Genome-Wide

Detection

of

Polymorphisms

at

Nucleotide

Resolution

with

a

Single

DNA

Microarray

David

Gresham

et

al.

Science

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REPORTS

Foundation

Sumitomo

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Tokyo

Biochemical

Research

Foundation

Uehara

Memorial

Foundation

-LRB-

N.S.

-RRB-

Boninchi

Foundation

-LRB-

T.C.

-RRB-

Terry

Fox

Foundation

from

National

Cancer

Institute

of

Canada

-LRB-

NCIC

-RRB-

-LRB-

T.C.

-RRB-

the

Canadian

Institutes

of

Health

Research

-LRB-

W.-C.Y.

and

P.S.O.

-RRB-

and

a

Canadian

Network

for

Vaccines

and

Immunotherapeutics

-LRB-

CANVAC

-RRB-

Network

Centres

of

Excellence

grant

-LRB-

P.S.O.

-RRB-

SOM

Text

Figs

to

References

December

accepted

February

science

Supporting

Online

Material

Materials

and

Methods

Genome-Wide

Detection

of

Polymorphisms

at

Nucleotide

Resolution

with

a

Single

DNA

Microarray

David

Gresham

Douglas

M.

Ruderfer

Stephen

C.

Pratt

Joseph

Schacherer

Maitreya

J.

Dunham

David

Botstein

Leonid

A

central

challenge

of

genomics

is

to

detect

simply

and

inexpensively

all

differences

in

sequence

among

the

genomes

of

individual

members

of

a

species

We

devised

a

system

to

detect

all

singlenucleotide

differences

between

genomes

with

the

use

of

data

from

a

single

hybridization

to

a

whole-genome

DNA

microarray

This

allowed

us

to

detect

a

variety

of

spontaneous

single

base

pair

substitutions

insertions

and

deletions

and

most

-LRB-

-RRB-

of

the

known

single-nucleotide

polymorphisms

between

two

Saccharomyces

cerevisiae

strains

We

applied

this

approach

to

elucidate

the

genetic

basis

of

phenotypic

variants

and

to

identify

the

small

number

of

single

base

pair

changes

accumulated

during

experimental

evolution

of

yeast

espite

the

ongoing

development

of

DNA

sequencing

technology

-LRB-

-RRB-

it

remains

technically

and

financially

infeasible

for

individual

laboratories

to

sequence

whole

genomes

Moreover

for

global

comparisons

of

genomes

within

species

where

one

expects

a

relatively

small

number

of

sequence

differences

throughout

the

genome

determining

the

entire

sequence

is

unnecessary

In

such

cases

it

is

sufficient

to

assess

the

extent

and

location

of

sequence

variation

in

a

manner

analogous

to

comparative

genomic

hybridization

which

compares

copy

number

changes

between

closely

related

genomes

at

genic

resolution

-LRB-

-RRB-

DNA

microarrays

of

short

oligonucleotides

designed

to

interrogate

each

base

individually

-LRB-

i.e.

resequencing

arrays

-RRB-

have

been

applied

to

the

analysis

of

individual

human

genes

-LRB-

-RRB-

and

small

genomes

such

as

the

human

mitochondrial

-LRB-

-RRB-

and

the

SARS

coronavirus

-LRB-

-RRB-

genomes

However

extension

of

this

approach

to

whole

genomes

of

most

organisms

is

currently

impractical

because

of

the

large

number

of

probes

required

for

complete

coverage

An

alternative

approach

uses

microarrays

that

detect

mismatches

exploiting

the

fact

that

hybridization

to

a

short

oligonucleotide

is

quantitatively

sensitive

to

the

number

and

position

of

mismatches

-LRB-

-RRB-

Sequence-level

differences

are

D

Lewis-Sigler

Institute

for

Integrative

Genomics

of

Molecular

Biology

of

Ecology

and

Evolutionary

Biology

Princeton

University

Princeton

NJ

USA

To

whom

correspondence

should

be

addressed

E-mail

leonid@genomics.princeton.edu

-LRB-

L.K.

-RRB-

dgresham@genomics

princeton.edu

-LRB-

D.G.

-RRB-

detected

without

allele-specific

probes

by

comparing

hybridization

intensities

of

individual

features

on

the

microarray

Ereferred

to

as

single-feature

polymorphisms

-LRB-

SFPs

-RRB-

-LRB-

-RRB-

This

method

has

been

successfully

applied

to

studies

of

genetic

diversity

-LRB-

-RRB-

and

gene

mapping

-LRB-

-RRB-

Until

recently

comprehensive

detection

of

single

base

pair

differences

has

been

limited

by

probe

density

across

the

genome

which

is

typically

a

few

oligonucleotides

per

gene

Even

complete

single-copy

coverage

of

the

genome

is

unlikely

to

be

sufficient

for

finding

all

mutations

because

statistically

detectable

decreases

in

hybridization

intensity

usually

require

that

a

variant

nucleotide

fall

within

the

central

bases

of

a

probe

-LRB-

-RRB-

We

used

high-density

Affymetrix

yeast

tiling

microarrays

-LRB-

YTMs

-RRB-

with

overlapping

oligomers

spaced

an

average

of

base

pairs

-LRB-

bp

-RRB-

apart

to

provide

complete

and

redundant

coverage

of

the

entire

S.

