Local

coexpression

domains

in

the

genome

of

rice

show

no

microsynteny

with

Arabidopsis

domains

Chromosomal

coexpression

domains

are

found

in

a

number

of

different

genomes

under

various

developmental

conditions

The

size

of

these

domains

and

the

number

of

genes

they

contain

vary

Here

we

define

local

coexpression

domains

as

adjacent

genes

where

all

possible

pair-wise

correlations

of

expression

data

are

higher

than

In

rice

such

local

coexpression

domains

range

from

predominantly

two

genes

up

to

and

make

up

of

the

genomic

neighboring

genes

when

examining

different

expression

platforms

from

the

public

domain

The

genes

in

local

coexpression

domains

do

not

fall

in

the

same

ontology

category

significantly

more

than

neighboring

genes

that

are

not

coexpressed

Duplication

orientation

or

the

distance

between

the

genes

does

not

solely

explain

coexpression

The

regulation

of

coexpression

is

therefore

thought

to

be

regulated

at

the

level

of

chromatin

structure

The

characteristics

of

the

local

coexpression

domains

in

rice

are

strikingly

similar

to

such

domains

in

the

Arabidopsis

genome

Yet

no

microsynteny

between

local

coexpression

domains

in

Arabidopsis

and

rice

could

be

identified

Although

the

rice

genome

is

not

yet

as

extensively

annotated

as

the

Arabidopsis

genome

the

lack

of

conservation

of

local

coexpression

domains

may

indicate

that

such

domains

have

not

played

a

major

role

in

the

evolution

of

genome

structure

or

in

genome

conservation

Introduction

The

fast-growing

data

sets

on

genome

annotation

and

genome-wide

gene

expression

facilitate

the

study

and

comparison

of

gene

activity

between

and

among

genomes

The

genomic

context

of

genes

is

supposed

to

play

an

important

role

in

the

regulation

of

gene

expression

-LRB-

van

Drunen

et

al.

-RRB-

Non-random

clusters

of

similarly

expressed

-LRB-

co-regulated

coexpressed

highly

expressed

and/or

broadly

expressed

-RRB-

genes

have

been

described

in

almost

all

organisms

ranging

from

prokaryotes

to

eukaryotes

In

eukaryotes

from

yeast

to

Arabidopsis

to

human

both

short-range

co-regulated/coexpressed

clusters

of

two

to

five

genes

-LRB-

Cohen

et

al.

Ren

et

al.

Zhan

et

al.

-RRB-

and

longer-range

coexpression

domains

of

up

to

genes

spanning

up

to

kb

and

more

-LRB-

Lercher

et

al.

Ma

et

al.

Spellman

and

Rubin

Zhan

et

al.

-RRB-

have

been

described

Duplicated

genes

-LRB-

Lercher

et

al.

-RRB-

shared

promoter

regions

-LRB-

Kruglyak

and

Tang

-RRB-

shorter

gene

distance

-LRB-

Cohen

et

al.

Roy

et

al.

Semon

and

Duret

Williams

and

Bowles

-RRB-

and/or

functional

relatedness

-LRB-

Cohen

et

al.

Lee

et

al.

Spellman

and

Rubin

Williams

and

Bowles

-RRB-

were

found

to

account

for

only

part

of

the

coexpression

between

genes

Therefore

most

studies

postulate

that

the

occurrence

of

coexpression

domains

small

or

large

is

regulated

on

the

level

of

higher-order

chromatin

structure

-LRB-

Cohen

et

al.

Hershberg

et

al.

Ren

et

al.

Spellman

and

Rubin

Williams

and

Bowles

-RRB-

although

alternative

views

exist

-LRB-

Semon

and

Duret

-RRB-

Previously

we

have

defined

and

demonstrated

the

existence

of

local

coexpression

domains

in

the

genome

of

Arabidopsis

-LRB-

Ren

et

al.

-RRB-

A

local

coexpression

domain

was

defined

as

any

set

of

physically

adjacent

genes

that

are

highly

coexpressed

with

a

pair-wise

Pearson

's

correlation

coefficient

larger

than

It

was

shown

that

a

small

-LRB-

-RRB-

yet

significant

fraction

of

genes

in

the

Arabidopsis

genome

is

organized

in

such

local

coexpression

domains

Genes

in

such

local

domains

were

for

the

major

part

not

categorized

in

the

same

functional

category

-LRB-

GOslim

-RRB-

Neither

tandemly

duplicated

genes

nor

shared

promoter

sequence

nor

gene

distance

explained

the

occurrence

of

coexpression

of

genes

in

such

chromosomal

domains

This

indicates

that

other

parameters

in

genes

or

gene

positions

are

important

to

establish

coexpression

in

local

domains

of

Arabidopsis

chromosomes

Here

it

is

analyzed

whether

a

similar

situation

exists

in

the

genome

of

the

monocotyledonous

model

plant

rice

-LRB-

Oryza

sativa

-RRB-

for

which

an

earlier

study

had

shown

the

existence

of

longer-range

domains

-LRB-

Ma

et

al.

-RRB-

We

combined

the

whole

genome

rice

annotation

data

-LRB-

TIGR

version

-RRB-

with

Massively

Parallel

Signature

Sequencing

-LRB-

MPSS

mpss.udel.edu/rice

-RRB-

expression

data

as

well

as

Affymetrix

array

expression

data

-LRB-

GEO

-RRB-

for

the

rice

cultivar

japonica

in

a

way

similar

to

the

analysis

performed

for

the

Arabidopsis

genome

-LRB-

Ren

et

al.

-RRB-

The

results

show

that

the

characteristics

of

the

two

genomes

with

respect

to

the

occurrence

and

configuration

of

local

expression

domains

are

remarkably

similar

Also

in

the

rice

genome

a

small

yet

significant

fraction

of

genes

is

organized

in

local

coexpression

domains

that

predominantly

consist

of

two

up

to

genes

that

are

not

categorized

in

the

same

functional

category

irrespective

of

the

expression

platform

used

for

analyses

The

presence

of

tandemly

duplicated

genes

shared

promoter

sequence

or

gene

distance

is

not

fully

explaining

the

occurrence

of

coexpression

of

genes

in

such

chromosomal

domains

Therefore

the

regulation

of

local

coexpression

domains

is

postulated

to

be

at

the

level

of

higher-order

of

chromatin

structure

Given

the

similarities

in

the

characteristics

and

occurrence

of

coexpression

domains

between

Arabidopsis

and

rice

we

investigated

whether

the

genes

involved

showed

microsynteny

between

the

two

genomes

These

analyses

did

not

identify

the

presence

of

syntenic

local

coexpression

domains

between

Arabidopsis

and

rice

Material

and

methods

Genome

data

The

rice

-LRB-

Oryza

sativa

-RRB-

genome

was

obtained

from

the

website

of

The

Institute

of

Genomic

Research

-LRB-

TIGR

-RRB-

The

rice

TIGR

version

-LSB-

Jan.

