ofmatK primers for barcoding gymnosperms

ofSystematics and Evolution
Journal
49(3):169-175(201 1)
doi:10.1111/j.1759—6831.2011.00128.X
Research Article
High universality of matK primers for barcoding gymnosperms
1,2yan LI
l,一,4Ram C.POUDEL
ILian.Ming GA04
l,3De.Zhu Li
5Alan FORREST
1(KeyLaboratoryofBiodiversityandBiogeography,KunmingInstituteofBotany,ChineseAcademyofSciences,Kunming 650204,China)
2(Institute ofAlpineEconomicPlants,YunnanAcademyofAgriculturalSciences,Lijians 674100,China)
3(GermplasmBankoffieldSpeciesinSouthwestChina,KunmingInstitute ofBotany,ChineseAcademyofSciences,Kunmjn9650204,China)
4(The Graduate University ofChineseAcademy ofSciences,Beijing 100049,China)
5(Royal Botanic Garden Edinburgh.Edinburgh EH3 5LIL UK)
DNA barcoding is a tool to provide rapid and accurate taxonomic identification using a standard DNA
two-marker
combination of motK+rbcL was formally proposed as the core barcodc for land plants by
region.A
the Consortium for the Barcode of Life Plant Working Group.HoweveL廿1ere are currently no barcoding primers
Abstract
for matK showing high universality in gymnosperms.W-e used 57 gymnosperm species representing 40 genera.1l
families and four subclasses to evaluate the universality of nine candidate matK primers and one rbcL primer in this
as a universal primer for gymnosperms due to high universality.
One ofthe nine candidate matK primers(Gym_F1A/Gym_R1A)is proposed as the best‘、miversal”matK primer for
gyrrmosperms because ofhigh polymerase chain reaction success and routine generation ofhigh quality bidirectional
well on the
sequences.A specific mark primer for Ephedra was newly designed in this study,which
study.Primer f l F/724R)of而吐is proposed here
performed
sampled species.111e primers proposed here for rbcL and matK
be easily and successfully
can
amplified for most
gymnosperms.
Key words DNA barcoding。matK,gymnosperm,primer universality.
DNA barcoding is
technique to provide rapid and
a specific DNA
a
accurate taxonomic identifcation using
region(Hebert&Gregory,2005).it has become
a
useful
tool for species
identification(Kress&Erickson.2007;
Erickson et a1.,2008),and discovering new or cryptic
species(Newmaster&Ragupathy,2009;VralentiIli et a1..
2009).The mitochondrial cytochrome c oxidase subunit
1(coJ『1
has been used
as
titleation in many animal
a
barcode for species iden—
groups(Hebert
et
a1..2003).
ever,universality of
low in some
matK primers was reported to be
studies(Sass
et
limit
its
use
et
a
noristic study was reported
of matK primers for
a1.,2009).Improvement
increase universality in
angiosperms,and
recommendation as part of the
barcode(Lahaye et a1.,2008;CBOL Plant Work-
barcoding
can
was
reason
a
core
key
for its
Group.2009).Although high
ing
suitable for barcoding
to
the search
substitutions in matK make it
for alternative
et
a1.,2005;
Cowan et a1.。2006;Fazekas et a1.,2008;Hollingsworth
et
al。2009).Recently,a two—marker combination
of
matK+rbcL was recommended as the core barcode for
C:onsortium
land plants bv the
(CBOL)(CBOL Plant
for the Barcode of Lifc
Working Group,2009).
Universality ofprimers for polymerase chain Feac—
tion(PCRl and sequencing is one ofthe most important
criteria for DNA
a1.,2009),which may
barcodc.For example.69%success
for matK in
recovery
(Kress
as a
However,the low substitution rate of CDl makes it tin-
plants.and has led
barcoding regions(Kress
a1.,2007;Fazekas et a1.,
2008;Fbrd et a1.。2009;Kress et
a
levels of nucleotide
good barcode marker in
terms of species identification.it is diffcult
to
routinely
amplify and sequence across divergent lineages and as
such more studies to develop‘‘universal’’matK primers
are necessary(Kress&Efickson.2007;Hollingsworth,
Plant Working Group。2009;Ford et a1.,
2008;CBOL
2009).Recently,order-specific matK primers for an-
giosperms
were suggested by Dunning&Savolainen
(20 l O).Nevertheless.there are still significant chal.
