Coffee

and

tomato

share

common

gene

repertoires

as

revealed

by

deep

sequencing

of

seed

and

cherry

transcripts

An

EST

database

has

been

generated

for

coffee

based

on

sequences

from

approximately

cDNA

clones

derived

from

five

different

stages/tissues

with

a

special

focus

on

developing

seeds

When

computationally

assembled

these

sequences

correspond

to

unigenes

which

were

analyzed

with

respect

to

functional

annotation

expression

profile

and

evolution

Compared

with

Arabidopsis

the

coffee

unigenes

encode

a

higher

proportion

of

proteins

related

to

protein

modification/turnover

and

metabolism

an

observation

that

may

explain

the

high

diversity

of

metabolites

found

in

coffee

and

related

species

Several

gene

families

were

found

to

be

either

expanded

or

unique

to

coffee

when

compared

with

Arabidopsis

A

high

proportion

of

these

families

encode

proteins

assigned

to

functions

related

to

disease

resistance

Such

families

may

have

expanded

and

evolved

rapidly

under

the

intense

pathogen

pressure

experienced

by

a

tropical

perennial

species

like

coffee

Finally

the

coffee

gene

repertoire

was

compared

with

that

of

Arabidopsis

and

Solanaceous

species

-LRB-

e.g.

tomato

-RRB-

Unlike

Arabidopsis

tomato

has

a

nearly

perfect

gene-for-gene

match

with

coffee

These

results

are

consistent

with

the

facts

that

coffee

and

tomato

have

a

similar

genome

size

chromosome

karyotype

-LRB-

tomato

n

coffee

n

-RRB-

and

chromosome

architecture

Moreover

both

belong

to

the

Asterid

I

clade

of

dicot

plant

families

Thus

the

biology

of

coffee

-LRB-

family

Rubiacaeae

-RRB-

and

tomato

-LRB-

family

Solanaceae

-RRB-

may

be

united

into

one

common

network

of

shared

discoveries

resources

and

information

Introduction

Coffee

is

an

important

international

commodity

ranking

among

the

five

most

valuable

agricultural

exports

from

developing

countries

-LRB-

Food

and

Agriculture

Organization

-RRB-

Moreover

production

and

processing

of

coffee

employs

more

than

million

people

worldwide

-LRB-

O'Brien

and

Kinnaird

-RRB-

Despite

its

economic

importance

coffee

has

received

little

attention

with

respect

to

molecular

genetics

and

genomics

research

As

of

December

only

nucleotide

and

protein

sequences

from

coffee

had

been

deposited

in

GenBank

with

the

majority

of

those

sequences

derived

from

leaf

ESTs

Many

of

the

remaining

sequences

correspond

to

enzymes

in

the

caffeine

biosynthesis

pathway

the

most

extensively

studied

pathway

in

coffee

-LRB-

Moisyadi

et

al.

Ogawa

et

al.

Mizuno

et

al.

Uefuji

et

al.

-RRB-

Commercial

coffee

production

relies

mainly

on

two

closely

related

species

Coffea

arabica

and

Coffea

canephora

accounting

for

approximately

and

of

worldwide

coffee

production

respectively

-LRB-

Herrera

et

al.

-RRB-

Although

C.

canephora

accounts

for

a

lower

total

proportion

of

the

coffee

market

than

does

C.

arabica

it

is

the

main

source

for

soluble

coffee

which

is

consumed

widely

throughout

the

world

C.

canephora

is

a

diploid

-LRB-

-RRB-

outcrossing

and

highly

polymorphic

species

native

to

central

Africa

but

which

has

expanded

through

cultivation

especially

to

western

Africa

Indonesia

and

Vietnam

-LRB-

Wrigley

-RRB-

In

contrast

C.

arabica

is

believed

to

be

a

recently

derived

tetraploid

-LRB-

-RRB-

native

to

a

small

region

of

what

is

now

Ethiopia

C.

arabica

is

now

grown

widely

throughout

the

world

The

goal

of

the

current

project

was

to

increase

the

genetic

and

molecular

knowledge

of

coffee

through

the

generation

and

annotation

of

an

EST

database

using

high

throughput

single-pass

sequencing

of

cDNAs

derived

from

leaf

pericarp

and

seed

tissues

from

a

set

of

C.

canephora

varieties

Special

emphasis

was

given

to

sequencing

cDNAs

from

different

stages

of

seed

development

both

to

shed

light

on

this

important

but

not

well

understood

aspect

of

plant

development

and

to

capture

as

many

genes

as

possible

involved

in

determining

the

final

chemical

composition

of

seeds

which

constitute

the

commercial

product

As

a

result

the

EST

database

reported

herein

is

to

our

knowledge

the

largest

public

database

of

seed-derived

ESTs

-LRB-

White

et

al.

Suh

et

al.

-RRB-

The

EST

database

was

used

to

derive

a

coffee

unigene

build

which

was

subsequently

subjected

to

functional

annotation

The

coffee

unigene

build

was

compared

with

the

model

species

Arabidopsis

Arabidopsis

is

the

only

fully

sequenced

dicot

genome

and

hence

provides

a

complete

set

of

predicted

genes

against

which

to

compare

the

EST-derived

unigene

set

of

coffee

However

coffee

and

Arabidopsis

belong

to

different

plant

families

-LRB-

Rubiaceae

and

Brassicaceae

respectively

-RRB-

which

are

distantly

related

phylogenetically

and

which

diverged

from

their

last

common

ancestor

approximately

million

years

ago

-LRB-

MYA

-RRB-

-LRB-

Gandolfo

et

al.

Crepet

et

al.

-RRB-

-LRB-

Fig.

-RRB-

Fig.

depicting

phylogenetic

relationships

of

coffee

to

other

higher

plant

taxa

-LRB-

based

on

Chase

et

al.

-RRB-

The

plant

family

most

closely

related

to

coffee

in

which

extensive

sequencing

has

been

conducted

is

Solanaceae

-LRB-

Fig.

-RRB-

In

this

family

comprehensive

EST

databases

have

been

developed

for

tomato

potato

pepper

eggplant

and

petunia

-LRB-

-RRB-

-LRB-

Hoeven

et

al.

Ronning

and

Stegalkina

Lee

et

al.

-RRB-

Both

Rubiaceae

and

Solanaceae

belong

to

the

Asterid

I

clade

of

dicots

and

based

on

existing

fossil

evidence

are

thought

to

have

diverged

from

one

another

approximately

MYA

-LRB-

Gandolfo

et

al.

Crepet

et

al.

-RRB-

-LRB-

Crepet

personal

communication

-RRB-

-LRB-

Fig.

-RRB-

The

closer

taxonomic

affinities

of

coffee

and

Solanaceae

-LRB-

e.g.

tomato

-RRB-

are

paralleled

by

a

number

of

striking

botanic

and

genetic

similarities

including

the

production

of

fleshy

berries

a

similar

genome

content

-LRB-

C

and

Mb

for

tomato

and

coffee

respectively

-RRB-

-LRB-

Hoeven

et

al.

-RRB-

similar

basic

chromosome

number

-LRB-

x

for

tomato

and

most

other

Solanaceae

x

for

coffee

-RRB-

and

similar

chromosome

architecture

with

highly

condensed

pericentric

heterochromatin

and

decondensed

euchromatin

at

the

pachytene

stage

of

meiosis

-LRB-

Rick

Pinto-Maglio

and

Cruz

-RRB-

For

these

reasons

the

coffee

unigene

set

was

also

compared

against

a

series

of

Solanaceae

EST-derived

unigene

sets

Materials

and

methods

Library

construction

Source

of

tissues

C.

canephora

was

farm-grown

in

east

Java

Plant

tissue

was

frozen

in

liquid

nitrogen

transported

on

dry

ice

and

stored

at

°C

until

RNA

extraction

The

specified

tissues

-LRB-

leaf

pericarp

young

cherries

with

seed

and

pericarp

mixed

middle

stage

seed

and

late

stage

seed

-RRB-

were

collected

from

five

different

varieties

Table

-RRB-

The

maturation

period

of

the

C.

canephora

varieties

under

study

is

approximately

months

from

pollination

to

ripening

The

early

stage

cherry

middle

stage

seed

and

late

stage

seed

were

collected

between

to

weeks

weeks

and

to

weeks

after

pollination

respectively

Table

of

the

cDNA

libraries

used

to

develop

the

coffee

EST

databaseLibrary

nameTissueVarietiesAverage

insert

size

kbGood

quality

ESTsLeafLeaves

PericarpPericarp

all

developmental

Early

stage

cherryWhole

cherries

and

week

after

Middle

stage

seedEndosperm

and

perisperm

of

seeds

week

after

Late

stage

seedEndosperm

and

perisperm

of

seeds

and

week

after

RNA

and

mRNA

isolation

Total

RNA

was

extracted

using

phenol/chloroform

-LRB-

Rogers

et

al.

