWAS in this study. All of the 360 foxtail
millet accessions were grown in the field at Jinzhong, China (37 250 N,
112 340 E, Shanxi Agricultural University) in May of 2019. Grains were
collected manually at S4 stage (late grain filling stage), frozen
immediately in liquid nitrogen, and stored at
80 C until use. Each
replicate was obtained by pooling samples from at least three plants.
Total RNA was extracted using TRIzol reagent. RNA-seq libraries were
constructed using an Illumina NEBNext UltraTM RNA Library Prep Kit
(NEB, USA) according to the manufacturer’s protocol and sequenced to
generate 150-nucleotide paired-end reads on an Illumina NovaSeq 6000
platform. Illumina sequencing reads were mapped to the xiaomi reference
genome (Yang et al., 2020) using Hisat2-2.1.0 (Kim et al., 2015) with
default settings. The featureCounts (Liao et al., 2014) was used to
obtain raw counts and transcripts per million (Li et al., 2010) values were
calculated to measure the expression levels of genes. The TWAS was
performed on each chromosome with significant GWAS associations for
traits.
Data integration
We used O2PLS (Bouhaddani et al., 2016) to integrate transcriptomics
and metabolomics data and identify highly intercorrelated metabolites
and gene transcripts. To focus on the potential differential signals, only
the top 2000 (10%) highly variable genes were selected for further
analysis. Metabolomics and transcriptomics data were then scaled and
transformed as described previously (Bylesjo € et al., 2007). The loading
values for the joint covariance part from the O2PLS analysis results
were extracted to find highly inter-associated genes and metabolites.
The penalized regression method, lasso adjusted for the population struc-
ture (Q) matrix (Supplemental Table 1), were used to explore relationships
between metabolites and IL-1b, IL-6, and TNF-a scores of the cytokines.
Ten-fold cross-validation was performed to determine the penalty param-
eter. Once the tuning parameter, corresponding to the lasso penalty was
finalized, the lasso was refitted to the full dataset to obtain the final model.
Network construction
The haplotype network for a gene was constructed using the haploNet()
function in the pegas package (Paradis, 2010). To build the network
between genes and metabolites, we first selected the top 100
metabolites and genes with known annotations based on the absolute
loading values. Then the Spearman correlation coefficients were
calculated between genes and metabolites. Only the gene-metabolite
pair with the adjusted p value < 1E–5 were kept to construct the network.
To explore the potential associations based on lasso-selected metabo-
lites, the metabolites and IL-1b, IL-6, and TNF-a networks were con-
structed based on the lasso regression results.
Genetic transformation in foxtail millet and of SiPSY1
To construct the CRISPR/Cas9 vector for SiPSY1 gene editing, two target
sequences on the first and third exons were cloned according to the
methods described by Ma et al. (2015). Agrobacterium tumefaciens
strain EHA105 with pYLCRISPR-SiPSY1 vectors was then used to infect
embryogenic calli induced from Ci846 mature grains to generate
SiPSY1 gene-edited foxtail millet as described previously (Yang et al.,
2020). The transgenic plants were screened by PCR amplification. The
primers used for vector construction and transgenic plant identification
are listed in Supplemental Table 16.
1380 Molecular Plant 15, 1367–1383, August 1 2022 ª 2022 The Author.
Determination of carotenoid composition
Carotenoid composition was measured by MetWare (Wuhan, China). To
measure carotenoid composition, grains and fresh leaves from transgenic
foxtail millet and Ci864 were collected and kept at
80 C until use. The
endosperms were homogenized and ground into a fine powder, then dried
using a freeze vacuum dryer. Endosperm powder (50 mg) was extracted
with a mixture of n-hexane:acetone:ethanol (2:1:1, v/v/v) and an internal
standard was added. After two extractions, the supernatant was evapo-
rated to dryness under nitrogen, and redissolved in a solution of metha-
nol:MTBE. The solution was passed through a 0.22-mm filter and then
analyzed using an LC–APCI–MS/MS system (UHPLC, ExionLC AD; MS,
Applied Biosystems 6500 Triple Quadrupole). A YMC C30 (3 mm, 2 3
100 mm) column was used for HPLC analysis. Samples were eluted using
a gradient from solvent A, methanol:acetonitrile (3:1, v/v) with 0.01% BHT
and 0.1% formic acid, to solvent B, methyl tert-butyl ether (with 0.01%
BHT). The analysis was carried out at 28 C with a flow rate of 0.8 ml/
min. MS analysis was performed using the API 6500 Q TRAP LC–MS/
MS System, equipped with an APCI Turbo Ion-Spray interface, operating
in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex).