a1..2007;Kress
1enges that need to be overcome for high—throughput
&Erickson.2007;Ford et a1.。2009;Hollingsworth
et a1..2009).In the core barcode,the primers for rbcL
floristic studies using this DNA region for barcoding
show
a
barcoding(Chase
et
Gymnospenns are a group of seed-bearing plants
mat consist of four subclasses,Cycadidae,Ginkgoidae,
Accepted:23 February 201 1
correspondenee.E.mail:gaolrn国mail.kib.ac.ca;Tel.:86-871-
Gnetidae.and Pinidae,representing 8 orders,1 2
families,83 genera,and approximately 990 species
(Christenhusz et a1..20 1 1).Gymnosperms are all woody
Plant W-orking
Group.2009;Kress
et
Received:23 December 2010
+Author for
plants.
plants(CBOL
a1.,2009).How-
high level of universality in land
5223505;Fax:86・871-5217791.
@201 1 Institute ofBotany,Chinese Academy ofSciences
万方数据
trees。shrubs
or
lianas.They grow
throughout most ofthe
170
Journal ofSystematics and Evolution
world,and
V01.49
No.
the dominant vegetation in many colder
are
and arctic
regions(Judd et a1.,2007).Gymnosperms
have major economic uses:many are important wood
resources and omamental plants.In China.there are
3
201 1
of Botany,Chinese Academy of
Sciences(KUN).Leaf
material was collected in the feld
or
from Kunming
Botanic Garden.and immediately dried in silica gel
til laboratory analysis.
un-
about 250 gymnosperm species in 34 genera and l 0
families(Wu&Raven.1 999).For gymnosperms.there
is
no
universal specific matK primer for DNA barcoding
that has
performed
well to date.Several matK primers
1.2 Candidate primers screening strategy
The nine candidate matK primer pairs and
proposed for barcoding land plants show low levels of
PCR success in gymnosperms fCBOL Plant W6rking
nine matK primer pairs
Group。2009;Ford et a1.。2009;Liu et a1.201 11.For
matK primer pairs
example.the PCR
success
rate
is less than 50%in gym—
nosperms based on data compiled from severallabora-
toties(CBOL Plant working Group,2009),and only
33%PCR
success
tor
pnmer pair
et
was obtained with the best matK
sampled
a1.r2009).The
gymnosperm
fact that
have been developed
as
no
species by pord
specific matK primers
barcodes for gymnosperms may
not
have
are
those most commonly used for
species representing the 11 families for PCR amplifi—
Cupressaceae.two
samples from each were included
in this family—level
assessment(see
success
amplification is essential for its application
and
cation with aU candidate matK primers and one rbcL
primer as contr01.For the large families Pinaceae and
velopment to improve the overall
of matK
recently designed
barcoding land plants.We adopted two steps to test the
universality of candidate primers.First。we selected 14
candidate
rate
are
been tested systematically,and the remaining three
explain the low amplification of this region.Primer de.
success
sin—
a
gle而cL primer pair are outlined in Table 2.Six of the
1’able l 1.Second
matK primers showing more
than 50%PCR
were used for further assessment
all remain.
on
ing
ofthe nine matK primer pairs met this
universal plant
samples(five
requirement).
matK primer for the whole plant kingdom because of
1.3
DNA extraction.amplification,and sequencing
Genomic DNA was extracted from silica.dried leaf
as
part
ofthe
barcode(CBOL Plant Working Group.
2009).However,it is a challenge to develop a universal
an
extensive variation within the DNA region(Kress
&Erickson 2007).Further primer development efforts
are required in non—angiosperms for maⅨfCBOL Plant
material using the CTAB method
Wbrking Group.2009).
buffer r 1 0 mmol/L Tris.HCI。pH 8.0,1 mm01/L EDTA)
to a final concentration of 40一50 ne;/“L to avoid any
In this study,we used nine candidate matK primers
and one而cL primer to evaluate the PCR and sequencing
vailation in PCR
universality for gymnosperms.Six ofthe nine
differences.
matK primers
candidate
recently designed with five as yet an—
the remaining three were proposed as
“universal”primers for barcoding land plants.W色se.
