RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary...

25
RNASeq 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....

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 of RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary...

Page 1: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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....

Page 2: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 3: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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.

Page 4: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

Why blueberry?

• Popular food with health benefits - functional food.

• Antioxidant, thanks to anthocyanin (other compounds)

• Other pathways?

diabetes

heart disease

learning & memory

Page 5: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 6: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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)

Page 7: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 8: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 9: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 10: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

IGB and RNA-Seq

BioViz.org

Freefrom

Page 11: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

Click  to  open  blueberry

Page 12: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

12

Gene  models  load  automaTcally

keyword  search

open  data  sets

Page 13: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

gene models

pads

cups

mature green

pink

ripe

involved in splicing

Page 14: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 15: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 16: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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.

Page 17: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

Pathway  tools  (VcCyc)

Vikas Gupta

Page 18: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 19: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 20: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

• BADH1  has  VERY  high  expression

20

bixin  aldehyde  dehydrogenase

75%  scov81%  ident

zoom  in  on  lower  range

top  10%  of  genes

RPKM

Page 21: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

21

• Also  high  expression

bixin  methyl  transferase

100%  scov    80%  ident

Page 22: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 23: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

Bixin  candidate  enzymes  peak  here

Mature  Green  

image  credit:  Zi/in,  et  al,  2012  Plant  Physiology  (Peter  Constable  lab)

Image  from  NCstudy

S4 S5

Page 24: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

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

Page 25: RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism

25

Thank you for your attention.