De Novo Transcriptome Assembly of Isatis indigotica at Reproductive Stages and Identification of Candidate Genes Associated with Flowering Pathways

in Journal of the American Society for Horticultural Science

The appropriate timing of bolting and flowering is one of the keys to the reproductive success of Isatis indigotica. Several flowering regulatory pathways have been reported in plant species, but we know little about flowering regulatory in I. indigotica. In the present study, we performed RNA-seq and annotated I. indigotica transcriptome using RNA from five tissues (leaves, roots, flowers, fruit, and stems). Illumina sequencing generated 149,907,857 high-quality clean reads and 124,508 unigenes were assembled from the sequenced reads. Of these unigenes, 88,064 were functionally annotated by BLAST searches against the public protein databases. Functional classification and annotation assigned 55,991 and 23,072 unigenes to 52 gene ontology (GO) terms and 25 clusters of orthologous group (COG) categories, respectively. A total of 19,927 unigenes were assigned to 124 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 80 candidate genes related to plant circadian rhythm were identified. We also identified a number of differentially expressed genes (DEG) and 91 potential bolting and flowering-related genes from the RNA-seq data. This study is the first to identify bolting and flowering-related genes based on transcriptome sequencing and assembly in I. indigotica. The results provide foundations for the exploration of flowering pathways in I. indigotica and investigations of the molecular mechanisms of bolting and flowering in Brassicaceae plants.

Contributor Notes

This work was supported by the National Natural Science Foundation of China (Grant no. 31171486).

Corresponding author. E-mail: xqtang@njau.edu.cn.

Article Sections

Article Figures

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    The length distribution of assembled transcripts and unigenes in Isatis indigotica.

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    Species distribution of the top BLAST hits against the NCBI nonredundant protein (Polashock et al., 2010) database.

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    Gene ontology (Boyle et al., 2004) classification of the Isatis indigotica unigenes.

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    Clusters of orthologous groups (Natale et al., 2000) functional classification of the Isatis indigotica unigenes.

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    The correlation dendrogram of expression patterns among the leaves, flowers, fruit, stems, and roots of Isatis indigotica.

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    Venn diagram of differentially expressed unigenes numbers among pairwise comparisons of flower, fruit, leaf, and stem with root.

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    Gene ontology (Boyle et al., 2004) classification of differentially expressed unigenes in Flower_vs._Root.

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    The pathway of circadian rhythm in Isatis indigotica. CCA1 = circadian clock associated 1; CHS = chalcone synthase; CO = constant; CRY1 = cryptochrome 1; CRY2 = cryptochrome 2; ELF3 = early flowering 3; FKF1 = flavin-binding kelch domain F box 1; GI = gigantea; LHY = late elongated hypocotyl; LKP2 = lov kelch protein 2; PHY A = phytochrome A; PHY B = phytochrome B; PHY D = phytochrome D; PHY E = phytochrome E; PIF3 = phytochrome interacting factor 3; TOC1 = timing of cab expression 1; WNK1 = with no lysine kinase 1; ZTL = zeitlupe; SCF = Skp1-cullin-F-box complex; APR3 = adenosine 5′-phosphosulfate reductase 3; APR5 = adenosine 5′-phosphosulfate reductase 5; APR7 = adenosine 5′-phosphosulfate reductase 7; APR9 = adenosine 5′-phosphosulfate reductase 9; CK2α = Casein Kinase2 a-subunit; CK2β = Casein Kinase2 β-subunit; PAP1 = production of anthocyanin pigment 1; LKP2 = Lov Kelch Protein2; UV-B = ultraviolet-B; Pfr = far-red light absorption Phytochrome.

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    Quantitative real time polymerase chain reaction (qRT-PCR) validation of eight putative genes involved in flowering pathways; RPKM = reads per kilobase of transcript per million mapped reads.

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    Gene ontology [GO (Boyle et al., 2004)] classification of differentially expressed genes (DEGs) in Fruit_vs._Root.

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    Gene ontology [GO (Boyle et al., 2004)] classification of differentially expressed genes (DEGs) in Leaf_vs._Root.

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    Gene ontology [GO (Boyle et al., 2004)] classification of differentially expressed genes (DEGs) in Root_vs._Stem.

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