Full-length sequencing of Ginkgo biloba L. reveals the synthesis of terpenoids during seed development
Introduction
Ginkgo biloba L. known as a "living fossil", is a relict plant that originated about 280 million years ago (Gong et al., 2008). G. biloba, a long-lived dioecious gymnosperm, is now the sole existing member of the Ginkgopsida. This species is considered significant for linking non-flowering plants and angiosperms due to its special evolutionary status (Wu et al., 2018). Originally from China, G. biloba has important horticultural and economic values (Guan et al., 2016), and its medicinal application dates back thousands of years (Goh and Barlow, 2002). Ginkgo biloba extract (GBE) is currently a popular health care product, and its numerous chemical components and pharmacological effects have been extensively studied and reported. Ginkgo fruits are composed of fleshy episperm, hard mesosperm, membranous endopleura, endosperm, and embryo. While the endosperm and embryo are edible, they have a certain allergenicity (Sado et al., 2019). Ginkgo fruits have antioxidant, antibacterial and insecticide biological activity, and are used in Chinese medicine to treat asthma, cough, and other diseases (Khalil et al., 2020; Wang and Zhang, 2019).
Plant secondary metabolites are a class of small molecular organic compounds produced by plant secondary metabolic activities. These substances are stored in certain organs or tissues of plants, are species-specific, and participate in the process of plant stress resistance, interaction, and information transfer (Wang et al., 2013). The main terpenoids in ginkgo include bilobalide (sesquiterpene) and ginkgolides (diterpene), which are the only ones that have t-butyl [-C17(CH3)3] natural substances with functional groups (Geng et al., 2018), which play an important role in the protection of nerves (Hua et al., 2017; Sui et al., 2019) and the treatment of cardiovascular and cerebrovascular diseases (Cao and Li, 2019; Liu et al., 2019). All parts of the ginkgo seed contain bilobalide and ginkgolide. The part with the highest total terpenoid content is the embryo, followed by the endosperm (Zhang et al., 2015). It is generally believed that ginkgo seeds undergo a dormancy process after maturity (Singh et al., 2008), although this is controversial (Feng et al., 2018). The immature embryo and endosperm of Ginkgo seeds gradually mature during the release of physiological dormancy until germination. Therefore, the synthesis and metabolism of terpenoids during seed development are worthy of attention.
High-throughput sequencing based on the Illumina platform is an effective method for gene annotation and expression quantification (Wang et al., 2016), while the read produced by next-generation sequencing (NGS) technology is relatively short with only 100–150 bp, and usually cannot span the entire transcript, reducing the accuracy of sequence assembly. Single-molecule real-time sequencing technology (SMRT) is a recently developed third-generation sequencing (TGS) technology, which has the characteristics of longer reads and the detection of base modifications. The average length of SMRT reads reaches 10−15 kb, which can meet the requirements for obtaining full-length transcripts (Roberts et al., 2013). This method can be used to accurately detect transcript structure, to analyze transcript homology, and to identify alternative splicing events, variable polyadenylation events, and fusion genes. A variety of plants have been studied using SMRT such as sorghum (Sorghum bicolor L., Abdel-Ghany et al., 2016), wheat (Triticum aestivum L., Dong et al., 2015), Salvia miltiorrhiza Bunge (Xu et al., 2015), Ananas comosus var. bracteatus (Ma et al., 2019), and ornamental crabapple (Malus spp., Huang et al., 2020). Illumina sequencing is often used to correct full-length transcripts due to the high error rate of SMRT sequencing. The strategy of combining SMRT and NGS makes the transcripts obtained in the experiment more accurate and enables the quantification of expression through NGS data (Koren et al., 2012; Chen et al., 2019).
In this study, the buds, leaves, and seeds (kernels) of G. biloba were sequenced using SMRT and NGS. A full-length transcript dataset was obtained for the development of ginkgo seeds. At the same time, the structure and function of the novel genes and isoforms were annotated, which expanded the annotation information of genes related to the seed development of ginkgo. It was aimed to lay a foundation for the further molecular research of ginkgo seed development through this work. In addition, the DEGs in terpenoid-related pathways were enriched and analyzed, which improved the understanding of the anabolic activities of terpenoids in ginkgo seed development.