1ected 57 species.representing 40 genera of 1l families
tllat correspond to all four subclasses of
for trial in this study.Our aim here is to
universality
of the matK primers for
gymnosperm
evaluate
the
gymnosperms.
described by Doyle
due to
success
DNA concentration
A11 PCR reactions were carried out
are
published,and
as
&Doyle f1 987).The genomic DNA was dissolved in TE
PCR System 9700
ter
thermal
City,CA,USA)in
contained 10 tzL 2
a
on a
cycler(Perkin
GeneAmp
Elmer,Fos.
total volume of 20“L,which
Taq PCR Master Mix f0.1 U Taq
x
polymerase//zL.0.5 mmol/L each dNTE 20 mmol/L
Tris—HCl(pH 8.3),1 00 mmoI/L KCl,3 mmoI/L MgCl2:
Tiangen
Biotech,Beijing,China),0.2/zL
bovine serum
albumin(1 0肛g/uL),0.5/zL each of the forward and
reverse primers(1 0/zmoI/L),and l“L template DNA.
Material
and methods
Negative controls were
run
alongside all PCR reactions.
The matK thermocycling profile was:94。C for 3 min.35
1.1 Sampling strategy
A total of 57 species representing 40 genera.11
families,and 8 orders were selected in this study(Ta-
cycles of94。C for 30 s,52。C for 30 s,72。C for 45 s,with
ble 1),covering approximately 47%of genera,92%of
50 s.52。C for 1 min,72。C for 1 min,with
families。88%of orders。and 1 00%of the subclasses
sion of72。C for l 0 min.The PCR products were visual.
ized on 1%Tris-acetate_EDl'A agarose gel stained with
of
era
gymnosperms.These
and families
samples represent all the gen.
in China.To represent the
OCCu币ng
a final
extension of72。C for 1 0 min.The rbcL thermocy.
cling profile was:94。C for l min.30 cycles of94。C for
ethidium bromide alongside
a
a
final exten.
GeneRuler 1 00 bp DNA
wider diversity of larger genera rthose containing>20
ladder(rermentas。Glen Burnie,MD’USA).The
species occurring in China),more than one species per
genus were included in the analysis.Voucher specimens
products were purified using ExoSAP.IT fGE Health.
were deposited in the herbarium of Kunmmg Institute
carried out in
PCR
care,Cleveland,OH,USA).Sequencing reactions were
a
total
volume of 6“L containing 0.2“L
@201 1 Institute of Botany,Chinese Academy of Sciences
万方数据
LI et
a1.:marK primers for gymnosperms
171
Gym_F1A
Gym_F2A
Gym_FIB
Gym_F2B
P刚
Gym_R1A
Gym_R2A
Gym_RIB
Gym_R2B
PKRl
Glm-06032
++
++
++
+
+
Cycadidae
Cycadaceae
Cycadidae
Cycadaceae
Cycas guizhouens括t
Cycas micholitzii
Ly・012
++
++
++
+
++
Cycadidae
Zamiaceae
Encephalartos lehmann舻Ly-014
++
++
++
+
+
Ginkgoidae
Ginkgoaceae
Ginkgo
一
+
+
+
+
Gnetidae
Ephedraceae
Gnetidae
Ephedraceae
Ephedra gerardiana
Ephedra intermedia
Gnetidae
Gnetidae
Ephedraceae
Gnetaceae
Pinidae
Araucariaceae
Gnetum pendulumt
Araucarla bidwillii
Pinidae
Araucariaceae
Araucaria cunninghamiP
Pinidae
Araucariaceae
Araucaria heterophylla
Pinidae
Cupressaceae
Pinidae
bff0幻’
Ephedra likiangensis.I
Ly一028
090937—‘—。———。—。
Sunh-zx一1688
一
一
一
一
一
Ly-024———。—。—。—。
++
++
GrBOWS0801
++
一
一
Ly—Ol 5
++
一
++
+
++
Ly一016
++
一
++
+
++
++
一
++
+
++
Calocedrus macrolepis
Ly—004
Glm—103074
++
一
++
+
一
Cupressaceae
Chamaecyparis加rmosensis
Glm-103065
++
++
++
一
+
Pinidae
Cupressaceae
