De Novo Assembly and Comparative Transcriptome Analyses of Red and Green Morphs of Sweet Basil Grown in Full Sunlight

Abstract

Sweet basil (Ocimum basilicum), one of the most popular cultivated herbs worldwide, displays a number of varieties differing in several characteristics, such as the color of the leaves. The development of a reference transcriptome for sweet basil, and the analysis of differentially expressed genes in acyanic and cyanic cultivars exposed to natural sunlight irradiance, has interest from horticultural and biological point of views. There is still great uncertainty about the significance of anthocyanins in photoprotection, and how green and red morphs may perform when exposed to photo-inhibitory light, a condition plants face on daily and seasonal basis. We sequenced the leaf transcriptome of the green-leaved Tigullio (TIG) and the purple-leaved Red Rubin (RR) exposed to full sunlight over a four-week experimental period. We assembled and annotated 111,007 transcripts. A total of 5,468 and 5,969 potential SSRs were identified in TIG and RR, respectively, out of which 66 were polymorphic in silico. Comparative analysis of the two transcriptomes showed 2,372 differentially expressed genes (DEGs) clustered in 222 enriched Gene ontology terms. Green and red basil mostly differed for transcripts abundance of genes involved in secondary metabolism. While the biosynthesis of waxes was up-regulated in red basil, the biosynthesis of flavonols and carotenoids was up-regulated in green basil. Data from our study provides a comprehensive transcriptome survey, gene sequence resources and microsatellites that can be used for further investigations in sweet basil. The analysis of DEGs and their functional classification also offers new insights on the functional role of anthocyanins in photoprotection.

Fig 1. Gene Ontology assignments for RR-TIG transcriptome.

The results are summarized in terms of three functional categories: cellular component, molecular function and biological process. The GO terms were visualized using WEGO (http://wego.genomics.org.cn).

Fig 2. Top 20 identified KEGG pathways.

The top 20 distribution of categorization of basil transcripts to KEGG biochemical pathways.

Fig 3. Validation of RNA-Seq data by qRT-PCR.

Comparison of expression level of nine DEGs between RNA-Seq data and qRT-PCR assay. PCR primers and unigene IDs are listed in S7 Table.

Fig 4. Enriched GO terms for RR-TIG DGEs.

The bar chart shows for each significant GO termthe amount (percentage) of annotated transcripts Blue bars correspond to the sequences of the test-set and red bars correspond to the reference transcriptome dataset.

Fig 5. MapMan-based visualization of the DEGs involved in “secondary metabolism”.

In the display, each BIN or subBIN is represented as a block where each transcript is displayed as a square, which is either colored red if this transcript is up-regulated in RR or green if this transcript is up-regulated in TIG.

Fig 6. KEGG map of the DEGs involved in the flavonoid biosynthesis for the sweet basil leaf.

Red boxes indicate overexpression in purple basil and green boxes overexpression in green basil.