RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes

BMC Genomics

Wei Wu⁴  Wenting Yang¹  Bin Yi⁴  Hongchang Liu³  Peng Qin²  Kai Hou⁴  Junwen Chen⁴ 

[1] Current address: Agricultural Bureau of Leshan, Sichuan 614000, China (WTY;Rice Research Institute of Sichuan Agricultural University, Wenjiang, Chengdu, Sichuan 611130, China;Current address: Agronomy College of Guizhou University, Guiyang Huaxi, Guizhou 550025, China (HCL;Agronomy College of Sichuan Agricultural University, Wenjiang, Chengdu, Sichuan 611130, China

关键词: RNA-seq;    Transcriptome;    Stevia rebaudiana;   

Others  :  855325
DOI  :  10.1186/1471-2164-15-571

 received in 2013-12-16, accepted in 2014-06-18,  发布年份 2014

【 摘 要 】

Background

Stevia (Stevia rebaudiana) is an important medicinal plant that yields diterpenoid steviol glycosides (SGs). SGs are currently used in the preparation of medicines, food products and neutraceuticals because of its sweetening property (zero calories and about 300 times sweeter than sugar). Recently, some progress has been made in understanding the biosynthesis of SGs in Stevia, but little is known about the molecular mechanisms underlying this process. Additionally, the genomics of Stevia, a non-model species, remains uncharacterized. The recent advent of RNA-Seq, a next generation sequencing technology, provides an opportunity to expand the identification of Stevia genes through in-depth transcript profiling.

Results

We present a comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent SG compositions characterized using RNA-seq. 191,590,282 high-quality reads were generated and then assembled into 171,837 transcripts with an average sequence length of 969 base pairs. A total of 80,160 unigenes were annotated, and 14,211 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Gene sequences of all enzymes known to be involved in SG synthesis were examined. A total of 143 UDP-glucosyltransferase (UGT) unigenes were identified, some of which might be involved in SG biosynthesis. The expression patterns of eight of these genes were further confirmed by RT-QPCR.

Conclusion

RNA-seq analysis identified candidate genes encoding enzymes responsible for the biosynthesis of SGs in Stevia, a non-model plant without a reference genome. The transcriptome data from this study yielded new insights into the process of SG accumulation in Stevia. Our results demonstrate that RNA-Seq can be successfully used for gene identification and transcript profiling in a non-model species.

