全 文 ：裸子植物 DNA条形码 ITS2 高通用性引物的筛选*
李摇 燕1,2, 吴摇 丁3, 高连明1**
(1 中国科学院昆明植物研究所生物多样性与生物地理学重点实验室, 云南 昆明摇 650201; 2 云南省农业科学院
高山经济植物研究所, 云南 丽江摇 674100; 3 景德镇学院, 江西 景德镇摇 333000)
摘要: DNA条形码技术是利用标准 DNA片段进行准确快速鉴定物种的一种方法, 理想的 DNA条形码片段
应具有高通用性。 虽然核糖体 DNA内部转录间隔区 II ( ITS2) 被建议作为种子植物有效的 DNA 条形码,
但目前裸子植物还没有通用性高的引物可用。 为获得高通用性的 ITS2 引物, 本研究基于裸子植物 55 个属
的 5. 8S基因的保守序列区设计了 3 个正向引物, 与已有的 ITS 反向引物组合, 组成了 7 对 ITS2 引物进行
通用性的评价。 选取了裸子植物 8 目、 12 科和 40 属的 56 个种用于本文的研究。 引物组合 5. 8SR / ITS4、
5. 8SRa / ITS4 和 5. 8SF2 / S3R因为在科水平评价中通用性低或者产生的 PCR产物有双带, 因而排除在全部
物种水平上进一步评价。 其余 4 对引物 (GYM_5. 8SF1 / ITS4、 GYM_5. 8SF1 / S3R、 GYM_5. 8SF2 / ITS4 和
S2F / S3R) 在 56 个物种的 PCR检测中, 均有 100%的扩增率。 基于 PCR 产物的亮度、 序列质量和正反向
引物覆盖率的综合评价, 建议引物 GYM_5. 8SF2 / ITS4 作为裸子植物条形码片段 ITS2 最好的通用引物。
关键词: DNA条形码; 裸子植物; ITS2; 引物通用性; 物种鉴定
中图分类号: Q 781摇 摇 摇 摇 摇 摇 文献标识码: A摇 摇 摇 摇 摇 摇 摇 文章编号: 2095-0845(2013)06-751-10
Highly Universal DNA Barcoding Primers of
ITS2 for Gymnosperms*
LI Yan1,2, WU Ding3, GAO Lian鄄Ming1**
(1 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650201, China; 2 Institute of Alpine Economic Plants, Yunnan Academy of Agricultural Sciences,
Lijiang 674100, China; 3 Jingdezhen University, Jingdezhen 333000, China)
Abstract: DNA barcoding is an approach for rapid and accurate species identification using a standard DNA region.
Ideal barcodes should have high level of universality in PCR and sequencing. Although the nuclear ribosomal DNA
(nrDNA) internal transcribed spacers 2 (ITS2) was proposed as potential DNA barcode for seed plants, including
gymnosperms, no suitable ITS2 primer pair for gymnosperms available shows high universality in either aspect. To
obtain a highly universal ITS2 primer pair, we newly designed three forward primers for ITS2 based on sequences in
the 5. 8S gene greatly conserved among 55 genera of gymnosperms. Combined with previously published reverse
primers of ITS, seven candidate primer combinations for ITS2 were assessed for their universality. A total of 56 spe鄄
cies, representing 40 genera from all the 12 families of the eight orders of gymnosperms, were sampled in this study.
Three ITS2 primer combinations, 5. 8SR / ITS4, 5. 8SRa / ITS4, and 5. 8SF2 / S3R, were excluded due to their low
universality or because of yielding double bands in the PCR in the family鄄level assessment. The remaining four prim鄄
er combinations, GYM_5. 8SF1 / ITS4, GYM_5. 8SF1 / S3R, GYM_5. 8SF2 / ITS4, and S2F / S3R, showed high PCR
universality (100% ) on all 56 sampled species. Based on our overall assessment of PCR and sequencing universali鄄
植 物 分 类 与 资 源 学 报摇 2013, 35 (6): 751 ~ 760
Plant Diversity and Resources摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 DOI: 10. 7677 / ynzwyj201313190
*
**
Funding: The Ministry of Science and Technology of the People忆s Republic of China (No. 2012FY110800, 2013FY112600), the Large鄄
scale Scientific Facilities of the Chinese Academy of Sciences (No. 2009鄄LSFGBOWS鄄01)
Author for correspondence; E鄄mail: gaolm@ mail. kib. ac. cn
Received date: 2013-09-20, Accepted date: 2013-09-30
作者简介: 李摇 燕 (1976-) 女, 副研究员, 主要从事高山经济植物的引种驯化和 DNA条形码研究。 E鄄mail: enleiyin@ 163. com
ty, brightness of PCR bands, and sequence quality and coverage, we propose the primer combination GYM_5. 8SF2 /
ITS4 as ITS2 barcode for gymnosperms because it was shown to be the most “universal冶 .
