Physiological and transcriptome analysis reveal molecular mechanism in Salvia miltiorrhiza leaves of near-isogenic male fertile lines and male sterile lines

doi:10.21203/rs.2.9785/v1

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Physiological and transcriptome analysis reveal molecular mechanism in Salvia miltiorrhiza leaves of near-isogenic male fertile lines and male sterile lines

https://doi.org/10.21203/rs.2.9785/v1

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published 26 Oct, 2019

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Background: Our previous study found that male sterility in Salvia miltiorrhiza could result in stunted growth, decrease biomass, inhibit primary metabolism, and promote secondary metabolism, but their molecular mechanisms have not yet been elucidated. In this article, we investigated the underlying mechanism of plant growth and metabolism by using physiological analysis and mRNA sequencing (RNA-Seq). Results: In this study, transcriptomic and physiological analyses were performed to identify the effect on plant growth and metabolic production in male sterile mutants. Through GO and KEGG analysis it was found that the pathways were mainly enriched in processes including organ development, primary metabolic process and secondary metabolic process. Physiological analyses showed that the chloroplast structure of male sterile mutants of Salvia miltiorrhiza was abnormally developed, which could result in decrease in leaf gas exchange (A, E and gs), chlorophyll fluorescence (Fv, Fm and Fv/Fm), and the chlorophyll content. Transcriptomic analyses indicated that disproportionating enzyme 1 (DPE1) catalyzed the degradation of starch, while sucrose synthase 3 (SUS3) and cytosolic invertase 2 (CINV2) catalyzed the degradation of sucrose in S. miltiorrhiza. The results suggested that phenylalanine ammonialyase (PAL) played an important role in the biosynthesis of rosmarinic acid and salvianolic acid B, and flavone synthase (FLS) was an important enzyme catalyzing steps of flavonoid biosynthesis. High expression level of these enzyme genes in male sterile mutants resulted in high content of secondary metabolites. Conclusions: Our results from the physiological and transcriptome analyses reveal underlying mechanism of plant growth and metabolism in male sterile mutants, and provide insight into the crop production of S. miltiorrhiza.

Epigenetics & Genomics

Salvia miltiorrhiza

Male sterile mutants

Active ingredients

Transcriptome

Chloroplast structure

Photosynthetic characteristics

Phenylpropanoid pathway

Male sterility is the result of abnormal development of stamens of flowering plants, which cannot produce normal pollen, but their pistils can still develop normally and receive pollen and fertilize [1, 2]. Male sterility can be attributed to many other causes. For example, anthers cannot grow and differentiate normally; Normal microspores do not emerge; mature pollen grains cannot be released; mature pollen grains cannot germinate on the surface of stigma [3]. Male sterility systems have been widely used in crops such as maize, wheat, and rice [4-6]. Male sterility plays an extremely important role in heterosis utilization and hybrid seed production of crops and its use has successfully increased production [7-9].

In recent years, researchers have shown that cardiovascular and cerebrovascular diseases have become a main cause of death in the world [10, 11]. In addition, cardiovascular and cerebrovascular diseases are no longer restricted to the elderly, and more and more young working people have the diseases. Salvia miltiorrhiza is one of the largest medicinal herbs in China and has been widely used to treat cardiovascular and cerebrovascular diseases, hyperlipidemia, and acute ischemic stroke [12-14]. There are many traditional Chinese medicines such as Fufang Danshen Tablets and Danshen injections, etc., which contain active ingredients from S. miltiorrhiza (named as Danshen in Chinese) [15]. Due to the increasing demand in recent years, its planting area has expanded rapidly. The S. miltiorrhiza cultivation is, however, affected by the factors such as climate and region, which lead to unstable quality of active ingredients. Fortunately, Shu et al. discovered a natural male sterile mutant of S. miltiorrhizain 2002. It provides us with the possibility to achieve stable and controllable quality of active ingredients. Since the discovery of the male sterile mutant of S. miltiorrhiza, we have created and developed near-isogenic male fertile lines (MF) and near-isogenic male sterile lines (MS) through multiple consecutive testcrosses and backcrosses [16]. The genetic linkage map between male fertile lines and male sterile lines of the S. miltiorrhiza mutant was constructed by amplified fragment length polymorphism (AFLP). At present, the research on male sterility mainly focuses on the regulation and restoration of fertility, but little attention has been paid to the effect on plant growth and metabolism. The molecular mechanism of plant growth and metabolism in the male sterile mutant of S. miltiorrhiza has not yet been elucidated.