-RRB-

and

further

treated

with

DNase

I

-LRB-

RNase-free

-RRB-

and

purified

using

an

RNeasy

Kit

-LRB-

Qiagen

Valencia

CA

-RRB-

Messenger

RNA

was

extracted

from

total

RNA

with

PolyTrack

mRNA

Isolation

Systems

-LRB-

Promega

Madison

WI

-RRB-

cDNA

libraries

Directional

cDNA

libraries

were

constructed

with

μg

of

mRNA

with

the

ZAP-cDNA

Gigapack

III

Gold

Cloning

Kit

-LRB-

Stratagene

La

Jolla

CA

-RRB-

The

average

insert

length

was

estimated

by

PCR

in

randomly

selected

clones

from

each

library

and

ranged

kb

-LRB-

Table

-RRB-

Sequencing

Bacteria

containing

coffee

cDNAs

were

cultured

in

plates

and

cDNA

inserts

subjected

to

end

sequencing

at

the

BioResource

Center

at

Cornell

University

-LRB-

-RRB-

The

average

size

of

quality

reads

was

bp

with

a

maximum

of

bp

Sequence

quality

processing

EST

sequences

were

base-called

and

screened

for

vector

sequences

using

PHRED

software

-LRB-

Ewing

et

al.

-RRB-

The

longest

stretch

of

overall

high

quality

-LRB-

PHRED

score

over

which

corresponds

to

over

confidence

-RRB-

of

each

sequence

was

identified

PolyA

repeats

were

trimmed

to

at

most

bp

and

any

sequence

past

the

PolyA

-LRB-

mostly

low

quality

sequence

-RRB-

was

discarded

After

the

trimming

the

sequences

were

screened

against

the

E.coli

genome

to

remove

any

bacteria

contamination

The

remaining

sequences

were

screened

for

minimum

length

-LRB-

bp

-RRB-

and

maximum

allowed

ambiguity

-LRB-

-RRB-

and

low

complexity

-LRB-

of

the

sequence

are

of

the

same

nucleotide

or

of

same

two

nucleotides

which

indicate

error

in

sequencing

-RRB-

Unigene

assembly

Unigene

sets

were

built

by

combining

the

sequences

from

all

five

coffee

cDNA

libraries

Clustering

was

performed

using

a

program

developed

at

the

Sol

Genomics

Network

-LRB-

SGN

at

-RRB-

which

relied

on

a

custom

pre-clustering

algorithm

and

on

the

program

for

contig

generation

-LRB-

Huang

and

Madan

-RRB-

The

preclustering

algorithm

clustered

sequences

using

a

Smith

Waterman

type

algorithm

with

initial

word

matching

The

command

line

settings

for

were

as

follows

e

p

d

b

The

e

d

and

b

options

are

set

such

that

the

assembler

disregards

them

or

minimizes

their

effect

The

p

option

increases

the

sequence

identity

necessary

on

overlaps

to

from

a

default

of

which

were

found

to

be

not

stringent

enough

Sequences

were

also

checked

for

length

complexity

and

contamination

The

builds

were

uploaded

to

the

database

where

each

unigene

was

assigned

a

unique

unigene

ID

Annotation

Protein

prediction

The

most

likely

coding

frame

and

the

corresponding

transcribed

peptide

for

each

unigene

were

generated

using

ESTScan

a

program

that

detects

coding

regions

in

EST

sequences

and

corrects

some

sequencing

errors

-LRB-

nucleotide

substitution

deletion/insertion

erroneous

stop

codon

etc

-RRB-

-LRB-

Iseli

et

al.

-RRB-

As

a

hidden

Markov

Model

-LRB-

HMM

-RRB-

based

program

ESTScan

requires

a

training

set

of

high

quality

coding

sequences

to

generate

a

model

However

very

few

high

quality

coffee

coding

sequences

are

publicly

available

-LRB-

less

than

full

length

in

GenBank

-RRB-

Therefore

we

used

as

a

training

set

of

nuclear

genes

from

the

closely

related

species

tomato

-LRB-

-RRB-

Table

compares

the

coffee

unigene

assembly

and

translation

-LRB-

using

ESTScan

-RRB-

with

that

of

tomato

using

the

same

training

tomato

set

and

parameters

The

fact

that

the

two

give

such

similar

results

gives

confidence

that

the

coffee

EST-derived

unigenes

are

being

efficiently

translated

via

the

ESTScan

software

using

the

tomato

training

set

-LRB-

Table

-RRB-

Table

of

the

coffee

and

tomato

EST

databases

derived

from

use

of

ESTScan

calibrated

with

the

same

tomato

training

set

-LRB-

see

Materials

and

methods

for

details

-RRB-

TomatoCoffeeTotal

Average

unigene

length

with

coding

Average

length

-LRB-

bp

-RRB-

of

predicated

ESTScan

BLAST

matches

between

coffee

unigenes

and

other

sequence

databases

Sequence

matches

for

coffee

unigenes

against

other

sequence

databases

were

obtained

using

BLAST

-LRB-

Altschul

et

al.

-RRB-

-LRB-

see

Results

and

discussion

section

for

details

of

each

BLAST

analysis

-RRB-

The

target

databases

were

The

GenBank

non-redundant

-LRB-

NR

-RRB-

protein

and

dbest

dataset

-LRB-

NCBI

-RRB-

The

predicted

Arabidopsis

proteome

and

cDNA

databases

which

were

downloaded

from

the

Arabidopsis

Information

Resource

-LRB-

TAIR

and

-RRB-

The

Solanaceae

EST-derived

unigene

sets

including

tomato

-LRB-

Solanum

lycopersicum

-RRB-

-LRB-

ESTs

unigenes

-RRB-

potato

-LRB-

Solanum

tuberosum

-RRB-

-LRB-

ESTs

unigenes

-RRB-

pepper

-LRB-

Capsicum

annuum

-RRB-

-LRB-

ESTs

unigenes

-RRB-

petunia

-LRB-

Petunia

hybrida

-RRB-

-LRB-

ESTs

unigenes

-RRB-

and

eggplant

-LRB-

Solanum

melongena

-RRB-

-LRB-

ESTs

unigenes

-RRB-

all

of

which

can

be

accessed

at

SGN

-LRB-

-RRB-

To

estimate

ribosomal

chloroplast

and

mitochondrial

contamination

the

coffee

unigene

set

was

also

screened

against

the

Arabidopsis

complete

mitochondrial

genome

-LRB-

-RRB-

tobacco

ribosomal

rRNA

gene

-LRB-

s

and

-RRB-

and

the

complete

tobacco

chloroplast

genome

-LRB-

-RRB-

sequences

from

GenBank

Functional

annotation

based

on

predicted

peptides

ESTScan-predicted

coffee

peptides

were

subjected

to

InterPro

Scan

annotation

which

integrates

the

most

commonly

used

protein

signature

databases

-LRB-

PROSITE

PRINTS

Pfam

ProDom

etc.

-RRB-

together

with

their

associated

scanning

methods

for

protein

domain

analysis

-LRB-

Apweiler

et

al.

