Recognition of candidate transcription factors related to bilberry fruit ripening by de novo transcriptome and qRT-PCR analyses

Nga Nguyen,Jaana Vuosku,Hely Häggman,Katja Karppinen,Marko Suokas,Laura Jaakola

doi:10.1038/s41598-018-28158-7

Abstract

Bilberry (Vaccinium myrtillus L.) fruits are an excellent natural resource for human diet because of their special flavor, taste and nutritional value as well as medical properties. Bilberries are recognized for their high anthocyanin content and many of the genes involved in the anthocyanin biosynthesis have been characterized. So far, neither genomic nor RNA-seq data have been available for the species. In the present study, we de novo sequenced two bilberry fruit developmental stages, unripe green (G) and ripening (R). A total of 57,919 unigenes were assembled of which 80.2% were annotated against six public protein databases. The transcriptome served as exploratory data to identify putative transcription factors related to fruit ripening. Differentially expressed genes (DEGs) between G and R stages were prominently upregulated in R stage with the functional annotation indicating their main roles in active metabolism and catalysis. The unigenes encoding putative ripening-related regulatory genes, including members of NAC, WRKY, LOB, ERF, ARF and ABI families, were analysed by qRT-PCR at five bilberry developmental stages. Our de novo transcriptome database contributes to the understanding of the regulatory network associated with the fruit ripening in bilberry and provides the first dataset for wild Vaccinium species acquired by NGS technology.

Highlights

Bilberry (Vaccinium myrtillus L.) is a perennial dwarf shrub growing native at low temperature regions in northern hemispheres, most abundantly from the west coast of Northern Europe to Caucasus toward the northern Asia Pacific coast[1]
Studies have been conducted in tomato, in which RIPENING INHIBITOR (RIN), COLORLESS NON-RIPENING (CNR), and NON-RIPENING (NOR) transcription factors (TFs) are well characterized as key regulators in fleshy fruit ripening process[16,18,19]
The application of Ion Torrent (Ion PGMTM system) technology enabling a 400-base chemistry is a relevant choice to perform RNA sequencing for non-model organism under the absence of reference genome[35]

Summary

Introduction

Bilberry (Vaccinium myrtillus L.) is a perennial dwarf shrub growing native at low temperature regions in northern hemispheres, most abundantly from the west coast of Northern Europe to Caucasus toward the northern Asia Pacific coast[1]. The biosynthesis of anthocyanins and other flavonoid compounds is well understood and the key structural genes and transcription factors (TFs) controlling their biosynthesis have been characterized in many species. Addition, abscisic acid (ABA) and auxin, which both play crucial roles in plant growth and development as well as environmental stress responses, have recently been proposed to have an important role in fleshy fruit ripening[13,14]. The ripening process of non-climacteric bilberry includes morphological, biochemical and physiological modifications, accumulation of anthocyanin pigments as well as changes in texture, taste and flavor as in many fleshy fruits[16,17]. Studies have been conducted in tomato, in which RIPENING INHIBITOR (RIN), COLORLESS NON-RIPENING (CNR), and NON-RIPENING (NOR) TFs are well characterized as key regulators in fleshy fruit ripening process[16,18,19]. Neither the genomic nor the transcriptomic data of bilberry are currently available in public databases hampering these studies

Methods

Results

Conclusion