-RSB-

annotation

has

gene

loci

In

case

of

alternative

splicing

the

longest

variant

of

the

gene

was

used

The

genes

along

each

chromosome

were

sorted

based

on

ascending

start

coordinates

and

were

numbered

consecutively

These

rank

numbers

-LRB-

rank

ID

-RRB-

helped

to

eliminate

any

discontinuity

in

the

unique

Os

gene

identifiers

of

the

annotated

genes

and

facilitated

analyzing

physically

adjacent

genes

In

case

of

overlapping

gene

loci

the

smaller

one

of

the

overlapping

genes

was

removed

from

the

data

set

This

way

the

order

of

both

gene

and

rank

ID

numbers

was

maintained

Expression

data

The

MPSS

expression

data

for

rice

-LRB-

cv

Japonica

-RRB-

were

obtained

from

the

rice

MPSS

database

-LRB-

-RRB-

Only

the

unique

MPSS

tags

-LRB-

mapping

to

the

genome

only

once

-RRB-

and

those

mapping

to

unique

gene

identifiers

in

TIGR

were

used

in

the

analyses

The

MA

expression

data

were

obtained

from

the

Gene

Expression

Omnibus

repository

-LRB-

GEO

-RRB-

The

data

with

accession

number

were

chosen

for

analysis

because

they

represent

the

only

Affymetrix

chip-based

data

set

in

the

public

domain

with

a

reasonable

coverage

of

the

Oryza

sativa

cv

japonica

transcriptome

under

four

different

experimental

conditions

-LRB-

Walia

et

al.

-RRB-

The

earlier

analysis

in

Arabidopsis

also

concerned

Affymetrix

chip

data

-LRB-

Ren

et

al.

-RRB-

The

MA

expression

data

were

mapped

to

the

TIGR

annotation

based

on

gene

accession

numbers

The

expression

values

in

libraries

representing

biological

replicates

i.e.

the

same

tissues

under

the

same

experimental

conditions

were

averaged

This

way

for

the

MPSS

data

different

libraries

were

generated

that

cover

expression

in

tissues

-LRB-

callus

panicle

leaves

root

germinating

seed

and

seedling

meristem

ovary

and

stigma

pollen

stem

-RRB-

under

different

experimental

treatments

or

in

different

developmental

stages

From

the

microarray

-LRB-

MA

-RRB-

data

only

the

expression

data

from

cv

japonica

were

used

to

allow

comparison

with

the

MPSS

data

Here

different

libraries

were

generated

a

sensitive

japonica

genotype

under

control

conditions

and

under

salt

stress

and

a

tolerant

japonica

genotype

under

control

conditions

and

under

salt

stress

-LRB-

Walia

et

al.

-RRB-

The

expression

data

is

from

crown

and

growing

point

tissue

Identification

of

local

coexpression

domains

Pearson

's

correlation

coefficient

-LRB-

R

-RRB-

was

calculated

between

all

adjacent

pairs

-LRB-

duplets

-RRB-

of

genes

using

the

expression

data

from

all

libraries

If

R

was

higher

than

the

gene

pair

concerned

was

considered

to

be

coexpressed

The

value

of

R

is

generally

considered

a

rule-of-thumb

threshold

-LRB-

see

for

example

bbc.botany.utoronto.ca

affydb/BAR

instructions

-RRB-

and

is

used

in

various

analyses

-LRB-

Cohen

et

al.

Lee

et

al.

Ren

et

al.

-RRB-

The

number

of

coexpressed

adjacent

pairs

was

counted

To

evaluate

the

statistical

significance

of

these

numbers

they

were

compared

with

the

number

of

coexpressed

pairs

from

randomizations

of

the

population

of

expressed

genes

using

the

cumulative

binomial

distribution

-LRB-

Cohen

et

al.

-RRB-

Previous

analyses

indicated

that

more

than

randomizations

did

not

result

in

significant

changes

in

the

numbers

obtained

-LRB-

Ren

et

al.

-RRB-

In

each

round

of

randomization

non-adjacent

pairs

of

genes

were

randomly

selected

with

replacement

from

the

list

of

expressed

genes

that

have

expressed

neighbors

till

the

same

total

number

of

pairs

was

obtained

Similarly

coexpressed

adjacent

triplets

quadruplets

and

pentaplets

were

identified

as

series

of

genes

with

consecutive

IDs

in

which

all

possible

-LRB-

that

is

-LRB-

n

-LRB-

n

-RRB-

-RRB-

where

n

is

the

number

of

genes

involved

-RRB-

pair-wise

R

's

should

be

above

the

cut-off

of

The

significance

of

results

was

evaluated

with

randomizations

equivalent

to

the

procedure

used

in

case

of

duplets

Duplicated

genes

Duplicated

genes

were

identified

by

local

pair-wise

protein

BLAST

-LRB-

BLASTP

-LSB-

-RSB-

-LRB-

Altschul

et

al.

-RRB-

-RRB-

on

all

gene

pairs

in

the

rice

genome

A

gene

pair

was

considered

to

be

duplicated

-LRB-

dup

-RRB-

if

BLASTP

yielded

an

E-value

-LRB-

Fukuoka

et

al.

Lercher

et

al.

Williams

and

Bowles

-RRB-

To

determine

duplicated

triplets

quadruplets

and

pentaplets

it

was

required

that

any

pair

of

the

genes

concerned

had

a

BLASTP

E-value

Analyses

of

gene

orientation

and

gene

distance

Adjacent

gene

pairs

were

separated

into

tandemly

divergently

and

convergently

transcribed

pairs

according

to

their

relative

direction

of

transcription

The

number

of

coexpressed

pairs

in

each

orientation

group

was

expressed

as

percentage

relative

to

the

total

number

of

adjacent

pairs

in

that

group

Random

pairs

were

made

by

randomly

picking

two

non-adjacent

genes

from

the

list

of

expressed

genes

represented

in

pairs

analyzed

for

their

orientation

and

compared

with

the

real

genome

using

a

variant

of

the

two-sample

t

test

for

proportions

for

determining

the

significance

of

a

difference

between

two

population

proportions

-LRB-

Ott

and

Longnecker

-RRB-

The

test

statistic

is

based

on

the

z

statistic

from

the

normal

distribution

and

is

given

by

-LRB-

-RRB-

-LRB-

-LRB-

-RRB-

-LRB-

-RRB-

-RRB-

with

and

the

two

sample

proportions

and

the

two

sample

sizes

under

the

condition

that

-LRB-

-RRB-

and

-LRB-

-RRB-

are

all

larger

than

The

z

value

is

converted

to

a

p

value

using

standard

normal

tables

To

determine

the

gene

distance

the

intergenic

distance

is

used

This

distance

is

defined

as

the

length

in

nucleotides

from

the

annotated

end

of

one

gene

to

the

annotated

start

of

the

next

gene

including

the

UTRs

when

known

otherwise

the

translation

start

and

stop

sites

were

taken

The

data

sets

excluding

the

duplicated

gene

pairs

were

analyzed

For

each

data

set

gene

pairs

were

sorted

based

on

gene

distance

from

short

to

long

and

bins

of

pairs

were

taken

and

analyzed

excluding

the

last

bin

with

less

than

pairs

The

advantage

of

using

equal

pair

bin

is

that

it

avoids

unequal

number

of

gene

pairs

in

different

distance

categories

Per

bin

gene

distance

was

calculated

as

the

average

over

all

pairs

For

each

bin

the

fraction

of

coexpressed

pairs

relative

to

the

total

number

of

pairs

in

each

orientation

group

in

each

bin

was

calculated

and

plotted

Functional

categorization

of

genes

TAIR

's

GOslim

the

Gene

Ontology

-LRB-

GO

-RRB-

developed

for

plants

-LRB-

Berardini

et

al.