Identification of the underlying mechanisms of plant growth and metabolism is very important to help the crop improvement. Our previous study found that male sterility in Salvia miltiorrhiza resulted in stunted growth and decreased biomass. Here, we analyzed the primary and secondary metabolism in S. miltiorrhiza leaves of male fertile lines and male sterile lines. It was shown that primary metabolism in male sterile mutants was inhibited but secondary metabolism was promoted. In order to elucidate the molecular mechanism, we used the transcriptome and physiological analyses of the S. miltiorrhiza leaves of male fertile lines and male sterile lines. Our results from the physiological and transcriptomic analyses reveal underlying mechanism of plant growth and metabolism in the plant leaves, and provide insight into crop production of S. miltiorrhiza.

Male sterility inhibiting primary metabolism

Since the discovery of the natural male sterile mutant of S. miltiorrhiza in 2002, a lot of laboratory work has been done to create and develop near-isogenic lines. After many generations of testcrossing, backcrossing and genotyping, our team obtained genetically stable near-isogenic male fertile (MF) and near-isogenic male sterile (MS) lines. Previous results showed that the height of MF S. miltiorrhiza was 1.71 times higher than that of MS ones. In this study, we measured the content of primary metabolites including polysaccharide, sucrose, starch, and protein (Figure 1A). The results showed very significant differences in content of primary metabolites between MF and MS S. miltiorrhiza leaves, and the MF S. miltiorrhiza leaves had higher content of primary metabolites than MS S. miltiorrhiza leaves did. Specifically, the content of polysaccharide, sucrose, starch and proteins were respectively 27.30 %, 14.91 %, 15.72 % and 18.90 % higher in MF than MS.

Male sterility promoting secondary metabolites

In previous studies, some phenolic acids have been perceived as the major active ingredients of the aqueous extracts of S. miltiorrhiza, including rosmarinic acid and salvianolic acids B. In all analyzed extracts, salvianolic acid B and rosmarinic acid were most plentiful, both recognized as antioxidants. These compounds are cause of the antioxidant activity in both root and leaf extracts, indicating that they are present in the whole plant of S. miltiorrhiza, regardless of their tissue type and location. The flavonoids and polyphenols could also play important roles in antioxidant activity, since they are particularly rich in S. miltiorrhiza leaves [17]. In conclusion, the S. miltiorrhiza leaf extract can be used as powerful herbal antioxidants [18]. As we know, the leaf biomass of S. miltiorrhiza makes up a significant fraction of the whole plant. Nevertheless, during the harvest, the leaves of S. miltiorrhiza are disposed as wastes. The results indicated that the leaves of S. miltiorrhiza could make a natural material for drug, nutritious foods, cosmetics and industries.

In this paper, we identified some of the mentioned active ingredients in MF and MS S. miltiorrhiza leaves (Figure 1B). The results showed that the content of secondary metabolites was higher in MS S. miltiorrhiza leaves than MF ones. In Figure 1B, we found that the relative content of salvianolic acid B, rosmarinic acid, total phenolic and total flavonoid were respectively 12.62 %, 23.59 %, 18.55 %, and 23.06 % higher in MS S. miltiorrhiza leaves than in MF ones. The result indicated that the active ingredients in the MS leaves were more abundant. In all, we infer that male sterility mutation inhibited the primary metabolites but promoted the secondary metabolites in S. miltiorrhiza leaves. However, the underlying mechanisms of plant growth and metabolism in the male sterile mutant were still unclear.

