De novo sequencing and comparative analysis of the blueberry transcriptome to discover putative genes related to antioxidants
Highlights
► Using RNA-seq, we have generated an extensive map of the blueberry transcriptome. ► 34,464 unique sequences were found to be expressed during the fruit development. ► 1,236 genes related to antioxidants were identified. ► Comparative analysis of the differentially expressed genes in different tissues.
Introduction
Blueberry (Vaccinium spp.) is an economically and nutritionally important fruit that is grown in many different parts of the world. Commercial success is driven by their novel consumer traits including taste, nutrition and health benefits (Wu et al., 2004). Blueberries have been shown to have one of the highest antioxidant activities compared to other fruits tested, such as cranberries, apples, and grapes (Prior et al., 1998, Sellappan et al., 2002, Wolfe and Liu, 2007). These antioxidant metabolites can provide effective protection by neutralizing free radicals which are unstable molecules that are linked to the development of a number of degenerative diseases and conditions (Zheng and Wang, 2003). The content of the anthocyanin antioxidants in blueberry is considered to be of primary importance. Anthocyanins may control diabetes (Prior et al., 2008), reduce obesity and slow the effects of aging (Grace et al., 2009), and anthocyanins have been shown to function in preventing coronary heart disease (Neto, 2007) and cancer (Faria et al., 2010). Therefore, it is important to understand better the molecular mechanisms that trigger biosynthesis and the accumulation of antioxidant metabolites in berries.
However, the blueberry genome is large (1216 Mbp) (Costich et al., 1993) and genomic information is lacking. As of May 2012, 361 nucleotide sequences, 22,402 expressed sequence tags (ESTs) and 213 proteins from blueberry (Vaccinium corymbosum) have been deposited in the National Center for Biotechnology Information (NCBI) GenBank. Before 2011, most blueberry research was focused on the analysis of their cold hardiness (Dhanaraj et al., 2004, Dhanaraj et al., 2007, Naik et al., 2007, Rowland et al., 2008). Recently, Zifkin et al. (2012) have developed a blueberry EST database and profiled the expression of flavonoid and regulatory genes to obtain an integrated view of ripening. EST sequencing has long been the core technology for reference transcript discovery. Currently, however, traditional sequencing methods for the generation of ESTs require costly and time-consuming approaches involving cDNA library construction, cloning, and labor intensive Sanger sequencing (Morozova et al., 2009, Simon et al., 2009). The inherent limitations of EST sequencing and the small number of available ESTs suggest that our understanding of the blueberry transcriptome is not enough.
Because of the massive amount of information buried in the genome-scale expression data, large-scale Illumina sequencing technology (RNA-Seq) provides a new gateway to identify the level of gene expression, new transcripts, splice variants and expressed SNPs. The information that is generated can be used to evaluate a wide variety of genetic characteristics of a species, including how to target the genes responsible for specific agronomic traits at the systemic level. To broaden the scope of pigment diversity in blueberry breeding, we performed the first global analysis of the blueberry transcriptome during berry development using the Illumina RNA-Seq method. This study has generated a large set of blueberry transcript sequences that can be used to discover tissue-specific functions and the mechanisms of secondary metabolism. The dataset will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes.
Section snippets
Plant material and tissue collection
Five-year-old blueberries (V. corymbosum ‘Northland’) were grown in a green house with a constant day temperature of 25 °C and a night temperature of 20 °C; humidity was controlled at 80%. Tissue samples were collected from flowers on 10 May 2010, from green fruit 25 days after full bloom, from pink fruit 45 days after full bloom, and from blue fruit 50 days after full bloom. The separated skin and pulp from the blue fruits were immediately frozen in liquid nitrogen and shipped on dry ice to BGI
Short-read de novo sequencing and assembly
Fruits from cv Northland were chosen for our study because this cultivar is known to have superior antioxidant capacity and anthocyanin content (Prior et al., 1998). Two pools of mRNA samples, one from blueberry skin and one from pulp, were used to build libraries for high-throughput parallel sequencing using Illumina sequencing technology on the Genome Analyzer IIx platform. After the adapter sequences were trimmed and sequences shorter than 75 bp were removed, 33,149,588 clean paired-end reads
Conclusions
Using Illumina sequencing technology, we surveyed the poly (A)+ transcriptome of blueberry at an unprecedented depth (4.8 Gb) and obtained 34,464 assembled unigenes, and 1236 transcripts and 862 TFs involved in the main antioxidant biosynthesis pathway were identified. 92 DEGs related to the anthocyanins were discovered in the skins of the blueberry fruit. This study demonstrates that the Illumina sequencing technology can be applied as a rapid and cost-effective method to define the metabolic
Acknowledgments
This work was supported by grants from the Special Fund for Agro-scientific Research in the Public Interest of China (grant no. 201103037), the Special Program for Research of Transgenic Plants (grant no. 2008ZX08010-002) and the National Natural Science Foundation of China (grant nos. 30400300 and 30971804).
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These authors contributed equally to this work.