Zdobnov

and

Apweiler

-RRB-

Based

on

the

domain

annotation

GO

accession

of

the

unigenes

were

assigned

using

conversion

file

from

the

GO

consortium

-LRB-

also

available

at

-RRB-

Functional

categorization

based

on

gene

ontology

GO

annotations

were

formatted

for

input

into

the

GOSlim

program

and

the

output

parsed

to

count

the

occurrence

of

each

GO

category

GOSlims

are

slimmed

down

versions

of

the

ontologies

that

allow

a

high-level

view

of

gene

functions

The

GOSlim

file

and

program

were

obtained

from

the

Gene

Ontology

Consortium

at

Gene

family

analysis

The

predicted

protein

sequences

for

the

coffee

unigene

set

and

the

Arabidopsis

protein

set

were

combined

into

a

single

file

formatted

as

a

blast

database

using

formatdb

and

run

with

BLASTP

-LRB-

protein

vs.

protein

sequence

-RRB-

against

itself

with

option

for

output

The

resulting

file

was

used

as

the

input

for

the

tribeML

program

TribeML

-LRB-

Enright

et

al.

-RRB-

formats

the

clusters

such

that

each

cluster

was

given

in

a

tab

delimited

file

one

cluster

per

line

Simple

scripts

were

used

to

parse

the

information

to

detect

the

largest

gene

families

coffee

specific

families

and

families

that

showed

large

expansions

in

coffee

Results

and

discussion

Generation

of

coffee

EST

database

and

unigene

set

A

total

of

cDNA

clones

derived

from

mRNA

from

five

different

tissues

were

subjected

to

sequencing

After

quality

evaluation

-LRB-

see

Materials

and

methods

for

details

-RRB-

the

database

was

reduced

to

high

quality

ESTs

averaging

bp

in

length

This

corresponded

to

approximately

high

quality

ESTs

from

each

of

the

five

cDNA

libraries

-LRB-

Table

-RRB-

These

high

quality

ESTs

were

assembled

into

unigenes

with

average

length

of

bp

-LRB-

ranging

bp

-RRB-

among

which

-LRB-

-RRB-

were

singletons

and

-LRB-

-RRB-

were

contigs

-LRB-

Fig.

-RRB-

Of

the

contigs

the

majority

-LRB-

-RRB-

was

represented

by

two

to

ten

ESTs

while

some

-LRB-

-RRB-

were

comprised

of

more

than

ten

ESTs

-LRB-

Fig.

-RRB-

All

trace

files

sequences

and

derived

unigenes/annotation

can

be

found

at

the

Solanaceae

Genomics

Network

-LRB-

SGN

-RRB-

website

-LRB-

-RRB-

and

will

be

submitted

to

GenBank

at

the

time

of

publication

Fig.

depicting

the

distribution

of

EST

content

for

all

coffee

unigenes

Numbers

above

bars

equals

the

number

of

unigenes

represented

in

each

Differentiating

between

paralogs

and

alleles

The

coffee

ESTs

were

derived

from

five

C.

canephora

varieties

-LRB-

Table

-RRB-

however

unigene

assembly

for

EST

reads

does

not

automatically

distinguish

between

polymorphic

alleles

and

highly

similar

paralogs

C.

canephora

is

known

to

be

a

highly

allogamous

heterozygous

and

polymorphic

species

raising

the

possibility

that

some

of

the

EST-derived

unigenes

might

actually

represent

different

allelic

forms

of

the

same

gene

In

order

to

evaluate

how

well

paralogs

were

distinguished

from

allelic

polymorphism

in

the

unigene

assembly

BLASTN

-LRB-

nucleotide

vs.

nucleotide

sequences

-RRB-

was

performed

amongst

all

of

the

coffee

unigenes

and

the

results

parsed

to

identify

the

best

non-self

matches

As

a

control

BLASTN

was

performed

in

a

similar

manner

on

a

complete

set

of

Arabidopsis

predicted

genes

including

both

coding

and

UTR

regions

obtained

from

TAIR

-LRB-

-RRB-

Since

the

Arabidopsis

database

was

derived

from

the

complete

genome

sequence

of

a

single

inbred

stock

-LRB-

Columbia

-RRB-

the

entire

gene

complement

is

present

without

the

complication

of

allelic

variation

-LRB-

initiative

-RRB-

For

both

coffee

and

Arabidopsis

pair-wise

combinations

for

which

the

homology

extended

over

more

than

of

the

sequence

were

recorded

The

sequence

similarity

for

each

of

these

combinations

was

plotted

for

both

coffee

and

Arabidopsis

-LRB-

Fig.

-RRB-

Fig.

depicting

the

sequence

identify

of

the

most

similar

match

for

each

coffee

unigene

as

compared

with

all

other

coffee

unigenes

As

a

control

a

similar

analysis

is

shown

for

Arabidopsis

genes

-LRB-

see

Results

for

details

-RRB-

The

Arabidopsis

plot

showed

two

peaks

one

with

low

identity

-LRB-

-RRB-

and

the

other

with

higher

identity

-LRB-

over

-RRB-

with

and

of

genes

falling

into

the

low

and

high

identity

peaks

respectively

Like

Arabidopsis

the

coffee

plot

also

showed

two

peaks

one

with

lower

identity

-LRB-

around

-RRB-

and

the

other

with

high

identity

-LRB-

over

-RRB-

These

two

peaks

corresponded

to

and

of

the

total

unigenes

respectively

ESTs

corresponding

to

ten

pairs

of

coffee

unigenes

from

the

peak

were

used

as

probes

on

genomic

southern

hybridizations

to

determine

whether

the

matching

pairs

were

truly

duplicated

in

the

coffee

genome

-LRB-

paralogs

-RRB-

or

rather

allelic

-LRB-

single

copy

-RRB-

For

eight

of

the

ten

pairs

the

paired

ESTs

hybridized

the

same

single

copy

gene

on

southerns

-LRB-

data

not

show

-RRB-

Thus

a

significant

number

-LRB-

approximately

-RRB-

of

the

unigenes

in

this

peak

are

likely

to

be

allelic

However

this

category

represented

only

a

small

portion

of

the

coffee

unigenes

-LRB-

-RRB-

A

similar

experiment

was

performed

with

ESTs

from

pairs

of

unigenes

in

the

second

lower

homology

peak

-LRB-

around

identity

see

Fig.