cerevisiae

genome

This

array

design

was

previously

used

to

discover

novel

expressed

sequences

and

to

precisely

map

sites

of

transcription

in

humans

-LRB-

-RRB-

This

design

provides

five

to

seven

measurements

of

a

given

effect

on

hybridization

efficiency

which

we

exploited

to

predict

the

presence

and

location

of

SNPs

and

deletion

breakpoints

throughout

the

entire

yeast

genome

Each

YTM

has

million

perfect

match

-LRB-

PM

-RRB-

probes

and

million

corresponding

mismatch

-LRB-

MM

-RRB-

probes

We

modeled

the

decrease

in

PM

probe

intensity

caused

by

a

single

SNP

as

a

function

of

the

position

within

the

probe

the

GC

content

the

VOL

SCIENCE

Fig.

Nucleotide-level

comparison

with

a

genome

divergent

from

the

sequenced

reference

genome

We

applied

our

approach

to

test

how

many

of

known

SNPs

in

the

yeast

strain

we

were

able

to

detect

Numbers

on

the

graph

indicate

prediction

signal

thresholds

On

the

basis

of

data

from

a

single

hybridization

experiment

we

were

able

to

correctly

identify

as

many

as

SNPs

at

a

prediction

signal

of

At

prediction

signals

of

the

number

of

falsepositive

predictions

is

reduced

to

in

a

test

of

the

reference

genome

and

of

true

positives

are

still

predicted

MARCH

Downloaded

from

on

May

nucleotide

sequence

surrounding

the

SNP

and

the

hybridization

intensity

obtained

using

a

nonpolymorphic

reference

-LRB-

-RRB-

genome

Estrain

-LRB-

-RRB-

To

fit

the

model

we

used

hybridization

data

for

a

training

set

of

nearly

high-quality

SNPs

in

strain

all

identified

by

direct

comparison

of

the

genomic

sequences

-LRB-

-RRB-

The

model

predicts

the

intensity

of

a

probe

in

the

presence

of

a

specified

SNP

-LRB-

-RRB-

-LRB-

figs

and

-RRB-

and

is

used

in

our

algorithm

SNPscanner

which

calculates

the

log

of

the

likelihood

ratio

-LRB-

the

Bprediction

signal

-LSB-

-RRB-

for

the

presence

of

a

SNP

at

each

nucleotide

position

in

the

genome

using

measurements

from

all

probes

that

cover

that

site

By

scanning

the

entire

genome

we

identify

SNPs

as

regions

of

elevated

signal

in

which

the

position

of

the

peak

value

is

considered

the

predicted

polymorphic

site

We

tested

the

performance

of

SNPscanner

on

a

set

of

high-quality

SNPs

from

that

were

not

included

in

the

training

set

We

assessed

the

false-positive

rate

by

using

SNPscanner

to

predict

SNPs

from

an

independent

hybridization

of

the

reference

strain

where

no

true

polymorphisms

are

expected

At

a

prediction

signal

of

we

detected

-LRB-

-RRB-

known

SNPs

in

and

called

false

positives

in

the

reference

strain

-LRB-

fig

-RRB-

By

increasing

the

prediction

signal

to

and

applying

a

heuristic

filter

-LRB-

-RRB-

we

elim

REPORTS

inated

all

false

positives

and

retained

-LRB-

-RRB-

of

real

SNPs

Analysis

of

this

set

of

correctly

predicted

SNPs

showed

the

sequenceconfirmed

SNP

to

be

within

bp

of

the

predicted

site

of

the

time

-LRB-

-RRB-

Fig.