The analysis of RNA-seq data

In order to explore the molecular mechanisms of plant growth and metabolism in male sterile S. miltiorrhiza leaves, we used transcriptomics for further research. Six libraries (male fertility: F1, F2 and F3; male sterility: S1, S2 and S3) were prepared and the libraries for each sample were repeated 3 times. We used an Illumina HiSeq™ 2500 platform to sequence. The transcriptomic profile of filtration, reads, ribosomal alignment, and genes coverage were shown in Additional file 1-5: Table S1, Table S2, Table S3, Figure S1 and Figure S2, respectively. These results demonstrated that the transcriptional profiling datasets had satisfactory reliability for further analysis. After data filtering, reads were aligned to the reference genome and the statistical results were shown in Table 1. The ratio of mapped reads to the reference genome were 95.04 %, 91.35 %, 92.71 %, 91.38 %, 94.66 %, 90.84 %, respectively.

Analysis of GO and KEGG classification and enrichment of DEGs

After parameter screening and optimization (p < 0.05, |log₂FC| > 1), GO analysis identified a total of 1369 DEGs between MF and MS lines. Figure 2A shows 853 differentially expressed genes were up-regulated and 516 were down-regulated. Through GO Analysis of DEGs, 30 significantly represented terms were displayed in Figure 2B. The results showed that metabolic process, cellular process, single-organism process were the most represented GO terms in biological process category. Cell, cell part, organelle, organelle part and macromolecular complex were markedly represented in cellular component category. Catalytic activity and binding were significantly represented in molecular function category.

This paper dealt with enrichment analysis on gene sets and how GO terms were represented using annotations for those gene sets. Through the deep mining analysis of the data, we selected several GO enrichment pathways that were represented in the data, as shown in Figure 3A. It was found that these pathways were mainly enriched in processes related to organ development, primary metabolic process and secondary metabolic process.

According to the KEGG orthology classification, DEGs were annotated into 90 pathways (Additional file 6: Table S4) and divided into five categories (Additional file 7: Figure S3). Assignment of DEGs to metabolic pathways provided insight into the biological functions and gene interactions and we found the most represented category was metabolism. The results showed that the metabolism category was the most annotated, including carbohydrate metabolism, biosynthesis of other secondary metabolites, etc. Analysis of bubble maps showed KEGG metabolic pathways were enriched (only the top 20 metabolic pathways shown in Figure 3B), our results found that the metabolic pathways with significant enrichment were phenylpropanoid biosynthesis pathway, protein processing in endoplasmic reticulum pathway, flavonoid biosynthesis pathway, tyrosine metabolism pathway and carbon fixation in photosynthetic organisms pathway, etc. In Figure 3, the enrichment results of GO and KEGG were analyzed, and we found that their pathways enrichment results were basically consistent, such as flavonoid biosynthesis and phenylpropanoid biosynthesis.

Abnormal development of chloroplast structure in male sterile mutants

From the phenotype observed, it was found that the male sterile leaves were significantly smaller than male fertile leaves. (Figure 4A and Figure 4B). Differences in organ development were found in the GO annotation results of the transcriptomic data. Further, the subcellular structure of leaves was observed by transmission electron microscopy. Interestingly, we found that the chloroplast structure of the two samples was significantly different, which was shown in Figure 4C and Figure 4D. The results showed that the chloroplast structure of male fertile leaves was fully developed and the thylakoid structure could be clearly seen. However, thylakoid structures could not be observed in the leaves of male sterile mutants. In addition, there was no spindle-like chloroplast in the male sterile leaves. Therefore, the results suggested that the chloroplast development of male sterile leaves might be incomplete.

Reduction of leaf gas exchange characteristics, chlorophyll fluorescence parameters and photosynthesis pigments in male sterile mutants

In Figure 4C, we found structural abnormalities in the chloroplasts in male sterile mutants. Chloroplasts are highly specialized organelles in higher plants where photosynthesis including important metabolic pathways takes place [19]. Photosynthesis is the basic source of plant biomass production. The difference in photosynthetic characteristics between varieties is often one of the direct causes of difference in growth. In this paper, we measured the photosynthetic parameters of S. miltiorrhiza leaves of the male fertile lines and male sterile lines, and found that all the parameters including the net photosynthetic rate (A), transpiration rate (E) and stomatal conductance (g_s) of the male fertile lines were higher than those of the male sterile lines except intercellular CO₂ concentration (C_i) in S. miltiorrhiza (Figure 5A). The results showed that the chloroplast structure was abnormal and the performance of photosynthesis was consistent with other research results [20]. The results also showed that the net photosynthetic rate was positively correlated with the stomatal conductance, which agrees with the statistical analysis of 14 species [21]. 90 % - 95 % plants᾽ dry matter of the above-ground parts comes from photosynthesis. Therefore, the most important thing to increase production was to increase the utilization efficiency of light energy, enhance the photosynthetic rate of plants, and increase the yield per unit area.