-RRB-

In

this

case

the

majority

-LRB-

out

of

-RRB-

was

determined

to

represent

true

paralogs

-LRB-

two

or

more

copies

in

the

genome

-RRB-

-LRB-

data

not

shown

-RRB-

Thus

for

further

discussions

it

is

assumed

that

the

majority

coffee

EST-derived

unigenes

do

in

fact

correspond

to

unique

coffee

genes

Functional

annotation

of

coffee

EST-derived

unigenes

Predicted

coffee

proteins

ESTScan

-LRB-

see

Materials

and

methods

for

details

-RRB-

was

able

to

identify

protein-coding

sequences

in

coffee

unigenes

-LRB-

of

total

unigenes

-RRB-

among

which

-LRB-

-RRB-

were

putatively

full-length

-LRB-

starting

with

ATG

and

ending

with

a

stop

codon

-RRB-

Due

to

the

cDNA

library

construction

method

the

unigenes

were

biased

for

the

end

of

the

unigenes

covered

the

end

-LRB-

ending

with

a

stop

codon

-RRB-

while

only

covered

the

end

-LRB-

starting

with

ATG

-RRB-

Of

the

of

unigenes

from

which

a

protein

sequence

could

not

be

predicted

were

singletons

and

the

majority

-LRB-

-RRB-

did

not

match

to

any

Arabidopsis

GenBank

non-redundant

-LRB-

NR

-RRB-

or

Solanaceae

unigene

sequences

suggesting

that

they

are

not

bona

fide

gene

transcripts

Protein

domain

annotation

Peptides

translated

from

ESTScan

predicted

coding

sequences

were

subjected

to

InterPro

Scan

-LRB-

see

Materials

and

methods

for

details

-RRB-

for

protein

domain

analysis

A

total

of

distinct

domains

were

identified

in

the

database

which

were

present

in

-LRB-

-RRB-

unigenes

Table

lists

the

most

represented

InterPro

domains

found

in

the

coffee

genes

as

well

as

comparative

statistics

for

tomato

and

Arabidopsis

genes

Coffee

and

tomato

were

very

similar

with

regard

to

the

most

frequent

protein

domains

however

a

number

of

striking

differences

were

noted

when

comparing

coffee

and

Arabidopsis

Most

notably

the

following

five

domains

were

much

more

common

in

coffee

encoded

proteins

than

in

Arabidopsis

proline-rich

regions

tyrosine

protein

kinases

glucose/ribitol

dehydrogenases

ubiquitins

and

ubiquitin-conjugating

enzymes

The

latter

two

categories

involving

ubiquitinization

suggest

a

more

active

and/or

complex

system

for

protein

turnover

in

coffee

versus

Arabidopsis

Table

most

abundant

InterPro

domains

identified

in

coffee

unigene

set

and

comparative

statistics

for

tomato

and

Arabidopsis

genesInterPro

accessionDescription

of

unigenes

-LRB-

ranking

-RRB-

CoffeeTomato

Arabidopsis

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

protein

-LRB-

-RRB-

protein

-LRB-

-RRB-

-LRB-

-RRB-

protein

kinase

active

-LRB-

-RRB-

-LRB-

-RRB-

region

-LRB-

RNA

recognition

motif

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

beta

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

GTPase

-LRB-

-RRB-

-LRB-

-RRB-

small

GTPase

Rab

-LRB-

-RRB-

-LRB-

II

-RRB-

oxygenase

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

group

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

DNA-binding

-LRB-

-RRB-

-LRB-

-RRB-

GTP-binding

protein

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

repeat

plant

-LRB-

-RRB-

-LRB-

-RRB-

Gene

ontology

annotation

Gene

ontology

-LRB-

GO

-RRB-

annotations

were

made

for

each

coffee

unigene

based

on

InterPro

domain

annotation

-LRB-

see

Materials

and

methods

for

details

-RRB-

A

total

of

unigenes

-LRB-

of

total

unigenes

and

of

unigenes

with

InterPro

domain

match

-RRB-

could

be

assigned

a

GO

annotation

In

order

to

obtain

an

overview

of

representation

of

biological

processes

by

the

coffee

unigene

set

the

GO

annotations

were

mapped

to

the

plant

GOSlim

a

group

of

selected

higher-level

categories

of

the

GO

ontology

-LRB-

see

Materials

and

methods

for

details

-RRB-

Figure

depicts

the

biological

process

GOSlim

categories

for

each

of

which

at

least

of

the

coffee

unigenes

were

assigned

Metabolism

is

the

most

abundant

category

both

in

terms

of

number

of

subcategories

-LRB-

subgroups

-RRB-

and

number

of

unigenes

in

the

subcategories

-LRB-

unigenes

and

of

the

GO

annotated

unigenes

-RRB-

In

addition

signal

transduction

cell

growth

and/or

maintenance

response

to

stress

and

response

to

endogenous

stimulus

were

among

the

most

abundant

categories

Fig.

of

the

gene

ontology-based

gene

annotation

categories

for

the

coffee

EST-derived

unigene

set

tomato

EST-derived

unigene

set

and

the

Arabidopsis

proteome

Figure

contains

only

categories

in

which

more

than

of

the

coffee

unigenes

were

assigned

Categories

for

which

coffee

differs

most

significantly

from

Arabidopsis

are

shown

in

underline

bold

-LRB-

-RRB-

Cellular

processes

other

than

signal

transduction

and

cell

growth

and/or

maintenance

-LRB-

-RRB-

Nucleobase/nucleoside/nucleotide

and

nucleic

acid

metabolism

other

than

DNA

metabolism

and

transcription

-LRB-

-RRB-

Protein

metabolism

other

than

protein

biosynthesis

and

protein

modification

-LRB-

-RRB-

Metabolism

other

than

amino

acid

and

derivative

metabolism

biosynthesis

carbohydrate

metabolism

catabolism

electron

transport

lipid

metabolism

nucleobase/nucleoside/nucleotide

and

nucleic

acid

metabolism

and

protein

metabolism

-LRB-

-RRB-

Cell

growth

and/or

maintenance

other

than

cell

cycle

and

cell

organization

and

biogenesis

-LRB-

-RRB-

Physiological

processes

other

than

photosynthesis

response

to

stress

response

to

endogenous

stimulus

response

to

external

stimulus

and

metabolism

A

comparison

was

made

between

the

GOSlim

biological

process

of

Arabidopsis

tomato

and

coffee

-LRB-

Fig.

-RRB-

For

both

the

tomato

and

coffee

unigene

sets

the

GO

annotations

were

based

on

InterProScan

results

and

approximately

of

both

unigene

sets

were

assigned

a

GO

annotation

In

Arabidopsis

the

genes

are

of

full

length

giving

a

higher

chance

of

finding

functional

domains

Moreover

extensive

experimental

research

and

manual

annotation

has

been

carried

out

in

Arabidopsis

resulting

in

a

higher

proportion

of

genes

with

assigned

GO

annotation

Therefore

of

the

Arabidopsis

genes

are

assigned

GO

annotations

No

significant

differences

were

observed

in

the

annotated

categories

for

coffee

versus

tomato

possibly

reflecting

their

close

taxonomic

affinity

However

for

a

number

of

categories

coffee

had

significantly

different

proportions

of

genes

than

Arabidopsis

The

categories

with

the

largest

significant

differences

-LRB-

P

based

on

Chi-square

test

-RRB-

are

carbohydrate

metabolism

other

metabolism

biosynthesis

catabolism

protein

biosynthesis

protein

modification

and

energy

pathways

In

all

cases

coffee

had

a

significantly

higher

proportion

of

genes

in

these

categories

than

Arabidopsis

-LRB-

Fig.

-RRB-

It

is

interesting

to

note

that

many

of

these

categories

center

around

the

synthesis

breakdown

or

modification

of

compounds

One

of

the

hallmarks

of

coffee

is

its

high-level

diversity

of

primary

and

secondary

compounds

which

contributes

to

the

sensory

quality

of

brewed

coffee

beans

The

Rubiaceae

family

in

general

contains

some

of

the

most

diverse

species

with

regard

to

secondary

metabolism

and

is

an

especially

rich

source

of

alkaloids

a

number

of

which

have

pharmacological

and/or

psychotropic

properties

-LRB-

Kutchan

Facchini

-RRB-

In

fact

the

most

widely

used

psychotropic

drug

caffeine

comes

mainly

from

coffee

One

can

speculate

that

this

metabolic

diversity

is

reflected

in

the

relatively

high

proportion

of

coffee

genes

with

putative

functions

related

to

metabolism

In

silico

analysis

of

unigene

expression

Complexity

and

uniqueness

of

different

stages/tissues

In

an

effort

to

determine

which

tissues

gave

rise

to

the

more

redundant

or

more

complex

pools

of

gene

expression

three

library/tissue

parameters

were

calculated

-LRB-

-RRB-

the

percentage

of

total

coffee

unigenes

represented

by

each

library/tissue

-LRB-

-RRB-

the

percentage

of

more

highly

expressed

unigenes

-LRB-

represented

by

ten

or

more

ESTs

from

the

library

-RRB-

in

each

library/tissue

-LRB-

-RRB-

the

percentage

of

unigenes

from

each

tissue/stage

which

are

unique

to

that

library/tissue

-LRB-

not

found

in

any

other

cDNA

library

-RRB-

The

results

from

these

calculations

are

depicted

in

Fig.

The

pericarp

and

leaf

libraries

represented

more

unigenes

contributed

to

more

unique

unigenes

and

contained

fewer

highly

abundant

unigenes

than

did

the

early

cherry

middle

seed

and

late

seed

libraries

perhaps

reflecting

the

overall

lower

complexity

of

these

latter

stages/tissues

-LRB-

Fig.

-RRB-

Among

the

seed

stages

the

middle

stage

of

development

gave

more

novel

genes

than

did

the

early

or

late

stages

of

seed

development

-LRB-

Fig.