Key words: DNA barcoding; Gymnosperms; ITS2; Universal primer; Species identification
摇 DNA barcoding is an approach to identify spe鄄
cies and / or discover new species using one or a few
standardized DNA regions from different genomes
(Hebert et al., 2003; Hollingsworth, 2011; Gao et
al., 2012), which has been widely used in the field
of taxonomy, ecological surveys and assessment of
biodiversity (Hebert et al., 2004; Liu et al., 2011;
Lahaye et al., 2008; Valentini et al., 2009;
Garc侏a鄄Robledo et al., 2013). The ideal DNA bar鄄
codes should satisfy the criterion of an appropriately
short sequence length (300-800 base pairs, bp) to
facilitate PCR amplification with a single primer pair
(universality), be amenable to PCR and bidirec鄄
tional Sanger sequencing with little requirement for
manual editing of the sequence trace files (sequence
quality and coverage), and provide maximal dis鄄
crimination among the species (Kress et al., 2005;
CBOL Plant Working Group, 2009). The mitochon鄄
drial gene CYTOCHROME OXIDASE 1 (COI) has
been proven to be a remarkably effective “ animal
barcode冶 for discriminating species of birds, fishes,
and insects (Hebert et al., 2004; Holmes et al.,
2009; Linares et al., 2009; Hollingsworth, 2011).
However, it is more difficult to find a similarly ro鄄
bust and effective barcode for plants ( CBOL Plant
Working Group, 2009; Hollingsworth et al., 2011;
Li et al., 2011a). Although many studies have com鄄
pared the performance of a range of candidate DNA
regions as barcodes (e. g. Sass et al., 2007; Fazek鄄
as et al., 2008; Hollingsworth et al., 2009; Seberg
and Petersen, 2009; Li et al., 2011b), no single can鄄
didate DNA locus evaluated fitted all criteria for seed
plants. Thus, combinations of different DNA loci
were suggested as barcodes for plants (Chase et al.,
2007; Kress and Erickson, 2007; Hollingsworth et
al., 2009; CBOL Plant Working Group, 2009).
Kress et al. (2005) initially recommended the
cpDNA trnH鄄psbA intergenic spacer and the nuclear
ribosomal DNA (nrDNA) internal transcribed spacer
(ITS) region as barcodes for flowering plants. In
2009, the Consortium for the Barcode of Life
(CBOL) Plant Working Group proposed the combi鄄
nation of the coding regions of rbcL +matK as the
core barcode for land plants. Thereafter, the combi鄄
nation of trnH鄄psbA and ITS2 ( internal transcribed
spacer 2) was demonstrated to represent an effective
barcode for species identification of medicinal plants
in several studies (e. g. Chen et al., 2010; Pang et
al., 2010; Gao et al., 2010). ITS2 was proposed to
be used as a universal DNA barcode complementing
CO1 to identify animal species (Yao et al., 2010),
and as complementary barcode to the core barcode of
rbcL+matK for plants (Hollingsworth et al., 2011).
Subsequently, based on the assessment of a large
scale dataset, the China Plant BOL Group ( Li et
al., 2011b) backed the incorporation of ITS or ITS2
into the core barcode for seed plants.
Though many researchers have proposed the use
of nrDNA ITS as a standard barcode for plants, there
is a residual nervousness stemming from three major
potential problems: i. e. fungal contamination, pres鄄
ence of paralogous copies, and recovery (simply dif鄄
ficult to amplify and sequence ) ( Hollingsworth,
2011). Of these, the main limitation is the some鄄
times great difficulty to amplify and sequence the ITS
region (Kress et al., 2005; Li et al., 2011b), per鄄
haps due to non鄄conserved primer sites ( M觟ller,
2000). For instance, the recovery rate of 76. 5% was
some 10% - 15% lower than for the other barcode
markers based on a 6286 sample dataset (Li et al.,
2011b). A high level of primer universality for poly鄄
merase chain reaction (PCR) and sequencing is one of
the most important criteria for DNA barcoding (Chase
et al., 2007; Kress and Erickson, 2007; Ford et
al., 2009; Hollingsworth et al., 2009). Thus, it is
very important to select highly efficient primer pairs
257摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
for the PCR amplification of ITS or ITS2.
