RNA-Seq analysis and transcriptome assembly for blackberry (Rubus sp. Var. Lochness) fruit

BMC Genomics

Beatriz Ramos-Solano¹  Cathie Martin²  Francisco J Gutierrez-Mañero¹  Yang Zhang²  Daniel Garcia-Seco¹ 

[1] Facultad de Farmacia, Universidad CEU San Pablo, Ctra. Boadilla del Monte km 5.3, Boadilla del Monte 28668, Madrid, Spain;John Innes Center, Norwich Research Park, Norwich NR4 7UH, UK

关键词: Chimera;    Alternative splicing;    SNP;    Transcriptome;    de novo;    RNA-seq;    Blackberry;   

Others  :  1118258
DOI  :  10.1186/s12864-014-1198-1

 received in 2014-06-30, accepted in 2014-12-22,  发布年份 2015

【 摘 要 】

Background

There is an increasing interest in berries, especially blackberries in the diet, because of recent reports of their health benefits due to their high content of flavonoids. A broad range of genomic tools are available for other Rosaceae species but these tools are still lacking in the Rubus genus, thus limiting gene discovery and the breeding of improved varieties.

Results

De novo RNA-seq of ripe blackberries grown under field conditions was performed using Illumina Hiseq 2000. Almost 9 billion nucleotide bases were sequenced in total. Following assembly, 42,062 consensus sequences were detected. For functional annotation, 33,040 (NR), 32,762 (NT), 21,932 (Swiss-Prot), 20,134 (KEGG), 13,676 (COG), 24,168 (GO) consensus sequences were annotated using different databases; in total 34,552 annotated sequences were identified. For protein prediction analysis, the number of coding DNA sequences (CDS) that mapped to the protein database was 32,540. Non redundant (NR), annotation showed that 25,418 genes (73.5%) has the highest similarity with Fragaria vesca subspecies vesca. Reanalysis was undertaken by aligning the reads with this reference genome for a deeper analysis of the transcriptome. We demonstrated that de novo assembly, using Trinity and later annotation with Blast using different databases, were complementary to alignment to the reference sequence using SOAPaligner/SOAP2. The Fragaria reference genome belongs to a species in the same family as blackberry (Rosaceae) but to a different genus. Since blackberries are tetraploids, the possibility of artefactual gene chimeras resulting from mis-assembly was tested with one of the genes sequenced by RNAseq, Chalcone Synthase (CHS). cDNAs encoding this protein were cloned and sequenced. Primers designed to the assembled sequences accurately distinguished different contigs, at least for chalcone synthase genes.

Conclusions

We prepared and analysed transcriptome data from ripe blackberries, for which prior genomic information was limited. This new sequence information will improve the knowledge of this important and healthy fruit, providing an invaluable new tool for biological research.

