De novo sequencing analysis of the Rosa roxburghii fruit transcriptome reveals putative ascorbate biosynthetic genes and EST-SSR markers
Introduction
Rosa roxburghii Tratt. (Rosaceae), a perennial rosebush native to China, is widely distributed in the southwestern provinces of China. The fruits of this species are known for their nutritional and medicinal components, such as ascorbic acid (AsA), superoxide dismutase, flavonoids, polysaccharides, amino acids, organic acids, and mineral elements (He et al., 1984, Fan et al., 1997, Fan et al., 2004, An et al., 2011). The fruits are therefore believed to have valuable senescence-retarding and cancer-preventing effects (Wen et al., 2007). Compared with fruits such as kiwifruit, strawberry and orange, R. roxburghii fruit has very high AsA content (1100–3000 mg per 100 g of fresh weight) (Liu et al., 2013). AsA is of vital importance to humans because of its roles in collagen synthesis and protection against oxidative stress (Padayatty et al., 2003). AsA is also crucial to the function of plant cells, where it is involved in anti-oxidative reactions, regulation of cell division and expansion, and processing of defense responses (Noctor and Foyer, 1998, Davey et al., 2000, Conklin, 2001, Conklin and Barth, 2004). Although several AsA biosynthetic pathways have been proposed in higher plants, and some genes associated with AsA biosynthesis have been identified (Wheeler et al., 1998), details of the molecular mechanisms triggering AsA biosynthesis remain unknown. In addition, the biosynthetic mechanisms operating in the AsA-overproducing fruit of R. roxburghii may have distinctive features.
Genomic information is currently unavailable for R. roxburghii. As of December 2013, only 167 partial expressed sequence tag (EST) sequences and several complete mRNA sequences have been deposited in the National Center for Biotechnology Information (NCBI) database. Most of these ESTs were submitted in association with studies on vitamin C biosynthesis and resistance to rose powdery mildew. These data are insufficient to determine transcriptome complexity and the molecular mechanisms of specific traits. In addition, simple sequence repeat (SSR) markers have not yet been developed for R. roxburghii. These markers are needed for an in-depth understanding of the natural diversity of R. roxburghii and to develop strategies for its sustainable use. The generation of extensive EST collections will aid in the development of molecular markers for further genetic research on R. roxburghii and closely related species, and will help determine the molecular mechanisms related to AsA biosynthesis.
In recent years, next-generation high-throughput DNA sequencing techniques have dramatically improved the efficiency and speed of gene discovery (Ansorge, 2009). For example, Illumina sequencing technology offers millions of sequence reads from a single instrument run. ESTs derived from Illumina sequencing can be used to develop SSR markers, which are commonly used to construct linkage maps of nuclear genomes (Gai et al., 2012). In this study, we generated a normalized cDNA library prepared from R. roxburghii fruit and established a substantial EST dataset using high-throughput Illumina RNA sequencing. Using these data, we analyzed the R. roxburghii fruit transcriptome and identified candidate genes involved in AsA biosynthesis. We also designed a set of SSRs to help genetic diversity analysis and marker-assisted breeding of R. roxburghii and closely related species.
Section snippets
Plant materials
Plants of R. roxburghii ‘Guinong 5’ (Fan et al., 2011) were grown in the fruit germplasm repository of Guizhou University, Guizhou, China. Fruits were collected at three different developmental stages: 20 days after anthesis (DAA), 60 DAA, and 100 DAA. The fruits were immediately frozen in liquid nitrogen and stored at − 70 °C until use.
cDNA preparation and sequencing
Total RNA was isolated from the harvested fruit using the Trizol reagent (Invitrogen), according to the manufacturer's instructions. Equal volumes of RNA from 20-,
Illumina paired-end sequencing and de novo assembly
The total cDNA library prepared from the fruit of R. roxburghii was sequenced using an Illumina HiSeq 2000 platform, resulting in 70,407,116 raw reads. The dataset is available at the NCBI Short Read Archive (SRA) with the accession number SRX731258. After cleaning and quality checking, 53,535,304 clean reads with 95.75% Q20 bases (base quality > 20) were generated from the cDNA libraries. Using the high-quality reads, 263,892 contigs with an average length of 208 bp were assembled (Table 1). The
Sequencing, assembly, and annotation
Transcriptome sequencing is an effective method to obtain EST sequences, which are essential for molecular marker development and novel gene identification. De novo sequencing and assembly of transcriptomes and genomes have been successfully used for both model (Cheung et al., 2006, Trick et al., 2009) and non-model (Li et al., 2012, Lai and Lin, 2013, Zheng et al., 2013) plants.
From R. roxburghii fruit, we obtained 106,590 unigenes, which was 600-fold greater than the number that had been
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (31360475), the National Key Technology R&D Program Topics of China (2011BAC09B01-11), and the Special Project of Major Science and Technology in Guizhou Province, P.R. China (20136006-1).
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2019, Scientia HorticulturaeCitation Excerpt :However, to obtain more precise results, further work still needs to be done. For no-model plant studies, developing an expressed sequence tag-simple sequence repeat database or single nucleotide polymorphism database from RNA-seq data is a good way to give more comprehensive information with a much lower cost (Yan et al., 2015), and would specially benefit the quantitative trait locus studies focusing on these bioactive compounds. Also, some recent studies employed multiple molecular markers to validate each other, which also helped to get a better result (Brake et al., 2014; Wang et al., 2017a,b).