title: De novo assembly and functional annotation of Myrciaria dubia fruit transcriptome reveals multiple metabolic pathways for L-ascorbic acid biosynthesis.
abstract: BACKGROUND
  Myrciaria dubia is an Amazonian fruit shrub that produces numerous bioactive phytochemicals, but is best known by its high L-ascorbic acid (AsA) content in fruits. Pronounced variation in AsA content has been observed both within and among individuals, but the genetic factors responsible for this variation are largely unknown. The goals of this research, therefore, were to assemble, characterize, and annotate the fruit transcriptome of M. dubia in order to reconstruct metabolic pathways and determine if multiple pathways contribute to AsA biosynthesis.
  
  
  RESULTS
  In total 24,551,882 high-quality sequence reads were de novo assembled into 70,048 unigenes (mean length = 1150 bp, N50 = 1775 bp). Assembled sequences were annotated using BLASTX against public databases such as TAIR, GR-protein, FB, MGI, RGD, ZFIN, SGN, WB, TIGR_CMR, and JCVI-CMR with 75.2 % of unigenes having annotations. Of the three core GO annotation categories, biological processes comprised 53.6 % of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1 %, respectively. Based on the KEGG pathway assignment of the functionally annotated transcripts, five metabolic pathways for AsA biosynthesis were identified: animal-like pathway, myo-inositol pathway, L-gulose pathway, D-mannose/L-galactose pathway, and uronic acid pathway. All transcripts coding enzymes involved in the ascorbate-glutathione cycle were also identified. Finally, we used the assembly to identified 6314 genic microsatellites and 23,481 high quality SNPs.
  
  
  CONCLUSIONS
  This study describes the first next-generation sequencing effort and transcriptome annotation of a non-model Amazonian plant that is relevant for AsA production and other bioactive phytochemicals. Genes encoding key enzymes were successfully identified and metabolic pathways involved in biosynthesis of AsA, anthocyanins, and other metabolic pathways have been reconstructed. The identification of these genes and pathways is in agreement with the empirically observed capability of M. dubia to synthesize and accumulate AsA and other important molecules, and adds to our current knowledge of the molecular biology and biochemistry of their production in plants. By providing insights into the mechanisms underpinning these metabolic processes, these results can be used to direct efforts to genetically manipulate this organism in order to enhance the production of these bioactive phytochemicals. The accumulation of AsA precursor and discovery of genes associated with their biosynthesis and metabolism in M. dubia is intriguing and worthy of further investigation. The sequences and pathways produced here present the genetic framework required for further studies. Quantitative transcriptomics in concert with studies of the genome, proteome, and metabolome under conditions that stimulate production and accumulation of AsA and their precursors are needed to provide a more comprehensive view of how these pathways for AsA metabolism are regulated and linked in this species.
authors.affiliation: Unidad Especializada de Biotecnología, Centro de Investigaciones de Recursos Naturales de la Amazonía (CIRNA), Universidad Nacional de la Amazonía Peruana (UNAP), Pasaje Los Paujiles S/N, San Juan Bautista, Iquitos, Perú. juanccgomez@yahoo.es.|Círculo de Investigación en Plantas con Efecto en Salud (FONDECYT N° 010-2014), Lima, Perú. juanccgomez@yahoo.es.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Castro
authors.affiliation: Pritzker Laboratory for Molecular Systematics and Evolution, The Field Museum of Natural History, Chicago, IL, USA. jdylanmaddox@gmail.com.
authors.forename: J Dylan
authors.initials: JD
authors.lastname: Maddox
authors.affiliation: Laboratorio de Biotecnología y Bioenergética, Universidad Científica del Perú (UCP), Av. Abelardo Quiñones km 2.5, San Juan Bautista, Iquitos, Perú. marianela_cobosr@yahoo.es.
authors.forename: Marianela
authors.initials: M
authors.lastname: Cobos
authors.affiliation: Laboratorio de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo (LID), Facultad de Ciencias, Universidad Peruana Cayetano Heredia (UPCH), Av. Honorio Delgado 430, San Martín de Porres, Lima, Perú. d.requena.a@gmail.com.|FARVET S.A.C. Carretera Panamericana Sur N° 766 Km 198.5, Chincha Alta, Ica, Perú. d.requena.a@gmail.com.
authors.forename: David
authors.initials: D
authors.lastname: Requena
authors.affiliation: Laboratorio de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo (LID), Facultad de Ciencias, Universidad Peruana Cayetano Heredia (UPCH), Av. Honorio Delgado 430, San Martín de Porres, Lima, Perú. mirko.zimic@upch.edu.pe.|FARVET S.A.C. Carretera Panamericana Sur N° 766 Km 198.5, Chincha Alta, Ica, Perú. mirko.zimic@upch.edu.pe.
authors.forename: Mirko
authors.initials: M
authors.lastname: Zimic
authors.affiliation: Department of Horticulture, Virginia Tech, Blacksburg, VA, 24061, USA. aurebg@vt.edu.
authors.forename: Aureliano
authors.initials: A
authors.lastname: Bombarely
authors.affiliation: Área de Conservación de Recursos Fitogenéticos, Instituto Nacional de Innovación Agraria (INIA), Calle San Roque 209, Iquitos, Perú. siman@inia.gob.pe.
authors.forename: Sixto A
authors.initials: SA
authors.lastname: Imán
authors.affiliation: Unidad Especializada de Biotecnología, Centro de Investigaciones de Recursos Naturales de la Amazonía (CIRNA), Universidad Nacional de la Amazonía Peruana (UNAP), Pasaje Los Paujiles S/N, San Juan Bautista, Iquitos, Perú. cerdeiragutierrez88@gmail.com.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Cerdeira
authors.affiliation: Unidad Especializada de Biotecnología, Centro de Investigaciones de Recursos Naturales de la Amazonía (CIRNA), Universidad Nacional de la Amazonía Peruana (UNAP), Pasaje Los Paujiles S/N, San Juan Bautista, Iquitos, Perú. amedinabardales@gmail.com.
authors.forename: Andersson E
authors.initials: AE
authors.lastname: Medina
mesh_terms: D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000235:genetics; D053898:Biosynthetic Pathways; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D059566:M Phase Cell Cycle Checkpoints; D058977:Molecular Sequence Annotation; D027822:Myrtaceae / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods; D059467:Transcriptome
chemical_list: D010940:Plant Proteins; D001205:Ascorbic Acid