-RRB-

was

used

to

classify

the

genes

present

in

local

coexpression

domains

The

three

aspects

of

GOslim

molecular

function

biological

process

and

cellular

component

were

analyzed

in

parallel

With

Python

scripts

the

number

of

pairs

of

which

both

members

could

be

classified

in

GOslim

was

determined

and

the

number

of

pairs

of

which

both

members

fall

into

the

same

well-defined

GOslim

category

was

also

determined

The

GOslim

categories

of

unknown

and

other

were

not

included

into

well-defined

categories

because

they

give

less

-LRB-

or

no

-RRB-

information

about

functional

categorization

The

percentage

of

coexpressed

pairs

falling

into

the

same

well-defined

category

was

compared

with

that

of

non-coexpressed

pairs

to

determine

whether

coexpressed

genes

are

more

enriched

in

the

same

functional

category

than

non-coexpressed

genes

Assessing

synteny

between

Arabidopsis

and

rice

The

Inparanoid

Eukaryotic

Orthologous

groups

database

-LRB-

inparanoid.cgb.ki.se

O'Brien

et

al.

-RRB-

was

used

to

download

all

known

orthologous

and

inparalogous

clusters

between

Arabidopsis

and

rice

Inparanoid

defines

inparalogs

as

paralogs

that

arose

through

gene

duplication

after

speciation

Inparalogs

can

form

a

group

of

genes

that

together

are

orthologous

to

a

gene

in

another

species

There

are

orthologous

clusters

between

Arabidopsis

-LRB-

from

Ensemble

-RRB-

and

rice

-LRB-

from

the

Model

Organism

database

-RRB-

and

all

of

them

were

taken

into

account

These

clusters

were

downloaded

on

Dec.

In

the

orthologous

clusters

sequences

-LRB-

proteins

-RRB-

from

Arabidopsis

are

inparalogs

and

sequences

-LRB-

proteins

-RRB-

from

rice

are

inparalogs

More

than

half

of

Arabidopsis

and

rice

inparalogs

are

many-to-many

or

many

to-one

orthology

cases

Less

than

half

of

the

cases

are

one-to-one

orthology

cases

The

Ensembl

protein

IDs

-LRB-

for

Arabidopsis

-RRB-

and

the

Model

organism

database

protein

IDs

-LRB-

for

rice

-RRB-

in

Inparanoid

were

first

translated

to

their

unique

gene

identifiers

in

the

respective

TIGR

annotation

by

BLASTP

using

an

E-value

e

This

yielded

unique

Arabidopsis

genes

and

unique

rice

genes

as

inparalogs

The

pairs

of

genes

in

local

coexpression

domains

were

analyzed

to

determine

which

genes

in

a

rice

local

coexpressed

pair

have

orthologs

in

an

Arabidopsis

local

coexpressed

pair

and

vice

versa

Because

of

the

larger

coverage

of

genes

and

tissues

only

the

MPSS

expression

data

were

used

for

this

analysis

As

coexpressed

triplets

and

quadruplets

are

always

combinations

of

coexpressed

pairs

they

were

not

further

analyzed

For

comparison

the

pairs

of

genes

that

are

not

coexpressed

were

analyzed

to

determine

how

many

non-coexpressed

pairs

or

one

of

their

member

genes

have

orthologs

in

the

other

plant

species

The

numbers

were

then

compared

between

coexpressed

pairs

and

non-coexpressed

pairs

to

determine

the

significance

of

occurrence

of

syntenic

local

coexpression

domains

Results

Local

coexpression

domains

consist

of

two

to

four

neighboring

genes

The

TIGR

version

of

the

rice

genome

has

predicted

genes

This

is

about

twice

the

number

of

genes

predicted

for

Arabidopsis

-LRB-

genes

annotation

-RRB-

The

coverage

of

the

MPSS

expression

data

for

the

rice

genome

is

The

expression

coverage

in

the

Affymetrix

array

data

set

we

used

is

-LRB-

Table

-RRB-

This

is

about

half

or

less

of

the

expression

coverage

for

the

Arabidopsis

genome

-LRB-

in

the

update

Ren

et

al.

-RRB-

This

difference

can

reflect

the

more

advanced

annotation

of

the

Arabidopsis

genome

at

this

time

and/or

the

more

complex

-LRB-

duplicated

-RRB-

organization

of

the

rice

genome

The

rice

genome

annotation

has

more

genes

that

are

physically

overlapping

than

the

Arabidopsis

genome

Excluding

the

smaller

overlapping

genes

from

the

analyses

we

were

able

to

identify

gene

pairs

with

MPSS

expression

data

and

pairs

with

MA

expression

data

in

rice

-LRB-

Table

see

also

Materials

and

methods

-RRB-

Of

these

-LRB-

-RRB-

in

MPSS

and

-LRB-

-RRB-

in

MA

were

identified

to

represent

a

local

coexpression

domain

as

defined

as

being

coexpressed

with

a

pair-wise

Pearson

's

correlation

coefficient

larger

than

-LRB-

Table

-RRB-

This

percentage

is

similar

to

what

we

have

found

previously

for

Arabidopsis

-LRB-

Ren

et

al.

-RRB-

and

agrees

well

with

other

findings

that

of

a

genome

is

tightly

coexpressed

-LRB-

Semon

and

Duret

-RRB-

Table

of

rice

expression

data

used

for

whole-genome

local

coexpression

analysisMPSSMAGenes

with

expressionExcluding

overlapping

Without

expressed

neighbor

-LRB-

s

-RRB-

represented

in

Adjacent

Tandemly

duplicated

pairs

-LRB-

td

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

bTotal

excluding

Coexpressed

excluding

td

-LRB-

-RRB-

-LRB-

-RRB-

cCoexpressed

adjacent

duplicated

-LRB-

-RRB-

-LRB-

-RRB-

dTandemly

duplicated

-LRB-

-RRB-

-LRB-

-RRB-

eaPercentage

of

tandemly

duplicated

pairs

relative

to

the

total

number

of

adjacent

pairsbPercentage

of

coexpressed

adjacent

pairs

relative

to

the

total

number

of

adjacent

pairscPercentage

of

coexpressed

adjacent

pairs

excluding

td

relative

to

the

total

number

of

adjacent

pairs

excluding

tandemly

duplicated

pairsdPercentage

of

coexpressed

tandemly

duplicated

pairs

relative

to

the

total

number

of

coexpressed

adjacent

pairsePercentage

of

coexpressed

tandemly

duplicated

pairs

relative

to

the

total

number

of

tandem

duplicated

pairs

Notably

duplicated

genes

are

supposed

to

influence

coexpression

statistics

due

to

their

common

origin

-LRB-

Lercher

et

al.

-RRB-

although

a

surprising

finding

for

the

Arabidopsis

coexpression

domains

was

that

only

a

minor

fraction

of

duplicated

genes

were

actually

coexpressed

-LRB-

Ren

et

al.

