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De Novo Comparative Transcriptome Analysis Provides New Insights Into Sucrose Induced Somatic Embryogenesis in Camphor Tree (Cinnamomum Camphora L.)

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De Novo Comparative Transcriptome Analysis Provides New Insights Into Sucrose Induced Somatic Embryogenesis in Camphor Tree (Cinnamomum Camphora L.)

Xueping Shi et al. BMC Genomics.

Abstract

Background: Somatic embryogenesis is a notable illustration of cell totipotency, by which somatic cells undergo dedifferentiation and then differentiate into somatic embryos. Our previous work demonstrated that pretreatment of immature zygotic embryos with 0.5 M sucrose solution for 72 h efficiently induced somatic embryo initiation in camphor tree. To better understand the molecular basis of somatic embryogenesis induced by osmotic stress, de novo transcriptome sequencing of three tissues of camphor tree (immature zygotic embryos, sucrose-pretreated immature zygotic embryos, and somatic embryos induced from sucrose-pretreated zygotic embryos) were conducted using Illumina Hiseq 2000 platform.

Results: A total of 30.70 G high quality clean reads were obtained from cDNA libraries of the three samples. The overall de novo assembly of cDNA sequence data generated 205592 transcripts, with an average length of 998 bp. 114229 unigenes (55.56 % of all transcripts) with an average length of 680 bp were annotated with gene descriptions, gene ontology terms or metabolic pathways based on Blastx search against Nr, Nt, Swissprot, GO, COG/KOG, and KEGG databases. CEGMA software identified 237 out of 248 ultra-conserved core proteins as 'complete' in the transcriptome assembly, showing a completeness of 95.6 %. A total of 897 genes previously annotated to be potentially involved in somatic embryogenesis were identified. Comparative transcriptome analysis showed that a total of 3335 genes were differentially expressed in the three samples. The differentially expressed genes were divided into six groups based on K-means clustering. Expression level analysis of 52 somatic embryogenesis-related genes indicated a high correlation between RNA-seq and qRT-PCR data. Gene enrichment analysis showed significantly differential expression of genes responding to stress and stimulus.

Conclusions: The present work reported a de novo transcriptome assembly and global analysis focused on gene expression changes during initiation and formation of somatic embryos in camphor tree. Differential expression of somatic embryogenesis-related genes indicates that sucrose induced somatic embryogenesis may share or partly share the mechanisms of somatic embryogenesis induced by plant hormones. This study provides comprehensive transcript information and gene expression data for camphor tree. It could also serve as an important platform resource for further functional studies in plant embryogenesis.

Figures

Fig. 1
Fig. 1
Length distribution of unigenes in the assembled transcriptomes. The x axis shows the lengths of unigenes and the y axis shows the number of unigenes
Fig. 2
Fig. 2
Further analyse of the BLAST results in Nr database. a Similarity distribution; b E-value distribution; c Best hit species distribution; d Best hit species classification
Fig. 3
Fig. 3
GO classifications of assembled unigenes by Blast2GO in Camphor tree. Unigenes were annotated in three main categories: biological process, cellular component and molecular function. The x-axis indicates the sub-categories and the y-axis indicates the number of unigenes
Fig. 4
Fig. 4
KOG classifications of assembled unigenes in Camphor tree. Out of 114229 de novo assembled unigenes, 11281 were annotated and seperated into 26 categories
Fig. 5
Fig. 5
Distribution of the number of genes expressed in various metabolic pathway. a Cellular processes; b Invironmental information processing; c Genetic information processing; d Metabolism; e Organismal systems
Fig. 6
Fig. 6
Venn diagram and Histogram of gene numbers differentially expressed during sucrose treatment and somatic embryo formation in camphor tree. a Histogram diagram showing the number of DEGs up- or down-regulated between different libraries; b A Venn diagram for analysis of the number of differentially expressed genes from IZE_Suc vs IZE, SE_5w vs IZE, and SE_5w vs IZE_Suc
Fig. 7
Fig. 7
Cluster analysis of differentially expressed genes during somatic embryo induction in camphor tree based on K-means method
Fig. 8
Fig. 8
Functional categorization of genes differentially expressed during sucrose pretreatment and somatic embryo iniation based on biological process of Gene Ontology
Fig. 9
Fig. 9
Expression patterns of 52 somatic embryogenesis-related DEGs in different tissues. The bar represents the scale of relative expression levels of DEGs, and the colors indicate relative signal intensities of DEGs. R roots, S stems, L young leaves, FL young flowers, FR young fruits, IZE immature zygotic embryos, IZE_Suc IZE pretreated with 1.0 M sucrose solution, SE_5w somatic embryos obtained from induction medium after culture for 5 weeks
Fig. 10
Fig. 10
Correlation between RNA-seq and quantitative real-time PCR (qRT-PCR). Comparison of log2 fold change of 52 DEGs related to somatic embryogenesis obtained by RNA-seq and qRT-PCR for IZE_Suc vs IZE (a) SE_5w vs IZE_Suc (b) and SE_5w vs IZE (c) respectively

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