-RRB-

Fig.

of

each

coffee

cDNA

library

in

comparison

to

the

entire

coffee

EST-derived

unigene

set

The

total

unigene

and

highly

expressed

unigene

categories

sum

to

greater

since

the

same

unigene

may

contain

ESTs

from

more

than

one

library

Differential

expression

of

genes

across

stages/tissues

The

coffee

cDNA

libraries

were

non-normalized

and

deeply

sampled

and

thus

could

be

used

to

compare

gene

expression

amongst

the

five

cDNA

libraries

using

statistical

method

-LRB-

Audic

and

Claverie

-RRB-

In

all

the

pair-wise

comparison

between

the

cDNA

libraries

only

a

small

proportion

of

the

overall

unigenes

-LRB-

approximately

-RRB-

showed

significantly

different

-LRB-

P

-RRB-

expression

levels

between

two

or

more

libraries

-LRB-

Table

-RRB-

The

leaf

and

the

early

stage

cherry

libraries

showed

the

highest

divergence

in

gene

expression

-LRB-

unigenes

which

is

of

total

unigenes

showed

significant

differential

expression

-RRB-

while

the

leaf

and

the

pericarp

libraries

had

the

lowest

number

of

differentially

expressed

unigenes

-LRB-

unigenes

and

of

total

-RRB-

Interestingly

the

three

stages

of

seed

development

showed

as

much

differential

expression

relative

to

each

other

as

they

did

to

non-seed

tissues

-LRB-

leaf

and

pericarp

-RRB-

These

results

provide

strong

evidence

that

throughout

the

approximately

months

from

pollination

to

mature

seed

a

continuously

changing

cascade

of

molecular

events

is

unfolding

in

the

developing

seed

Table

of

coffee

unigenes

showing

significantly

-LRB-

P

-RRB-

different

expression

in

pairwise

comparisons

of

cDNA

librariesLibraryPericarpEarly

stage

cherryMiddle

stage

seedLate

stage

stage

stage

Highly

expressed

genes

Table

lists

the

blast

annotation

library

composition

and

the

best

Arabidopsis/Solanaceae

gene

match

for

the

most

highly

expressed

unigenes

from

the

coffee

EST

database

Some

of

these

highly

expressed

genes

are

common

to

and

highly

expressed

in

all

plant

species

a

good

example

of

this

is

Unigene

which

encodes

the

small

subunit

of

ribulose

bisphosphate

carboxylase

a

key

enzyme

in

photosynthesis

and

carbon

fixation

-LRB-

Table

-RRB-

However

a

number

of

these

highly

expressed

genes

are

unique

to

coffee

and

many

have

expression

patterns

confined

to

particular

tissue/stages

providing

a

glimpse

into

some

of

the

potential

unique

aspects

of

coffee

biology

A

brief

description

of

some

of

these

is

given

below

Table

most

highly

expressed

coffee

unigenes

functional

annotation

and

most

similar

Arabidopsis

and

Solanaceae

homologsCoffee

unigene

annotationBest

match

-LRB-

e

value/score

-RRB-

EST

countArbidopsisSolanaceae

ESTsLeafPericarpEarly

stage

cherryMiddle

stage

seedLate

stage

putative

seed

storage

-LRB-

-RRB-

seed

storage

-LRB-

-RRB-

-LRB-

-RRB-

unknown

-LRB-

-RRB-

-LRB-

-RRB-

unknown

-LRB-

-RRB-

-LRB-

-RRB-

unknown

-LRB-

-RRB-

-LRB-

-RRB-

photoassimilate-responsive

-LRB-

-RRB-

-LRB-

-RRB-

unknown

-LRB-

-RRB-

ADP-ribosylation

-LRB-

-RRB-

-LRB-

-RRB-

secretory

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

unknown

-LRB-

-RRB-

-LRB-

-RRB-

SAM

-LRB-

-RRB-

-LRB-

-RRB-

transcription

-LRB-

-RRB-

-LRB-

-RRB-

unknown

-LRB-

-RRB-

-LRB-

-RRB-

AdoMet

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

plasmodesmal

-LRB-

-RRB-

-LRB-

-RRB-

rubiso

small

-LRB-

-RRB-

-LRB-

-RRB-

BLAST

match

values

are

given

in

parenthesesBold

numbers

indicate

library

for

which

the

highest

number

of

ESTs

were

observed

for

each

gene

Italic

numbers

indicate

library

for

which

the

number

of

EST

is

significantly

lower

-LRB-

P

-RRB-

than

the

highest

Seed

storage

protein

genes

Unigene

a

putative

seed

storage

protein

Unigene

is

the

most

highly

expressed

gene

across

the

entire

coffee

unigene

set

-LRB-

ESTs

-RRB-

and

was

the

dominant

transcript

in

the

middle

stage

seed

library

accounting

for

of

the

ESTs

at

this

stage

-LRB-

Table

-RRB-

This

gene

shows

high

homology

to

a

tomato

unigene

derived

from

a

developing

seed

cDNA

library

but

has

no

detectable

homolog

in

Arabidopsis

-LRB-

Table

-RRB-

Other

than

the

match

with

tomato

weak

homology

was

also

detected

for

seed

storage

proteins

from

sesame

sunflower

and

Brazil

nut

-LRB-

in

decreasing

order

of

similarity

-RRB-

It

is

interesting

to

note

that

coffee

tomato

sesame

and

sunflower

are

fairly

closely

related

taxonomically

All

belong

to

the

Asterid

I/II

clade

of

Eudicots

-LRB-

Fig.

-RRB-

This

close

phylogenetic

relationship

may

explain

why

Unigene

has

homologous

matches

only

in

these

species

Moreover

since

Unigene

shows

homology

to

the

seed

storage

proteins

in

these

related

species

we

conjecture

that

unigene

its

tomato

unigene

match

the

sesame

Brazil

nut

and

sunflower

storage

protein

gene

all

encode

orthologous

seed

storage

proteins

This

is

the

first

time

that

a

seed

storage

protein

has

been

identified

in

coffee

or

any

Solanaceae

species

Finally

a

BLAST

search

of

Unigene

against

the

coffee

unigene

set

revealed

additional

putative

copies

of

the

seed

storage

protein

However

on

close

examination

all

appear

to

be

splicing

variants

or

low

quality

sequences

Moreover

southern

hybridization

with

a

cDNA

probe

on

genomic

DNA

confirmed

that

the

gene

is

single

copy

in

the

coffee

genome

-LRB-

data

not

shown

-RRB-

Unigene

seed

storage

protein

Unigene

is

the

second

most

abundant

unigene

containing

ESTs

-LRB-

Table

-RRB-

This

gene

is

preferentially

expressed

during

middle

and

late

stage

seed

development

and

shares

high

similarity

-LRB-

over

identity

-RRB-

with

a

previously

cloned

C.

arabica

seed

storage

protein

-LRB-

Marraccini

et

al.

Rogers

et

al.

-RRB-

This

unigene

also

has

a

highly

significant

match

to

the

Arabidopsis

storage

protein

and

to

a

tomato

unigene

derived

from

seed

ESTs

-LRB-

Pang

et

al.

-RRB-

-LRB-

Table

-RRB-

Given

these

results

we

conclude

that

unigene

is

allelic

with

the

previously

described

seed

storage

protein

gene

from

C.

arabica

and

orthologous

to

seed

storage

proteins

in

both

tomato

and

Arabidopsis

A

BLAST

search

of

Unigene

against

the

coffee

unigene

set

revealed

additional

putative

copies

of

the

seed

storage

protein

However

like

the

seed

storage

protein

-LRB-

Unigene

-RRB-

all

appears

to

be

results

of

alternative

splicing

or

low

sequence

quality

Moreover

southern

hybridization

with

an

cDNA

probe

on

genomic

DNA

confirmed

that

the

gene

is

single

copy

in

the

coffee

genome

-LRB-

data

not

shown

-RRB-

Other

seed-specific

genes

Early

stage

seed

development

Unigenes

and

were

all

highly

expressed

and

specific

to

the

early

cherry

stage

The

early

cherry

library

was

derived

from

RNA

from

both

pericarp

and

seed

tissue

while

the

pericarp

library

was

derived

from

RNA

coming

from

all

stages

of

pericarp

development

Thus

if

the

above

genes

were

highly

expressed

in

the

pericarp

of

the

early

cherry

they

should

be

present

in

the

pericarp

library

as

well

The

fact

that

these

genes

showed

little

or

no

expression

outside

the

early

cherry

stage

suggests

that

they

are

probably

specific

to

early

developing

seed

tissues

and

not

pericarp

tissues

-LRB-

Table

-RRB-

Unigene

showed

high

homology

to

an

Arabidopsis

gene

annotated

as

encoding

the

enzyme

S-adenosyl-l-methionine

-LRB-

SAM

-RRB-

synthetase

-LRB-

Table

-RRB-

This

enzyme

synthesizes

S-adenosyl-l-methionine

from

l-methionine

and

ATP

and

is

often

represented

by

multiple

isozymes

in

plant

species

-LRB-

Schroder

et

al.

