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.

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. 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. 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. Why blueberry?
•
Popular food with health
benefits - functional food.
•
Antioxidant, thanks to
anthocyanin (other
compounds)
•
Other pathways?
diabetes
learning &
memory
heart
disease

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. 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. 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. 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. 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. IGB and RNA-Seq
Free
from
BioViz.org

11. Click
to
open
blueberry

12. Gene
models
load
automaTcally
open
data
sets
keyword
search
12

13. ripe
pink
mature green
cups
pads
involved in splicing
gene
models

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. 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. 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. Pathway
tools
(VcCyc)
Vikas Gupta

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. 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. bixin
aldehyde
dehydrogenase
75%
scov
81%
ident
top
10%
of
genes
RPKM
• BADH1
has
VERY
high
expression
zoom
in
on
lower
range
20

21. bixin
methyl
transferase
• Also
high
expression
100%
scov
80%
ident
21

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. 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. 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.
25