【 授权许可】

   
2015 Garcia-Seco et al.; licensee Biomed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Kalkman C: Rosaceae. In Flowering plants · dicotyledons. Springer, Berlin Heidelberg; 2004:343-86.
• [2]Martin C, Zhang Y, Tonelli C, Petroni K: Plants, diet, and health. Annu Rev Plant Biol 2013, 64:19-46.
• [3]Kaume L, Howard LR, Devareddy L: The blackberry fruit: a review on its composition and chemistry, metabolism and bioavailability, and health benefits. J Agric Food Chem 2012, 60(23):5716-27.
• [4]Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D: The genome of the domesticated apple (Malus domestica Borkh.). Nat Genet 2010, 42(10):833-9.
• [5]Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher AL, Jaiswal P, Mockaitis K, Liston A, Mane SP: The genome of woodland strawberry (Fragaria vesca). Nat Genet 2010, 43(2):109-16.
• [6]Verde I, Abbott AG, Scalabrin S, Jung S, Shu S, Marroni F, Zhebentyayeva T, Dettori MT, Grimwood J, Cattonaro F: The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat Genet 2013, 45(5):487-94.
• [7]Pindo M, Montanari S, Cestaro A, Velsaco R: A draft genome sequence of European pear (Pyrus communis L.‘Bartlett’). 6th Rosaceous Genomics Conference (RGC6), Mezzocorona (TN), 30th September-4th October 2012: 2012 2012.
• [8]Genome Database for Rosaceae (GDR). [http://www.rosaceaeorg].
• [9]Focke WO: Species ruborum. Schweizerbart, Stuttgart; 1910.
• [10]Monasterio-Huelin E: Revisión taxonómica del género Rubus L (Rosaceae) en la Península Ibérica e Islas Baleares. Universidad Complutense de Madrid, Madrid, Spain; 1992.
• [11]Alice LA, Campbell CS: Phylogeny of Rubus (rosaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences. Am J Bot 1999, 86(1):81-97.
• [12]Brooks RM: The brooks and olmo register of fruit & nut varieties. Ashs Press, Alexandria, VA; 1997.
• [13]Kassim A, Poette J, Paterson A, Zait D, McCallum S, Woodhead M, Smith K, Hackett C, Graham J: Environmental and seasonal influences on red raspberry anthocyanin antioxidant contents and identification of quantitative traits loci (QTL). Mol Nutr Food Res 2009, 53(5):625-34.
• [14]Zheng D, Schröder G, Schröder J, Hrazdina G: Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus. Plant Mol Biol 2001, 46(1):1-15.
• [15]Zheng D, Hrazdina G: Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.). Arch Biochem Biophys 2008, 470(2):139-45.
• [16]Graham J, Smith K, MacKenzie K, Jorgenson L, Hackett C, Powell W: The construction of a genetic linkage map of red raspberry (Rubus idaeus subsp. idaeus) based on AFLPs, genomic-SSR and EST-SSR markers. Theor Appl Genet 2004, 109(4):740-9.
• [17]Zheng D, Hrazdina G: Cloning and characterization of an expansin gene, RiEXP1, and a 1-aminocyclopropane-1-carboxylic acid synthase gene, RiACS1 in ripening fruit of raspberry (Rubus idaeus L.). Plant Sci 2010, 179(1):133-9.
• [18]Kumar A, Ellis BE: A family of polyketide synthase genes expressed in ripening Rubus fruits. Phytochemistry 2003, 62(3):513-26.
• [19]Woodhead M, Weir A, Smith K, McCallum S, MacKenzie K, Graham J: Functional markers for red raspberry. J Am Soc Hortic Sci 2010, 135(5):418-27.
• [20]Varshney RK, Graner A, Sorrells ME: Genic microsatellite markers in plants: features and applications. Trends Biotechnol 2005, 23(1):48-55.
• [21]Simon SA, Zhai J, Nandety RS, McCormick KP, Zeng J, Mejia D, Meyers BC: Short-read sequencing technologies for transcriptional analyses. Annu Rev Plant Biol 2009, 60:305-33.
• [22]Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF: Complementary DNA sequencing: expressed sequence tags and human genome project. Science 1991, 252(5013):1651-6.
• [23]Lister R, Gregory BD, Ecker JR: Next is now: new technologies for sequencing of genomes, transcriptomes, and beyond. Curr Op Plant Biol 2009, 12(2):107-18.
• [24]Wang Z, Gerstein M, Snyder M: RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 2009, 10(1):57-63.
• [25]Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnol 2010, 28(5):511-5.
• [26]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. Nature Biotechnol 2011, 29(7):644-52.
• [27]Li R, Li Y, Kristiansen K, Wang J: SOAP: short oligonucleotide alignment program. Bioinformatics 2008, 24(5):713-4.
• [28]Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, Lee Y, White J, Cheung F, Parvizi B: TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics 2003, 19(5):651-2.
• [29]Altschul SF, Madden TL, Schäffer 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):3389-402.
• [30]Iseli C, Jongeneel CV, Bucher P: ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. ISMB 1999, 1999:138-47.
• [31]Pruitt KD, Tatusova T, Maglott DR: NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 2007, 35(suppl 1):D61-5.
• [32]Bairoch A, Boeckmann B: The SWISS-PROT protein sequence data bank. Nucleic Acids Res 1991, 19(Suppl):2247.
• [33]Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000, 28(1):27-30.
• [34]Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN: The COG database: an updated version includes eukaryotes. BMC Bioinformatics 2003, 4(1):41. BioMed Central Full Text
• [35]Consortium GO: The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res 2004, 32(suppl 1):D258-61.
• [36]Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21(18):3674-6.
• [37]Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L: WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 2006, 34(suppl 2):W293-7.
• [38]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5(7):621-8.
• [39]Legendre M, Pochet N, Pak T, Verstrepen KJ: Sequence-based estimation of minisatellite and microsatellite repeat variability. Genome Res 2007, 17(12):1787-96.
• [40]Castillo NR, Reed BM, Graham J, Fernández-Fernández F, Bassil NV: Microsatellite markers for raspberry and blackberry. J Am Soc Hortic Sci 2010, 135(3):271-8.
• [41]Nakasugi K, Crowhurst R, Bally J, Waterhouse P: Combining transcriptome assemblies from multiple de novo assemblers in the allo-tetraploid plant nicotiana benthamiana. PLoS One 2014, 9(3):e91776.
• [42]Ramos-Solano B, Garcia-Villaraco A, Gutierrez-Mañero F, Lucas J, Bonilla A, Garcia-Seco D: Annual changes in bioactive contents and production in field-grown blackberry after inoculation with Pseudomonas fluorescens. Plant Physiol Bioch 2014, 74:1-8.
• [43]Martin JA, Wang Z: Next-generation transcriptome assembly. Nat Rev Genet 2011, 12(10):671-82.
• [44]de Bruijn NG, Erdos P: A combinatorial problem. K Ned Akad Van Wet-P 1946, 49(49):758-64.
• [45]Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA: Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007, 35(suppl 2):W71-4.
• [46]Sokal RR, Rohlf F: Biometry. 2nd edition. WH Feeman and Company, New York; 1981.