-RRB-

The

occurrence

of

duplicated

pairs

in

the

rice

set

was

determined

with

pair-wise

protein

BLAST

using

a

cut-off

of

E

-LRB-

Fukuoka

et

al.

Lercher

et

al.

Williams

and

Bowles

-RRB-

This

identified

-LRB-

-RRB-

duplicated

gene

pairs

in

the

MPSS

gene

pair

data

set

and

-LRB-

-RRB-

duplicated

gene

pairs

in

the

MA

gene

pair

data

set

Of

these

only

-LRB-

-RRB-

in

the

MPSS

data

and

-LRB-

-RRB-

in

the

MA

data

were

coexpressed

-LRB-

Table

-RRB-

Although

this

percentage

is

somewhat

higher

than

the

percentage

of

coexpression

in

non-duplicated

pairs

-LRB-

-RRB-

the

majority

of

all

duplicated

pairs

-LRB-

in

MPSS

data

and

in

MA

data

-RRB-

are

not

coexpressed

This

shows

that

also

in

rice

gene

duplication

does

not

correlate

well

with

coexpression

and

suggests

that

expression

divergence

is

a

common

phenomenon

after

duplication

-LRB-

Williams

and

Bowles

-RRB-

Excluding

the

duplicated

pairs

from

the

coexpressed

sets

there

are

gene

pairs

in

the

MPSS

data

and

gene

pairs

in

the

MA

data

coexpressed

in

rice

This

accounts

for

-LRB-

-RRB-

in

MPSS

and

-LRB-

-RRB-

in

MA

of

all

coexpressed

pairs

Therefore

also

in

rice

the

occurrence

of

duplicated

genes

can

not

explain

the

occurrence

of

local

coexpression

domains

Extending

the

size

of

the

local

coexpression

domain

to

triplets

quadruplets

pentaplets

and

on

requiring

that

all

pair-wise

combinations

of

genes

have

a

tightly

correlated

expression

shows

that

few

larger

local

coexpression

domains

exist

-LRB-

Table

-RRB-

No

quadruplet

domains

could

be

identified

when

tandemly

duplicated

genes

were

excluded

-LRB-

Table

-RRB-

To

assess

the

significance

of

the

occurrence

of

the

various

local

coexpression

domains

we

compared

the

number

of

coexpressed

pairs

triplets

and

quadruplets

with

the

average

of

such

domains

in

randomly

generated

genomes

using

the

cumulative

binomial

distribution

-LRB-

Cohen

et

al.

-RRB-

Such

comparisons

revealed

that

local

coexpression

pairs

occur

in

the

rice

genome

significantly

more

often

than

expected

by

chance

alone

-LRB-

Table

-RRB-

However

when

excluding

the

duplicated

genes

triplets

and

quadruplets

do

not

occur

significantly

more

often

than

by

chance

-LRB-

at

P

-RRB-

in

both

expression

datasets

Local

coexpression

domains

therefore

consist

of

at

most

genes

when

duplicated

genes

are

not

taken

into

consideration

This

number

appears

smaller

than

in

the

Arabidopsis

genome

-LRB-

Ren

et

al.

-RRB-

but

this

may

reflect

the

lesser

coverage

of

the

annotation

of

the

rice

genome

Table

coexpression

domains

in

the

rice

genomeRice

genomeRandom

genome

-LRB-

-RRB-

TotalaCoexpressedbAveragecP-valuedPairsMPSS

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

TripletsMPSS

-LRB-

-RRB-

-LRB-

-RRB-

MA

-LRB-

-RRB-

-LRB-

-RRB-

n.s.QuadrupletsMPSS

-LRB-

-RRB-

-LRB-

-RRB-

n.s.MA

-LRB-

-RRB-

number

of

pairs

triplets

quadruplets

in

each

data

setbCoexpressed

pairs

triplets

quadruplets

in

each

data

set

Percentages

in

brackets

are

coexpressed

relative

to

the

totalcAverage

plus/minus

standard

deviation

from

randomizationsdP-value

according

to

the

cumulative

binomial

distribution

-LRB-

Cohen

et

al.

-RRB-

for

obtaining

such

a

result

by

chance

P

is

considered

significant

n.s.

not

significanteMPSS

data

set

including

tandemly

duplicated

genesfMPSS

data

set

excluding

tandemly

duplicated

genesgMA

data

set

including

tandemly

duplicated

geneshMA

data

set

excluding

tandemly

duplicated

genesiNot

determined

Local

coexpression

domains

seem

randomly

distributed

over

the

genome

-LRB-

Fig.

MPSS

data

only

-RRB-

Only

coexpressed

pairs

are

common

between

the

MPSS

and

MA

coexpressed

sets

out

of

a

total

of

common

pairs

-LRB-

excluding

tandemly

duplicated

pairs

-RRB-

Detailed

information

about

locations

orientations

expressions

and

gene

distances

-LRB-

only

for

the

pairs

-RRB-

for

all

pairs

triplets

and

quadruplets

in

both

the

MPSS

and

MA

data

sets

are

given

in

the

supplementary

data

All

subsequent

analyses

were

focused

on

domains

consisting

of

non-duplicated

gene

pairs

unless

stated

differently

Fig.

of

local

coexpression

domains

over

all

rice

chromosomes

Rectangles

are

schematic

representation

of

chromosomes

from

top

to

bottom

The

numbers

on

the

top

show

the

scale

in

million

bases

along

the

chromosomes

Each

gene

in

a

local

coexpression

domain

is

depicted

with

a

black

bar

Only

MPSS

datasets

excluding

tandemly

duplicated

genes

are

shown

The

orders

of

the

drawings

in

each

rectangle

are

first

lane

coexpressed

pairs

second

lane

coexpressed

triplets

third

lane

coexpressed

quadruplets

fourth

lane

partially

syntenic

coexpression

domains

-LRB-

PSCDs

-RRB-

between

Arabidopsis

and

rice

Orientation

and

distance

do

not

solely

explain

the

occurrence

of

local

coexpression

In

yeast

there

are

several

examples

that

divergently

transcribed

promoter

regions

are

the

cause

of

co-regulated

neighboring

genes

-LRB-

Korbel

et

al.

Kruglyak

and

Tang

-RRB-

If

promoter

sharing

is

an

important

mechanism

for

coexpression

in

the

rice

genome

divergently

transcribed

gene

pairs

should

be

over-represented

in

the

sub-population

of

coexpressed

pairs

compared

to

coexpressed

pairs

that

are

tandemly

or

convergently

transcribed

For

all

three-orientation

groups

the

number

of

pairs

and

the

number

of

coexpressed

pairs

in

the

rice

genome

were

determined

-LRB-

Table

-RRB-

For

each

orientation

group

the

fraction

of

coexpressed

pairs

relative

to

the

total

number

of

pairs

in

that

group

was

calculated

-LRB-

Table

-RRB-

of

the

fractions

are

significantly

different

from

each

other

using

a

statistical

test

for

comparing

population

proportions

-LRB-

Ott

and

Longnecker

-RRB-

The

fraction

of

coexpressed

divergent

pairs

in

the

MPSS

data

is

the

lowest

of

the

three

groups

-LRB-

Table

-RRB-

Therefore

shared

promoter

regions

can

not

solely

explain

the

coexpression

of

adjacent

genes

Table

of

coexpressed

gene

pairsOrientation

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

atan-td

div-td

con-td

respectively

are

the

sub-groups

of

tandemly

divergently

convergently

transcribed

pairs

excluding

tandem

duplicatesbTotal

number

of

pairs

in

each

direction

groupcNumber

of

coexpressed

pairs

in

each

direction

group

Percentages

in

the

brackets

are

number

of

coexpressed

pairs

relative

to

the

total

number

of

pairs

of

the

proportions

are

significantly

different

from

each

other

according

to

the

z

test

for

comparing

population

proportions

The

physically

closer

two

genes

are

the

higher

the

likelihood

is

that

they

are

coexpressed

due

to

either

cis

or

trans-activation

-LRB-

Hershberg

et

al.