SNPscanner

accurately

predicts

SNPs

in

for

independent

CANR

mutants

-LRB-

A

-RRB-

Multiple

overlapping

probes

cover

each

nucleotide

A

mutation

at

the

site

indicated

in

red

perturbs

hybridization

of

the

sample

to

all

probes

-LRB-

B

-RRB-

The

decrease

in

observed

hybridization

is

used

to

estimate

the

log

of

the

likelihood

ratio

of

the

presence

of

a

polymorphism

versus

the

absence

of

a

polymorphism

-LRB-

the

prediction

signal

-RRB-

The

presence

of

a

SNP

typically

results

in

a

region

of

positive

prediction

signal

with

a

peak

defined

as

the

predicted

SNP

for

the

confirmed

mutation

indicated

in

red

text

in

-LRB-

A

-RRB-

the

entire

sequence

in

green

has

a

positive

prediction

signal

shown

in

-LRB-

B

-RRB-

-LRB-

C

-RRB-

Using

this

approach

we

detected

single

base

pair

substitutions

and

a

deletion

in

four

independent

spontaneous

CANR

mutants

isolated

in

a

reference

genome

background

-LRB-

each

color

represents

a

different

experiment

-RRB-

-LRB-

D

-RRB-

SNPscanner

accurately

predicts

mutations

and

SNPs

in

a

nonreference

genome

The

results

of

nine

independent

CANR

mutants

in

the

CEN.PK

strain

background

are

shown

for

the

entire

gene

We

confirmed

unique

nucleotide

substitutions

for

seven

of

the

mutants

as

well

as

a

single-base

insertion

in

one

mutant

and

a

singlebase

deletion

in

another

At

common

polymorphisms

indicated

in

red

text

the

SNPscanner

signal

is

highly

reproducible

across

multiple

samples

allowing

intrastrain

comparisons

of

nonreference

genomes

To

test

our

ability

to

predict

a

large

number

of

SNPs

we

analyzed

the

highly

diverged

sequenced

strain

originally

recovered

from

an

AIDS

patient

-LRB-

-RRB-

We

selected

a

set

of

sequence-confirmed

SNPs

in

that

were

isolated

from

each

other

by

at

least

bp

and

were

covered

by

probes

on

the

YTM

Analysis

of

a

single

hybridization

with

SNPscanner

yielded

-LRB-

-RRB-

correctly

predicted

SNPs

at

a

prediction

sig

SCIENCE

VOL

MARCH

Downloaded

from

on

May

REPORTS

nal

threshold

of

-LRB-

Fig.

-RRB-

At

a

prediction

signal

threshold

of

we

detected

of

known

SNPs

and

called

only

eight

false

positives

in

a

similar

analysis

of

the

reference

genome

These

false

positives

were

readily

excluded

by

our

heuristic

filter

To

test

our

ability

to

detect

accurately

a

very

small

number

of

sequence

differences

that

distinguish

two

genomes

we

analyzed

spontaneous

mutants

in

the

strain

Independent

clones

from

the

same

archival

isolate

were

grown

and

mutants

in

the

and

genes

were

selected

on

plates

containing

canavanine

sulfate

D-serine

and

D-histidine

or

respectively

-LRB-

-RRB-

For

each

mutant

we

hybridized

total

genomic

DNA

to

a

single

YTM

and

analyzed

the

data

with

SNPscanner

-LRB-

Fig.

A

and

B

-RRB-

In

each

of

four

mutants

we

detected

a

single

peak

at

the

locus

that

fulfilled

our

prediction

criteria

for

a

SNP

-LRB-

Fig.