Section snippets
Plant materials
Ginkgo grows on the campus of Nanjing Forestry University (32°4′43.45″ N, 118°48′52.01″ E), located in Nanjing, Jiangsu Province, China. Ginkgo seeds collected in June (early development period), October (fruit ripening, fertilization process completed), and January of the following year (after stratification treatment, physiological post-ripening completed) were selected as the research materials for differential analysis related to seed development. Based on the above plant tissues, male
PacBio sequencing and error correction
A total of 252,049 circular consensus sequences (CCS) were sequenced from the mixed library, including 70.84 % (178,548) of the FLNC sequences with an average length of 1311 bp (Table 1). The 12 samples used in RNA-seq sequencing produced more than 12,100 million clean reads, with an average of 10 Gb per sample. RNA-seq sequencing data were filtered for quality control, which revealed that the base content distribution, base sequencing quality, and GC content distribution were all normal. The
PacBio sequencing enriched the genomic annotations of G. Biloba
G. biloba is one of the five major gymnosperms. It has no close relatives and has a unique evolutionary status. Fossil evidence shows that since the age of dinosaurs, modern ginkgo has hardly changed, and are therefore important tree species for the study of plant evolution (Zhou and Zheng, 2003). G. biloba has become the focus of scientific research because of its high utilization, and its medicinal, ornamental, and edible values. Compared with the huge genome size of G. biloba, the existing
Conclusions
In this study, PacBio SMRT and Illumina RNA sequencing were used to sequence the full-length transcriptome of ginkgo flowers, leaves, and seeds at different developmental stages. Some novel ginkgo isoforms together with according lncRNAs were identified, which were confirmed to be related to seed development and terpenoid biosynthesis through functional annotation. Furthermore, the DEGs at the developmental stages of seeds were analyzed, and it was found that in ginkgo seeds, synthase family
Funding
This research was funded by the National Natural Science Foundation of China (31971689), the Guangdong Basic and Applied Basic Research Foundation (2019A1515111150), and the China Postdoctoral Science Foundation (2015T80557).
Author contributions
L.X. and M.X. conceived and designed the project; X.H. and Y.X. undertook the molecular biology experiment; X.H. and B.H. participated in the data analysis; X.H. drafted the manuscript; L.X., B.H., and M.X. modified the manuscript. All authors have read and approved the manuscript for publication.
CRediT authorship contribution statement
Xin Han: Conceptualization, Investigation, Writing - original draft, Visualization. Bing He: Validation, Formal analysis, Writing - review & editing. Yue Xin: Resources, Data curation. Meng Xu: Conceptualization, Methodology. Li-an Xu: Conceptualization, Validation, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no competing interests.
References (47)
- et al.
Long noncoding RNAs in cell-fate programming and reprogramming
Cell Stem Cell
(2014) - et al.
Antioxidant capacity in Ginkgo biloba
Food Res. Int.
(2002) - et al.
Phylogeography of a living fossil: pleistocene glaciations forced Ginkgo biloba L. (Ginkgoaceae) into two refuge areas in China with limited subsequent postglacial expansion
Mol. Phylogenet. Evol.
(2008) - et al.
Ginkgolide B and bilobalide ameliorate neural cell apoptosis in alpha-synuclein aggregates
Biomed. Pharmacother.
(2017) - et al.
Ginkgo biloba
Medicinal Plants of South Asia
(2020) - et al.
Antioxidant effects of ginkgolides and bilobalide against cerebral ischemia injury by activating the Akt/Nrf2 pathway in vitro and in vivo
Cell Stress Chaperones
(2019) - et al.
Concentrations of various forms of vitamin B6 in ginkgo seed poisoning
Brain Dev.
(2019) - et al.
Biology and chemistry of Ginkgo biloba
Fitoterapia
(2008) - et al.
Protective and therapeutic role of Bilobalide in cuprizone-induced demyelination
Int. Immunopharmacol.
(2019) - et al.
De novo transcriptome analysis revealed genes involved in flavonoid biosynthesis, transport and regulation in Ginkgo biloba
Ind. Crops Prod.
(2018)
A survey of the sorghum transcriptome using single-molecule long reads
Nature Commun.
Isoform Sequencing and. State-of-Art Applications for Unravelling Complexity of Plant Transcriptomes
Genes (Basel).
Sensitive protein alignments at tree-of-life scale using DIAMOND
Nat. Methods
Bilobalide protects H9c2 cell from oxygen-glucose-deprivation-caused damage through upregulation of miR-27a
Artif. Cells Nanomed. Biotechnol.
The developmental dynamics of the Populus stem transcriptome
Plant Biotechnol. J.
Fastp: an ultra-fast all-in-one FASTQ preprocessor
Bioinformatics
Transcriptome analysis based on a combination of sequencing platforms provides insights into leaf pigmentation in Acer rubrum
BMC Plant Biol.
Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research
Bioinformatics
Single-molecule real-time transcript sequencing facilitates common wheat genome annotation and grain transcriptome research
BMC Genomics
Embryo Development, Seed Germination, and the Kind of Dormancy ofGinkgo biloba L
Forests
HMMER web server: interactive sequence similarity searching
Nucleic Acids Res.
Research development of ginkgo terpene lactones
Zhongguo Zhong Yao Za Zhi
Draft genome of the living fossil Ginkgo biloba
Gigascience
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These authors contributed equally.