Using chlorophyll fluorescence, a natural probe emitted from plants, can detect a lot of information about the state of plant growth [22]. By comparing the chlorophyll fluorescence detected from male fertile and male sterile plant leaves, it was found that there were significantly variations in F_m, F_v and F_v/F_m between the two lines except F₀ (Figure 5B), and they were positively correlated with their growth and photosynthesis. This indicated that a higher growth rate was closely related to higher PSII potential viability and higher light conversion efficiency. These studies had fully demonstrated that chlorophyll fluorescence could sensitively reflect the growth and development of plants, so it might become one of the indicators for predicting plant growth potential.

Further, we determined the photosynthetic pigment content of the S. miltiorrhiza leaves of male fertile and sterile lines. Our results found that the chlorophyll a content and chlorophyll a+b content of male fertile and sterile leaves was significantly different, but there was no significant difference found in chlorophyll b content (Figure 5C). These results implied that chlorophyll a played a greater role than chlorophyll b in photosynthetic electron transport. It was found that the chlorophyll content was high and the photosynthetic activity was also higher. The results also showed that the morphological structure and physiological function of the plants corresponded closely, which was confirmed by the fact that the photosynthetic processes were correlated to the morphological characteristics of the photosynthetic organs of the leaves. Therefore, the results indicated that one of the causes for the stunted growth, decreased biomass and inhibited primary metabolism in the male sterile S. miltiorrhiza mutant might be due to the abnormal chloroplast structure, which reduced gas exchange and chlorophyll fluorescence and decreased chlorophyll content.

Male sterile mutants promoting the degradation of sucrose - starch metabolism

In order to further explore the metabolic pathways, we used MapMan software (http://mapman.gabipd.org/) to analyze the transcriptomic data. MapMan is specialized in the classification of plant gene functions. It focuses on the functional classification of genes in metabolic pathways and biological processes in organisms [23, 24]. The software is excellent at visualizing gene function classification and gene expression data. Mapping files were obtained from Mercator Automated Sequence Annotation using nucleic acid sequences ofS. miltiorrhiza (Additional file 8: Excel S1). We could find that there were significant differences (Figure 6) in sucrose synthase 3 (SUS3), cytosolic invertase 2 (CINV2), alpha amylase (AMY) and disproportionating enzyme 1 (DPE1) in the sucrose-starch metabolism pathway. It was inferred that the male sterile mutation in S. miltiorrhiza reduced the relative content of sucrose and starch. In Figure 6, the DEGs were mainly involved in the degradation process of sucrose and starch. Therefore, we selected the genes (SUS3, CINV2 and DPE1) that were significantly up-regulated in the sucrose-starch metabolism pathway as candidate genes. In most of higher plants, sucrose is main sugar that moves pass phloem from leaves to support other tissues. Sucrose then needs to be hydrolyzed into hexoses by either INV or SUS [25], and our results just confirmed this conclusion. The researchers found that the lack of DPE1 in plants causes slightly more accumulation of starch in the leaves than in the control plants [26], which agrees with our results. Therefore, the results suggested that one of the reasons for the decrease in sucrose and starch in male sterile mutants of S. miltiorrhiza might be due to the degradation of starch catalyzed by DPE1, while the degradation of sucrose catalyzed by SUS3 and CINV2.