Gymnosperms are a class of seed鄄bearing plants,
which are the dominant vegetation in many colder
and arctic regions and have major economic uses as
important wood resources and as ornamental plants.
The class consists of four subclasses, i. e. Cycadi鄄
dae, Ginkgoidae, Gnetidae, and Pinidae, represen鄄
ting eight orders, 12 families, 83 genera, and ap鄄
proximately 990 species (Christenhusz et al., 2011).
There are about 250 gymnosperm species in 34 gene鄄
ra and 10 families in China (Fu et al., 1999). The
great variation in length of the ITS region (including
ITS1, ITS2 and 5. 8S) in gymnosperms is possibly a
limit for the use of the region as DNA barcode.
However, length variation was predominantly ob鄄
served for ITS1 (e. g. ranging from 630 to 3 125 bp
in length in Pinus, Liston et al., 1999), while the
ITS2 and 5. 8S gene are more length conserved (Lis鄄
ton et al., 1996, 1999; Gernandt and Liston, 1999).
Therefore, ITS2 seems a more likely candidate for a
gymnosperm DNA barcode, when obtaining full ITS
sequences is problematic. Another detrimental aspect
of is the sometimes low universality of ITS when using
universal gymnosperm PCR primers ( Li et al.,
2011b), also reported for gymnosperm universal
primers for ITS2 (Chiou et al., 2007; Chen et al.,
2010). Thus, a highly PCR universal primer pair
for ITS2 is required if this region is even to be con鄄
sidered suitable for gymnosperms.
In the present study, we therefor newly designed
and tested three forward primers, GYM _5. 8SF1,
GYM_5. 8SF2, and 5. 8SRa, based on sequences in
the 5. 8S gene greatly conserved over 55 gymnosperm
genera. We selected 56 species representing 42 gene鄄
ra of all 12 gymnosperm families to test the new prim鄄
ers. A total of seven candidate primer combinations
for ITS2 were evaluated to determine their universality
in PCR amplification across the 56 gymnosperm spe鄄
cies and Sanger sequencing of seven species from the
four subclasses of gymnosperms here. The ultimate
aim was to be able to propose the most universal
primer pairs of ITS2 for barcoding gymnosperms.
Materials and methods
Sample strategy
To represent the large diversity across gymno鄄
sperms, a total of 56 species representing 42 genera
from 12 families of eight orders in gymnosperms were
sampled in this study ( Table 1), covering around
49% of genera, and all families, orders, and sub鄄
classes of gymnosperms. All gymnosperm species oc鄄
curring in China were covered. Voucher specimens
were deposited at the herbarium of Kunming Institute
of Botany, Chinese Academy of Sciences (KUN).
For some larger genera, more than one species per
genus were selected.
DNA extraction
Total DNA was extracted from silica鄄gel dried
leaf tissues using the CTAB method ( Doyle and
Doyle, 1987). The concentration of total genomic
DNA was check using spectrophotometer (ND鄄1000,
NanoDrop, Wilmington, USA), and then was dis鄄
solved in TE buffer (10 mmol·L-1 Tris鄄HCl, pH 8. 0,
1 mmol·L-1 EDTA) to a final concentration of 40-
50 ng·滋L-1 to avoid any variation in PCR success
due to DNA concentration differences.
Primer design and primer combination screening
strategy
Sequences of the 5. 8S gene and ITS2 for a total
of 74 species, representing 55 genera from all the 12
families of gymnosperms, were obtained from Gen鄄
Bank. These sequences were aligned in Clustal X
(Larkin et al., 2007). Three new forward primers
(GYM_5. 8SF1, GYM_5. 8SF2, and 5. 8SRa, see
Table 2) were designed in regions of the 5. 8S gene
largely conserved across the gymnosperms included
here with the help of Oligo 6. 0 (Offerman and Rych鄄
lik, 2003). A further forward primer, 5. 8SR (de鄄
signed by Vilgalys lab: http: / / biology. duke. edu /
fungi / mycolab / primers. htm) was selected for the
universality assessment in our study as well. In com鄄
bination with the reverse primers ITS4 and S3R, we
had six ITS2 primer combinations to test. The primer
pair, S2F / S3R for ITS2 (Chiou et al., 2007), pro鄄
posed for plant barcoding in several studies previous鄄
3576 期摇 摇 摇 摇 摇 摇 摇 LI and GAO: Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms摇 摇 摇 摇 摇 摇 摇 摇
ly (Chen et al., 2010; Gao et al., 2010; Pang et
al., 2010, 2012), was used as a bench mark in
comparison to primer pairs including our newly de鄄
signed primers (Table 2).