Ly-007
++
++
++
+
++
Pinidae
Cupressaceae
Cunni,29hamia lanceolata
Cupressus duclouxiana
一
++
+
+
Cupressaceae
Cryptomeriajaponica
Ly一01l
Glm一103142
++
Pinidae
++
一
十+
+
++
Pinidae
Cupressaceae
Folaenia hodginsii
Ly-006
++
++
++
++
++
Pinidae
Cupressaceae
Ly-005
++
++
++
++
++
Pinidae
Cupressaceae
Glyptostrobuspensilis
dun由eru¥pingii vaL wiIsonii
一
++
一
一
Cupressaceae
++
一
十+
一
一
Pinidae
Cupressaceae
dunit,erus squamatat
Metasequoia glyptostroboidest
Ly一026
G1m-082126
++
Pinidae
Ly—002
++
++
++
+
++
Pinidae
Cupressaceae
Platyctadus orientalis
Ly一020
++
一
++
+
一
Pinidae
Cupressaceae
Ly一021
Glm-l 03046
++
++
++
+
+
++
++
++
+
++
Glm-09241 1
++
++
++
++
++
Ly一009
++
+
++
+
++
Ly・019
孤“缸suwhuenens括Ly-01 8
++
++
++
+
++
++
++
++
++
++
Glm-102904
++
++
++
++
++
Ly-017
++
+
++
++
++
Ly_0口9
++
++
++
++
++
Pinidae
Cupressaceae
Sequoia sempervirens
Taiwania cryptomerioides
Pinidae
Cupressaceae
Taiwania砌usiana
Pinidae
Cupressaceae
Taxo硪um distichum
Pinidae
Cupressaceae
Thujopsis dolabrata
Pinidae
Pinidae
Cupressaceae
Pinaceae
Pinidae
Pinaceae
Pinidae
Pinaceae
Cathaya argyrophylla
Cedrus deodara
Pinidae
Pinaceae
Keteleeria davidiana
Ly-001
++
+
++
+
++
Pinidae
Pinaceae
Kete彪eria evelyniana
Ly一003
++
+
++
++
++
Pinidae
Pinaceae
Keteleeriafortunei
++
+
++
+
十十
Pinidae
Pinaceae
++
一
十+
++
++
Pinidae
Pinaceae
Lar/xpotaninii var.chinensis
Larixhimalaica
Ly・010
G1m-06081
Glm-081604
++
一
++
+
++
Pinidae
Pinaceae
Pinidae
Abies george:
Glm一08912
++
一
++
+
++
Pinaceae
Picea liMangens括
Picea smithiana
GIm-08 1 533
++
一
十+
+
++
Pinidae
Pinaceae
Picea spinulosa
Glm-08 l 883
++
一
++
++
++
Pinidae
Pinaceae
Pitills arman硪f
Ly・027
++
一
++
++
++
Pinidae
Pinaceae
Pinus wa肼chiana
81471
++
一
++
+
++
Pinidae
Pinaceae
Pinus yunnanens捃t
Ly一025
++
+
++
++
++
Pinidae
Pinaceae
Pseudolarix amabilis
++
+
++
++
++
Pinidae
Pinaceae
++
一
++
一
十
Pinidae
Pinaceae
Pseudotsuga sinensis
Tsuga dumosa
Ly一008
Glm—l 03063
Glm一081797
++
一
++
一
++
Pinidae
P0docarpaeeae
Dacrycarpus imbricatust
Ly一023
++
++
++
++
+
Pinidae
Podocarpaceae
Nageiafleuryi
++
++
++
++
++
Pinidae
Podocarpaceae
Nageia nagi
Ly一022
Ly-01 3
++
++
++
++
一
Pinidae
Podocarpaceae
Podocarpus macrophyllus
GBOWS0294
++
++
++
++
一
Pinidae
Podocarpaceae
Podocarpus
Glm062 11
++
++
++
+
一
Pinidae
SciadopitVS verticiff4衄t
Amentotaxus argotaenia
G1m-09224l
++
+
++
+
+
Pinidae
Sciadopitvac七ae
Taxaceae
Pinidae
Taxaceae
Cephalotaxus
Pinidae
Pinidae‰【aceae
Taxaceae
Cephaiotaxus wUsonianat
Pseudotaxus chienii
Pinidae
Taxus wfifllichianat
mannii
1 82
++
++
++
++
十
ZSD001
++
+十
++
++
++
Glm-103119
++
++
++
++
++
Glm-07549
++
+十
++
++
++
RC l 289
++
++
++
+
++
++
++
++
++
++
Torreya力rgesll var.yunnanensis GLM一092567—18
evaluatir
PCR
success using
SUCCESS
"to
to evaluate e
and coverage
and
em
in this
this study.伶pccles
selected tor
selected
for
ties
s
inn bold were
e
selected for
i
sec
c
p
S
study._|-Species
ni
s
evaluating I"UK
u
sequencing
sequence quality
Pinidae
St
Taxaceae
neriifolius
Taxaceae
the nine candidate matK primer
sets at
family-level.一,no band;+,weak band;++,strong band.