PAL plays an important role in the phenylpropanoid metabolism pathway of male sterile mutants in S. miltiorrhiza

Phenylpropanoid metabolism pathway has important physiological significance in plants. And its intermediate products and its further transformed products were closely related to physiological activities such as differentiation of cells in plant development, resistance to pathogen infection, and formation of pigmentation [27]. We also used MapMan to analyze the phenylpropanoid metabolism pathway, and the results were shown in Figure 7 and Additional file 9: Figure S4. It could be seen that there were significant differences in the key enzymes of the phenylpropanoid metabolism pathway, such as phenylalanine ammonialyase (PAL) in general phenylpropanoid pathway, caffeic acid O-methyltransferase (COMT) and ferulic acid-5-hydroxylase (F5H) in lignin biosynthesis pathway, flavonoid 3᾽-hydroxylase (F3᾽H), flavonol synthetase (FLS), dihydroflavonol 4-reductase (DFR) and flavonol 3-O-glucosyltransferase (F3oGT) in flavonoid biosynthesis pathway. PAL, an enzyme in the phenylpropanoid pathway, is involved in physiological processes such as anthocyanin accumulation, lignification, flavonoid synthesis, and pathogen defenses. The results showed that most of the genes in the male sterile mutants were significantly up-regulated. In Figure 7, we found that there was a significant up-regulation of PAL in S. miltiorrhiza leaves of the male sterile mutant. Researchers found that PAL had a significant positive correlation with rosmarinic acid and salvianolic acid B content, and our results confirmed this conclusion [28]. Therefore, PAL played an important role in the synthesis of phenolic acid compounds inS. miltiorrhiza. In this study, it was also found that male sterile mutants had higher relative content of total flavonoid and total phenolic (Figure 1B). Flavonoids and polyphenols are widespread secondary metabolites in higher plants and play an important role in diverse activities. For example, they could protect plants from pathogenic infection, provide flowers with pigment to attract pollinators, and reduce the risk of oxidative damage to human health [29-31]. In Figure 7, we selected the FLS gene, whose expression level was significantly up-regulated, as a candidate gene which was involved in the flavonoid biosynthesis pathway. The expression of FLS gene required activation of the MYB transcription factor [32]. Overexpression of FLS gene affected the accumulation of flavonoids [33]. Therefore, FLS gene could be a key enzyme encoding gene affecting the synthesis of flavonoids. In addition, current research on the genes related to the metabolic pathways could provide useful information for metabolic engineering strategies to control the overall metabolic flux of potent antioxidants that have beneficial implications for human health [34]. To validate the reliability of transcriptomic data, the sequences of seven core DEGs related to phenylpropanoids pathway and sucrose-starch metabolism pathway were analyzed by RT-qPCR. The results of RT-qPCR analysis showed a close similarity to the results of RNA-Seq, as shown in Additional file 10: Figure S5.

Conclusions

In conclusion, our results showed that the male sterile S. miltiorrhiza mutant had reduced primary metabolism, and increased secondary metabolism in leaves. In order to understand the molecular mechanism of plant growth and metabolism in the male sterile mutant on, we used the transcriptome and physiological analyses of the S. miltiorrhiza leaves of male fertile lines and male sterile lines. Based on GO and KEGG analysis of transcriptomic data, we explored the causes of stunted growth, decreased biomass, inhibited primary metabolism and promoted secondary metabolism in the male sterile mutant. One of the reasons why the male sterile mutant had stunted growth, decreased biomass and inhibited primary metabolism could be due to the abnormal chloroplast structure, which altered the leaf gas exchange (A, E and g_s) and chlorophyll fluorescence (F_v, F_m and F_v/F_m) and reduced the chlorophyll content. Reduced primary metabolite contents in the male sterile mutant could be attributed to the differential expression of some enzymes in sucrose-starch metabolism pathway. The results indicated that DPE1 catalyzed the degradation of starch, while SUS3 and CINV2 catalyzed the degradation of sucrose in S. miltiorrhiza. At the same time, the results also suggested that PAL played a vital role in the biosynthesis of rosmarinic acid and salvianolic acid B, and FLS was essential for the biosynthesis of flavonoids. The results showed that high expression level of these enzyme genes in male sterile mutants resulted in high content of secondary metabolites. Our results from the physiological and transcriptomic analyses reveal underlying mechanisms of plant growth and metabolism in the male sterile mutant. These results may also be useful for optimizing quality assisted selection breeding.