-RRB-

Amplification

and

sequencing

of

the

locus

identified

a

single-base

substitution

in

each

of

three

mutants

-LRB-

Y

T

Y

G

Y

C

-RRB-

and

deletion

of

a

single

thymine

in

a

run

of

four

thymines

in

the

fourth

-LRB-

-RRB-

Although

the

prediction

signal

for

this

deletion

was

comparatively

low

its

detection

is

noteworthy

because

no

insertions

or

deletions

-LRB-

indels

-RRB-

were

included

in

the

set

of

SNPs

used

to

train

the

model

Analysis

of

DNA

from

a

mutant

resistant

to

D-histidine

and

D-serine

predicted

a

mutation

in

-LRB-

chromosome

XI

-RRB-

which

we

confirmed

as

a

Y

G

substitution

by

sequence

analysis

-LRB-

fig

-RRB-

Similarly

we

accurately

predicted

a

mutation

in

-LRB-

chromosome

XVI

-RRB-

for

a

mutant

resistant

to

-LRB-

Y

T

fig

-RRB-

Thus

we

were

able

to

detect

a

variety

of

single-base

changes

including

a

single-base

deletion

at

several

different

loci

in

the

genome

and

map

them

to

within

bp

of

the

verified

substitution

-LRB-

table

-RRB-

In

addition

to

the

anticipated

mutations

our

analysis

yielded

to

additional

predictions

per

genome

-LRB-

table

-RRB-

We

identified

two

main

causes

of

experimental

noise

-LRB-

i

-RRB-

false

positives

that

fell

within

repetitive

genomic

features

such

as

retrotransposons

and

telomeres

which

we

subsequently

excluded

-LRB-

table

-RRB-

and

-LRB-

ii

-RRB-

manufacturing

defects

in

microarrays

which

we

computationally

removed

-LRB-

-RRB-

-LRB-

fig

-RRB-

We

ranked

the

remaining

predictions

on

the

basis

of

signal

strength

for

each

mutant

and

found

the

expected

mutation

in

the

top

five

predictions

for

all

mutants

except

the

one

resulting

from

an

indel

-LRB-

table

-RRB-

One

SNP

prediction

-LRB-

chromosome

IV

position

sequence

confirmed

as

Y

C

-RRB-

was

common

to

all

samples

suggesting

an

early

mutation

event

that

preceded

later

experiments

-LRB-

perhaps

during

single-colony

purification

from

the

archived

stock

culture

-RRB-

Sequence

confirmation

of

high-quality

predictions

passing

our

filtering

criteria

identified

additional

unique

mutations

in

three

of

the

six

spontaneous

mutants

-LRB-

table

-RRB-

Thus

our

algorithm

is

sufficiently

sensitive

to

detect

a

small

number

of

base

changes

that

distinguish

two

genomes

with

no

a

priori

knowledge

of

the

variants

location

These

results

indicate

that

only

a

small

number

of

mutations

-LRB-

-RRB-

are

associated

with

the

generation

of

spontaneous

drug

resistance

mutants

We

extended

our

approach

to

characterize

the

genome

of

the

unsequenced

laboratory

yeast

strain

CEN.PK

commonly

used

in

continuous

culture

experiments

CEN.PK

shares

ancestry

with

the

reference

strain

but

some

genes

are

absent

in

CEN.PK

-LRB-

-RRB-

We

obtained

a

nucleotide-resolution

comparison

with

the

reference

sequence

by

analyzing

data

with

SNPscanner

from

a

single

hybridization

of

CEN.PK

DNA

CEN.PK

has

a

strikingly

mosaic

structure

with

large

portions

of

the

genome

sharing

essentially

complete

sequence

identity

with

interspersed

with

regions

of

sequence

divergence

and

large

deletions

-LRB-

fig

-RRB-

We

investigated

whether

we

could

detect

single

mutations

on

a

genome-wide

scale

in

a

nonreference

genome

this

was

expected

to

be

a

more

difficult

statistical

problem

-LRB-

-RRB-

We

selected

spontaneous

CanR

mutants

in

the

CEN.PK

strain

background

and

hybridized

genomic

DNA

to

the

YTM

SNPscanner

Fig.

Genome-wide

mutation

detection

facilitates

a

genomic

approach

to

genetics.Whole-genome

analysis

of

a

strain

in

which

was

deleted

but

that

failed

to

demonstrate

the

expected

nonclumpy

phenotype

predicted

the

presence

of

a

deletion

-LRB-

defined

by

the

outermost

peak

values

in

prediction

signal

-RRB-

in

-LRB-

shown

in

its

entirety

-RRB-

Sequence

analysis

confirmed

the

deletion

which

spans

bp

and

is

flanked

by

the

nucleotide

sequence

CTG

and

mapped

the

breakpoints

to

nucleotides

to

Prediction

signal

-LRB-

-RRB-

Chromosomal

coordinate

x

Table

Predicted

SNPs

detected

in

a

yeast

strain

subjected

to

experimental

evolution

Strain

and

Generations

under

sulfur

limitation

Number

of

SNPscanner

predictions

unique

SNPs

unique

SNPs

shared

SNPs

Sequence-confirmed

mutation

Chromosome

IV

Y

A

in

-LRB-

-RRB-

Chromosome

VII

Y

C

in

-LRB-

-RRB-

MARCH

VOL

SCIENCE

Downloaded

from

on

May

tions

correctly

identified

a

mutation

in

of

mutants

as

well

as

three

polymorphic

sites

present

in

the

wild-type

CEN.PK

background

-LRB-

Fig.