@201 1 Institute ofBotany,Chinese Academy ofSciences
万方数据
172
Journal of
Systematics and Evolution
V01.49
No.
3
201 1
Polymerase chain reaction primers used for screening universal primers for gymnosperms in this study
Table 2
Gene
Primer name
rflatK
Gym—R1A
Gym FlA
Direction
f
5’.TCA YCC GGA RAT TTT GGT TCG.3
r
5’一ATYGYRCTTTTATGTTTACARGC.3’
Gym—R2A
f
57-ACYTTTCGYYRCTGGATCCAAG.3’
This study,designed by Alan Forrest
This study,designed by Alan Forrest
This study,designed bv Alan Forrest
GvIll F2A
r
5’一GTTTTAGCRC—汀GRRAGTCGAAG.3’
This
Gym—RlB
f
57一TCA TCC RGA AAT TTT GGT KCG.3’
r
5’.ATMGTACTTTTATGTTTACARGC.3’
f
57-AyYTTTCGTCGCTGGATCCGAG.3
GⅧ_F2B
r
5’.GTT 11A GCR CAT
NY552F
f
5’一CTGGATYCAAGATGCTCCTT-3’
NYll50R
r
5’.GGT CTT TGA GAA GAA CGG AGA.37
Damon Linle,unpublished
Damon Little,unpublished
PKF4
f
5’-CCCTATTCTATTCAYCCNGA.3
Fazekas
et
PKRl
r
57-CGTATCGTGCTTTTRTGY丁丁.37
Fazekas
et
5R
f
57-G11 CTA GCA CAA GAA AGT CG一3
XF
Gym_FlB
Gvll-1 R2B
study,designed by Alan Forrest
This study,designed by Alan Forrest
This study,designed by Alan Forrest
This study,designed by Alan Forrest
GRW AIrl’RRA AG.37
This
7
study,designed by Alan Fotiest
Ford
et
Ford
et
al r20081
al f2008)
a1.(2009)
a1.(2009)
r
57.TAA TTT ACG ATC AAT TCA TTC.3’
3F_KIM
1R KIM
f
57一CGT ACA GTA CTT TTG TGT TTA CGA G.3
r
5'-ACC CAG TCC ATC TGG
1326R
f
5'-TCT AGC ACA CGA AAG TCG AAG T-3’
Ki-Joong Kim,unpublished
Cu6noud et a1.2002
r
5'-CGA TCT ATT CAT TCA A1-A TTT C.37
Cu6noud
f
5'-ATG TCA CCA CAA ACA GAA AC.3’
Fay
r
5'-TCG CAT GAT CCT GCA GTA GC.3’
Fay et a1.1997
390F
rbcL
Source
Sequence(5'-3 7)
lF
724R
AAA
7
Ki—Joong Kim,unpublished
TCT TGG TTC.3’
et
et
a1.2002
a1.1997
e forward;r’reverse.Damon Little is based at New York Botanic Garden,New№rk.Ki—Joong Kiln is based at Korea University,Seoul
purified PCR product,0.1 5雎L
BigDye terminator
sequencing
pied 1 4 species at family level(Table 1 1.The universal.