Plant materials

This work used S. miltiorrhiza leaves of near-isogenic male fertile lines and near-isogenic male sterile lines as experimental materials, which were collected from a botanical gardenof S. miltiorrhiza at Northwest A&F University in Yangling, Shaanxi Province, China. The leaves of MF and MS were processed under sterile conditions, immediately frozen and stored in liquid nitrogen at - 80 ℃ before the RNA isolation. Leaf subcellular structures and features were observed by transmission electron microscopy (TEM).

Determination of primary metabolites

Polysaccharide, starch, sucrose and proteins were identified and estimated by an anthrone-sulfuric acid assay, a perchloric acid hydrolysis, a resorcinol-based assay and a Coomassie Brilliant Blue G-250-based assay.

Determination of secondary metabolites

The dried leaves of S. miltiorrhisa were weighed 0.1 g, and 4 ml 70 % methanol aqueous solution was added. The leaves stood extracted overnight, followed by ultrasonic extraction for 45 min and centrifugation 10000 g for 10 min. The supernatant was taken as the test solution for determination the content of total phenolic, total flavonoid and phenolic acids.

Total phenolic was determined using a modified method [35]. Test samples (1.0 mL) were mixed with 1 mL Folin-Ciocalteu and incubated at room temperature for 3 min. After the addition of 4.0 mL of 7.5 % Na₂CO₃ and 4.0 mL distilled water, the reaction tubes were incubated for 1 h at 40 ℃ and the absorbance readings were taken at 765 nm. The measurements were done with a calibration curve plotted against gallic acid to establish the content of total phenolic in extracts, whose results were shown as milligrams of gallic acid equivalents per gram of dry extract. The curve equation Y = 0.0128 X + 0.0001 (R² = 0.9994) was used, where Y was absorbance and X was total phenolic content.

Total flavonoid content was measured using a modified aluminums chloride colorimetric assay [36]. Test samples (1.0 mL) were mixed with 6.4 mL 30 % ethanol, 0.3 mL 0.5 M NaNO₂ and 0.3 mL 0.3 M AlCl₃ and kept in the dark for 5 minutes at room temperature. After the addition of 2.0 mL of 1 M NaOH, the absorbance readings were taken at 506 nm. The same measurements and calculations were done to establish TF content. And the gallic acid curve equation was shown as Y = 0.0007 X + 0.0071 (R² = 0.9991).

The extraction of phenolic acids was done according to the protocol for total phenolic and total flavonoid. The quantitative and qualitative analysis of the phenolic acids were carried out according to reference procedures [37]. All chromatographic isolation was performed by using HPLC system equipped with an autoinjector, a UV detector, and LC-solution software (Waters, UK). The column type, column temperature, mobile phase flow rate, mobile phase solvent ratio and program settings are set according to the reference. The injection volume was 10 μl. Spectral data were collected at 280 nm in the whole run. Sample and mobile phase filtration were done at a 0.22 μm filter before a HPLC injection. Analysis of each extract was conducted in triplicate.

RNA extraction, library preparation and mRNA-seq

Total RNA was extracted with RNA pure Plant kit (Tiangen Biotech Co., Ltd., Beijing, China). The total RNA concentration, RIN value, 28S/18S and fragment size were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies Co. Ltd., Santa Clara, CA, USA) and Agilent RNA 6000 Nano Kit. The purity measurement of the samples was made using an ultraviolet spectrophotometer NanoDrop^TM. After the isolation and fragmentation of the total RNA, the eukaryotic mRNA was enriched by using Oligo (dT) coupled to magnetic beads. PCR was used to construct cDNA libraries according to the following protocol. The first strand cDNA was manufactured from the fully spliced mRNA as a template, and then a second strand reaction system was used to make double-stranded cDNA. The second strand cDNA was synthesized and purified by a system kit. When the cDNA ends were repaired, an ‘A’ nucleobase hybridized to the 3᾽ ends of the cDNA. Then the cDNA fragments of different sizes were selected and then amplified. The qualities of amplified cDNA libraries were checked and tested. At last, the sequencing was run on an Illumina HiSeq™ 2500 platform.

