High throughput sequencing and assembly
In the present study, two C7 tolerant and MO17 susceptible parents were studied to evaluate the transcriptome of maize plant in grain tissue in response to Fusarium rot rot disease of maize. From four samples (two sensitive and tolerant parents in control conditions and 96 hours after inoculation), the library was prepared and sequencing was performed by Illumina HiSeq 2500. 10470567 readings were obtained from a total of four samples. After removing the adapters and low quality readings (less than equal to Q20) 102061029 (96.767%) High quality readings were obtained (Table 1). After data processing and elimination of poor quality readings, out of 22 million read sequences, 5117 sequences with significant differential expression were obtained. The results of the raw sequencing data of this research at the Internet address
ftp://Novo_WTNHHW161317-05_175032:[email protected]:2300 Novogen Company is available.
Table 1
Summary of Illumina sequencing data and mapped reads for the samples.
sample | C7 0h | C7 96h | MO17 0h | MO17 96h |
Raw reads | 26178434 | 22944925 | 28654220 | 24283450 |
Clean reads | 26021603 | 22813438 | 28465424 | 24170102 |
Raw base (G) | 7.85 | 6.88 | 8.60 | 7.73 |
Clean base (G) | 7.81 | 6.84 | 8.54 | 7.68 |
Effective rate (%) | 99.40 | 99.43 | 99.34 | 99.53 |
Error rate (%) | 0.01 | 0.01 | 0.01 | 0.01 |
Sequencing depth (%) (Q20) | 96.5 | 96.57 | 96.68 | 96.64 |
GC content (%) | 55.71 | 55.22 | 57.2 | 57 |
To facilitate the identification of genes respondents, the level of gene expression of two genotypes C7 (tolerance) and MO17 (sensitive) in both treatment and control conditions with F. verticillioides the B73 maize reference genome and to quantify the abundance of each transcript expression with FPKM index was performed by Cuflinks 50. Genes with significant FDR levels of 0.05 and Log2 ≥ 1 changes were defined as differential genes 2. After data processing and removal of unreadable from 102061029 million read, 5117 differential expression genes were identified with significant. REST software was used to calculate the expression ratio of target molecule and reference molecule in each sample. In this model, the reference gene is used to normalize the expression of the target gene.
Transcriptome sequencing genotypes C7 and MO17 in inoculated and non-inoculated fungus F. verticillioides in corn
Comparison results showed that inoculated with Fusarium fungi of 1296 differentially expressed genes, 863 genes in the parent had increased expression and 354 genes in the sensitive parent in response to the disease. In terms of treatment and control in tolerant genotype F.verticillioides, 145 unique differential genes were identified, expression of 46 genes were reduce and 99 genes were upregulated.In order to better understand, differential gene analysis was performed separately based on treatments and cultivars. In response to corn ear rot disease at 96 hours post-infection, a total of 1462 genes with differential expression of the increased genes expressed in the tolerant parent were more susceptible than the parent (863 vs. 599 genes). Comparing the two tolerant (C7) and sensitive (MO17) parents, the results showed that the number of genes with differential expression in F.verticillioides treatment was higher than control conditions. In addition, in both control and treated conditions in all comparisons between the two parents, the number of genes with differential expression was higher in genotype C7. Under controlled conditions of seed filling stage, the total number of genes with differential expression between the two genotypes was 1253, of which 753 were differentially expressed in the tolerant parent and 500 differential in the sensitive parent. Van diagrams were used to identify genes number specifically expressed in F.verticillioides treatment between the two C7 and MO17 genotypes (Fig. 1). In the two resistant and susceptible genotypes and by comparison between green and pink ellipses (96 h post-infection and control conditions), 1108 genes (745 genes expressed in parent C7 and 363 genes in parent MO17), 885 genes (624 Genes in parent C7 and 261 genes in parent MO17) and 715 genes, respectively, were related to F.verticillioides responsive genes, control and common conditions between the two treatments and finally, putting two parents together (blue and orange oval), 160 stress-specific genes in parent C7, 259 stress-specific genes in parent MO17, and 39 disease-responsive genes in common. Both parents were expressed. The four genes were common in all comparisons.