【 授权许可】

   
2014 Chen et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Yadav AK, Singh S, Dhyani D, Ahuja PS: A review on the improvement of stevia [Stevia rebaudiana (Bertoni)]. Can J Plant Sci 2011, 91(1):1-27.
• [2]Brandle J, Telmer P: Steviol glycoside biosynthesis. Phytochemistry 2007, 68(14):1855-1863.
• [3]Wölwer-Rieck U: The leaves of Stevia rebaudiana (Bertoni), their constituents and the analyses thereof: a review. J Agric Food Chem 2012, 60(4):886-895.
• [4]Gardana C, Scaglianti M, Simonetti P: Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry. J Chromatogr A 2010, 1217(9):1463-1470.
• [5]Corcuff JB, Brossaud J: Rebaudioside A and cortisol metabolism: sweet news for consumers. Clin Chimica Acta 2014, 431:276-277.
• [6]Brandle J, Starratt A, Gijzen M: Stevia rebaudiana: its agricultural, biological, and chemical properties. Can J Plant Sci 1998, 78(4):527-536.
• [7]Goyal SK, Samsher , Goyal RK: Stevia (Stevia rebaudiana) a bio-sweetener: a review. Int J Food Sci Nutr 2010, 61(1):1-10.
• [8]Prakash Chaturvedula VS, Prakash I: A new Diterpene Glycoside from Stevia rebaudiana. Molecules 2011, 16(4):2937-2943.
• [9]Totté N, Ende WV, Van Damme EJ, Compernolle F, Baboeuf I, Geuns JM: Cloning and heterologous expression of early genes in gibberellin and steviol biosynthesis via the methylerythritol phosphate pathway in Stevia rebaudiana. Can J Bot 2003, 81(5):517-522.
• [10]Chaturvedi P, Misra P, Tuli R: Sterol glycosyltransferases—the enzymes that modify sterols. Appl Biochem Biotech 2011, 165(1):47-68.
• [11]Madan S, Ahmad S, Singh G, Kohli K, Kumar Y, Singh R, Garg M: Stevia rebaudiana (Bert.) Bertoni-a review. Indian J Nat Prod Resour 2010, 1(3):267-286.
• [12]Chatsudthipong V, Muanprasat C: Stevioside and related compounds: therapeutic benefits beyond sweetness. Pharmacol Ther 2009, 121(1):41-54.
• [13]Chaturvedula VS, Prakash I: A new diterpene glycoside from Stevia rebaudiana. Molecules 2011, 16(4):2937-2943.
• [14]Ekblom R, Galindo J: Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity 2010, 107(1):1-15.
• [15]Yang SS, Tu ZJ, Cheung F, Xu WW, Lamb JF, Jung H-JG, Vance CP, Gronwald JW: Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems. BMC Genomics 2011, 12(1):199.
• [16]Haas BJ, Zody MC: Advancing RNA-seq analysis. Nat Biotechnol 2010, 28(5):421.
• [17]Ilut DC, Coate JE, Luciano AK, Owens TG, May GD, Farmer A, Doyle JJ: A comparative transcriptomic study of an allotetraploid and its diploid progenitors illustrates the unique advantages and challenges of RNA-seq in plant species. Am J Bot 2012, 99(2):383-396.
• [18]Iyer MK, Chinnaiyan AM: RNA-Seq unleashed. Nat Biotechnol 2011, 29(7):599.
• [19]Henschel R, Nista PM, Lieber M, Haas BJ, Wu L-S, LeDuc RD: Proceedings of the 1st Conference of the Extreme Science and Engineering Discovery Environment: bridging from the eXtreme to the campus and beyond. 2012, 45. ACM
• [20]Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q: Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 2011, 29(7):644-652.
• [21]Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25(17):3389-3402.
• [22]Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT: Gene ontology: tool for the unification of biology. Nat Genet 2000, 25(1):25-29.
• [23]Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M: KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 2012, 40(D1):109-114.
• [24]Tanksley SD: Molecular markers in plant breeding. Plant Mol Biol Rep 1983, 1(1):3-8.
• [25]Xu Y, Crouch JH: Marker-assisted selection in plant breeding: from publications to practice. Crop Sci 2008, 48(2):391-407.
• [26]Varshney RK, Graner A, Sorrells ME: Genic microsatellite markers in plants: features and applications. Trends Biotechnol 2005, 23(1):48-55.
• [27]Li R, Li Y, Fang X, Yang H, Wang J, Kristiansen K, Wang J: SNP detection for massively parallel whole-genome resequencing. Genome Res 2009, 19(6):1124-1132.
• [28]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5(7):621-628.
• [29]Wang L, Feng Z, Wang X, Wang X, Zhang X: DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 2010, 26(1):136-138.
• [30]Tang Q, Ma X, Mo C, Wilson IW, Song C, Zhao H, Yang Y, Fu W, Qiu D: An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis. BMC Genomics 2011, 12(1):343.
• [31]Kumar H, Kaul K, Bajpai-Gupta S, Kaul VK, Kumar S: A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni). Gene 2012, 492(1):276-284.
• [32]Ross J, Li Y, Lim E, Bowles DJ: Higher plant glycosyltransferases. Genome Biol 2001, 2(2):1-6. REVIEWS3004
• [33]Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG: Primer3—new capabilities and interfaces. Nucleic Acids Res 2012, 40(15):e115.
• [34]Li R, Yu C, Li Y, Lam T-W, Yiu S-M, Kristiansen K, Wang J: SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 2009, 25(15):1966-1967.
• [35]Langmead B, Schatz MC, Lin J, Pop M, Salzberg SL: Searching for SNPs with cloud computing. Genome Biol 2009, 10(11):134.
• [36]Storey JD, Tibshirani R: Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 2003, 100(16):9440-9445.
• [37]Young MD, Wakefield MJ, Smyth GK, Oshlack A: Method gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 2010, 11:R14.
• [38]Mao X, Cai T, Olyarchuk JG, Wei L: Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 2005, 21(19):3787-3793.
• [39]Wichert S, Fokianos K, Strimmer K: Identifying periodically expressed transcripts in microarray time series data. Bioinformatics 2004, 20(1):5-20.
• [40]Storey JD: A direct approach to false discovery rates. J R Stat Soc Series B Stat Methodol 2002, 64(3):479-498.
• [41]Woelwer-Rieck U, Lankes C, Wawrzun A, Wüst M: Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana. Eur Food Res Technol 2010, 231(4):581-588.