RNA-­‐Seq	
  Analysis	
  of	
  
Blueberry	
  Fruit	
  Identifies	
  
Candidate	
  Genes	
  Involved	
  in	
  
Ripening	
  a...
Archana Raja

Ra'ad
Gharabaih
Nate Watson

Rob Reid

April
Estrada

Stig Andersen
Stig Andersen
Plants for Health Institut...
Transcriptome
project goals
•

Identify genes expressed in blueberry
during fruit development and ripening.

•

Identify g...
Why blueberry?
•

Popular food with health
benefits - functional food.

•

Antioxidant, thanks to
anthocyanin (other
compou...
454 sequencing
2009 Harvest
green
ripe

2010 Harvest
green
ripe

2.5 million 454 ESTs, average
size around 300 bases
... n...
Illumina RNA-Seq - 5 stages, 3 bioreps
pad

cup

green

pink

April 19

April 28

May 20

ripe

3 plants

2-41

2-42

3-33...
Data	
  Processing,	
  Data	
  Analysis
• Align	
  to	
  genome,	
  and	
  then	
  annotate.
• May	
  2013	
  Assembly	
  ...
Annotate:	
  GABox	
  pipeline
RNASeq
Illumina

TopHat,	
  
CuffLinks

RNASeq
454
Align	
  w/	
  
GMAP

60K	
  protein-­‐
c...
Blueberry	
  Resources
• Integrated	
  Genome	
  Browser	
  with
– Illumina	
  RNA-­‐Seq	
  alignments,	
  coverage	
  gra...
IGB and RNA-Seq

Free
from

BioViz.org
Click	
  to	
  open	
  blueberry
Gene	
  models	
  load	
  automaTcally

open	
  data	
  
sets

keyword	
  
search

12
ripe

pink

mature green

cups

pads

involved in splicing

gene
models
DifferenTal	
  gene	
  expression
11	
  days

9	
  days

22	
  days

460
DE	
  
genes

1,049
DE	
  
genes

5,117
DE	
  
gen...
FuncTonal	
  AnnotaTon
• Gene	
  Ontology	
  annota<on	
  -­‐	
  Blast2GO
• Enzyme	
  annota<on	
  -­‐	
  PlantCyc
Biologi...
FuncTonal	
  AnnotaTon	
  -­‐	
  PlantCyc
• Searched	
  berry	
  sequences	
  against	
  PlantCyc
• Filtered	
  hits	
  
–...
Pathway	
  tools	
  
(VcCyc)

Vikas Gupta
Focused	
  analysis
• Which	
  pathways	
  are	
  up-­‐	
  or	
  down-­‐regulated	
  
during	
  berry	
  fruit	
  developm...
Bixin	
  biosynthesis	
  (PWY-­‐5305)	
  
superpathway of
carotenoid biosynthesis
all-trans-lycopene

• Apo-­‐carotenoid
•...
bixin	
  
aldehyde	
  

dehydrogenase
75%	
  scov
81%	
  ident

top	
  10%	
  
of	
  genes

RPKM

• BADH1	
  has	
  VERY	
...
bixin	
  
methyl	
  

transferase
• Also	
  high	
  
expression

100%	
  scov
	
  	
  80%	
  ident

21
Bixin	
  biosynthesis	
  (PWY-­‐5305)	
  
superpathway of
carotenoid biosynthesis
all-trans-lycopene

not	
  found	
  
as	...
Bixin	
  candidate	
  enzymes	
  peak	
  here

S4

S5

image	
  credit:	
  Zi/in,	
  et	
  al,	
  2012	
  Plant	
  Physiol...
Summary
• 	
  New	
  resources

• GAbox	
  -­‐	
  Annota<on
• IGB	
  Blueberry	
  Browser
• GO	
  &	
  Pathway	
  annota<o...
Thank you
for your
attention.

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

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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.