Functional grouping of genes with differential expression
Ontology (GO) gene analysis was performed for functional grouping of 5117 genes with differential expression based on three main groups: cell position, molecular role and biological processes.. The path of the correlation network between the enhanced genes expressed in the three functional groups versus the Locus ID background information (ZmB73_RefGen_v2) is schematically shown in Figures (S1 Fig), (S2 Fig) and (S3 Fig). The largest difference in expression is related to the subgroups of cellular process, metabolic process, cell, cell part, response to stressbinding, which showed the highest percentage of differential genes in the two groups of metabolic processes and catalytic activity in control conditions.
On the other hand, the highest frequency of transcripts involved in molecular function in genotype C7 was enriched with the highest number of genes in the cell component group, cell subgroups and cell component, while in the binding and catalytic activity groups the highest number of genes were enriched. They played a molecular role in the group. In the group of biological processes, metabolic and cellular processes were significantly enriched (S3 Fig, S5 Fig, S6 Fig).
Differential expression of genes related to metabolic pathways
metabolic pathways analysis to evaluate the metabolic pathways between the two genotypes tolerant (C7) and sensitive (MO17) 96 h after inoculation with F.verticillioides was performed (Fig. 2). Thus genes to differentiate between the two genotypes were evaluated under the conditions of insemination. In response to inoculation F.verticillioides, with 397 genes in metabolic pathways were identified 85 genotypes (respectively 283 and 114 genes in the parent C7 and MO17). Twelve metabolic pathways were identified only in the parent MO17, 41 pathways in the parent C7 and 32 pathways in both genotypes. Metabolic pathways of biosynthesis of secondary metabolites, carbohydrate metabolism, energy metabolism, nucleotide metabolism and hormone signaling with the greatest number of genes in the parent suffered C7 were rich. It should be noted biological pathway enrichment analysis of gene ontology analysis was in order.
Gene ontology analysis of the expression of specific genes seed after inoculation with F.verticillioides showed the exception of the genes involved in the cell wall such as GO (Zm00001d016896), metabolic process lignin (Zm00001d023617), the evolution of the secondary cell wall (Zm00001d004929 ), xyloglucosyltransferase activity (Zm00001d038924), response to biotic stimulus (Zm00001d018229) are correlated (Fig. 3-a) all these results show distinct pathway resistance in the resistant line C7 F.verticillioides will be activated after the inoculation.
Analysis Global genotypes in non-inoculated condition showed that most genes expressed by GO different, such as the cellular response to abscisic acid (Zm00001d004843), transfer membrane (Zm00001d011083), active signal transmission (Zm00001d049741) and metabolic process cellulose in plant cell walls (Zm00001d020531) are correlated. 1,200 specific genes expressed in samples 96 hours after infection by defensive response genes (Zm00001d009296), serine / threonine kinase protein (Zm00001d003824), phosphorylation proteins (Zm00001dald0000100m19m948), kinase proteins and peroxidase (Zm00001D022456) have been rich (Fig. 3 (b)),
the expression of these genes indicates the activation of the PTI pathway and other multiple pathways of kinase proteins that have been induced after seed contamination with F.verticillioides. Analysis of the GO pathway also showed that some genes associated with the activation of transcription factors associated with specific DNA sequences (Zm00001d005886) activated lignin catabolic processes (Zm00001d0023617), transcription factors associated with disease resistance and pathogen attack late on but power is expressed, resulting in activation of downstream genes. So confluence of hormone signaling pathways and interactions of the cell wall thickening (Zm00001d0047125) and apoptosis and induces the hypersensitive.