ity for the nine matK primers showed a much more
and
variable PCR
mixture(V3.1),1.2雎L sequence buffer,
1.4“L“mol几primer.The cycle sequencing pro—
file was 32 cycles of96。C for 10 s.50。C for 5 s.and
60。C for 4 min.The purified sequencing products were
run on an ABI 3730xl automated sequencer(Applied
RlA。Gym
1.4 Sequence quality and coverage analysis
Sequences were trimmed using a window size of
20 bp’with segments with 2 bp showing<20
fQV
an
established metric
for
Qual.
determining
quality sequencing data)trimmed.Sequence quality
was assessed using Sequencing Analysis 5.3.1 software
(Applied Biosystems).The
sequence quality recovered
was defined such that both the forward and
reverse
lengths should be>50%ofthe original read length and
the assembled contigs should have>50%overlap in the
alignment of the forward and reverse reads with<1%
low—quality bases(<20 QV)and<1%internal gaps
and substitutions when aligning the forward and reverse
reads.The percentage ofbases with>20 QV>30 QV
and>40 OV was determined using Sequencher 4.9
Codes.Ann
ities for
l
。
Arbor,MI,USA).Error
PCR universality at genus
R l,
probabil.
sequences were calculated after base
pair calling using Phred as implemented in Aligner 5.3.6
consensus
level(50.0%一92.9%、and
family level(75%一1 00%),respectively.The remaining
four matK primers(3F KIM/lRⅪM,390F/1326R,
XF/5R.and NY552F仆TY“50R)showed low PCR
SUC.
cess(0.0%一1 4.3%1.These four primers with low PCR
success were rejected for further evaluation on the ex—
panded sample set.
On all 57 sampled
reads
should have high length of read:after trimming to an
average Qv>20 in a 20.bp window,the post.trim
(Gene
14
..F2A/Gym..R2AKFG4/PymKRFl)B/Gym
P myG/B2showed
Band
2R.
F_myGhigh
B
Biosystems,F(Ister City'CA,USA).
ity Value
rate(0.0%一92.9%)on the
success
selected species in the family level assessment.Five
of the nine candidate matK primers(Gym
F.1 A/Gym
1 00%PCR
with
species.而吐performed
universality(Table
well
1 1.For the five
candidate matK primers used for further evaluation.
Gym_F1A/Gym_R1A showed the highest PCR success
with 94.7%.followed by Gym FlB/Gym
RlB with
93.0%。Gym F2B/Gym R2B with 80.7%.KF4/PKRl.P
with 77.2%.and Gym F2A/Gym R2A with 57.9(Ta—
ble l 1.However,three primers Gym F2A/Gym I也A.
Gym
P BKf
4/P
B2and
R_myG/
2F_。
lRK
.
yielded weak
PCR bands of25.7%,53.1%,and 21.3%.respectively,
for the
57 species.A11 the sampled species of
sampled
family Ephedraceae failed
to
amplify using these five
matK primers(1'able 1).
(CodonCode,Dedham,MA,USA).
2 Results
2.1
Primer universality
The universality ofprimer for rbcL(1 F/724R)per-
formed well with 1 00%PCR SUCCESS on a11 the sam.
2.2 Sequence quality and coverage
To assess the quality of generated
traces
sequence
for matK primers Gym_F 1 A/Gym_R 1 A and
Gym_F 1 B/Gym_R 1 B,which
success
and
showed
PCR
high
PCR
selected
nine
bands,we
s仃ong
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@201 1 Institute ofBotany,Chinese Academy ofSciences
万方数据
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PF,Y4q*I诅I.K¥p州1wM
for sequencing,representingthe nine sampledfamilies
except for Ephedraccae,Gnemceac.and Ginkgoaceae
due to PCR failure A¨the generated 36 bidirectional
coverability,sequence quality,andlevel ofspaciesdis-
criminationfCBOL PlantⅦbrkingGroup,2009)Hl曲
sequencetraces shoⅥed high sequence quality.withfew
PCR amplification success of rb吐for gyrrmosparms
has been reportedln some studies rCBOL PlantWork—
Iow-quality bases trimined oul The amplicon sizes of
ing Group,2009,Liu
the
primer pairs were 876 bp including primer
sites
Length of read for sequence u'dces generated
by primer Oym_FIA/Gym—R】A ranged from 799 to
low in gymnosperms
versalily
Inthis study,PCR universality ofrbcLhereperformed
best with 100%PCR success thr all the 57 sampled
829 bp with high QV f120J.and 779 8i6 bp with
mghQV(120Jforthe sequences generated by primer
Gym F1B,Oym—R【B Thcre wcrc 93 7%97 2%and
species representingl】of】2fmmiliesofgymnosparms
SoⅥproposcdprimerlF/724R asthe“universal”rbcL
DA'O
82 8%-92 5%ofbases(OV)30).and 9l 3%一96 2%
and 83 7即904%of bases(Qv)401向r primer
pairsGym—FIA/Gym—RlAandGym FIB/Gym_RlR,
respectively.On the whole the sequence quality of
Gym F1~日ym—RlA
a】itflc
ls
better than that
of
Gym
FiB,Oym—RlB The assembled contigs sfioⅥed
>70%overlapmthe alignment oftheforward and re—
verse
readswitfioutIow<lualiⅣbases amonggenerated
Position and orientationofmark primen
Position
and orientation of the m-e candi.