Table 1摇 Voucher information and sample code of the species included in this study, and polymerase
chain reaction success for four ITS2 primer combinations
Subclass Family Taxon Voucher Code
Primer
GYM_
5. 8SF1
ITS4
GYM_
5. 8SF1
S3R
GYM_
5. 8SF2
ITS4
S2F
S3R
Cycadidae Cycadaceae Cycas guizhouensis* Glm鄄06032 G1 +++ +++ +++ +++
Cycadidae Cycadaceae Cycas micholitzii Ly鄄012 G2 +++ +++ +++ +++
Cycadidae Zamiaceae Encephalartos lehmannii* Ly鄄014 G3 +++ +++ +++ +++
Ginkgoidae Ginkgoaceae Ginkgo biloba* Ly鄄028 G4 + + +++ ++
Gnetidae Ephederaceae Ephedra intermedia 12384 G57 +++ +++ +++ +++
Gnetidae Ephederaceae Ephedra likiangensis* Ly鄄024 G55 +++ +++ +++ +++
Gnetidae Gnetaceae Gnetum pendulum* GBOWS0801 G54 +++ +++ +++ +++
Gnetidae Welwitschiaceae Welwitschia mirabilis* RBGE19941311 G59 +++ +++ +++ +++
Pinidae Araucariaceae Agathis dammara Gy058 G58 +++ +++ +++ +++
Pinidae Araucariaceae Araucaria bidwillii Ly鄄015 G46 +++ +++ +++ +++
Pinidae Araucariaceae Araucaria cunninghamii* Ly鄄016 G45 +++ +++ +++ +++
Pinidae Araucariaceae Araucaria heterophylla Ly鄄004 G47 +++ +++ +++ +++
Pinidae Cupressaceae Calocedrus macrolepis var. formosana Glm鄄103074 G35 +++ +++ +++ +++
Pinidae Cupressaceae Chamaecyparis formosensis Glm鄄103065 G33 +++ +++ +++ +++
Pinidae Cupressaceae Cunninghamia lanceolata Ly鄄007 G22 ++ ++ ++ ++
Pinidae Cupressaceae Cupressus duclouxiana Ly鄄011 G32 +++ +++ +++ +++
Pinidae Cupressaceae Cryptomeria japonica Glm鄄103142 G25 +++ +++ +++ +++
Pinidae Cupressaceae Fokienia hodginsii Ly鄄006 G31 +++ +++ +++ +++
Pinidae Cupressaceae Glyptostrobus pensilis* Ly鄄005 G26 +++ +++ +++ +++
Pinidae Cupressaceae Juniperus pingii var. wilsonii Ly鄄026 G38 +++ +++ +++ +++
Pinidae Cupressaceae Juniperus squamata* Glm鄄082126 G30 +++ +++ +++ +++
Pinidae Cupressaceae Metasequoia glyptostroboides Ly鄄002 G29 +++ +++ +++ +++
Pinidae Cupressaceae Platycladus orientalis Ly鄄020 G37 +++ +++ +++ +++
Pinidae Cupressaceae Sequoia sempervirens Ly鄄021 G28 +++ +++ +++ +++
Pinidae Cupressaceae Taiwania flousiana Glm鄄092411 G24 +++ +++ +++ +++
Pinidae Cupressaceae Taxodium distichum Ly鄄009 G27 +++ +++ +++ +++
Pinidae Cupressaceae Thujopsis dolabrata Ly鄄019 G34 +++ +++ +++ +++
Pinidae Cupressaceae Thuja sutchuenensis Ly鄄018 G36 +++ +++ +++ +++
Pinidae Pinaceae Abies georgei Glm鄄102904 G12 +++ +++ +++ +++
Pinidae Pinaceae Cathaya argyrophylla* Ly鄄017 G13 +++ +++ +++ +++
Pinidae Pinaceae Cedrus deodara Ly鄄029 G8 +++ +++ +++ +++
Pinidae Pinaceae Keteleeria davidiana Ly鄄001 G14 +++ +++ +++ +++
Pinidae Pinaceae Keteleeria evelyniana Ly鄄003 G15 +++ +++ +++ +++
Pinidae Pinaceae Keteleeria fortunei Ly鄄010 G16 +++ +++ +++ +++
Pinidae Pinaceae Larix himalaica Glm鄄081604 G10 +++ +++ +++ +++
Pinidae Pinaceae Larix potaninii var. chinensis Glm鄄06081 G9 +++ +++ +++ +++
Pinidae Pinaceae Picea likiangensis Glm鄄08912 G19 +++ +++ +++ +++
Pinidae Pinaceae Picea smithiana Glm鄄081533 G18 +++ +++ +++ +++
Pinidae Pinaceae Pinus armandi Ly鄄027 G7 +++ +++ +++ +++
Pinidae Pinaceae Pinus wallichiana Glm鄄081471 G5 +++ +++ +++ +++
Pinidae Pinaceae Pinus yunnanensis* Ly鄄025 G6 +++ +++ +++ +++
Pinidae Pinaceae Pseudolarix amabilis Ly鄄008 G11 +++ +++ +++ +++
457摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