FPKM calculation and DESeq2 differential expression analysis

We used alignmentpackage Bowtie2 (v2.2.5) to align clean reads back to reference genomes [38]. Then the gene expression level of every sample was calculated by RSEM (v1.2.12), a software package designed to estimate geneand isoform expressionlevels from RNA-Seq data [39]. FragmentsPer Kilobase per Millionmapped reads (FPKM) values were calculated by mapping the reads to fragments, which could be used to quantify the abundance of the transcripts and so to analyze differential gene expression in the samples. Differential expression analyses were performed with DESeq2 package [40]. Differentially expressed genes (DEGs) were selected according to p-value (p ≤ 0.05) and fold change analysis (FC ≥ 2.00).

GO and KEGG enrichment analysis of DEGs

According to the official classification categories and based on the GO and KEGG annotation results [41], the DEGs are classified into functional and biological pathways, and the R function phyper isused for enrichment analysis. A p-value of 0.05 (p ≤ 0.05) implies that the accepted false discovery rate (FDR) is 5%.

RT-qPCR validation and DEGs analysis

In order to validate internal control genes for expression analysis, ABI StepOnePlus^TM RT-PCR System (Applied Biosystems, USA) was used to perform RT-qPCR and investigate differential expression of gene sets. GenScript Real-time PCR online software was adopted to build sequencing primers, shown in Additional file 11: Table S5. RT-qPCR were carried out by PrimeScript^TM RT reagent Kit and SYBR Green^TM Premix Ex Taq^TMto transcribe RNA into complementary DNA (cDNA) (Takara Biotech Co., Ltd., Japan) with β-actin was used as an internal control. Three independent technical replicates and three biological replicates for each sample were used to validate the performance of RT-qPCR.

Determination of leaf photosynthesis and pigment content

The photosynthetic parameters were determined between 08:00 am to 12:00 am by the portable photosynthesis system (LI-6400, LI-COR Inc., USA) [42]. Fluorescence kinetic parameters of chlorophyll were determined by FluorCam fluorometer acquisition system (PDA-100 Heinz Walz, Germany). Chlorophyll fluorescence analysis was used to assess the photosynthetic performance of S. miltiorrhiza. Before the measurements, the leaves were kept in the dark for 30 min using cuvettes on ice. A 5 s light pulse at 400 μmol m^-2s^-1 was employed.

0.1 g of the fresh sample was extracted with 80 % acetone, and then the chlorophyll content of the leaves was determined by an ultraviolet spectrophotometer. Absorbance of the chlorophyll extractions and blank (80 % acetone) were measured at 663 nm and 645 nm. Finally, chlorophyll a, b and total chlorophyll were calculated by Arnon᾽ equations.

Statistical analysis

Each experiment was carried out in triplicates. The results were shown as means ± standard error and dealt with using SPSS (version 21.0). ANOVA and Tukey᾽sHonestlySignificant Difference were used to determine significant differences among the samples. A 0.05 significance level was considered.

MF: Near-isogenic male fertile lines; MS: Near-isogenic male sterile lines; DEGs: Differentially expressed genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; N50: Covering 50% of all the nucleotide sequences of the largest unigene length; FDR: False discovery rate; FPKM: Fragments Per Kilobase of transcript per Million mapped reads; FC: fold change; Q30 percentage: Percentage of bases with sequencing error rate lower than 1‰; RNA-Seq: RNA sequencing; RT-qPCR: Real-time quantitative polymerase chain reaction; TEM: Transmission electron microscopy; F5H: Ferulic acid-5-hydroxylase; AMY: Alpha amylase; DPE1: Disproportionating enzyme 1; SUS3: Sucrose synthase 3; CINV2: Cytosolic invertase 2. PAL: Phenylalanine ammonialyase; C4H: Cinnamate 4-hydroxylase; 4CL: 4-coumarate-CoA ligase; RAS: Rosmarinic acid synthase; CHS: Chalcone synthase; COMT: Caffeic acid O-methyltransferase; CHI: Chalcone isomerase; CCR: Cinnamoyl-CoA reductase; F3H: Flavanone 3-Hydroxylase; FLS: Flavonol synthetase; F3᾽H: Flavonoid 3᾽-hydroxylase; F3’5’H: Flavonoid-3’,5’-hydroxylase; F3oGT: Flavonol 3-O-glucosyltransferase; ANS: Anthocyanidin synthase; DFR: Dihydroflavonol 4-reductase; LAR: Leucoanthocyanidin reductase; ANR: Anthocyanidin reductase.