al,2011).wfiereas PCR uni・
primer forbareoding gymnosperms
The hi曲ly vail曲ie,删坼region has Iower PCR
amplification SUCCESS than the mote conSUl%.ed rbcL
genc fKress&Erick,^m
Ing Group,2009)It
D“fy m口tK with
a
1s
2007 CBOL PIam Work-
currently
not
possible
to
am-
single universal primer pair across
the whole plant kingdom to ncilitate high throughout、
rapid,automated,and
cost-e舵ctive
species ldentifiea・
tion fKress&Erickson
2007:Dunning&Savolainen.
20lOl Three,M,K primer secs namely 390F/l 326R
(Cednoud
2.3
et
ofmarKⅫrelatively
et
aI.2【)02I XF‘5R fFord c【al
2009)and
”一KIⅥlR—KIM(K—J Kim.KoreaUniversi眦Seoul.
unpubl data,also Sec Hollingsworth 2008)werc pro・
fo㈨nfK with(Fnhnl{}|…k?n
posed as currently the best“universal”脚tK primers
mr land plant fhgp://www.barcoding si ed“PDⅣ
template ls Illustrated m Fig l Primer
Gym Fl~bym RIA and Gym—FlB/G),xn RIB Io—
calcd at the same position with a length of832 gp be.
twcen primer sets Primers Gym F2A/Gym—R2A and
performed well for most groups of angiosperm they
shcm'edlow PCR SUCCESSfor sampled species ofgym-
date
primers
lune,as
Gym F2B’|G)m—R2B also share
an
Idenfenl position
wIth a length of 782 bp between primer se【s(Fig l h
Forward primerPKF4 S Latted alpositloff 537 bp andthe
w㈣primerPKR4
at
1
bctwccn primer scts(Fig
327bpwith alel39th of770 bp
al
0
as
the ptant colt:barcode based on its
201IInstilBte ofBotan*Chme"Academy ofSciences
万方数据
three,w,K
unsuitabfe as“universal”mdtiC primersfor
gymrlosperms
Two primers ofthe nine eandidale mdfK primers.
and
Gym_FIB/Gym—RIB,
Gym—FIA/Gym—RlA
produczs岛r sampled species Ofthetwomd,K primer
pairs,primer Gym F】丸'Gym—RlA,performed bene^
successfully amplifyingallthcll sampledfamifies and
Primer uni、ersalgy is the mosl important crite-
for DNA baK(mmg in the^nl instance(Chase
ommcnded
primers
are
shoued highIevels
2007;Kress&Erickson 2007;Ford et al 2009;
fIo[Iingsworth et al 2009)rbcL+matK has bccn mc-
el
nospcrm m this study.1ndicaring that these
of时R universality.high sequence
and
ty
qua
coverage.and could a[so yield swong PCR
11
3 Discussion
rion
Informaoononbarcodeloci pdn Althou曲theseprimers
re・
39 oul ofthe 40 sampledgeneraf97 5%1
or如hedra(Gnelales:EphedmceacI
Only species
ampli竹
fTabIe”For primer pair G)wa—FIB,Gvm_RIB.37
falIed to
of 40 genera f92 5%l”m ampllfled successfully.
but species of two sampled families fEphedraeeae
174
ofSystematics and Evolution
Journal
and
Ginkgoaceae)could
1).Based
not
study(Table
be
No.