摇 Table 1 continued
Subclass Family Taxon Voucher Code
Primer
GYM_
5. 8SF1
ITS4
GYM_
5. 8SF1
S3R
GYM_
5. 8SF2
ITS4
S2F
S3R
Pinidae Pinaceae Pseudotsuga sinensis Glm鄄103063 G17 +++ +++ +++ +++
Pinidae Pinaceae Tsuga dumosa Glm鄄081797 G21 +++ +++ +++ +++
Pinidae Podocarpaceae Dacrycarpus imbricatus Ly鄄023 G39 +++ +++ +++ +++
Pinidae Podocarpaceae Nageia fleuryi* Ly鄄022 G40 +++ +++ +++ +++
Pinidae Podocarpaceae Nageia nagi Ly鄄013 G41 +++ +++ +++ +++
Pinidae Podocarpaceae Podocarpus macrophyllum GBOWS0294 G42 +++ +++ +++ +++
Pinidae Podocarpaceae Podocarpus neriifolius Glm06211 G43 +++ +++ +++ +++
Pinidae Sciadopityaceae Sciadopitys verticillata* Glm鄄092241 G44 +++ +++ +++ +++
Pinidae Taxaceae Amentotaxus argotaenia 182 G48 +++ +++ +++ +++
Pinidae Taxaceae Cephalotaxus mannii ZSD001 G53 +++ +++ +++ +++
Pinidae Taxaceae Cephalotaxus wilsoniana Glm鄄103119 G52 +++ +++ +++ +++
Pinidae Taxaceae Pseudotaxus chienii* Glm鄄07549 G51 +++ +++ +++ +++
Pinidae Taxaceae Taxus wallichiana RC1289 G50 +++ +++ +++ +++
Pinidae Taxaceae Torreya fargesii var. yunnanensis Glm鄄092567 G49 +++ +++ +++ +++
Species in bold were selected for sequencing to evaluate sequence quality and coverage in this study. * Species were selected for evaluating PCR
success using the seven candidate ITS2 primer combinations at family鄄level. +++, strong band; ++, medium band; +, weak band
Table 2摇 The seven combinations of ITS2 primers tested for universality in this study
Primer name Direction Sequence (5忆鄄3忆) Length/ bp
Optimum annealing
temperature / 益 Source
GYM_5. 8SF1
ITS4
F
R
TTGYAGAATCCCGTGARTC
TCCTCCGCTTATTGATATGC
19
20 48
Designed in this study
White et al., 1990
GYM_5. 8SF1
S3R
F
R
TTGYAGAATCCCGTGARTC
GACGCTTCTCCAGACTACAAT
19
21 50
Designed in this study
Chiou et al., 2007
GYM_5. 8SF2
ITS4
F
R
GYAGAATCCCGTGARTCATC
TCCTCCGCTTATTGATATGC
20
20 52
Designed in this study
White et al., 1990
GYM_5. 8SF2
S3R
F
R
GYAGAATCCCGTGARTCATC
GACGCTTCTCCAGACTACAAT
20
21 52
Designed in this study
Chiou et al., 2007
5. 8SR
ITS4
F
R
TCGATGAAGAACGCAGCG
TCCTCCGCTTATTGATATGC
18
20 52
Vilgalys lab
White et al., 1990
5. 8SRa
ITS4
F
R
WCGATGAAGAACGYAGCG
TCCTCCGCTTATTGATATGC
18
20 52
Designed in this study
White et al., 1990
S2F
S3R
F
R
ATGCGATACTTGGTGTGAAT
GACGCTTCTCCAGACTACAAT
20
21 52
Chiou et al., 2007
Chiou et al., 2007
F, forward; R, reverse. R-A / G, W-A / T, Y-C / T; bp-base combinations
摇 摇 To determine the optimum annealing tempera鄄
ture, gradient PCR ( annealing temperature ranging
from 46 益 to 58 益 with a 2 益 gradient) was per鄄
formed using the seven primer combinations on four
species, Cycas guizhouensis, Ginkgo biloba, Pinus
yunnanensis, and Gnetum pendulum, representing
the four gymnosperm subclasses ( Table 1 ). The
gradient PCR reactions were carried out twice to con鄄
firm the optimum annealing temperatures. We then
adopted a two鄄step approach to determine the univer鄄
sality of the six primer combinations ( Li et al.,
2011). First, we selected 14 species representing
all the 12 families for PCR amplification with all the
candidate ITS2 primer combinations. For the large
families Pinaceae and Cupressaceae, two species
were used in the family鄄level assessment (Table 1).