-RRB-

We

confirmed

the

sequences

of

all

mutations

and

polymorphisms

in

the

CanR

mutants

Whereas

seven

of

the

nine

detected

mutants

had

base

substitutions

in

one

mutant

contained

a

insertion

and

another

had

a

deletion

All

mutations

were

confirmed

as

lying

within

bp

of

the

predicted

site

-LRB-

table

-RRB-

SNPscanner

prediction

signals

were

highly

reproducible

across

multiple

experiments

We

compared

genome-wide

SNP

predictions

for

each

CEN.PK

mutant

to

SNP

predictions

for

CEN.PK

wild-type

DNA

and

applied

our

heuristic

filter

-LRB-

-RRB-

This

resulted

in

the

prediction

of

fewer

than

SNPs

genome-wide

that

were

not

predicted

to

exist

in

wild-type

CEN.PK

for

of

mutants

-LRB-

table

-RRB-

In

most

cases

excluding

those

predictions

that

fell

in

repetitive

regions

further

reduced

the

total

number

By

using

this

approach

we

retained

the

identified

mutation

for

seven

of

nine

mutants

We

ranked

the

remaining

predictions

and

observed

that

the

sequence-confirmed

mutation

was

in

the

top

predictions

for

all

seven

mutants

So

even

in

this

somewhat

more

challenging

case

our

system

succeeded

in

detecting

most

of

the

single-nucleotide

sequence

differences

and

mapping

them

within

a

few

nucleotides

Mutations

predicted

in

our

REPORTS

collection

of

CEN.PK

and

spontaneous

mutants

corresponded

to

of

the

possible

base

substitutions

that

resulted

in

six

of

the

eight

possible

mismatches

between

probe

and

sample

Thus

our

method

can

detect

single

base

pair

indels

in

addition

to

virtually

all

base

substitutions

We

sought

to

apply

our

genome-wide

mutation

detection

approach

to

biological

questions

that

had

remained

refractory

to

traditional

genetic

techniques

We

complemented

a

positional

cloning

project

to

predict

and

confirm

mutations

in

a

peripheral

mitochondrial

inner

membrane

protein

-LRB-

-RRB-

that

are

causative

of

a

growth

defect

on

a

nonfermentable

carbon

source

-LRB-

-RRB-

-LRB-

table

-RRB-

We

also

used

our

method

to

determine

the

genetic

basis

of

an

unusual

phenotype

Deletion

of

results

in

upregulation

of

daughter-specific

genes

and

a

nonclumpy

growth

phenotype

-LRB-

-RRB-

However

when

we

deleted

in

an

strain

-LRB-

-RRB-

we

recovered

a

transformant

that

displayed

low

expression

of

daughter-specific

genes

and

a

clumpy

phenotype

-LRB-

strain

-RRB-

Deletion

of

in

was

confirmed

by

sequence

analysis

and

independent

deletions

of

in

both

and

yielded

the

expected

phenotype

which

suggested

the

presence

of

a

suppressor

mutation

in

Preliminary

genetic

analysis

tended

to

indicate

the

presence

of

an

unlinked

suppressor

mutation

We

hybridized

genomic

DNA

from

to

the

YTM

Analysis

using

SNPscanner

confirmed

the

deletion

of

-LRB-

-RRB-

and

identified

an

additional

deletion

on

chromosome

XII

-LRB-

Fig.

-RRB-

The

predicted

deletion

spans

kb

and

includes

the

majority

of

the

coding

region

of

Subsequent

sequence

analysis

confirmed

that

the

predicted

breakpoints

were

within

bp

of

the

actual

sites

-LRB-

Fig.

-RRB-

The

deletion

of

provides

a

plausible

explanation

for

both

aspects

of

the

aberrant

phenotype

of

encodes

a

transcription

factor

that

is

thought

to

drive

the

transcription

of

genes

with

daughter-specific

expression

-LRB-

-RRB-

Its

absence

in

probably

causes

a

low

expression

of

the

daughter

cell

specific

genes

some

of

which

are

required

for

cell

separation

after

budding

-LRB-

e.g.

