rna-seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary...
DESCRIPTION
I presented these slides at the Plant Metabolic Network workshop held at the Plant Animal Genome Conference (PAG) XXII, January, 2014. The main goals of the talk were to describe RNA-Seq based annotation of a blueberry genome assembly and explain how we used PlantCyc enzyme data to associate blueberry genes with metabolic pathways.TRANSCRIPT
RNA-‐Seq Analysis of
Blueberry Fruit Identifies Candidate Genes Involved in
Ripening and Secondary
Metabolism
Ann Loraine presenting work of: Vikas Gupta, University of Aarhus
April Roberts, Ivory Clabaugh, Ketan Patel, Nate Watson, University of North Carolina, Charlotte
and many more....
Mike Wang
Allan Brown
Mark Burke
Cory Brouwer
Rob ReidApril
Estrada
Ketan Patel
Vikas GuptaIvory
Clabaugh
Stig AndersenStig AndersenPlants for Health Institute
Ann Loraine
Ra'adGharabaih
Nate Watson
Mary Ann Lila
Archana Raja
Funded by University of North Carolina GeneralAdministration
Transcriptome project goals
• Identify genes expressed in blueberry during fruit development and ripening.
• Identify genes encoding enzymes responsible for synthesis of bioactive compounds.
• Resources for breeding, genomics.
Why blueberry?
• Popular food with health benefits - functional food.
• Antioxidant, thanks to anthocyanin (other compounds)
• Other pathways?
diabetes
heart disease
learning & memory
454 sequencing
green ripe
Ketan Patel
2009 Harvestgreen ripe
2010 Harvest
2.5 million 454 ESTs, average size around 300 bases... not enough to form
complete transcript models
Illumina RNA-Seq - 5 stages, 3 bioreps3
plan
ts
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
Stages similar to Zifkin, 2012 (Constable lab)
Data Processing, Data Analysis
• Align to genome, and then annotate.• May 2013 Assembly –Work of Rob Reid, Allan Brown, et al– Predicted: 600 Mb (flow cyto)– Assembly: ~400 Mb (aDer removing N's)• ~13K Scaffolds• N50 145 Kb
Rob Reid
Allan Brown
Cory Brouwer
Mike Wang
Mark Burke
RNASeqIllumina
60K protein-‐coding genes 70K transcripts
RNASeq454
Ab iniTopredicTon
GenewiseAugustusGlimmer
Annotate: GABox pipeline
Align w/ GMAP
TopHat, CuffLinks
Filter based on expression, structure ,etc.
nr gene set
Developed by: Vikas GuptaUsed it to annotate 3 genomes:
MUCH alternaTve splicing,some of it developmental regulated
Blueberry Resources
• Integrated Genome Browser with– Illumina RNA-‐Seq alignments, coverage graphs, splice juncTons features (alternaTve splicing)
– 454, Sanger EST alignments (GMAP)– Gene models (GABox pipeline)
• Func7onal annota7ons for data analysis– Gene Ontology annotaTons– Pathway enzyme annotaTons PlantCyc
IGB and RNA-Seq
BioViz.org
Freefrom
Click to open blueberry
12
Gene models load automaTcally
keyword search
open data sets
gene models
pads
cups
mature green
pink
ripe
involved in splicing
DifferenTal gene expression13 days
22 days11 days 9 days
5,117DE
genes
22DE
genes
1,049DE
genes
460DE
genes
early high MG high ripe high
FuncTonal AnnotaTon• Gene Ontology annota<on -‐ Blast2GO• Enzyme annota<on -‐ PlantCyc
Biological process (Filtered by #Seqs: cutoff=5)
transport (1,544)translation (412)
signal transduction (337)secondary metabolic process (58)
response to stress (435)response to extracellular stimulus (10)response to endogenous stimulus (25)
response to biotic stimulus (28)response to abiotic stimulus (60)
regulation of gene expression, epigenetic (10)
pollen-pistil interaction (46)photosynthesis (48)
multicellular organismal development (21)lipid metabolic process (475)
generation of precursor metabolites and energy (137)
DNA metabolic process (634)
anatomical structure morphogenesis (5)
carbohydrate metabolic process (771)
catabolic process (863)
cell cycle (59)
cell death (26)
cellular component organization (362)
cellular homeostasis (97)cellular protein modification process
(1,437)
Vikas Gupta
FuncTonal AnnotaTon -‐ PlantCyc
• Searched berry sequences against PlantCyc• Filtered hits – % 65 subj. coverage, % 60 idenTty
• Iden<fied hits for:– 6000 genes – Annotated to 426 pathways Similar to grape -‐ grape and blueberry sequences very similar.
Pathway tools (VcCyc)
Vikas Gupta
Focused analysis
• Which pathways are up-‐ or down-‐regulated during berry fruit development and ripening? (Gene enrichment analysis, GOSeq)
• Which pathways are new (not well-‐studied in blueberry)?
• Which pathways have high expression? • Next slide -‐ an example
18
all-trans-lycopene
OO
bixin aldehyde
O-
O
O-
O
norbixin
O
O
OO
bixin dimethyl ester
1.13.12.-
1.2.1.-
2.1.1.-
2.1.1.-
superpathway ofcarotenoid biosynthesis
O-
O
O
O
bixin
Bixin biosynthesis (PWY-‐5305)
3 isozymesbixin aldehyde dehydrogenase
3 isozymesbixin/norbixin methyl transferase
????• Apo-‐carotenoid• Abundant in annato– Food colorant– TradiTonal medicine
Ivory Clabaugh
• BADH1 has VERY high expression
20
bixin aldehyde dehydrogenase
75% scov81% ident
zoom in on lower range
top 10% of genes
RPKM
21
• Also high expression
bixin methyl transferase
100% scov 80% ident
22
all-trans-lycopene
OO
bixin aldehyde
O-
O
O-
O
norbixin
O
O
OO
bixin dimethyl ester
1.13.12.-
1.2.1.-
2.1.1.-
2.1.1.-
superpathway ofcarotenoid biosynthesis
O-
O
O
O
bixin
Bixin biosynthesis (PWY-‐5305)
3 isozymesbixin aldehyde dehydrogenase
3 isozymesbixin/norbixin methyl transferase
not found as yet
• High expression in mature green fruit
Bixin candidate enzymes peak here
Mature Green
image credit: Zi/in, et al, 2012 Plant Physiology (Peter Constable lab)
Image from NCstudy
S4 S5
Summary• New resources
•GAbox -‐ Annota<on• IGB Blueberry Browser• GO & Pathway annota<ons
• IdenTfied new pathways for blueberry• What's next:• Refine and improve the annotaTon• Import VcCyc into PlantCyc/PMN
25
Thank you for your attention.