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Candidate genes of flavonoid biosynthesis in Selaginella bryopteris (L.) Baker identified by RNA-Seq

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Abstract

In the present study, de novo transcriptome analysis of Selaginella bryopteris in frond and root was performed to understand the regulation of flavonoid (FL) biosynthesis. High-quality data of 5.84 and 5.86 Gb was generated for frond and root, respectively, that assembled into 94,713 and 81,567 transcripts. A total of 87,471 and 73,395 unigenes were obtained from frond and root, respectively. A total of 41,267 and 31,048 CDS of frond and root, respectively, were annotated by BLASTX, which showed maximum hits against S. moellendorffii. Out of 11,285 differentially expressed genes, a total of 5639 genes were found to be down-regulated and 5628 genes up-regulated in frond as compared to those in root. In silico analysis of expression of genes in frond as compared to that in root was done for those related to phenylpropanoid (PP)/FL biosynthesis along with transcription factors (TFs) after DESeq and MapMan-based information. Results showed that genes of PP/FL biosynthesis pathway namely SbCHS, SbCHI, SbF3H, SbF3’H, SbDFR, SbUF3GT, SbCCOAMT, and SbCATOMT and TFs (SbMYB1, SbMYB2, SbMYB3, SbBHLH1, and SbWD40-5) were up-regulated in frond in comparison to those in root. Further, this in silico expression data was validated by RT-PCR analysis which showed predominant expression of most of these genes in frond and indicated their importance in the biosynthesis of flavonoids in S. bryopteris. A total of 9074 simple sequence repeats (SSRs) were also identified for frond and 3811 SSRs for root; these can be used for experimental validation.

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Acknowledgements

Authors thank and acknowledge the SERB-DST, New Delhi, Board of Research in Nuclear Sciences-Department of Atomic Energy, Mumbai and Bihar Agricultural University, Sabour for all the supports and facilities. M/s Xcelris Labs Ltd., Ahmedabad, India, is acknowledged for their sequencing services. The manuscript represents Bihar Agricultural University Communication No. 259/2017.

Funding

Grants received from the SERB-DST (SB/YS/LS-209/2013) and the Board of Research in Nuclear Sciences-Department of Atomic Energy (2013/35/39/BRNS/1362) for the study on Selaginella bryopteris.

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Authors and Affiliations

  1. Department of Plant Breeding and Genetics, Bihar Agricultural College, Bihar Agricultural University, Sabour, Bhagalpur, Bihar, 813210, India

    Ravi S. Singh, Ravi Kesari, Ujjwal Kumar, Vikash Kumar Jha, Anjani Kumar, Tribhuwan Kumar, Awadhesh K. Pal & Prabhash K. Singh

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  1. Ravi S. Singh
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  2. Ravi Kesari
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  3. Ujjwal Kumar
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Contributions

Conceived the idea and designed the experiment: RSS; performed the experiments and gene expression analysis: RSS, UJ, VJ, and AK; result/data interpretation: RSS and RK; prepared the manuscript: RSS; edited the manuscript: RSS, RK, AKP, TK, and PKS.

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Correspondence to Ravi S. Singh.

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Electronic supplementary material

Supplementary Fig. 1

Unigene length distribution in frond and root transcriptome. (PPT 214 kb)

Supplementary Fig. 2

Transcript length distribution in frond and root transcriptome. (PPT 219 kb)

Supplementary Fig. 3

CDS length distribution in frond and root transcriptome. (PPT 180 kb)

Supplementary Fig. 4

MapMan visualization of the metabolic pathways. (A) Total frond CDS mapped, (B) Top 500 frond CDS mapped. (PPT 288 kb)

Supplementary Fig. 5

Distribution of gene expression for root and frond is shown with Volcano plot. DEseq was performed to show the differentially expressed genes. Green, red, and gray correspond to genes with p-value <0.05 and logFC <0, p-value <0.05 and logFC >0, p-value >0.05 (Not DEG) respectively (Panel a); Digital gene expression between root Sample and frond sample. Each point represents a gene. The x- and y-axis are the log2 of the normalized gene expression level of control and experimental sample. Red and green points indicate significant change at the absolute value of log2 (FPKM ratio in two groups) and p-value<0.05 Red points indicate up-regulated genes and green points indicate down-regulated genes in the two groups which its expression level is represented by the y-axis (Panel b). (PPT 673 kb)

Supplementary Table 1

Primer sequences and PCR conditions used in RT-PCR based expression analysis (DOC 38 kb)

Supplementary Table 2

Frond transcriptome Annotation (XLS 13456 kb)

Supplementary Table 3

Root transcriptome Annotation (XLS 10336 kb)

Supplementary Table 4

Frond transcriptome GO annotation (XLS 3414 kb)

Supplementary Table 5

Root transcriptome GO annotation (XLS 2479 kb)

Supplementary Table 6

Frond genes KEGG pathway annotation (XLS 741 kb)

Supplementary Table 7

Root genes KEGG pathway annotation (XLS 686 kb)

Supplementary Table 8

Top 500 frond CDS mapped for secondary metabolites by MapMan (XLS 45 kb)

Supplementary Table 9

All frond CDS mapped for secondary metabolism by MapMan (XLS 125 kb)

Supplementary Table 10

Differentially expressed genes in fronds and root (XLS 21379 kb)

Supplementary Table 11

SSR in frond transcriptome of S. bryopteris (XLS 3223 kb)

Supplementary Table 12

SSR in root transcriptome of S. bryopteris (XLS 1496 kb)

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Singh, R.S., Kesari, R., Kumar, U. et al. Candidate genes of flavonoid biosynthesis in Selaginella bryopteris (L.) Baker identified by RNA-Seq. Funct Integr Genomics 18, 505–517 (2018). https://doi.org/10.1007/s10142-018-0603-2

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