-RRB-

Therefore

we

determined

the

intergenic

distance

defined

as

the

sequence

length

in

nucleotides

from

the

annotated

end

of

one

gene

to

the

annotated

start

of

the

neighboring

gene

including

UTRs

when

known

otherwise

taking

the

start

and

stop

site

for

translation

This

distance

was

used

to

investigate

whether

it

would

explain

the

characteristics

of

local

coexpression

domains

In

Fig.

the

fraction

of

coexpressed

pairs

is

plotted

for

each

orientation

and

for

each

bin

after

sorting

based

on

intergenic

distance

The

results

show

that

the

fraction

of

coexpressed

pairs

irrespective

of

gene

orientation

does

not

decrease

with

larger

gene

distance

When

gene

distance

is

defined

as

the

sequence

length

from

the

start

of

one

gene

till

the

start

of

the

next

gene

-LRB-

Ren

et

al.

-RRB-

the

result

is

similar

-LRB-

data

not

shown

-RRB-

As

a

consequence

increasing

intergenic

distances

do

not

seem

to

be

a

barrier

for

the

occurrence

of

local

coexpression

and

short

intergenic

distances

do

not

favor

coexpression

Therefore

intergenic

distance

does

not

solely

explain

local

coexpression

in

the

rice

genome

as

it

did

not

in

the

Arabidopsis

genome

-LRB-

Ren

et

al.

-RRB-

Fig.

distance

does

not

solely

explain

the

occurrence

of

coexpression

Gene

distance

defined

as

the

length

in

nucleotides

from

the

annotated

end

of

one

gene

to

the

annotated

start

of

the

next

gene

relative

to

the

strand

the

genome

that

is

given

with

annotated

start

always

smaller

than

the

annotated

end

X-axis

is

the

averaged

gene

distance

-LRB-

in

base

pair

-RRB-

in

each

bin

The

Y-axis

depicts

the

number

of

pairs

-LRB-

A

D

-RRB-

number

of

coexpressed

pairs

-LRB-

B

E

-RRB-

and

the

fraction

of

coexpressed

pairs

-LRB-

C

F

-RRB-

relative

to

the

total

number

of

pairs

in

each

orientation

-LRB-

tan

tandem

pairs

div

divergent

pairs

con

convergent

pairs

-RRB-

in

each

bin

Functional

categorization

of

coexpressed

genes

To

characterize

the

kind

of

genes

that

are

present

in

the

rice

coexpression

domains

the

gene

ontology

-LRB-

GO

-RRB-

developed

for

plants

-LRB-

GOslim

Berardini

et

al.

-RRB-

was

used

The

GOslim

ontology

provides

a

controlled

vocabulary

to

describe

gene

and

gene

product

attributes

in

plants

focusing

on

three

aspects

of

annotation

molecular

function

biological

process

and

cellular

component

Each

aspect

has

categories

with

categories

having

terms

like

unknown

or

other

To

all

pairs

of

genes

each

aspect

of

the

GOslim

annotation

was

assigned

For

each

aspect

the

number

of

pairs

was

determined

for

which

both

member

genes

were

covered

by

a

GOslim

assignment

In

addition

the

number

of

pairs

for

which

both

member

genes

fall

into

the

same

well-defined

categories

-LRB-

excluding

the

unknown

and

other

subcategories

-RRB-

was

determined

The

fraction

of

the

latter

was

compared

between

coexpressed

pairs

and

non-coexpressed

pairs

to

determine

whether

coexpressed

pairs

were

enriched

in

the

same

categories

-LRB-

Table

-RRB-

In

the

MPSS

data

the

GOslim

annotation

coverage

for

both

member

genes

in

a

pair

is

for

molecular

function

for

biological

process

and

only

for

cellular

component

In

the

MA

data

these

percentages

are

and

respectively

Comparing

these

figures

with

the

GOslim

coverage

of

the

Arabidopsis

genes

-LRB-

data

from

Ren

et

al.

but

updated

to

the

annotation

-RRB-

which

is

for

all

aspects

shows

that

currently

the

rice

genome

is

considerably

less

well

annotated

than

the

Arabidopsis

genome

When

comparing

the

coexpressed

and

non-coexpressed

pairs

in

rice

for

the

fraction

of

gene

pairs

falling

into

the

same

well-defined

GOslim

category

there

is

no

significant

difference

-LRB-

Table

-RRB-

Therefore

also

in

the

rice

genome

coexpressed

gene

pairs

are

not

enriched

for

the

same

functional

category

Table

of

gene

pairs

over

GOslim

categories

-LRB-

Non-duplicated

pairs

-RRB-

AllaCoexpressedbNon-coexpressedcP-valuedMPSS

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

MA

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aNumber

of

neighboring

pairs

excluding

td

All

other

pairs

are

all

duplicate-free

unless

stated

otherwisebNumber

of

coexpressed

pairscNumber

of

non-coexpressed

pairsdP

value

from

the

standard

normal

tables

of

the

z

statistic

for

the

difference

of

the

population

proportion

between

coexpressed

pairs

and

non-coexpressed

pairs

in

the

rice

genome

significant

-LRB-

two-tailed

P

-RRB-

The

P

value

is

the

probability

under

the

hypothesis

that

the

two

population

proportions

are

the

sameeNumber

of

pairs

of

which

both

members

are

assigned

-LRB-

covered

-RRB-

with

GOslim

categoriesfNumber

of

pairs

of

which

both

members

fall

into

the

same

known

GOslim

category

-LRB-

excluding

the

categories

with

the

indications

unknown

and

other

-RRB-

Percentage

is

the

number

of

pairs

relative

to

the

number

of

pairs

covered

Microsynteny

of

local

coexpression

domains

between

rice

and

Arabidopsis

The

structural

characteristics

of

local

coexpression

domains

in

rice

and

in

Arabidopsis

-LRB-

Ren

et

al.

-RRB-

are

remarkably

similar

This

prompts

the

question

whether

such

domains

also

share

functional

characteristics

and

possibly

consist

of

the

same

or

related

genes

Microsynteny

in

local

expression

domains

of

these

two

genomes

would

reflect

conservation

of

such

domains

The

Inparanoid

Eukaryotic

Orthologous

database

-LRB-

O'Brien

et

al.