which

encodes

chitinase

-RRB-

Moreover

deletion

of

alone

results

in

a

clumpy

phenotype

-LRB-

-RRB-

and

clumpiness

segregates

with

the

locus

in

a

cross

between

and

-LRB-

-RRB-

Previous

studies

have

shown

the

occurrence

of

characteristic

gene

expression

patterns

-LRB-

-RRB-

and

large-scale

gene

duplication

and

deletion

-LRB-

-RRB-

in

yeast

cultures

that

are

experimentally

evolved

under

a

nutrient-limiting

condition

However

the

extent

and

nature

of

nucleotide

changes

that

occur

during

this

process

have

remained

completely

unknown

We

sought

to

assess

the

degree

of

sequence

variation

that

had

accumulated

in

a

strain

of

yeast

subjected

to

experimental

evolution

under

sulfur

limitation

in

continuous

culture

We

compared

the

SNPscanner

signals

obtained

from

DNA

of

the

ancestral

strain

CEN.PK

to

those

signals

obtained

from

DNA

of

two

clones

from

the

same

population

that

had

undergone

experimental

evolution

under

sulfur

limitation

for

-LRB-

-RRB-

and

-LRB-

-RRB-

generations

We

compared

our

set

of

predictions

to

those

made

for

CEN.PK

CANR

mutants

to

exclude

common

predictions

that

were

the

result

of

systematic

error

SNP

predictions

that

fell

within

repetitive

regions

were

considered

to

be

unreliable

and

were

excluded

from

further

analysis

At

a

prediction

signal

of

we

called

a

small

number

of

predicted

SNPs

in

strains

and

of

which

were

common

to

the

two

strains

-LRB-

Table

-RRB-

We

confirmed

the

sequences

of

single

strain-specific

mutations

found

in

and

-LRB-

Table

-RRB-

The

relatively

small

number

of

mutations

strongly

suggests

that

the

events

associated

with

adaptive

evolution

in

chemostats

do

not

involve

even

transient

genome-wide

mutagenesis

this

number

is

also

consistent

with

the

experience

that

in

yeast

evolved

strains

are

rarely

if

ever

found

to

have

mutator

phenotypes

-LRB-

-RRB-

This

is

in

contrast

to

studies

of

Escherichia

coli

grown

in

batch

conditions

in

which

mutator

phenotypes

have

been

observed

in

numerous

independent

cultures

-LRB-

-RRB-

The

small

number

of

mutations

identified

in

our

experiments

means

that

it

will

be

feasible

to

comprehensively

identify

and

experimentally

verify

mutations

that

are

important

for

adaptation

during

studies

of

experimental

evolution

On

the

basis

of

a

single

experimental

hybridization

we

are

able

to

accurately

detect

the

single-nucleotide

changes

that

distinguish

two

genomes

Recently

a

similar

microarray

design

has

been

used

as

a

preliminary

screen

to

identify

possible

mutations

in

the

pathogen

Helicobacter

pylori

-LRB-

-RRB-

In

this

method

the

initial

screen

is

followed

by

the

manufacture

of

targeted

resequencing

microarrays

Our

method

relies

on

only

a

single

experiment

to

derive

a

statistical

measure

of

the

likelihood

of

a

polymorphism

at

a

particular

site

Our

approach

is

optimal

when

direct

comparisons

are

made

to

the

reference

strain

represented

on

the

microarray

However

we

are

also

able

to

compare

two

nonreference

genomes

and

identify

the

SNPs

that

distinguish

them

with

only

minimal

added

cost

in

terms

of

false

negatives

and

false

positives

Although

our

algorithm

is

trained

on

a

set

of

known

base

substitutions

we

found

that

it

also

detected

single-base

deletions

and

insertions

as

well

as

large

deletions

with

nearnucleotide

accuracy

in

the

prediction

of

breakpoints

Any

genomic

variation

that

results

in

novel

sequence

-LRB-

such

as

inversions

or

retrotransposon

insertions

-RRB-

should

in

principle

be

detectable

by

SNPscanner

We

expect

that

the

simplicity

and

affordability

of

this

method

will

enable

individual

SCIENCE

VOL

laboratory

groups

to

devise

and

use

new

and

truly

comprehensive

genomic

approaches

to

Mendelian

and

complex

genetics

and

to

the

characterization

of

mutants

obtained

through

genetic

and

suppressor

screens

In

addition

complete

knowledge

of

nucleotide

diversity

will

allow

us

to

address

questions

regarding

the

mutagenic

effect

of

phenomena

such

as

aging

and

recombination

on

a

genome-wide

scale

By

representing

entire

genomes

of

other

organisms

on

oligonucleotide

microarrays

with

a

similar

redundant

design

it

is

likely

that

our

approach

may

be

extended

to

higher

organisms

Although

increased

genome

complexity

presents

a

challenge

reports

of

successful

SFP-based

genotyping

in

Arabidopsis

-LRB-

-RRB-

which

has

a

genome

of

Mb

suggest

that

genome-wide

prediction

of

all

sequence

variants

may

be

possible

in

larger

genomes

including

those

of

model

organisms

such

as

Caenorhabditis

elegans

and

Drosophila

melanogaster

We

analyzed

haploid

genomes

and

a

single

homozygous

diploid

genome

as

with

all

sequencing

technologies

identifying

heterozygosity

in

diploid

genomes

represents

the

ultimate

challenge

References

and

Notes

M.

Margulies

et

al.

Nature

-LRB-

-RRB-

J.

Shendure

et

al.

Science

-LRB-

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published

online

August

-LRB-

science

-RRB-

J.

R.

Pollack

et

al.

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

J.

G.

Hacia

L.

C.

Brody

M.

S.

Chee

S.

P.

Fodor

F.

S.

Collins

Nat

Genet

-LRB-

-RRB-

A.

Maitra

et

al.

Genome

Res

-LRB-

-RRB-

C.

W.

Wong

et

al.

Genome

Res

-LRB-

-RRB-

U.

Maskos

E.

M.

Southern

Nucleic

Acids

Res

-LRB-

-RRB-

E.

A.

Winzeler

et

al.

Science

-LRB-

-RRB-

T.

L.

Turner

M.

W.

Hahn

S.

V.

Nuzhdin

PLoS

Biol

-LRB-

-RRB-

S.

K.

Volkman

et

al.

Science

-LRB-

-RRB-

E.

A.

Winzeler

et

al.

Genetics

-LRB-

-RRB-

J.

O.

Borevitz

et

al.

Genome

Res

-LRB-

-RRB-

R.

B.

Brem

G.

Yvert

R.

Clinton

L.

Kruglyak

Science

-LRB-

-RRB-

published

online

March

-LRB-

science

-RRB-

A.

M.

Deutschbauer

R.

W.

Davis

Nat

Genet

-LRB-

-RRB-

J.

Ronald

R.

B.

Brem

J.

Whittle

L.

Kruglyak

PLoS

Genet

-LRB-

-RRB-

L.

M.

Steinmetz

et

al.

Nature

-LRB-

-RRB-

G.

Yvert

et

al.

Nat

Genet

-LRB-

-RRB-

J.

Ronald

et

al.

Genome

Res

-LRB-

-RRB-

J.

Cheng

et

al.

Science

-LRB-

-RRB-

published

online

March

-LRB-

science

-RRB-

See

supporting

material

on

Science

Online

Z.

Gu

et

al.

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

P.

Daran-Lapujade

et

al.

FEMS

Yeast

Res

-LRB-

-RRB-

T.

