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The leaf transcriptome of fennel (Foeniculum vulgare Mill.) enables characterization of the t-anethole pathway and the discovery of microsatellites and single-nucleotide variants

Palumbo, Fabio, Vannozzi, Alessandro, Vitulo, Nicola, Lucchin, Margherita, Barcaccia, Gianni·2018·Scientific Reports
DOI 10.1038/s41598-018-28775-2PMC PMC6041299
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Abstract

Fennel is a plant species of both agronomic and pharmaceutical interest that is characterized by a shortage of genetic and molecular data. Taking advantage of NGS technology, we sequenced and annotated the first fennel leaf transcriptome using material from four different lines and two different bioinformatic approaches: de novo and genome-guided transcriptome assembly. A reference transcriptome for assembly was produced by combining these two approaches. Among the 79,263 transcripts obtained, 47,775 were annotated using BLASTX analysis performed against the NR protein database subset with 11,853 transcripts representing putative full-length CDS. Bioinformatic analyses revealed 1,011 transcripts encoding transcription factors, mainly from the BHLH, MYB-related, C2H2, MYB, and ERF families, and 6,411 EST-SSR regions. Single-nucleotide variants of SNPs and indels were identified among the 8 samples at a frequency of 0.5 and 0.04 variants per Kb, respectively. Finally, the assembled transcripts were screened to identify genes related to the biosynthesis of t-anethole, a compound well-known for its nutraceutical and medical properties. For each of the 11 genes encoding structural enzymes in the t-anethole biosynthetic pathway, we identified at least one transcript showing a significant match. Overall, our work represents a treasure trove of information exploitable both for marker-assisted breeding and for in-depth studies on thousands of genes, including those involved in t-anethole biosynthesis.

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Figure 1 Gene ontology (GO) level distribution chart for the fennel leaf transcriptome according to the Biological Process (BP), Molecular Function (MF) Cellular Component (CC) categories.
Figure 1 Gene ontology (GO) level distribution chart for the fennel leaf transcriptome according to the Biological Process (BP), Molecular Function (MF) Cellular Component (CC) categories.CC BY · PubMed Central
Figure 2 Gene ontology (GO) classification of assembled transcripts. The results of BLASTX searches against the Pentapetalae clade subset of the NR protein database were used for GO term mapping and annotation. The numbers of sequences assigned to level 5 GO terms for GO subcategories including molecular function (a), cellular component (b), and biological process (c), are shown.
Figure 2 Gene ontology (GO) classification of assembled transcripts. The results of BLASTX searches against the Pentapetalae clade subset of the NR protein database were used for GO term mapping and annotation. The numbers of sequences assigned to level 5 GO terms for GO subcategories including molecular function (a), cellular component (b), and biological process (c), are shown.CC BY · PubMed Central
Figure 3 Transcription factor family analysis. The transcription factors determined within the fennel clustered leaf transcriptome assembly are grouped by transcription factor family.
Figure 3 Transcription factor family analysis. The transcription factors determined within the fennel clustered leaf transcriptome assembly are grouped by transcription factor family.CC BY · PubMed Central
Figure 4 Reconstruction of the t-anethole biosynthetic pathway based on the annotation of the fennel clustered transcriptome assembly. All enzymes involved in the pathway are schematically reported together with their enzyme classification (EC) and abbreviated name. The main BLAST results are summarized in the lower table, including: i) a first TBLASTN approach used to find the best match between the fennel transcriptome and each enzyme of the t-anethole pathway ii) a BLASTX approach used to find the best match between each result of the TBLASTN approach and the Pentapetalae database iii) a BLASTN approach used to find the best match between each result of the TBLASTN approach and the D. carota transcriptome.
Figure 4 Reconstruction of the t-anethole biosynthetic pathway based on the annotation of the fennel clustered transcriptome assembly. All enzymes involved in the pathway are schematically reported together with their enzyme classification (EC) and abbreviated name. The main BLAST results are summarized in the lower table, including: i) a first TBLASTN approach used to find the best match between the fennel transcriptome and each enzyme of the t-anethole pathway ii) a BLASTX approach used to find the best match between each result of the TBLASTN approach and the Pentapetalae database iii) a BLASTN approach used to find the best match between each result of the TBLASTN approach and the D. carota transcriptome.CC BY · PubMed Central
Figure 5 Summary statistics related to EST-SSR regions: (a) distribution of the EST-SSR motif repeat numbers from di- to hexa-nucleotide types (the vertical axis shows the abundance of microsatellites with different motif repeat numbers, from 5 to >11, which are discriminated by different colours as reported in the legend); (b) most common types of EST-SSR motif repeats.
Figure 5 Summary statistics related to EST-SSR regions: (a) distribution of the EST-SSR motif repeat numbers from di- to hexa-nucleotide types (the vertical axis shows the abundance of microsatellites with different motif repeat numbers, from 5 to >11, which are discriminated by different colours as reported in the legend); (b) most common types of EST-SSR motif repeats.CC BY · PubMed Central
Figure 6 Heat map representing genetic similarity as calculated using the simple matching coefficient in all possible pairwise comparisons based on SNPs identified in the 8 individuals (belonging to 4 breeding lines analysed in two biological replicates) under study.
Figure 6 Heat map representing genetic similarity as calculated using the simple matching coefficient in all possible pairwise comparisons based on SNPs identified in the 8 individuals (belonging to 4 breeding lines analysed in two biological replicates) under study.CC BY · PubMed Central

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Cited by

Metadata

Authors
Palumbo, F., Vannozzi, A., Vitulo, N., Lucchin, M., & Barcaccia, G.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2018-07-11
Volume
8
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-018-28775-2
PMC
PMC6041299
Cited by
24
References
0
Language
en
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