-RRB-

Thus

Unigene

appears

to

be

an

SAM

synthetase

specific

to

early

seed

development

-LRB-

Table

-RRB-

Unigene

shows

high

homology

to

the

highly

conserved

WRKY

transcription

factor

family

The

WRKY

transcription

factor

is

a

large

gene

family

having

more

than

members

in

the

Arabidopsis

genome

-LRB-

Dong

et

al.

-RRB-

Previous

studies

showed

that

it

is

related

to

wounding

stress

pathogen

infection

and

senescence

in

many

plant

species

In

some

recent

studies

the

WRKY

protein

family

was

found

to

be

involved

in

sugar

signaling

in

barley

and

seed

development

in

Arabidopsis

-LRB-

Johnson

et

al.

Sun

et

al.

-RRB-

However

the

function

of

the

best

Arabidopsis

match

to

Unigene

-LRB-

-RRB-

has

not

been

determined

Hence

understanding

the

function

of

this

highly

expressed

WRKY-like

coffee

gene

awaits

further

study

Unigene

is

highly

homologous

to

a

gene

in

Arabidopsis

annotated

as

encoding

a

protein

proteins

are

found

in

virtually

all

eukaryotes

While

they

are

conjectured

to

be

involved

in

cell

cycle

control

there

is

still

little

experimental

evidence

demonstrating

biological

function

-LRB-

Pon

-RRB-

Thus

it

seems

premature

to

conjecture

what

role

Unigene

might

have

that

is

specific

to

the

early

development

of

coffee

seeds

Middle

stage

seed

development

As

described

earlier

Unigene

is

a

putative

seed

storage

protein

with

peak

expression

during

middle

seed

development

Also

showing

preferential

expression

during

this

same

stage

were

Unigenes

and

Unigene

is

a

gene

of

unknown

function

with

high

homology

matches

both

in

Arabidopsis

and

Solanaceae

EST-derived

unigenes

-LRB-

Table

-RRB-

Unigene

is

homologous

to

an

Arabidopsis

gene

classified

as

a

photoassimilate-responsive

protein

which

is

related

to

pathogenesis

-LRB-

Herbers

-RRB-

Finally

Unigene

which

is

also

specific

to

middle

stage

seed

development

is

apparently

a

gene

unique

to

coffee

which

will

be

discussed

more

in

the

following

section

Late

stage

seed

development

As

discussed

previously

Unigene

corresponds

to

the

seed

storage

protein

which

is

largely

expressed

late

in

seed

development

Other

genes

with

preferential

expression

in

late

stage

seeds

are

Unigenes

and

Unigene

likely

encodes

a

chitinase

and

is

further

discussed

in

the

next

section

Unigene

shows

high

homology

to

genes

in

both

Arabidopsis

and

Solanaceae

EST-derived

unigene

sets

however

have

known

function

Unigene

gives

a

strong

match

to

an

Arabidopsis

gene

annotated

as

a

plasmodesmatal

receptor

Two

highly

expressed

genes

with

homology

to

chitinase

Unigenes

and

show

high

sequence

similarity

to

a

number

of

genes

classified

as

chitinases

in

other

organisms

Chitinases

are

a

large

and

diverse

class

of

proteins

some

of

which

have

been

implicated

in

resistance

to

fungi

in

various

plant

species

including

coffee

-LRB-

Rojas-Herrera

Chen

et

al.