P.

Ellis

K.

G.

Helfenbein

A.

Tzagoloff

C.

L.

Dieckmann

J.

Biol

Chem

-LRB-

-RRB-

D.

Gresham

et

al.

data

not

shown

A.

Colman-Lerner

T.

E.

Chin

R.

Brent

Cell

-LRB-

-RRB-

W.

J.

Racki

A.

M.

Becam

F.

Nasr

C.

J.

Herbert

EMBO

J.

-LRB-

-RRB-

T.

L.

Ferea

D.

Botstein

P.

O.

Brown

R.

F.

Rosenzweig

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

M.

J.

Dunham

et

al.

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

P.

D.

Sniegowski

P.

J.

Gerrish

R.

E.

Lenski

Nature

-LRB-

-RRB-

T.

J.

Albert

et

al.

Nat

Methods

-LRB-

-RRB-

MARCH

Downloaded

from

on

May

REPORTS

S.

P.

Hazen

et

al.

Plant

Physiol

-LRB-

-RRB-

We

thank

E.

Foss

for

creation

of

strain

E.

Smith

for

analysis

of

mapping

data

and

D.

Storton

and

J.

Matese

for

technical

support

Supported

by

National

Institute

of

General

Medical

Sciences

grant

and

center

grant

-LRB-

D.B.

-RRB-

NIH

grant

and

James

S.

McDonnell

Foundation

grant

-LRB-

L.K.

-RRB-

and

Pew

Charitable

Trusts

award

-LRB-

S.C.P.

-RRB-

The

raw

data

and

software

are

available

at

SNPscanner

Figs

to

Tables

to

References

December

accepted

February

Published

online

March

science

Include

this

information

when

citing

this

paper

Supporting

Online

Material

Materials

and

Methods

SOM

Text

Rice

Domestication

by

Reducing

Shattering

Changbao

Li

Ailing

Zhou

Tao

Sang

Crop

domestication

frequently

began

with

the

selection

of

plants

that

did

not

naturally

shed

ripe

fruits

or

seeds

The

reduction

in

grain

shattering

that

led

to

cereal

domestication

involved

genetic

loci

of

large

effect

The

molecular

basis

of

this

key

domestication

transition

however

remains

unknown

Here

we

show

that

human

selection

of

an

amino

acid

substitution

in

the

predicted

DNA

binding

domain

encoded

by

a

gene

of

previously

unknown

function

was

primarily

responsible

for

the

reduction

of

grain

shattering

in

rice

domestication

The

substitution

undermined

the

gene

function

necessary

for

the

normal

development

of

an

abscission

layer

that

controls

the

separation

of

a

grain

from

the

pedicel

ereals

the

primary

food

were

domesticated

from

wild

grass

species

Because

wild

grasses

naturally

shed

mature

grains

a

necessary

early

step

toward

cereal

domestication

was

to

select

plants

that

could

hold

on

to

ripe

grains

to

allow

effective

field

harvest

-LRB-

-RRB-

-LRB-

fig

-RRB-

The

selection

process

might

have

been

mainly

unconscious

because

grains

that

did

not

fall

as

easily

had

a

better

chance

of

being

harvested

and

planted

in

the

following

years

Consequently

nonshattering

alleles

had

an

increased

frequency

and

eventually

replaced

the

shattering

alleles

during

domestication

The

finding

that

one

locus

accounted

for

most

phenotypic

variance

of

grain

shattering

between

a

cereal

crop

and

its

wild

progenitor

suggested

that

the

domestication

process

could

have

been

initiated

quickly

by

selection

at

the

locus

-LRB-

-RRB-

The

molecular

genetic

basis

of

the

selection

however

has

not

been

characterized

Rice

-LRB-

Oryza

sativa

-RRB-

was

domesticated

from

one

or

both

of

two

closely

related

species

O.

nivara

and

O.

rufipogon

distributed

from

southeastern

Asia

to

India

-LRB-

-RRB-

Our

recent

genetic

analysis

of

an

population

derived

between

O.

sativa

ssp

indica

and

the

wild

annual

species

O.

nivara

identified

three

quantitative

trait

loci

-LRB-

QTL

-RRB-

and

responsible

for

the

reduction

of

grain

shattering

in

cultivated

rice

-LRB-

-RRB-

Of

these

QTL

explained

of

phenotypic

variance

and

the

other

two

explained

and

of

phenotypic

variance

The

allele

of

the

wild

species

caused

shattering

and

was

dominant

C

Department

of

Plant

Biology

Michigan

State

University

East

Lansing

MI

USA

To

whom

correspondence

should

be

addressed

E-mail

sang@msu.edu

Two

previous

QTL

studies

using

crosses

between

O.

sativa

ssp

indica

and

the

wild

perennial

species

O.

rufipogon

detected

four

and

five

shattering

QTL

-LRB-

-RRB-

Both

studies

identified

a

QTL

at

the

same

location

of

with

either

the

largest

or

nearly

largest

phenotypic

effect

among

the

detected

QTL

Moreover

genetic

analyses

between

O.