Acknowledgments

We would like to thank Yun Jiang for helping with the check of English writing.

Funding

This study was supported by National Natural Science Foundation of China (grant No. 31670301) and National Natural Science Foundation of Shaanxi (grant No. 2016JM8108).

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Availability of data and materials

The datasets supporting the conclusions of this article are included with in the article and its additional files.

Authors᾽ contributions

RW drafted the manuscript and was responsible for the data analysis. JD and HG assisted to manuscript revision. HJ did some bioinformatics analysis. ZZ collected the sample and performed RT-qPCR. All authors read and approved the final version of the manuscript.

Author details

¹College of Life Sciences, Northwest A&F University, Yangling 712100, China. ²College of Chemistry and Pharmacy, Northwest A&F University, Yangling 712100, China.

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Table 1 The statistics of reference genome alignment results.

Sample	All Reads Num	Mapped Reads	Unmapped Reads
F1	10219982	9713429 (95.04%)	506553 (4.96%)
F2	10465470	9560268 (91.35%)	905202 (8.65%)
F3	44454092	41211575 (92.71%)	3242517 (7.29%)
S1	10038332	9173391 (91.38%)	864941 (8.62%)
S2	10506634	9946024 (94.66%)	560610 (5.34%)
S3	46055644	41835694 (90.84%)	4219950 (9.16%)

Note: The three biological replicates of male fertility are F1, F2 and F3, and the three biological replicates of male sterility are S1, S2 and S3.

Additional file 1: Table S1. The base information before and after filtration. (DOC 33 kb)

Additional file 2: Table S2. The reads information before and after filtering. (DOC 34 kb)

Additional file 3: Table S3. The statistics of ribosomal alignment results. (DOC 34 kb)

Additional file 4: Figure S1. The distribution of genes coverage. The three biological replicates of male fertility are F1, F2 and F3, and the three biological replicates of male sterility are S1, S2 and S3. In the figure, A, B, and C represent F1, F2, and F3, respectively. And D, E, and F represent S1, S2, and S3, respectively. (PDF 1, 619 kb)

Additional file 5: Figure S2. The classification of raw reads. The three biological replicates of male fertility are F1, F2 and F3, and the three biological replicates of male sterility are S1, S2 and S3. (PDF 111 kb)

Additional file 6: Table S4. Pathway enrichment of DEGs between MF and MS in S. miltiorrhiza leaves. (DOC 127 kb)

Additional file 7: Figure S3. KEGG classified into five largest pathways between near-isogenic male fertile and male sterile lines in S. miltiorrhiza leaves. (PDF 110 kb)

Additional file 8: Excel S1. Mapping files of nucleic acid sequences in S. miltiorrhiza. (EXCEL 4,647 kb)

Additional file 9: Figure S4. Partial phenylpropanoids pathway between MF and MS in S. miltiorrhiza leaves. The figure showed the result of MS compared against MF. Blue represents down-regulated and red represents up-regulated. PAL: Phenylalanine ammonialyase; C4H: Cinnamate 4-hydroxylase; 4CL: 4-coumarate-CoA ligase; CCR: Cinnamoyl-CoA reductase; COMT: Caffeic acid O-methyltransferase; F5H: Ferulic acid-5-hydroxylase. (PDF 361kb)

Additional file 10: Figure S5. Expression verification of seven candidates from MF and MS in S. miltiorrhiza leaves. β-actin was used as an internal control. Each gene has three biological replicates and three technical replicates. (PDF 218 kb)

Additional file 11: Table S5. Primer sequences of DEGs between MF and MS in S. miltiorrhiza leaves. (DOC 42 kb)

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Physiological and transcriptome analysis reveal molecular mechanism in Salvia miltiorrhiza leaves of near-isogenic male fertile lines and male sterile lines

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