-RRB-

The

two

unigenes

differ

in

that

Unigene

is

expressed

in

leaves

pericarp

and

early

stage

cherries

but

not

in

mid

or

late

stage

seed

development

-LRB-

Table

-RRB-

Unigene

on

the

other

hand

was

found

to

be

exclusively

expressed

in

late

stage

developing

seeds

and

pericarp

tissue

As

previously

mentioned

early

stage

cherries

contained

both

pericarp

and

seed

tissues

The

fact

that

Unigene

was

not

found

in

the

middle

and

late

stages

of

seed

development

suggests

that

this

gene

may

not

be

expressed

in

seeds

but

rather

in

the

maternally

derived

pericarp

and

leaf

tissues

Based

on

these

results

one

can

speculate

that

these

two

putative

chitinase

genes

may

be

involved

in

pathogen

defense

in

developing

CHERRIES

with

Unigene

being

expressed

in

early

developing

post

pollination

pericarp

and

leaf

tissues

and

Unigene

being

expressed

primarily

late

in

seed

development

just

prior

to

maturity

Highly

expressed

genes

unique

to

coffee

Unigene

This

highly

expressed

unigene

had

no

significant

matches

in

the

Arabidopsis

proteome

Solanaceae

EST-derived

databases

GenBank

NR

databases

or

GenBank

dbest

Moreover

the

predicted

protein

encoded

by

Unigene

has

no

recognizable

domains

which

might

give

clues

to

its

function

ESTs

for

this

unigene

were

detected

in

all

five

libraries

with

highest

expression

being

observed

in

the

pericarp

-LRB-

Table

-RRB-

Unigene

Unigene

also

has

no

significant

match

in

any

of

the

tested

databases

with

the

possible

exception

of

a

very

weak

match

in

the

Solanaceae

unigene

sets

-LRB-

the

best

hit

was

from

pepper

with

an

e

value

of

Table

-RRB-

Like

Unigene

its

predicted

protein

has

no

recognizable

domains

Unigene

was

highly

and

exclusively

expressed

in

the

middle

stage

of

developing

seeds

-LRB-

Table

-RRB-

The

fact

that

neither

Unigene

nor

Unigene

have

counterparts

in

any

other

databases

suggests

that

they

may

represent

coffee-specific

genes

or

genes

that

have

been

evolving

at

such

a

rapid

rate

that

they

no

longer

bear

any

recognizable

homologies

with

proteins

from

other

plants

including

the

closely

related

Solanaceous

plants

We

speculate

that

these

genes

may

be

related

to

chemical

or

morphological

features

unique

to

coffee

Gene

families

unique

or

significantly

expanded

in

coffee

The

predicted

protein

sequences

for

the

coffee

unigene

set

and

the

Arabidopsis

proteome

were

combined

and

organized

into

protein

families

using

tribeML

software

-LRB-

see

Materials

and

methods

for

details

-RRB-

In

most

cases

the

number

of

gene

family

members

was

higher

in

arabidopsis

than

in

coffee

-LRB-

data

not

shown

-RRB-

However

this

is

to

be

expected

since

the

entire

Arabidopsis

genome

has

been

sequenced

-LRB-

thus

revealing

all

genes

-RRB-

whereas

in

coffee

the

EST-derived

unigene

set

is

by

nature

incomplete

and

unlikely

to

contain

all

members

of

any

given

gene

family

However

despite

the

incomplete

nature

of

the

coffee

EST-derived

unigene

set

several

cases

was

encountered

in

which

coffee

has

more

gene

family

members

than

does

Arabidopsis

To

further

investigate

this

topic

a

list

was

made

of

the

gene

families

for

which

coffee

has

significantly

more

members

than

Arabidopsis

-LRB-

Table

-RRB-

We

refer

to

these

as

coffee-expanded

gene

families

In

addition

gene

families

found

in

coffee

but

for

which

no

counterparts

exist

in

Arabidopsis

were

also

compiled

-LRB-

Table

-RRB-

We

refer

to

these

as

coffee-unique

gene

families

All

gene

families

listed

in

both

Tables

and

were

subjected

to

manual

examination

to

remove

any

members

determined

to

be

results

of

alternative

splicing

and/or

poor

sequence

quality

both

of

which

could

inflate

the

number

of

putative

gene

family

members

in

coffee

Hence

the

differences

in

copy

number

reported

are

likely

to

be

due

to

true

differences

in

gene

copy

number

and

not

artifacts

of

EST-based

gene

predictions

Table

families

expanded

in

coffee

relative

to

Arabidopsis

Family

Arabidopsis

family

member

Coffee

family

memberLongest

coffee

gag

protein

class

isoenzyme

beta

subunit

with

BURP

protein

band

v

I

hair

defective

Unknown

Trypsin

inhibitor

KunitzTable

families

unique

to

coffee

in

comparison

to

Arabidopsis

Gene

family

Family

memberLongest

memberSolanaceae

potatoRetrotransposon

gag

protein

class

tomatoThaumatin

pathogenesis

potatoZn-finger

CCHC

potatoDisease

resistance

protein

-LRB-

TIR-NBS-LRR

class

-RRB-

tomatoRetrotransposon

gag

protein

classs

tomatoDisease

resistance

tomatoLeucine-rich

repeat

disease

resistance

noneUnknown

noneUnknown

tomatoUnknown

tomatoABA/WDS

induced

tomatoProline-rich

region

extension-like

tomatoUnknown

potatoLeucine-rich

repeat

plant

specific

receptor-related

protein

kinase

Coffee-expanded

gene

families

The

most

expanded

gene

family

in

coffee

corresponds

to

a

retrotransposon

gag

protein

-LRB-

Table

-RRB-

This

result

has

two

implications

First

the

retroelement

encoding

this

gag

protein

occurs

at

a

higher

frequency

in

coffee

compared

with

Arabidopsis

although

we

can

not

determine

whether

this

difference

is

due

to

a

true

expansion

of

this

element

in

coffee

subsequent

to

divergence

from

Arabidopsis

or

rather

a

loss

of

the

element

in

the

Arabidopsis

lineage

Second

the

fact

that

this

retrotransposon

gag

protein

element

was

discovered

in

an

EST-database

indicates

that

this

particular

retroelement

is

being

transcribed

in

the

coffee

genome

and

hence

may

represent

an

active

retrotransposon

Another

gene

family

for

which

coffee

has

significantly

more

members

than

Arabidopsis

encodes

proteins

annotated

in

Arabidopsis

as

acid

endochitinases

and

photoassimilate-responsive

proteins

-LRB-

Table

-RRB-

As

noted

earlier

chitinases

are

associated

with

fungal

resistance

and

are

among

the

most

highly

expressed

genes

in

coffee

The

fact

that

chitinases

are

both

highly

expressed

and

represented

by

an

expanded

gene

family

in

coffee

may

reflect

a

greater

need

for

fungal

resistance

engendered

both

by

the

perennial

nature

of

coffee

and

the

fact

that

it

is

a

tropical

species

for

which

a

multiplicity

of

fungal

pathogens

is

common

The

reasons

for

the

putative

expansions

of

the

other

gene

families

listed

in

Table

remain

for

future

studies

to

determine

Coffee-unique

gene

families

Table

lists

the

top

gene

families

-LRB-

based

on

copy

number

-RRB-

which

occur

in

coffee

but

not

in

Arabidopsis

Of

the

gene

families

listed

four

are

of

unknown

function

For

those

that

could

be

functionally

annotated

five

-LRB-

-RRB-

have

putative

functions

related

to

disease

resistance

such

as

TIR-NBS-LRR

disease

resistance

proteins

LRR

proteins

and

thaumatin

pathogenesis-related

proteins

-LRB-

Table

-RRB-

These

finding

are

consistent

with

rapid

evolution

of

genes/gene

families

related

to

disease

resistance

likely

driven

by

selection

pressure

from

continuously

changing

pathogens

and/or

pathogens

unique

to

the

particular

environments

of

a

species

-LRB-

Meyers

Michelmore

and

Meyers

-RRB-

Also

included

in

this

list

of

coffee-unique

gene

families

are

two

which

encode

retrotransposon

gag-proteins

-LRB-

Table

-RRB-

Comparison

of

the

coffee

gene

repertoire

with

that

of

Arabidopsis

and

Solanaceae

species

Each

coffee

unigene

was

screened

against

the

Arabidopsis

proteome

using

BLASTX

which

compares

all

six

translated

frames

of

the

query

with

the

target

In

total

of

the

coffee

unigenes

had

no

clear

match

in

Arabidopsis

-LRB-

match

score

-RRB-

-LRB-

Fig.

-RRB-

These

coffee

genes

which

lack

an

Arabidopsis

counterpart

may

have

evolved

rapidly

since

coffee

and

Arabidopsis

diverged

from

their

last

common

ancestor

and

hence

their

counterparts

in

each

genome

are

no

longer

recognizable

by

standard

string

searches

like

BLAST

To

investigate

this

topic

further

the

unigenes

with

no

clear

match

in

Arabidopsis

-LRB-

match

score

see

Fig.

-RRB-

were

screened

against

the

Solanaceae

EST-derived

unigene

sets

using

TBLASTX

which

compares

all

six

translated

frames

of

both

query

-LRB-

coffee

unigenes

-RRB-

and

target

-LRB-

Solanaceae

unigenes

see

Materials

and

methods

for

details

-RRB-

sequences

The

family

Solanaceae

is

much

more

closely

related

to

coffee

than

is

Arabidopsis

-LRB-

Fig.

-RRB-

and

hence

coffee

genes

without

a

match

in

Arabidopsis

may

well

have

recognizable

orthologs

in

the

Solanaceae

The

results

show

that

a

large

portion

-LRB-

-RRB-

of

the

coffee

genes

that

lack

a

match

in

Arabidopsis

do

have

a

match

in

Solanaceae

at

the

same

stringency

criteria

-LRB-

match

score

-RRB-

For

many

of

these

cases

however

the

coffee-Solanaceae

matches

are

still

quite

diverged

-LRB-

low

match

scores

-RRB-

suggesting

that

the

proteins

encoded

by

these

genes

have

been

evolving

at

a

rapid

rate

in

dicots

Fig.

showing

match

scores

for

each

coffee

unigene

as

compared

with

its

best

match

in

the

Arabidopsis

proteome

There

were

however

some

exceptions

to

this

trend

represented

by

coffee

genes

having

no

match

in

Arabidopsis

yet

a

strong

match

in

Solanaceae

These

latter

cases

may

represent

genes

that

have

been

under

differential

selection

pressure

since

the

time

that

the

Rosids

-LRB-

which

includes

Arabidopsis

-RRB-

diverged

from

the

last

common

ancestor

with

the

Asterids

-LRB-

which

includes

Solanaceae

and

coffee

-RRB-

-LRB-

Fig.

-RRB-

Alternatively

these

genes

may

have

been

lost

in

the

lineage

leading

to

Arabidopsis

but

retained

in

the

Asterids

In

either

case

such

genes

may

have

functional

roles

that

are

in

part

responsible

for

the

morphological

developmental

and

chemical

characteristics

that

differentiate

Arabidopsis

from

Rosid

species

-LRB-

e.g.

coffee

Solanaceae

-RRB-

In

an

effort

to

shed

more

light

on

this

issue

the

coffee

unigenes

having

the

highest

match

scores

with

Solanaceae

but

lacking

a

counterpart

in

Arabidopsis

were

parsed

and

annotated

-LRB-

Table

-RRB-

Seven

of

these

coffee

genes

have

no

match

either

in

GenBank

NR

or

dbest

hence

their

function

is

unknown

Another

unigene

-LRB-

Unigene

the

seed

storage

protein

see

above

discussion

-RRB-

had

weak

match

to

ESTs

of

sesame

a

species

also

belonging

to

the

Asterid

I

clade

-LRB-

Fig.

-RRB-

Thus

these

eight

genes

-LRB-

-RRB-

appear

to

be

specific

to

the

tomato/coffee/sesame

Asterid

I

clade

-LRB-

Fig.

