RNA-Seq Analysis of Blueberry Fruit Development and Ripening
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This document summarizes an RNA-Seq analysis of blueberry fruit development and ripening. Researchers sequenced RNA from five stages of fruit development to generate over 20 million reads per sample. Reads were aligned to the blueberry genome assembly to identify over 50,000 expressed genes and their expression profiles across stages. Analysis identified thousands of differentially expressed genes between stages and clusters of genes with similar expression patterns. Pathway analysis revealed metabolic pathways active during fruit development, including a potential new pathway for bixin biosynthesis with high expression during fruit maturation. Resources from the project include an online blueberry browser and gene expression data.
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RNA-Seq Analysis of Blueberry Fruit Development and Ripening
- 1. RNA-‐Seq Analysis of Blueberry Fruit Development and Ripening Ann Loraine presenting work of: Vikas Gupta, University of Aarhus Rob Reid, April Roberts, Ivory Clabaugh, Ketan Patel, Nate Watson, University of North Carolina, Charlotte and many more....
- 2. Blueberry genome project • Launched 2008 at North Carolina Research Campus in Kannapolis – Inaugural project, funding from UNC General AdministraDon (startup) • Genome -‐ Allan Brown, Rob Reid • Transcriptome -‐ Ann Loraine • Profiling bioacDves -‐ Mary Ann Lila
- 3. Why blueberry? • FuncDonal food – Popular food with health benefits – Major small fruit crop • AnDoxidant, thanks to polyphenolic compounds – flavonoids, esp. anthocyanins • Other pathways? diabetes heart disease learning & memory Anthocyanidin ring
- 4. Transcriptome project goals • Use data to annotate genome – Gene-‐finding – FuncDonal annotaDon • Profile gene expression in berry fruit – beWer understand berry biology – gain insights into secondary metabolism
- 5. Coordinated with USDA SCRI project • Sequenced DNA from diploid blueberry from Jeannie Rowland's group • USDA Specialty Crops Research project: – GeneraDng Genomic Tools for Blueberry Improvement (9/08-‐8/12) Nahla Bassil USDA-‐ARS Corvallis Jim Olmstead, UF PI, Jeannie Rowland USDA-‐ARS Beltsville, Md with Frank Drummond, Lee Beers, Julie Graham, Susan McCallam, Nadim Alkarouf, Emily Buck, Chad Finn and others
- 6. Illumina RNA-‐Seq -‐ 5 stages, 3 bioreps, ~20 million reads per sample 3 plants 2-‐41 2-‐42 3-‐33 pad cup green pink ripe April Estrada [row]-‐ [plant] April 4 April 19 April 28 May 20 June 2 ~ 2 months in 2011 Stages similar to Ziiin, 2012 (Constable lab)
- 7. Align reads to genome assembly • Used May 2013 Assembly – Predicted genome size: 600 Mb (flow cyto) – ~400 Mb (aler removing N's) • ~13K Scaffolds • N50 145 Kb • Work of Rob Reid, Allan Brown, others • Around 60% of reads aligned Rob Reid Allan Brown Cory Brouwer Mike Wang Mark Burke
- 8. RNASeq Illumina ~63K genes ~71K transcripts RNASeq 454 Ab iniDo predicDon Genewise Augustus Glimmer Annotate -‐ make gene models Align w/ GMAP TopHat, CuffLinks Filter based on expression, structure ,etc. nr gene set Developed by: Vikas Gupta Used it to annotate 3 genomes: GABox annotaDon pipeline
- 9. Gene model dataset -‐ 50,000 genes • Extensive alternaDve splicing predicted – developmentally regulated • Predicted protein sequence from longest ORFs 57K gene models with protein > 30 amino acids, lower bound length
- 10. Gene funcDon predicDon • Gene Ontology -‐ Blast2GO – Using InterproScan + BLAST search results • Enzyme classificaDons -‐ Blast vs PlantCyc curated databases of plant enzymes Molecular function (Filtered by #Seqs: cutoff=5) transporter activity (782) translation factor activity, nucleic acid binding (66) transcription regulator activity (59) structural molecule activity (335) signal transducer activity (120) sequence-specific DNA binding transcription factor activity (381) receptor binding (9) receptor activity (33) oxygen binding (5) nucleotide binding (3,674) DNA binding (1,375) carbohydrate binding (132) chromatin binding (206) enzyme regulator activity (228) ion binding (5) kinase activity (1,319) lipid binding (94) motor activity (84) nuclease activity (159)
- 11. BitBucket "git" repository • Contains analysis code, key data files • Can "clone" repository, repeat analyses hWp://www.bitbucket.org/lorainelab/blueberrygenome
- 13. • Click to open blueberry • Then choose May 2013 genome (newer Aug 2015 version loads first)
- 14. 14 Gene models load automaDcally keyword search open data sets
- 15. gene models pads cups green pink ripe conserved, unknown protein
- 16. RNA-‐Seq analysis goals • IdenDfy highly expressed genes at each stage. • Cluster genes by expression profile. • IdenDfy differenDally expressed genes. • IdenDfy metabolic pathways acDve in berry • Next slide -‐ highlights 16 13 days 22 days 11 days 9 days pad cup mature green pink ripe
- 17. DE genes and expression profiles 13 days 22 days 11 days 9 days 5,117 DE genes 22 DE genes 1,049 DE genes 460 DE genes early high Mature Green high ripe high leucoanthocyanidin reductase unknown funcDon transcripDon factor MYB113 Clusters from STEM (Short Time Series Expression Miner) hWp://www.cs.cmu.edu/~jernst/stem/ pad cup mature green pink ripe
- 18. New pathway for blueberry? Bixin biosynthesis 3 isozymes bixin aldehyde dehydrogenase 3 isozymes bixin/norbixin methyl transferase not found • Abundant in annato – Food colorant – TradiDonal medicine Ivory Clabaugh • High expression in mature green fruit
- 19. For more informaDon, see: • Gigascience, Feb 2015
- 20. Summary • New resources • IGB Blueberry Browser • GO & Pathway annotaDons • Gene expression profiles, new enzymes • What's next: • Repeat the study using newer assembly, newer tools • Contact Rob Reid, Ann Loraine for quesDons
- 21. Mike Wang Allan Brown Mark Burke Cory Brouwer Rob Reid April Estrada Ketan Patel Vikas Gupta Ivory Clabaugh SDg Andersen SDg Andersen Plants for Health InsDtute Ann Loraine Ra'ad Gharabaih Nate Watson Mary Ann Lila Archana Raja Funded by University of North Carolina General AdministraDon