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

  1. 1. 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....
  2. 2. Archana Raja Ra'ad Gharabaih Nate Watson Rob Reid April Estrada Stig Andersen Stig Andersen Plants for Health Institute Ann Loraine Ketan Patel Mary Ann Lila Mark Burke Vikas Gupta Ivory Clabaugh Funded by University of North Carolina Mike Wang General Administration Allan Brown Cory Brouwer
  3. 3. 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.
  4. 4. Why blueberry? • Popular food with health benefits - functional food. • Antioxidant, thanks to anthocyanin (other compounds) • Other pathways? diabetes learning & memory heart disease
  5. 5. 454 sequencing 2009 Harvest green ripe 2010 Harvest green ripe 2.5 million 454 ESTs, average size around 300 bases ... not enough to form complete transcript models Ketan Patel
  6. 6. Illumina RNA-Seq - 5 stages, 3 bioreps pad cup green pink April 19 April 28 May 20 ripe 3 plants 2-41 2-42 3-33 [row][plant] April 4 June 2 ~ 2 months Stages similar to Zifkin, 2012 (Constable lab) April Estrada
  7. 7. 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   Mark   Burke Mike   Wang Rob  Reid Allan   Brown Cory   Brouwer
  8. 8. Annotate:  GABox  pipeline RNASeq Illumina TopHat,   CuffLinks RNASeq 454 Align  w/   GMAP 60K  protein-­‐ coding  genes   70K  transcripts Ab  iniTo predicTon Genewise Augustus Glimmer Filter  based  on   expression,   structure  ,etc. nr  gene  set MUCH  alternaTve  splicing, some  of  it  developmental  regulated Developed  by:   Vikas  Gupta Used  it  to  annotate  3   genomes:
  9. 9. 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
  10. 10. IGB and RNA-Seq Free from BioViz.org
  11. 11. Click  to  open  blueberry
  12. 12. Gene  models  load  automaTcally open  data   sets keyword   search 12
  13. 13. ripe pink mature green cups pads involved in splicing gene models
  14. 14. DifferenTal  gene  expression 11  days 9  days 22  days 460 DE   genes 1,049 DE   genes 5,117 DE   genes early high MG high 13  days 22 DE   genes ripe high
  15. 15. 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
  16. 16. 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.
  17. 17. Pathway  tools   (VcCyc) Vikas Gupta
  18. 18. 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
  19. 19. Bixin  biosynthesis  (PWY-­‐5305)   superpathway of carotenoid biosynthesis all-trans-lycopene • Apo-­‐carotenoid • Abundant  in  annato ???? 1.13.12.- O O – Food  colorant bixin aldehyde – TradiTonal  medicine 1.2.1.- O O 2.1.1.- OO O OOO bixin 2.1.1.- 3  isozymes bixin/norbixin  methyl  transferase O O O O bixin dimethyl ester Ivory Clabaugh 3  isozymes bixin  aldehyde   dehydrogenase norbixin
  20. 20. bixin   aldehyde   dehydrogenase 75%  scov 81%  ident top  10%   of  genes RPKM • BADH1  has  VERY   high  expression zoom  in  on  lower  range 20
  21. 21. bixin   methyl   transferase • Also  high   expression 100%  scov    80%  ident 21
  22. 22. Bixin  biosynthesis  (PWY-­‐5305)   superpathway of carotenoid biosynthesis all-trans-lycopene not  found   as  yet 1.13.12.- • High  expression  in   mature  green  fruit O O bixin aldehyde 1.2.1.O 3  isozymes bixin  aldehyde   dehydrogenase O 2.1.1.- OO O OOO bixin 2.1.1.- 3  isozymes bixin/norbixin  methyl  transferase norbixin O O O O bixin dimethyl ester 22
  23. 23. Bixin  candidate  enzymes  peak  here S4 S5 image  credit:  Zi/in,  et  al,  2012  Plant  Physiology  (Peter  Constable  lab) Mature  Green   Image  from  NC study
  24. 24. 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. 25. Thank you for your attention. 25

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