Table

-RRB-

Elucidating

their

function

may

shed

light

on

the

molecular

processes

which

diversified

early

in

the

evolution

of

dicot

species

especially

those

species

belonging

to

the

Asterid

clade

of

dicots

Table

genes

not

found

in

Arabidopsis

but

with

conserved

counterparts

in

tomato

or

other

Solanaceous

speciesCoffee

unigeneSolanaceae

EST-derived

unigene

matchScoreGenBank

-LRB-

non-redundant

and

dbest

-RRB-

best

-LSB-

Brassica

napus

-RSB-

-LSB-

Oryza

sativa

-RSB-

-LSB-

Oryza

sativa

-RSB-

-LSB-

Oryza

sativa

-RSB-

basal

transcription

factor

-LSB-

Seasame

indicum

-RSB-

-LSB-

Drosophila

melanogaster

-RSB-

inositol

transfer

-LSB-

Rose

-RSB-

-LSB-

Populus

tremula

-RSB-

-LSB-

Oryza

sativa

-RSB-

-LSB-

Rattus

norvegicus

-RSB-

class

-LSB-

Vitis

vinifera

-RSB-

Unknown

-LSB-

Macaca

mulatta

-RSB-

Ribosomal

protein

-LSB-

Oryza

sativa

-RSB-

GenBanks

Best

match

exclude

those

from

Solanaceae

Coffea

and

Hedyotis

-LRB-

both

members

of

the

Rubiaceae

family

-RRB-

Solanaceae

EST-derived

Surprisingly

-LRB-

-RRB-

of

the

coffee

genes

having

no

match

in

Arabidopsis

did

have

matches

in

species

phylogenetically

distant

from

both

coffee

and

Arabidopsis

including

two

to

non-plant

species

-LRB-

Drosophila

and

rat

-RRB-

-LRB-

Table

-RRB-

Five

of

these

matches

were

to

rice

genes

which

is

a

monocot

and

highly

divergent

from

coffee

Arabidopsis

and

other

dicot

species

-LRB-

Table

-RRB-

The

fact

that

all

of

these

species

diverged

from

Arabidopsis

and

coffee

long

before

the

latter

diverged

from

each

other

suggests

that

these

genes

may

have

been

present

in

the

last

common

ancestor

of

Arabidopsis

and

coffee/Solanaceae

but

subsequently

lost

in

the

Arabidopsis

lineage

Coffee

genes

share

greater

similarity

to

genes

in

tomato/Solanaceae

than

to

Arabidopsis

As

discussed

earlier

coffee

is

much

more

closely

related

to

the

Solanaceae

than

to

Arabidopsis

-LRB-

Fig.

-RRB-

Hence

Solanaceae

species

may

be

better

models

for

coffee

genomics

than

Arabidopsis

The

results

for

fast-evolving

genes

presented

above

are

consistent

with

this

prediction

To

further

investigate

this

assertion

the

degree

to

which

each

coffee

unigene

matched

Arabidopsis

versus

Solanaceae

was

investigated

In

doing

this

analysis

one

has

to

keep

in

mind

that

the

entire

gene

repertoire

of

Arabidopsis

is

known

whereas

the

EST-derived

unigene

sets

for

Solanaceae

do

not

represent

the

entire

gene

repertoire

of

these

species

We

estimate

that

the

combined

EST-derived

unigene

sets

of

Solanaceae

species

represent

as

much

as

three-quarters

of

the

Solanaceae

gene

content

-LRB-

Hoeven

et

al.

-RRB-

Moreover

Arabidopsis

genes

are

of

full

length

while

Solanaceae

EST-derived

unigenes

are

not

necessarily

of

full

length

To

mitigate

against

these

factors

each

coffee

unigene

was

first

screened

via

TBLASTX

against

the

Solanaceae

EST-derived

unigene

databases

Only

those

coffee

unigenes

matching

a

Solanaceae

unigene

with

a

match

score

greater

than

were

compared

with

the

Arabidopsis

proteome

Moreover

the

match

to

Arabidopsis

was

confined

to

only

the

portion

of

the

coffee

unigene

matching

a

Solanaceae

EST-derived

unigene

The

ratio

of

the

Arabidopsis

match

score

divided

by

the

Solanaceae

match

score

was

used

as

a

measure

of

the

degree

to

which

the

Solanaceae

homolog

shows

greater

similarity

to

the

coffee

unigene

than

to

the

closest

Arabidopsis

homolog

The

results

of

this

analysis

are

shown

in

Fig.

The

ratio

of

the

Arabidopsis/Solanaceae

match

score

averaged

indicating

a

significantly

better

match

in

most

cases

with

Solanaceae

versus

Arabidopsis

Moreover

in

more

than

of

the

cases

the

ratio

was

less

than

one

indicating

that

in

most

instances

coffee

contains

genes

significantly

more

homologous

to

Solanaceae

than

to

Arabidopsis

For

those

cases

in

which

the

ratio

was

greater

than

-LRB-

-RRB-

we

can

not

rule

out

the

possibility

that

the

true

ortholog

was

missing

in

the

Solanaceae

EST-derived

unigene

sets

hence

the

comparison

was

between

paralogs

These

results

combined

with

the

results

on

fast

evolving

genes

presented

earlier

provide

clear

evidence

that

the

Solanaceae

provide

a

much

better

model

than

Arabidopsis

for

genomic

and

biological

studies

in

coffee

This

is

especially

relevant

as

sequencing

of

the

tomato

genome

is

currently

underway

-LRB-

-RRB-

Fig.

of

highest

Arabidopsis

match

score

to

highest

Solanaceae

match

score

for

individual

coffee

unigene

The

analysis

restricted

to

coffee

unigenes

with

a

Solanaceae

match

score

Conclusions

Herein

we

describe

the

development

and

analysis

of

a

large

EST

database

for

coffee

The

resulting

ESTs

correspond

to

unique

genes

-LRB-

unigenes

-RRB-

a

large

portion

of

which

are

expressed

during

seed

development

a

stage

important

to

coffee

as

a

crop

and

one

for

which

our

understanding

of

molecular

development

is

still

rudimentary

To

our

knowledge

this

is

the

largest

public

database

for

seed-derived

ESTs

Hence

this

EST

database

represents

a

new

public

resource

which

can

facilitate

a

better

understanding

of

seed

development

as

well

as

genomic

molecular

and

breeding

research

in

coffee

By

comparisons

with

Arabidopsis

and

Solanaceous

species

we

have

identified

the

two

major

seed

storage

proteins

of

coffee

-LRB-

and

-RRB-

and

demonstrated

that

these

proteins

are

expressed

at

different

times

during

seed

development

Through

in

silico

gene

expression

analysis

we

have

identified

a

number

of

highly

expressed

genes

that

show

high

specificity

for

different

stages

of

seed

development

as

well

as

for

the

pericarp

tissue

that

surrounds

the

seeds

Many

of

these

highly

expressed

genes

are

unique

to

coffee

and/or

the

Asterid

clade

of

higher

plants

While

the

functions

of

most

of

these

highly

expressed

tissue/stage

specific

genes

remain

to

be

determined

the

fact

that

they

have

been

identified

points

the

way

to

promoters

which

can

potentially

be

used

to

drive

gene

expression

in

specific

stages/tissues

of

the

coffee

plant

Many

of

these

genes

are

specific

to

defined

periods

of

seed

and/or

pericarp

development

both

critically

important

for

insect/pathogen

resistance

and

in

determining

the

quality

of

the

coffee

bean

with

respect

to

commercial

coffee

products

Coffee

as

a

member

of

the

family

Rubiaceae

is

distantly

related

to

the

model

species

Arabidopsis

A

computational

comparison

of

the

coffee

EST-derived

unigene

set

with

the

sequence

databases

for

Arabidopsis

and

Solanaceous

species

-LRB-

e.g.

tomato

pepper

-RRB-

indicate

that

the

latter

are

much

better

genomic

models

for

coffee

than

is

Arabidopsis

These

results

are

consistent

with

the

fact

that

coffee

and

solanaceous

species

share

very

similar

chromosome

architecture

and

are

closely

related

both

belonging

to

the

Asterid

I

clade

of

dicot

plant

family

Moreover

the

ability

to

identify

orthologous

genes

between

coffee

and

tomato

opens

the

door

to

eventually

developing

detailed

comparative

maps

for

these

two

species

and

to

the

sharing

of

genomic

and

biological

tools/discoveries

an

outcome

that

should

expedite

research

in

both

taxa

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

coffea canephora
rubiaceae
solanaceae
seed development