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. 2019 Mar 12;9(1):4230.
doi: 10.1038/s41598-019-40227-z.

Transcriptome Analysis of the Curry Tree (Bergera Koenigii L., Rutaceae) During Leaf Development

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Free PMC article

Transcriptome Analysis of the Curry Tree (Bergera Koenigii L., Rutaceae) During Leaf Development

Vikram S Shivakumar et al. Sci Rep. .
Free PMC article

Abstract

The curry tree (Bergera koenigii L.) is a widely cultivated plant used in South Asian cooking. Next-generation sequencing was used to generate the transcriptome of the curry leaf to detect changes in gene expression during leaf development, such as those genes involved in the production of oils which lend the leaf its characteristic taste, aroma, and medicinal properties. Using abundance estimation (RSEM) and differential expression analysis, genes that were significantly differentially expressed were identified. The transcriptome was annotated with BLASTx using the non-redundant (nr) protein database, and Gene Ontology (GO) terms were assigned based on the top BLAST hit using Blast2GO. Lastly, functional enrichment of the assigned GO terms was analyzed for genes that were significantly differentially expressed. Of the most enriched GO categories, pathways involved in cell wall, membrane, and lignin synthesis were found to be most upregulated in immature leaf tissue, possibly due to the growth and expansion of the leaf tissue. Terpene synthases, which synthesize monoterpenes and sesquiterpenes, which comprise much of the curry essential oil, were found to be significantly upregulated in mature leaf tissue, suggesting that oil production increases later in leaf development. Enzymes involved in pigment production were also significantly upregulated in mature leaves. The findings were based on computational estimates of gene expression from RNA-seq data, and further study is warranted to validate these results using targeted techniques, such as quantitative PCR.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
From top-left clockwise: (1) A cluster of immature leaves circled in red, (2) Immature leaves in a cluster at the top of the tree stem, (3) A fully mature leaf.
Figure 2
Figure 2
Average quality scores per base in single-end 50 bp sequencing reads from Illumina HiSeq 4000 sequence libraries; (A) Immature; (B) Mature.
Figure 3
Figure 3
The N50 statistic computed using subsets of highly expressed genes. Percentile expression represents the minimum percentile of expression for which to include a gene in the N50 calculation. The N50 value represents the contig length at which half the assembled bases in the assembly can be found. The peak N50 contig length is reached at the 90th percentile, indicating a high-quality transcriptome assembly.
Figure 4
Figure 4
A comparison of the completeness of the newly sequenced transcriptome to that of a previously published curry leaf transcriptome (Meena et al.). Using Benchmarking Universal Single-Copy Orthologs (BUSCO), 2121 single-copy orthologs standard to eudicots were searched against each transcriptome to determine if they were found complete (single-copy/duplicated), fragmented, or missing. The newly-assembled transcriptome was found to be more complete than the previously published transcriptome, and fewer duplications of the single-copy benchmark orthologs were found.
Figure 5
Figure 5
Volcano plot showing the spread of genes based on fold change and False Discovery Rate (FDR), representing statistical significance. Genes with an FDR significance <0.05 are labelled in red.
Figure 6
Figure 6
A heat map showing the expression patterns of significantly differentially expressed genes, defined as having greater than 6-fold (a factor of 64) change in expression and FDR <10−12, between immature and mature leaf tissue. Genes without a blast hit are listed with a unique transcript ID assigned at assembly. Coloring of rows represents the fold change [log2(exprA/exprB)] calculated from the Fragment Per Kilobase of transcript per Million mapped reads (FPKM) value.
Figure 7
Figure 7
The distribution of functional gene group expression in the leaf transcriptome of Bergera koenigii. Categories were based on GO terms assigned after functional annotation.
Figure 8
Figure 8
Volcano plot showing the spread of terpene synthase genes based on fold change and False Discovery Rate (FDR), representing statistical significance. Genes with an FDR significance <0.05 are labelled in red.
Figure 9
Figure 9
A heat map showing 25 transcripts involved in the terpene biosynthetic pathway that are most differentially expressed, identified by the GO term GO:0010333. All 25 transcripts have FDR values <0.05. Transcripts are labelled with the name of the closest homologous match from BLAST. Coloring of rows represents the fold change [log2(exprA/exprB)] calculated from the Fragment Per Kilobase of transcript per Million mapped reads (FPKM) value.

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