sativa

ssp

japonica

and

O.

rufipogon

and

two

other

closely

related

wild

species

O.

glumaepetula

and

O.

meridionalis

all

found

that

a

single

dominant

allele

from

each

of

the

three

wild

species

was

responsible

for

grain

shattering

-LRB-

-RRB-

This

locus

named

was

mapped

to

the

same

chromosomal

location

as

Our

QTL

analysis

located

between

simple

sequence

repeat

-LRB-

SSR

-RRB-

markers

and

-LRB-

-RRB-

which

had

a

physical

distance

of

about

kb

in

the

O.

sativa

genome

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

Because

of

the

large

and

dominant

effect

of

the

O.

nivara

allele

we

were

able

to

phenotypically

distinguish

individuals

that

were

homozygous

recessive

-LRB-

ss

-RRB-

from

those

that

had

at

least

one

O.

nivara

allele

of

-LRB-

ns

and

nn

-RRB-

regardless

of

the

genotypes

at

the

remaining

two

QTL

of

small

effect

After

evaluating

a

total

of

plants

genotyped

at

the

three

shattering

QTL

we

consistently

found

that

plants

with

the

ns

and

nn

genotypes

at

shed

all

mature

grains

when

hand

tapped

whereas

plants

with

the

ss

genotype

at

did

not

shed

grains

or

only

partially

shed

mature

grains

under

vigorous

hand

shaking

With

the

reliable

phenotyping

method

available

we

grew

seedlings

and

screened

for

recombinants

between

and

-LRB-

-RRB-

Plants

with

the

genotype

of

ss

at

one

marker

and

ns

at

the

other

were

selected

and

a

total

of

individuals

were

grown

for

phenotypic

VOL

SCIENCE

evaluation

By

progressively

examining

SSR

and

SNP

-LRB-

single-nucleotide

polymorphism

-RRB-

markers

between

and

we

finally

mapped

the

mutation

responsible

for

the

derivation

of

nonshattering

in

cultivated

rice

to

a

region

of

a

gene

with

a

previously

unknown

function

-LRB-

Fig.

and

table

-RRB-

The

gene

is

predicted

to

be

a

transcription

factor

and

its

coding

region

is

physically

located

between

and

base

pairs

-LRB-

bp

-RRB-

on

assembly

of

rice

chromosome

-LRB-

The

TIGR

Rice

Genome

Annotation

Database

-RRB-

The

comparison

of

the

sequences

between

the

mapping

parents

revealed

seven

mutations

-LRB-

Fig.

-RRB-

These

include

one

mutation

in

the

intron

-LRB-

a

-RRB-

a

substitution

three

mutations

in

the

first

exon

-LRB-

b

-RRB-

a

or

five

amino

acid

insertion/deletion

-LRB-

c

-RRB-

a

or

one

amino

acid

insertion/deletion

and

-LRB-

d

-RRB-

a

or

an

amino

acid

substitution

and

three

mutations

upstream

of

the

start

codon

-LRB-

e

-RRB-

a

substitution

at

site

-LRB-

f

-RRB-

a

insertion

deletion

between

sites

and

and

-LRB-

g

-RRB-

an

insertion/deletion

between

sites

and

To

assess

the

polymorphism

and

evolutionary

direction

of

these

mutations

we

sequenced

this

region

from

an

additional

rice

cultivars

representing

the

diversity

of

O.

sativa

-LRB-

-RRB-

accessions

of

O.

nivara

covering

the

distributional

range

of

the

wild

species

-LRB-

-RRB-

accessions

of

O.

rufipogon

and

accession

of

each

of

the

four

remaining

wild

A-genome

species

-LRB-

Fig.

and

table

-RRB-

The

cultivars

were

polymorphic

for

mutation

f

i.e.

some

of

the

cultivars

had

the

same

sequence

as

O.

nivara

At

the

remaining

six

mutation

sites

all

cultivars

shared

the

same

sequences

which

were

different

from

those

of

the

O.

nivara

parent

Surprisingly

three

accessions

of

O.

nivara

had

the

same

sequences

as

O.

sativa

at

these

six

sites

It

was

then

found

that

plants

grown

from

these

accessions

had

the

nonshattering

phenotype

Greenhouse

observations

indicated

that

these

accessions

had

additional

characteristics

of

cultivated

rice

that

were

not

found

in

O.

nivara

such

as

upright

tillers

short

awns

and

or

photoperiod

sensitivity

This

suggests

that

the

three

accessions

are

weedy

rice

that

has

received

and

fixed

the

allele

from

cultivars

The

remaining

accessions

of

the

wild

species

with

confirmed

shattering

differed

invariably

from

the

cultivars

by

one

mutation

d

which

was

a

nucleotide

substitution

of

G

for

T

or

an

amino

acid

substitution

of

MARCH

Downloaded

from

on

May

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O