Respone to F.verticillioides specific genes in corn
To further explore the mechanism of resistance to F.verticillioides should focuse detail the specific genes that increase or decrease the contamination by Fusarium. Compare sensitive and tolerant genotypes in these conditions showed differential expression of 211 and 288 genes were up and down-regulated, respectively, genes for differential analysis was used analysis of biological pathways enrichment. Multiple pathway represents the most a statistically significant, such as carbohydrate metabolism (Zm00001d032271), naringenin oxoglutarate (Zm00001d037487), cynamino acid metabolism (Zm00001d037849), nitrogen metabolism (Zm00001d029932), benzenediol (Zm00001d023617), biosynthesis of terpenes (Zm00001d049957), ribosomes (Zm00001d051280), sulfur (Zm00001d041804), phenylpropanoids (Zm00001d037849) and aspartate (Zm00001d004817) (Fig. 4). The results show that genes involved in the biosynthesis of secondary metabolites which are then F.verticillioides become infected with antimicrobial compounds and is expected to lead to resistance in the maize genotype C7.
Transcription factors (TFs), and transcription regulators (TR) with differential expression
The specific defense response in the plant is in the form of expression of R genes, expression of transcription factors such as MYB and WRKY, as well as signaling pathways and expression of PR proteins, thermal shock proteins, etc. Experimental results showed that after contamination of corn kernels with F.verticillioides genotype C7 activated the resistance system and specific hormones of their signaling pathways aimed at the PTI resistance mechanism. In this regard, they showed an increase in the significant expression of APR / ERF, MYB, WRKY, Bzip and ZF-HD transcription factors. Transcripts editing through protein phosphorylation is an important mechanism for the rapid modulation of transcription factors with the location of the response to extracellular signals and the important role it plays in signal transmission. This modulation is performed by kinase proteins and phosphatases such as tyrosine phosphatase protein (Zm00001d043447) and phosphatase protein 2C (Zm00001d0064886) and phosphatase 2A (Zm00001d043447) which were identified 96 hours after infection in the parent C7. The phosphatase 2C protein gene is involved in the absisic acid pathway.
Of the differentiated genes, a total of 75 differentiated genes were identified as transcription factors in stressful conditions with 29 members (Figs. 5 and 6). Most members were from the protein kinase family with 24 genes, followed by AP2 / ERF-ERF with 9 genes, NAC, Zf-HD, and C2C2-Dof, each with 3 different genes (Fig. 5 on the left). In order to identify transcription factors in response to Fusarium factor, differential gene derived from stressed Venn diagram of two genotypes were evaluated. Of the 75 genes encoding transcription factors, 46 genes (21 families) in the C7 genotype and 5 genes (5 families) in the MO17 genotype and 36 genes (5 families) in both parents showed an increase in expression. 75 gene encoding transcription factors into three groups of transcription factors (21 groups of 37 genes), regulators (4 to 9 gene) and protein kinase family (4 of 29 gene), respectively.
Transcription factors in both genotypes treated with F.verticillioides families ERF (24 percent), NAC, Zf-HD and C2C2-Dof (each 8.1 percent), WRKY, HB-HD-ZIP, TCP and MYB each 5.4%. The tolerant parent is upregulated C7(Fig. 6), a total of 9 gene transcription regulators (TR) with differential expression were identified belonged to four families. Of these, only two genes were related to sensitive parent MO17.
Compared the genotypes of 29 genes as genes encoding protein kinase in the gene differential were identified in four families, respectively (Fig. 5, left) in tolerant and sensitive to, respectively, 20 and 9 genes related to protein kinase with expression differential expression. Of the four members CAMK and RLK-Pelle differential protein kinase is overexpressed in both genotypes but more differentiation in parent suffered a number of genes were upregulated.
In general, the results of this study showed that the type and frequency of families of transcription factors in the treatment of Fusarium fungus depends on the genotype and transcription factors related to each family show different patterns of expression according to different conditions. For example, family members and Tify C3H exclusively expressed in both parents increased, while most upregulated transcription factors are stress related to parents resistance C7.
