Metabolite Profiling of Different Sweet sorghum cultivars Seeds

This study focused on the metabolic Diversity of three Sweet sorghum cultivars with white, red, and purple seeds to elucidate the factors responsible for the differences in Seed color. We found 651 metabolites were divided into 24 categories including Lipids_Fatty acids, Lipids_Glycerolipids, Flavonoid, Benzoic acid derivatives, Anthocyanins, Nucleotide and its derivates, etc. Through clustering analysis, principal component analysis (PCA), and orthogonal signal correction and partial least squares-discriminant analysis (OPLS-DA), different samples were clearly separated. It shows that contained metabolites of three groups are quite different. There are 217 significantly different metabolites between Z27 and HC4 (106 down-regulated, 111up-regulated), 240 between Z6 and HC4 (58 down-regulated, 182 up-regulated), 199 between Z6 and Z27 (54 down-regulated, 135 up-regulated). Venn diagram analysis indicated that 45 the differential metabolites were common to all three comparison groups. study new between

. Primary metabolites are necessary for life-sustaining activities and growth. Although their primary metabolites are high in content, it has very few varieties. Secondary metabolites are more involved in plant disease resistance, stress resistance and other environmental responses [ 4]. For example, the red color of strawberry is determined by anthocyan in the purple and color of blueberry is determined by flavonoids. The red color of tomato is determined by carotenoid, and the nitrogenous substance, such as alkaloid, has anti-tumor function, mainly due to biological Alkali substance. Flavonoids that involve flavones, flavonols, anthocyanins, flavanones, and Chalcones is a class of important plant secondary metabolites [ 5]. Many of colored fruits and flowers Contains a large amount of Flavonoids, which is conducive to protection against damage by dormancy, ultraviolet light and phytopathogens, and free from biotic and abiotic stresses [ 6]. research indicated that it is very helpful to some patients with chronic diseases which include cardiovascular disease, certain types of cancer [ 7], and inflammatory diseases. due to their antioxidant activities [ 8], recently, anthocyanins is a subgroup of flavonoids, has been paid more and more attention.Many plants, just like fruits, vegetables, and flowers, contains more than 600 anthocyanins which have been identified [9][10][11]. Purple and dark colors depend on the anthocyanin derivatives of delphinidin, petunidin, and malvidin. The derivatives of cyanidin and pelargonidin are sources of bright-redcolored fruits [ 12].
In order to better understand the mechanism of change of these metabolites, Metabolomics is increasingly favored by researchers. The main purpose of metabolomics analysis is to detect and screen biologically important Metabolites from biological samples [ 13]. Based on this, we hope clarify the metabolic processes and mechanisms of changes in organisms. Metabolomics analysis includes two major parts of metabolomics experiments and data analysis which mainly includes metabolite screening and metabolic pathway analysis. Based on experimental design, sample collection and processing, we can identify the metabolites and the quality control of the sample data. Some different metabolites were screened out so as to perform functional prediction on the metabolites.

Planting process
The three sweet sorghum cultivars have excellent quality characteristics and are local cultivars of Tongliao in Inner Mongolia. The color is divided into white, namely Z6, red namely Z27and black, namely HC4. Z6 is a homozygous material after 6 generations of self-fertilization, female parent Z591, male parent HT6. Z27 is a homozygous material after 6 generations of self-interest, female parent ZH1, male parent T27. HC4 is a homozygous material after 6 generations of self-fertilization, female parent H9, male parent C4. The seeds used in the experiment were provided by the Sorghum from Institute of the Tongliao Academy of Agricultural Sciences, however, voucher specimens of all materials were not deposited in a publicly available herbarium. The experiment was conducted at the Experimental Base of the Agricultural College of Inner Mongolia University for Nationalities, Tongliao City, Inner Mongolia Autonomous Region. The geographical position is between 42°15′-45°41′ north latitude and 119°15′-123°43′ east longitude. The number of days with winds above 8 can reach 20 to 30 days. The soil in the experimental field is gray meadow sand. The soil organic matter is 26g/kg and the alkali nitrogen is 62mg/kg. The quick-acting phosphorus is 38mg/kg, quick-acting potassium is 184mg/kg and the pH value is 8.3. The trial used a randomized block design with 3 replicates in Planting 16 rows per plot, with a length of 5m, a row spacing of 0.25m, a plot area of 20m2, and a spacing of 0.5m. Seeding is carried out by means of aerial seeding. The compound fertilizer was applied at the time of sowing with 750 kg/hm2, and the others were consistent with the production management of farmland. All sweet sorghum cultivars were sown on April 28, 2018. The bag is put at the flowering stage, and Mesh bag is alternative at the end of the flowering period. After ripening, three sweet sorghum seeds are harvested from each plant.