-RRB-

was

used

to

retrieve

the

current

list

of

genes

that

are

supposed

to

be

orthologous

between

Arabidopsis

-LRB-

genes

-RRB-

and

rice

-LRB-

genes

-RRB-

including

all

many-to-many

relationships

The

genes

establishing

coexpressed

pairs

based

on

MPSS

expression

data

in

either

Arabidopsis

-LRB-

pairs

including

duplicated

pairs

data

from

Ren

et

al.

but

updated

to

the

TIGR

annotation

-RRB-

or

rice

-LRB-

pairs

including

duplicated

pairs

-RRB-

were

searched

against

these

lists

This

way

we

aimed

to

identify

the

pairs

of

which

both

genes

in

the

pair

have

an

ortholog

in

the

other

plant

and

these

orthologs

are

also

coexpressed

The

analyses

showed

that

there

was

not

a

single

coexpressed

pair

in

either

Arabidopsis

or

rice

of

which

both

genes

are

orthologous

to

a

gene

of

a

coexpressed

pair

in

the

other

species

Therefore

given

the

current

annotation

of

the

two

genomes

there

are

no

syntenic

local

coexpression

domains

between

Arabidopsis

and

rice

Partially

syntenic

local

coexpression

domains

can

occur

by

chance

In

cases

though

one

gene

of

a

coexpressed

pair

in

one

plant

species

was

orthologous

to

at

least

one

gene

of

a

coexpressed

pair

in

the

other

plant

That

is

of

all

-LRB-

-RRB-

coexpressed

pairs

in

Arabidopsis

and

of

all

-LRB-

-RRB-

coexpressed

pairs

in

rice

We

will

refer

to

such

a

case

as

a

partially

syntenic

coexpression

domain

-LRB-

PSCD

-RRB-

To

assess

the

significance

of

such

partially

syntenic

domains

we

evaluated

all

the

genes

in

non-coexpressed

pairs

comparing

Arabidopsis

-LRB-

pairs

including

duplicated

pairs

-RRB-

and

rice

-LRB-

pairs

including

duplicated

pairs

-RRB-

to

establish

whether

PSCDs

are

more

enriched

in

the

genome

than

partially

syntenic

non-coexpressed

domains

-LRB-

PSND

-RRB-

We

identified

PSNDs

-LRB-

due

to

duplicated

pairs

-RRB-

between

all

non-coexpressed

pairs

of

genes

in

both

plant

genomes

This

is

of

all

Arabidopsis

non-coexpressed

pairs

and

of

all

rice

non-coexpressed

pairs

The

percentage

of

PSNDs

among

non-coexpressed

pairs

is

times

higher

than

that

of

PSCDs

from

coexpressed

pairs

Therefore

PSCDs

do

not

seem

to

occur

more

often

than

expected

by

chance

alone

A

complicating

issue

in

the

analysis

of

synteny

is

the

occurrence

of

many-to-many

orthologs

The

Inparanoid

database

defines

so-called

inparalogs

as

paralogs

arising

through

gene

duplication

after

speciation

These

can

form

a

group

of

genes

that

together

are

orthologous

to

a

gene

in

the

other

species

As

a

result

there

can

be

many

to

many

many

to

one

and

one

to

one

relationships

Individual

member

genes

in

many-to-many

or

many-to-one

relationships

may

not

be

the

main

orthologs

Interestingly

there

is

one

many-to-one

case

in

which

four

Arabidopsis

genes

are

all

orthologs

of

the

same

single

rice

gene

-LRB-

-RRB-

These

Arabidopsis

genes

are

and

The

first

two

form

a

local

coexpressed

pair

The

other

two

genes

and

are

not

more

than

ten

genes

away

from

the

previous

two

genes

on

the

same

chromosomal

region

The

latter

two

genes

are

separated

from

each

other

by

a

few

genes

Further

analysis

shows

that

gene

has

a

duplicated

neighbor

but

is

not

coexpressed

with

it

It

is

however

coexpressed

with

its

other

neighbor

but

is

not

duplicated

with

it

Orthology

is

established

between

and

the

rice

gene

but

not

between

any

of

the

neighbors

of

the

Arabidopsis

genes

The

rice

gene

is

also

coexpressed

with

one

of

its

neighboring

genes

but

it

is

not

a

duplicate

of

it

while

this

rice

gene

itself

is

a

duplicate

of

another

neighboring

gene

-LRB-

-RRB-

but

it

is

also

not

coexpressed

with

it

A

schematic

representation

of

the

resulting

gene

configuration

is

given

in

Fig.

Such

detailed

analyses

may

reveal

local

microsynteny

in

the

twilight

zone

of

statistical

significance

and

evolutionary

relevance

Fig.

representation

of

the

chromosomal

regions

covering

genes

involved

in

a

four-to-one

orthology

between

Arabidopsis

and

rice

Top

part

of

the

figure

is

the

chromosomal

region

from

rice

-LRB-

from

gene

locus

to

gene

locus

-RRB-

Bottom

part

of

the

figure

is

the

chromosomal

region

from

Arabidopsis

representing

genes

-LRB-

from

gene

locus

to

the

numbers

in

the

picture

do

not

carry

-RRB-

Black

arrows

represent

the

four

Arabidopsis

and

the

one

rice

gene

involved

in

this

orthology

and

dashed

curved

connecting

lines

show

the

orthology

relationships

Black

bracket-like

lines

depict

duplication

and

genes

connected

and

included

within

by

black

bracket

line

are

duplicated

to

each

other

Dotted

lines

depict

coexpression

relationship

and

genes

connected

and

included

by

dotted

line

are

coexpressed

with

each

other

Discussion

Local

coexpression

domains

represent

only

a

small

part

of

the

genome

Setting

stringent

criteria

for

coexpression

using

MPSS

and

microarray

expression

data

the

rice

genome

was

found

to

contain

a

small

but

significant

number

of

local

coexpression

domains

that

range

from

predominantly

two

up

till

genes

irrespective

of

the

expression

platform

used

for

analysis

This

is

similar

to

the

situation

in

Arabidopsis

-LRB-

Ren

et

al.

-RRB-

It

shows

that

a

genome

is

essentially

not

a

random

entity

with

respect

to

the

occurrence

of

local

coexpression

domains

Our

results

agree

with

other

coexpression

studies

where

strong

coexpression

was

shown

to

occur

only

within

close

proximity

of

several

genes

-LRB-

Cohen

et

al.

Hershberg

et

al.

Lercher

et

al.

Semon

and

Duret

-RRB-

Although

coexpression

was

shown

to

extend

to

regions

covering

up

to

genes

and

more

-LRB-

Spellman

and

Rubin

-RRB-

and

to

cover

chromosomal

regions

up

to

kb

-LRB-

Ma

et

al.

Spellman

and

Rubin

Williams

and

Bowles

-RRB-

and

more

there

appears

to

exist

a

decrease

in

the

strength

of

coexpression

with

increasing

distances

The

local

coexpression

domains

described

here

represent

of

the

potential

coexpression

fraction

in

the

whole

genome

as

found

in

other

studies

-LRB-

Semon

and

Duret

-RRB-

Larger

but

looser

coexpression

domains

might

cover

up

to

-LRB-

Cohen

et

al.

