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De novo transcriptome sequencing in Ficus hirta Vahl. (Moraceae) to investigate gene regulation involved in the biosynthesis of pollinator attracting volatiles

Abstract

Flower volatile organic compounds (VOCs) emitted from the receptive figs of Ficus (Moraceae) play important roles in maintaining the specificity of pollinator attraction and reproductive isolation between host species. The dominant components of VOCs are terpenoids, and while the genes involved in terpenoid biosynthesis are known, we know very little about the regulation of these genes and its relationship to pollinator attraction in Ficus. Thus, we selected pre-receptive (pre-VOC attractant-emitting) and receptive (VOC attractant-emitting) phase female flowers of a dioecious fig for transcriptome sequencing to investigate the differential activity of genes, including those related to terpenoid biosynthesis. We annoted 53,445 unigenes, of which 608 were found to be differently expressed in the two stages, with 242 unigenes upregulated and 366 unigenes downregulated in pre-receptive relative to receptive phase flowers. With respect to the production of VOCs, 80 unigenes encoded 34 enzyme-coding genes involved in terpenoid biosynthesis, including nearly all key enzymes of the MVA and MEP pathways forming the backbone of terpenoid biosynthesis. We designed pair primers for 16 of these 34 enzyme genes and validated 5 of them in 30 Ficus species representing 6 subgenera. In addition to the transcript sequences, we identified 35,558 putative microsatellite loci and designed primers for 11,468 of them. Our data and results will contribute to genetic research on terpene biosynthesis in figs and will aid in the understanding of the morphological and chemical changes that occur as the female flowers develop and produce pollinator-specific VOCs.

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Acknowledgments

The authors thank Prof. Zhang Mingyong, Dr. Xia Kuaifei, and Dr. Zeng Jiqing from South China Botanical Garden, the Chinese Academy of Sciences for their support with the transcriptome sequencing. We also thank three anonymous reviewers for extensive comments on this manuscript. This study was supported by the National Basic Research Program of China (973 Program) (2014CB954103), National Natural Science Foundation of China (31370409), Natural Science Foundation of Guangdong Province (S2013020012814), and the Applied Fundmental Research Foundation of Yunnan Province (2014GA003).

Conflict of interest

The authors declared that they have no competing interests.

Data archiving statement

Raw sequence reads are available from NCBI SRA (SRX951263 and SRX951272).

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Correspondence to Xuejun Ge.

Additional information

Communicated by C. Chen

This article is part of the Topical Collection on Genome Biology

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Fig. A1

Overview of the transcriptome assembly of female flowers in male syconia of Ficus hirta. a. The size distribution of the transcripts. b. The size distribution of the unigenes. (GIF 1663 kb)

(GIF 1398 kb)

High Resolution Image (TIFF 12947 kb)

High Resolution Image (TIFF 12947 kb)

Fig. A2

Characteristics of the homology search of Ficus hirta female flower unigenes. a. E-value distribution of the top BLASTx hits against the non-redundant (Nr) protein database for each unigene. The relative abundance of hits in each E-value range is represented as a percentage of the total number of hits. b. Species distribution of the top BLASTx hits in the NCBI nr database. (GIF 1637 kb)

High Resolution Image (TIFF 1560 kb)

Fig. A3

Distribution of most abundant gene ontology (GO) terms assigned to the Ficus hirta transcriptome. Proportions of annotated unigenes in the three GO categories 'Biological Process', 'Cellular Component' and 'Molecular Function' are reported for categories represented in at least the 0.1 % of the annotated unigenes. (PDF 6 kb)

Fig. A4

KOG function classification of the Ficus hirta transcriptome. In total, 8,376 unigenes were grouped into 25 classifications. (PDF 5 kb)

Fig. A5

KEGG function classification of the Ficus hirta transcriptome. In total, 3,990 unigenes are summarized into four main categories: A. cellular process; B. environmental information processing; C. genetic information processing; D. metabolism. (PDF 5 kb)

Fig A6

Functional categories of unigenes differentially expressed between the pre-receptive and receptive phase female flowers of Ficus hirta. Proportions of annotated unigenes in the three GO categories 'Biological Process' (BP), 'Cellular Component' (CC) and 'Molecular Function' (MF) are reported for categories represented in at least the 0.1 % of the annotated unigenes. (PDF 6 kb)

ESM 1

(PDF 69 kb)

Appendix 1

The putative SSR loci detected in the transcriptome sequence in Ficus hirta Vahl. Primer pair sequences are indicated for the subset of SSR loci for which Primer3 was able to design primers. For these loci, three pairs of primers were designed. (XLSX 3766 kb)

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Yu, H., Nason, J.D., Zhang, L. et al. De novo transcriptome sequencing in Ficus hirta Vahl. (Moraceae) to investigate gene regulation involved in the biosynthesis of pollinator attracting volatiles. Tree Genetics & Genomes 11, 91 (2015). https://doi.org/10.1007/s11295-015-0916-4

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  • DOI: https://doi.org/10.1007/s11295-015-0916-4

Keywords

  • Fig
  • Fig wasp
  • RNA-Seq
  • VOC
  • Terpenoid biosynthesis
  • Microsatellite