Sample preparation and extraction
Three different colored seeds were obtained base on color, which was divided in three goup, Z6 group in white, Z27 in red, HC4 in black and three replicates in every group. Every replicate, all of the sample, amixture of seeds from 5-6 plants for metabolomics, was washed and stored in -80 °C for further analysis.
We used a mixer mill (MM 400, Retsch), a zirconia bead for 1.5 min at 30 Hz, to crush the freeze-dried The pheatmap package, in R software (version 3.5.1), was used to draw Heatmap plots [ 15]. Before it, Log 2 transformation of metabolite relative response values was conducted.
R and SPSS19 software (IBM Corp., Armonk, NY, USA) were performed to calculate the area under the receiver operating characteristics (AUC) values, sensitivity (SE) and specificity (SP) of the potential biomarkers to evaluate the differential performance of metabolites [ 16].
The Kyoto Encyclopaedia of Genes and Genomes (KEGG) Pathway database (http://www.kegg.jp/kegg/pathway.html) is centered on metabolic reactions and make a connection metabolic pathways and corresponding proteins [ 17]. The enrichment analysis and topological analysis of the pathways where differential metabolites are present enables further screening to identify the key pathways that show the highest correlation with metabolite differences [ 18].

Results
Agronomic trait of three sweet sorghum cultivars In order to explore the expression trends of Metabolites in different groups, we then performed a heat map analysis. Based on the relative differences, Metabolites resulted in three main clusters based on the relative in accumulation patterns in three groups ( Figure 4A). Metabolites belonging to clusters 1Were higher quantity in HC4, while the Metabolites within cluster 3 and 4 were higher levels in z27 and z6 respectively. We then compared trend change of differential metabolite in any two groups. The differential metabolite obviously differentiated between between Z27 and HC4, 240 between Z6 and HC4, between Z6 and Z27 (Figure4B, Ffigure4C, Figure4D).
KEGG (Kyoto Encyclopedia of Genes and Genomesconduce) was conducted to integrate genes, expressions, and metabolites as a whole for research. The results of the annotation of the significant metabolite KEGG are classified according to the type of pathway in KEGG. The classification diagram is shown ( Figure 5). We found that more Differential metabolite were in metabolic pathways and biosynthesis of secondary metabolites.

Discussion
Three different cultivars was planted in same time and place, however, many of agronomic trait, Seeding leaf color, Seed Color, of three sweet sorghum cultivars have a great change. Based on this, we use metabolomics to explore Mechanism of phenotypic change.
Plant metabolomics, as new direction in the post-genome era [ 19], reveal mechanism of change to metabolites in different tissues. Products of Metabolites was a response to genetic and environmental changes [ 20]. Therefore, metabolomic analysis is a very useful tool can to clarify the relationship between biological processes and phenotypes; furthermore, we can find some intuitive changes at the metabolic level [ 21]. Up until now, more and more reaches have reported to explore mechanism of the relationship between biological processes and phenotypes [ 5,22]. In this study, we found 651 metabolite which was divided into 24 categories including Lipids_Fatty acids, Lipids_Glycerolipids, flavonoid, Benzoic acid derivatives, Anthocyanins, Nucleotide and its derivates, etc.(Table4s).
PCA and heat-map analyses of three groups showed a distinguished separation between the three cultivars. The results showed that there were large differences in the metabolites contained in the three groups. To further explore the mechanism of seed color differences between different cultivar, differentially expressed metabolites was analyzed between any two groups. There are 217 significantly different metabolites between Z27 and HC4, 240 between Z6 and HC4, 199 between Z6 and Z27. Although many substances, including Benzoic acid derivatives, Anthocyanins, Nucleotide and its derivates, have different variations between any two groups, the most different metabolites in comparison groups mainly are Anthocyanins and flavonoid. It is consistent with many reports [ 23,24]. As we all known, anthocyanins are a kind of flavonoids, which are water-soluble plant pigments, which are the main causes of different colors such as white, red, black and blue in vegetables, seeds, fruits and flowers. Anthocyanins are very useful for plants, and they can prevent damage from harsh environments such as cold and drought, avoiding damage from fungi, bacteria and viruses, and beneficial to pollination and seed dispersal [25][26][27].
After get differentially expressed metabolites, the differential metabolites between three cultivars were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database in order to obtain detailed pathway information. Results show that there are more differential metabolites were in metabolic pathways and biosynthesis of secondary metabolites. Secondary metabolites are mainly related to changes in the color of flowers and seeds, and also have a great effect on resistance to external aggression. The results also confirm this.
With the development of the technology of the sequencing, metabolomics method has been established and widely applied in much more research including Black Sesame and so on [ 28,29]. Many new metabolites have been detected and characterized. In our research, 651 metabolites were divided into 24 categories, was found in Sweet Sorghum.
Sweet sorghum is cultivated throughout China, but it is mostly in the south of the Yellow River Basin.
As the living standards of urban and rural people increase, it is no longer a staple food. Sweet sorghum is mainly used as feed in many countries such as China and Australia. Due to its high fiber density and homogenous flakes, it is well suited for use as a raw material for papermaking. It is also a new type of renewable and efficient energy crop [

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Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
All authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Authors' contributions WZ as the corresponding author contributed to the conception of the study; YZ as the first author contributed significantly to analysis and manuscript preparation; GZ as the second author performed the data analyses and helped to write the manuscript; YL as the third author wrote the manuscript and helped to prepare the material; ZL and HL as the fourth and fifth authors helped perform the analysis with constructive discussions. All authors read and approved the final manuscript. production of biogas from sweet sorghum and maize biomass. Environmental technology 2019:1-11. Tables   Table 1. Sweet sorghum sorghum agronomic traits statistics table   name Figure 1 Seed color of the three sweet sorghum cultivars

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