Carotenoid contents were detected by MetWare based on the AB Sciex
QTRAP6500 LC–MS/MS platform.
Statistical analyses
All the statistical analyses were carried out using R (4.0.2, http://www.
r-project.org). The values of the coefficient of variation were calculated
independently for each metabolite (using the mean of the biological repli-
cates of the untransformed m-trait data): SD/mean for each metabolite in
the population. Broad-sense heritability (H2) was calculated using the
following equation by treating accessions as a random effect and the bio-
logical replication as a replication effect using one-way analysis of
variance: H2 = var(G)/(var(G) + varV), where var(G) and V(E) are the variance
derived from genetic and environmental effects, respectively. The
nonparametric test was performed using a two-tailed Wilcoxon rank-
sum test in R to compare outcomes between two independent groups.
The best subsets regression is a model selection approach that consists
of testing all possible combinations of the predictor variables, and then se-
lecting the best model according to some statistical criteria. The R func-
tion regsubsets() in ‘‘leaps’’ package can be used to identify different
best models of different sizes.
ACCESSION NUMBERS
The raw sequencing data reported in this paper have been deposited in
the NCBI under BioProject accession no. PRJNA633413 (genome re-
sequencing of 398 accessions) and PRJNA633940 (RNA-seq of 312 ac-
cessions). These data are also available in the BIG Data Center under
the accession number PRJCA002634 (CRA002636 for genome re-
sequencing of 398 accessions, and CRA002635 for RNA-seq of 312 ac-
cessions). The metabolomics data have been deposited on the UCSD Me-
tabolomics Workbench under the accession no. ST002168.
SUPPLEMENTAL INFORMATION
Supplemental information is available at Molecular Plant Online.
FUNDING
This work was supported by the National Key R&D Program of China
(2019YFD1000700 and 2019YFD1000702); the Joint Funds of the National
Natural Science Foundation of China (U21A20216); the Key R&D Program
of Shanxi Province (201903D11006); the Major Special Science and Tech-
nology Projects in Shanxi Province (202101140601027); the National Nat-
ural Science Foundation of China (32001608 and 31771810); the Scientific
and Technological Innovation Programs of Shanxi Agricultural University
(2017YJ27); and Lundbeck Foundation (R346-2020-1546) grants. S.P.
also acknowledges the financial aid of an ARC Discovery grant
(DP19001941), Villum Investigator (25915), DNRF Chair (DNRF155),
Novo Nordisk Laureate (NNF19OC0056076), Novo Nordisk Emerging
Investigator (NNF20OC0060564).
Multi-omics insights into the domestication, metabolome, and anti-inflammatory of foxtail millet
AUTHOR CONTRIBUTIONS
Y.H. supervised the study. Y.H., X.L., P.C., X.W.W., S.P., and X.C.W.
conceived of the study idea. X.D. prepared the material for genotyping.
S.H., Y.K.Z., Y.Y., and J.T. carried out the sampling process and partici-
pated in the material preparation. X.L., J.G., J.S., and K.G. performed
most of the experiments. X.L., P.C., H.L.W., L.Z., and D.L. carried out
the metabolite analyses. J.S., H.W., and M.H. performed the anti-
inflammatory experiments. Q.H. and Y.Y.Z. performed the genetic trans-
formation. X.L., K.G., L.X., and Y.L. analyzed the data. X.L. drafted the
manuscript. All of the authors discussed the results and commented on
the manuscript.
ACKNOWLEDGMENTS
We appreciate the critical reading and helpful comments on the manu-
script made by Professor Jianxin Ma and Professor Yiwei Jiang (Purdue
University), Professor Don Grierson (University of Nottingham), and Pro-
fessor Ming D Li (Zhejiang University), We are grateful to Professor Qing-
chun Pan (China Agricultural University) for help in GWAS. No conflict of
interest declared.
Received: March 9, 2022
Revised: June 22, 2022
Accepted: July 6, 2022
Published: July 7, 2022
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