Williams

and

Bowles

-RRB-

till

-LRB-

Spellman

and

Rubin

-RRB-

of

the

genome

The

difference

in

occurrence

between

local

and

longer-range

weaker

but

still

statistically

significant

coexpression

domains

is

highly

dependent

on

the

method

used

-LRB-

Semon

and

Duret

-RRB-

Moreover

the

expression

platforms

used

focus

on

snapshots

of

gene

expression

at

the

RNA

level

Such

data

ignore

various

post-transcriptional

regulatory

mechanisms

that

can

for

example

result

in

poor

correlations

between

RNA

and

protein

levels

Although

we

have

shown

before

that

in

a

transgenic

set-up

the

creation

of

an

artificial

local

coexpression

domain

results

in

markedly

improved

RNA/protein

correlations

-LRB-

Mlynarova

et

al.

-RRB-

this

does

not

need

to

be

the

case

for

all

endogenous

local

coexpression

domains

now

identified

The

terms

cluster

or

chromosomal

domain

and

associated

terms

such

as

neighboring

are

generally

based

on

a

-LRB-

much

-RRB-

more

loose

definition

compared

to

the

definition

used

here

to

identify

local

coexpression

domains

Local

coexpression

domains

require

a

pair-wise

correlation

between

the

expressions

of

ALL

adjacent

genes

above

The

larger

domains

are

defined

on

the

basis

of

the

use

of

a

sliding

window

of

either

a

given

sequence

length

-LRB-

number

of

nucleotides

-RRB-

or

of

a

given

number

of

genes

-LRB-

Spellman

and

Rubin

Williams

and

Bowles

-RRB-

In

such

a

window

the

average

correlation

is

calculated

and

compared

with

simulated

sets

This

allows

for

the

presence

of

genes

within

a

domain

that

are

not

strongly

-LRB-

co

-RRB-

expressed

but

are

carried

along

for

a

ride

in

the

open

chromatin

domain

-LRB-

Spellman

and

Rubin

-RRB-

The

two

expression

data

sets

here

analyzed

show

very

little

overlap

in

coexpressed

pairs

-LRB-

only

out

of

all

common

pairs

-RRB-

The

low

number

of

shared

pairs

is

thought

to

reflect

the

biological

background

of

the

data

sets

The

MPSS

data

concern

a

broad

range

of

tissues

and

experimental

conditions

while

the

MA

data

only

cover

crown

and

growing

point

tissue

under

control

and

salt

stress

condition

-LRB-

Walia

et

al

et

al.

-RRB-

The

number

of

libraries

taken

into

account

as

well

as

their

biological

background

obviously

influences

the

possibility

of

identifying

significant

numbers

of

coexpression

domains

above

the

number

of

domains

expected

by

chance

Two

genes

evaluated

in

four

comparably

similar

conditions

-LRB-

MA

data

-RRB-

are

predicted

to

have

a

higher

likelihood

of

exhibiting

a

similar

expression

pattern

than

when

examined

in

different

-LRB-

MPSS

-RRB-

conditions

When

considering

genome-wide

local

coexpression

of

genes

a

wide

diversity

of

tissues

and/or

conditions

should

be

taken

for

analysis

Highly

tissue-specific

coexpression

may

be

masked

in

this

approach

and

should

be

analyzed

by

other

means

Parameters

shaping

local

coexpression

domains

The

existence

of

local

coexpression

domains

in

rice

could

not

be

explained

solely

by

gene

orientation

such

as

tandemly

divergently

or

convergently

oriented

gene

pairs

No

relative

enrichment

of

the

proportion

of

coexpressed

pairs

was

seen

The

fraction

of

coexpressed

genes

in

the

divergent

orientation

was

even

lower

than

for

the

other

two

orientations

-LRB-

Table

-RRB-

So

shared

promoter

regions

-LRB-

for

divergent

pairs

-RRB-

and

transcriptional

read-through

-LRB-

for

tandem

pairs

-RRB-

do

not

explain

the

local

coexpression

domains

in

rice

similar

to

what

we

have

concluded

for

Arabidopsis

-LRB-

Ren

et

al.

-RRB-

This

is

in

contrast

to

some

other

studies

in

which

shared

promoter

region

-LRB-

for

divergent

pairs

-RRB-

and

transcriptional

read-through

established

coexpression

domains

-LRB-

Semon

and

Duret

-RRB-

Whereas

we

do

not

detect

any

preferred

orientation

to

result

in

coexpression

in

rice

other

studies

show

a

higher

degree

of

coexpression

in

divergent

and

tandemly

oriented

gene

pairs

-LRB-

Williams

and

Bowles

Zhan

et

al.

-RRB-

The

differences

in

conclusions

are

most

likely

due

to

the

different

methods

used

such

as

the

definition

of

coexpression

of

neighboring

genes

as

well

as

the

dataset

and/or

expression

platform

used

Gene

distance

is

not

the

explanatory

factor

for

the

occurrence

of

local

coexpression

domains

in

rice

No

significant

decrease

in

the

fraction

of

coexpressed

genes

was

observed

with

increasing

intergenic

distance

-LRB-

Fig.

-RRB-

The

fraction

of

coexpressed

pairs

does

not

decrease

even

with

gene

distances

up

to

kb

It

shows

that

at

a

relatively

large

distance

neighboring

genes

can

still

be

coexpressed

Another

study

reported

that

when

genes

kb

apart

were

taken

into

account

the

negative

correlation

between

coexpression

and

gene

distance

was

gone

-LRB-

Williams

and

Bowles

-RRB-

In

a

comparative

study

of

eukaryotic

genomes

coexpression

was

shown

to

vary

at

chromosomal

distances

above

kb

-LRB-

Fukuoka

et

al.

-RRB-

This

suggests

that

considerable

coexpression

of

neighboring

genes

can

occur

even

at

large

gene

distance

although

the

coexpression

may

not

be

related

to

the

physical

distance

anymore

While

gene

distance

itself

is

not

predictive

for

coexpression

-LRB-

Cohen

et

al.

Kruglyak

and

Tang

-RRB-

the

likelihood

of

coexpression

would

favor

short

gene

distances

-LRB-

Hershberg

et

al.

Hurst

et

al.

Lercher

et

al.

Semon

and

Duret

-RRB-

It

should

be

kept

in

mind

that

the

rice

data

set

now

analyzed

is

far

from

complete

in

terms

of

its

annotation

so

what

are

now

far-apart

neighboring

genes

may

be

no

longer

directly

neighboring

the

moment

the

annotation

is

improved

Because

the

characteristics

of

the

local

coexpression

domains

in

Arabidopsis

and

rice

are

remarkably

alike

the

actual

numbers

and

possibly

even

genes

involved

in

local

coexpression

domains

may

be

subject

to

change

but

not

the

occurrence

of

such

domains

in

these

genomes

With

the

gene

ontology

developed

for

plants

-LRB-

GOslim

-RRB-

there

is

no

evidence

that

coexpressed

genes

are

more

enriched

in

the

same

functional

category

in

comparison

to

non-coexpressed

genes

-LRB-

Table

-RRB-

Previous

studies

suggested

that

clustering

of

functionally

related

genes

would

occur

in

all

metazoans

-LRB-

Cohen

et

al.

Lercher

et

al.

-RRB-

Recent

studies

demonstrated

a

significant

enrichment

for

coexpressed

genes

in

the

same

metabolic

pathway

-LRB-

Williams

and

Bowles

-RRB-

or

the

same

biological

processes

-LRB-

Zhan

et

al.

