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RNA-Seq analysis for transcriptome assembly, gene identification, and SSR mining in ginkgo (Ginkgo biloba L.)

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Abstract

Ginkgo (Ginkgo biloba L.) is a unique tree due to its distinctive classification in the plant kingdom and a long history as a medicinal plant, yet the genetic information and molecular processes in ginkgo have been largely lacking. In this study, 63.68 million paired-end reads from the asepsis seedling of ginkgo with lengths of 75 bp were assembled using Trinity, and 70,752 transcripts and 49,396 unique transcripts were generated. Among the 39,941 unique transcripts with coding regions, 24,645 unique transcripts had at least one hit in the public databases. And only 15,154 unique transcripts were annotated into 52 gene ontology (GO) functional categories. There were 50 and 43 sequences analyzed to be involved in the synthesis of flavonoids and ginkgolide/bilobalide, respectively, uncovering the putative enzymes catalyzing each step of the metabolic pathways. Sequence analysis identified a ginkgo chalcone isomerase gene (GbCHI1) encoding one of the key enzymes in the flavonoid biosynthesis pathway and exhibiting differences on protein sequences from the previously identified GbCHI. Phylogenetic analysis showed that GbCHI1 is close to GbCHI and the chalcone isomerase from Picea sitchensis, and the expression levels of GbCHI1 are coincided with the contents of flavonoids in different tissues. Furthermore, 4000 simple sequence repeats (SSRs) were found in the transcript library, and 1513 PCR primers of the SSR loci were designed. This study generated rich genetic and genomic information about the complex transcriptome of ginkgo, which would be valuable in hel** with molecular study in this unique species in the future.

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Abbreviations

COG:

Clusters of Orthologous Groups

GO:

Gene ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

KOG:

EuKaryotic Orthologous Groups

MEP:

2-C-Methyl-d-erythritol 4-phosphate

MVA:

Mevalonate pathway

NR:

NCBI non-redundant

RNA-Seq:

RNA sequencing

SRA:

Sequence read archive

SSRs:

Simple sequence repeats

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Acknowledgments

This work was supported in part by the National Natural Science Foundation of China (Grant no. 31270316 to W Yang and 31328004 to H Shi) and excellent doctorial dissertation cultivation grant from the Central China Normal University (Grant no. 2013YBZD24).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SH performed the statistical analysis. SH and ZW carried out the experiments. SH, ZW, and YJ drafted the manuscript. WY and HS revised the manuscript. WY and HS conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.

Data achieving statement

The original RNA-Seq data was deposited to the NCBI SRA under the accession number SRX087425 by Michigan State University (http://www.ncbi.nlm.nih.gov/sra/?term=SRX087425). The Transcriptome Shotgun Assembly project presented in this paper has been deposited at DDBJ/EMBL/GenBank under the accession GBYR00000000. The version described in this paper is the first version, GBYR01000000.

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Authors and Affiliations

  1. Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, 430079, People’s Republic of China

    Shiming Han, Zhenjiang Wu, Ye **, Wannian Yang & Huazhong Shi

  2. Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409, USA

    Huazhong Shi

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  1. Shiming Han
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  2. Zhenjiang Wu
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  3. Ye **
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  4. Wannian Yang
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Corresponding authors

Correspondence to Wannian Yang or Huazhong Shi.

Additional information

Communicated by Y. Tsumura

This article is part of the Topical Collection on Gene Expression

Electronic supplementary material

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Additional file 1

The gingko transcripts database showing significant similarity with the ESTs from NCBI. (XLS 3365 kb)

Additional file 2

KOG functional classification of the Ginkgo transcriptome. (GIF 379 kb)

High Resolution Image (TIFF 16438 kb)

Additional file 3

COG functional classification of the Ginkgo transcriptome. (GIF 70 kb)

High Resolution Image (TIFF 16480 kb)

Additional file 4

The information of the blast data against the public databases. (XLSX 5731 kb)

Additional file 5

Similarity of the transcripts sequences of Ginkgo with the CDS of Arabidopsis and three woody plants. (RAR 46586 kb)

Additional file 6

HSP of top-hit species and similarity and length distributions of the HSPs. (XLS 8487 kb)

Additional file 7

Putative unique putative transcripts involved in flavonoid biosynthesis. (DOC 460 kb)

Additional file 8

Candidate genes encoding key enzymes in the ginkgolide and bilobalide biosynthesis pathways. (DOC 1053 kb)

Additional file 9

Several group of proteins or transcription factors involved in regulation of the synthesis of secondary metabolites (XLSX 118 kb)

Additional file 10

The sequences encoding key isomerases, cytochrome P450 superfamily and transcription factors. (RAR 174 kb)

Additional file 11

The primers used in the experiments of Ginkgo and The sequences of Gb17001_c0_seq2_orf1 and GbCHI1. (DOCX 14 kb)

Additional file 12

The alignment nucleotide sequences between Gb17001_c0_seq2 _orf1 and GbCHI1. (GIF 78 kb)

High Resolution Image (TIFF 1742 kb)

Additional file 13

The alignment of protein sequences among GbCHI1, GbCHI and CHIs from other plant species. (PPT 530 kb)

Additional file 14

Primer pairs for SSRs. (OUT 714 kb)

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Han, S., Wu, Z., **, Y. et al. RNA-Seq analysis for transcriptome assembly, gene identification, and SSR mining in ginkgo (Ginkgo biloba L.). Tree Genetics & Genomes 11, 37 (2015). https://doi.org/10.1007/s11295-015-0868-8

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  • DOI: https://doi.org/10.1007/s11295-015-0868-8

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