V01.49
in
amplified
this
assessments of primer
on
universality and sequence quality and coverage,we
propose that primer Gym_F 1 A/Gym_R1 A currently
the
represents
best universal
primer
for
matK
in
gymnosperms-
of an appropriately short sequence
to facilitate DNA extraction and
length(300-800 bp)
amplification(Kress
and orienmtion of the five candi.
al。2005).Position
date marK primers showing high PCR
success
is given in
Fig.1.The length ofthe proposed matK barcode region
for primer Gym_F 1 A/Gym_R 1 A is 832 bp long using
fortunei matK sequence(AF228 1 09)
the Cephalotaxus
as
reference(excluding primer sequence).The
a
size
of mis product is amenable to the acquisition of bidi.
rectional sequence reads with a single primer pair,thus
meeting the criterion of DNA barcode length.
As none of the candidate matK primers amplified
samples ofEphedra,a specific matK primer for Ephedra
(Eph
F:
57一TCATTCAGAGCTGTTAGTTAG.3
5'-
.h17pCEGA.TCGAT CATG A:R
7.
3
newly designed in this study.This primer per-
formed very well on the three sampled species of
was
Ephedra
with
located at
a
Gym
l 00%PCR
success.This
similar position
the matK gene
on
primer is
as
primer
A/Gym.
A.
the ength of the
IF
R1,
1
ensuring that
gymnosperms
barcode region for
to
align.The primers
is similar and easy
proposed in this study for
barcode rbcL and marK
can
core
be easily and successfully
amplified for all the genera of gymnosperms in China.
We
hope our work will facilitate DNA barcoding of
gymnosperms worldwide
as
Wre
well.
to
Dr.Chun.Xia
Acknowledgements
grateful
ZENG.Mr.Jun.Bo YANG。Mrs Jing YANG,and other
are
colleagues for laboratory help
also thank Prof.Peter
structive comments
or
providing samples.We
HOLLINGSWoI订H for
his
con.
the manuscript.This study was
the
Research
Fund for the Large.Scale
supported by
Scientific Facilities of Chinese Academy of Sciences
on
(Grant No.2009.LSF.GBOWS.0 1 1.the West Light
Pro.
gramme of the Chinese Academy of Sciences(Grant
Project of Yunnan
Province,China(Grant No.2008PY064).Alan FOR.
1也ST is grateful to the Gordon and Betty Moore Foun.
No.9223 1l
201 1
Chase MW,Cowan RS,Hollingsworth PM,Van Den Berg C,
Madrifi{in S,Petersen G,Seberg O,Jorgsensen T,Cameron
KM,Carine M,Pedersen N,Hedderson TAJ,Conrad F。
Salazar GA,Richardson
JE,Hollingsworth ML,Barra-
TG.Kelly L,Wilkinson M.2007.A proposal for
standardised protocol to barcode allland plants.Taxon 56:
clough
a
295-299.
An ideal DNA barcode should sarisfy the criterion
et
3
lWll
and the
Talent
dation(#2535)for funding which supported the devel.
opment of matK primers described in this paper.
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gymnosperms
作者:
作者单位:
Yan LI, Lian-Ming GAO, RAM C.POUDEL, De-Zhu Li, Alan FORREST
Yan LI(Key Laboratory of Biodiversity and Biogeography, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650204, China;Institute of Alpine
Economic Plants, Yunnan Academy of Agricultural Sciences, Lijiang 674100, China)
, Lian-Ming GAO(Key Laboratory of Biodiversity and Biogeography, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China), RAM
C.POUDEL,De-Zhu Li(Key Laboratory of Biodiversity and Biogeography, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming 650204,
China;Germplasm Bank of Wild Species in Southwest China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650204, C), Alan FORREST(Royal
Botanic Garden Edinburgh, Edinburgh EH3 5LR, UK)
刊名:
植物分类学报
JOURNAL OF SYSTEMATICS AND EVOLUTION
2011,49(3)
英文刊名:
年,卷(期):
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