-RRB-

although

this

appeared

not

to

be

the

explanation

for

the

coexpression

of

neighboring

genes

-LRB-

Williams

and

Bowles

-RRB-

In

worm

clusters

of

similarly

expressed

genes

cover

similar

biological

functions

-LRB-

Roy

et

al.

-RRB-

In

human

coexpression

over

the

whole

genome

was

shown

to

correlate

with

functional

relationships

between

the

genes

-LRB-

Lee

et

al.

-RRB-

Our

study

found

no

enrichment

of

coexpressed

gene

pairs

in

the

same

functional

category

than

non-coexpressed

pairs

suggesting

that

it

is

not

necessarily

true

that

the

natural

selection

maintained

regional

coexpression

by

keeping

genes

with

similar

functions

in

adjacent

positions

-LRB-

Cohen

et

al.

Semon

and

Duret

-RRB-

The

genomic

context

of

genes

is

supposed

to

play

an

important

role

in

the

regulation

of

gene

expression

-LRB-

van

Drunen

et

al.

-RRB-

In

a

number

of

coexpression

studies

in

various

organisms

the

occurrence

of

coexpression

domains

whether

small

-LRB-

local

-RRB-

or

larger

-LRB-

global

-RRB-

sometimes

independent

of

gene

orientation

and

gene

distance

were

all

supposed

to

be

regulated

at

the

level

of

higher-order

chromosomal

structure

-LRB-

Cohen

et

al.

Hershberg

et

al.

Ren

et

al.

Spellman

and

Rubin

Williams

and

Bowles

Zhan

et

al.

-RRB-

It

will

be

difficult

to

formally

exclude

the

possibility

of

transcriptional

regulation

as

a

cause

of

local

coexpression

Co-regulated

transcription

could

occur

through

shared

promoter

elements

between

the

neighboring

genes

in

coexpression

domains

However

previous

experience

with

transgene

expression

data

indicated

that

the

particular

position

of

neighboring

genes

in

a

genome

affects

the

expression

of

that

gene

considerably

-LRB-

Mlynarova

et

al.

-RRB-

Two

physically

neighboring

transgenes

could

only

be

made

to

into

an

artificial

local

expression

domain

that

is

show

correlated

expression

when

chromatin-organizing

elements

were

placed

around

the

genes

-LRB-

Mlynarova

et

al.

-RRB-

Although

this

experimental

result

indicates

the

importance

of

chromatin

organization

in

establishing

local

coexpression

in

a

plant

genome

this

result

does

not

need

to

be

the

case

for

all

coexpression

domains

identified

With

this

caveat

this

study

of

local

coexpression

domains

in

rice

that

are

independent

of

duplication

gene

orientation

or

gene

distance

strengthen

the

notion

that

the

regulation

of

genes

in

such

domains

resides

at

the

level

of

higher-order

chromatin

structures

Future

studies

using

for

example

advanced

fluorescence

in

situ

hybridization

-LRB-

FISH

-RRB-

technology

on

interphase

chromosomes

-LRB-

Walter

et

al.

-RRB-

or

the

transgenic

approach

outlined

above

-LRB-

Mlynarova

et

al.

-RRB-

will

generate

further

experimental

support

for

the

location

and

characteristics

of

local

coexpression

domains

In

addition

the

new

approaches

in

and

insights

from

epigenetics

and

epigenomics

-LRB-

Henderson

and

Jacobsen

-RRB-

such

as

the

genome-wide

mapping

and

analysis

of

DNA

methylation

-LRB-

Zhang

et

al.

Zilberman

et

al.

-RRB-

and

future

histone

modification

maps

-LRB-

Esteller

-RRB-

of

plants

possibly

in

combination

with

genetics

-LRB-

Jansen

and

Nap

-RRB-

will

be

useful

to

get

more

insight

into

the

occurrence

and

function

of

local

coexpression

domains

in

plant

genomes

Lack

of

microsyntenic

coexpression

From

an

evolutionary

point

of

view

syntenic

regions

between

species

reveal

genes

for

conserved

and

important

traits

Macrosynteny

is

generally

not

easily

detectable

after

a

long

evolutionary

time

as

colinearity

erodes

by

various

mechanisms

such

as

transposon

activity

intra

or

inter-chromosomal

rearrangements

duplications

translocations

inversions

and/or

individual

divergence

after

speciation

-LRB-

Salse

et

al.

-RRB-

While

macrosynteny

may

not

be

detectable

any

more

for

genomes

that

diverged

more

than

million

years

ago

-LRB-

mya

-RRB-

microsynteny

i.e.

conservation

of

local

gene

order

and

orientation

may

still

exist

and

be

informative

-LRB-

Devos

et

al.

Salse

et

al.

-RRB-

Arabidopsis

and

rice

are

thought

to

have

diverged

about

mya

-LRB-

Salse

et

al.

-RRB-

Microsyntenic

local

coexpression

domains

between

Arabidopsis

and

rice

would

indicate

the

importance

of

the

evolutionary

conservation

of

regulatory

systems

beyond

sequence

similarity

after

the

divergence

of

dicotyledonous

and

monocotyledonous

plants

Analyses

show

that

there

is

not

a

single

coexpressed

pair

in

either

Arabidopsis

or

rice

of

which

both

genes

are

orthologous

to

a

gene

in

a

coexpressed

pair

in

the

other

species

Therefore

there

are

no

syntenic

local

coexpression

domains

between

Arabidopsis

and

rice

Although

the

analyses

were

performed

for

only

one

monocot

and

one

dicot

and

should

be

extended

to

many

more

genomes

the

results

could

be

taken

to

suggest

that

maintenance

of

coexpression

has

not

been

an

important

driving

force

in

genome

conservation

during

or

after

the

divergence

of

dicotyledonous

and

monocotyledonous

plants

Although

individual

genes

in

local

coexpression

domains

in

either

rice

or

Arabidopsis

may

have

an

ortholog

in

the

other

species

establishing

so-called

partially

syntenic

coexpression

domains

-LRB-

PSCDs

-RRB-

this

does

not

seem

to

occur

above

chance

in

the

context

of

whole-genome

configurations

Without

statistical

significance

the

occurrence

of

such

PSCDs

is

unlikely

to

have

any

evolutionary

relevance

on

a

genome-wide

scale

Detailed

analyses

of

individual

cases

and

gene

locations

may

suggest

the

occurrence

of

local

microsynteny

and

point

to

chains

of

evolutionary

events

in

which

the

conservation

of

coexpression

could

be

involved

However

more

detailed

studies

are

required

to

assess

the

functional

relevance

if

any

of

such

genomic

constitutions

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

-LRB-

XLS

KB

-RRB-

O O O O O O O B 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 O O O O O O O O O O O O O O O O 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 B O O O O O B O O O O O B O B O O O O O O B 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 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 B 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 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 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 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 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 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 B 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 B O B 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 B 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 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 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 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 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 B 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 B 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 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 B 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 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 B 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 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 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 B O O O O O O 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 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 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 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 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 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 B O O O O O B O B O O O O O O O O B 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 O O O O O O O O O O O O O O O B 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 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 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 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 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 B O O O O O O O O O O O B 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 B 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 B 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 B 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 B O B 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O 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 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 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 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 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 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 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 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 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 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 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 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

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