RNA-Seq results are confirmed by qRT-PCR
In order to confirm the profile of RNA-Seq results using qRT-PCR, some genes involved in the detoxification process of dioxinivanols, genes stored in the synthesis of coraloxins, antioxidant enzymes, developer The defense response involved in the mechanical properties of the cell and the active transcription factors in the PTI pathway were randomly selected. Therefore, the expression of eight genes of Cytokinin-n-Glycosyltransferase, PRX, Kaurene synthase 6, Glutathion transferase 18, Premnaspirodiene oxygenase, Phosphatase 2C, Myb transcription factor and Pectin Acethylesterase in response to ear rot disease were examined. The results of the study showed, the expression ratios of these genes in the C7 tolerant parent to MO17 increased at 96 hours after F. verticillioides infection and were consistent in RNA-Seq and qRT-PCR (Fig. 7).
The expression pattern of some genes involved in resistanc under treatment by Fusariom
Expression pattern analysis of Phosphatase 2c (Fig. 8a, b), Kaurene synthase (Fig. 8c, d), Pectin acetylesterase (Fig. 8e, f), and Glutathion transferase (Fig. 8g, h) genes in the seed and silk samples of the two tolerance C7 and MO17 susceptible genotypes in response to F.verticillioides compared to the control (0) at different time points (6, 12, 24, 48, 72, 96, and 144 hours after exposure to salt stress) were evaluated by qRT-PCR technique in contrast to α-tubulin as a house-keeping gene after inoculation with F. verticillioides (Fig. 8).
Phosphatase 2c gene expression pattern occurred in the seeds of C7 resistant parent compared to MO17 susceptible parent in 12 hours after infection, at which time the expression of gene was about 6 times higher than MO17 susceptible parent. While in susceptible parents, gene expression increased 24 hours after infection compared to resistant parents, at other times the resistant parent performed better in defense against infection. In silk tissue, comparing the expression of this gene in a resistant parent to a sensitive parent, we find that the greatest difference in expression is seen in 72 hours (about 6 times more in a resistant parent) after infection( Fig. 9a, b).
The ratio of kaurene synthase gene expression pattern in two C7 to MO17 parents is shown in Figure (Fig. 9c, d). The expression process in these two parents was different at different times after infection. In both parents, a significant decrease in expression is observed 12 hours after infection. It is likely that the plant was stressed in the early hours after the pathogen attack and tried to reduce the expression of many genes, including kaurene synthase. It copes with disease conditions through the expression of some other genes, and this reduction in expression can be the result of a sudden shock to the plant. In parent C7, the highest expression was observed at 48 hours after infection, then decreased and again at 96 hours increased expression. Expression results in MO17 parent showed an periodic trend of increase and decrease in expression and finally a decrease in expression was observed one week after infection.
Comparing the expression pattern of Pectin acetylesterase gene in C7 resistant parent compared to MO17 susceptible parent in grain, the highest expression difference was observed at 24 hours after infection, at which time gene expression was induced in seed of resistant parent in susceptible parent at high level ( Fig. 9e, f). Comparing the expression pattern in silk fibers at all times, the resistant parent had a higher rate of expression than the susceptible line. The trend of expression changes of Pectin acetylesterase gene in silk fibers of both parents follows the same pattern, while in grain tissue in both parents showed different expression patterns.
Comparison of Glutathion transferase gene expression pattern in C7 parent compared to MO17 parent in six stages of 0, 12, 24, 48, 72 and 96 hours and one week after inoculation with F. verticilliodes is shown in Figure Fig. 8g, h. Based on these results, the expression process of this gene was different at different time intervals in grain and silk tissue. The results of the expression pattern in silk tissue showed an increase in expression at all time intervals, while a decrease in the expression ratio was observed at 72 hours after infection. The trend of changes in the expression of this gene in C7 parent silk tissue was increasing from 24 hours onwards and the highest expression was observed at 72 hours (Fig. 9g, h). In the MO17 parent, gene expression in silkworms decreased at all time intervals compared to the zero hour (control) time except for 24 h.