5576 期摇 摇 摇 摇 摇 摇 摇 LI and GAO: Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms摇 摇 摇 摇 摇 摇 摇 摇
Second, the candidate primer combinations per鄄
formed well with a single PCR band and high PCR
success rate were selected for further assessment on
all the remaining sampled species.
PCR amplification and sequencing
All PCR reactions were carried out with a 2720
Thermal Cycler (Applied Biosystems, Foster City,
CA, USA). The final reaction volume of 20 滋L con鄄
tained 0. 5 滋L each of the forward and reverse prim鄄
ers (5 滋mol·L-1), 10 滋L 2伊Taq PCR Master Mix
(0. 1 U Taq polymerase·滋L-1, 0. 5 mmol·L-1 each
dNTP, 20 mmol·L-1 Tris鄄HCl (pH 8. 3), 100 mmol
·L-1 KCl, 3 mmol·L-1 MgCl2; Tiangen Biotech,
Beijing, China), 0. 2 滋L bovine serum albumin (10
滋g·滋L-1) and 1 滋L template DNA. Negative con鄄
trols were run alongside all PCR reactions. The ther鄄
mocycling profile for ITS2 was as follows: 94 益 for
3 min, then 35 cycles of 94 益 for 1 min, 46-58 益
(in the gradient PCR) or the primer pair specific
optimum annealing temperature (Table 2) for 45 s,
72 益 for 1 min, and a final extension step of 72 益
for 7 min. The PCR products were analyzed by elec鄄
trophoresis on 1. 2% Tris鄄acetate鄄EDTA agarose gels
stained with ethidium bromide alongside a GeneRul鄄
er 100 bp DNA ladder ( Fermentas, Glen Burnie,
MD, USA).
The PCR products were purified using ExoSAP鄄
IT ( GE Healthcare, Cleveland, OH, USA). Se鄄
quencing reactions were carried out on forward and
reverse strands in a total volume of 6 滋L containing
0. 2 滋L of the purified PCR product, 0. 15 滋L of
BigDye terminator sequencing mixture (V3. 1), 1. 2
滋L of sequence buffer, and 1. 4 滋M of primer).
The Sanger sequencing profile was 32 cycles of 96 益
for 10 s, 50 益 for 5 s, and 60 益 for 4 min. The pu鄄
rified sequencing products were run on an ABI
3730xl automated sequencer ( Applied Biosystems,
Foster City, USA).
Analysis of PCR and sequencing success, sequence
quality and coverage
Recording of the PCR and sequencing success
followed the methods of Li et al. (2011b). The se鄄
quence quality was assessed using the software Se鄄
quencing Analysis 5. 3. 1 (Applied Biosystems, Fos鄄
ter City, USA) and followed Li et al. (2011). The
percentage of bases with > QV30 ( QV, an estab鄄
lished metric for determining quality sequencing da鄄
ta) and the coverage of the bidirectional sequences
was determined using Sequencher 5. 0 (Gene Codes
Corporation, Ann Arbor, MI, USA).
Results
Optimum annealing temperature of the seven
candidate primer combinations
The optimum annealing temperature for the can鄄
didate primer combination was determined based on
the success rate of the PCR reactions and the bright鄄
ness of the PCR bands ( assumed to reflect amplifi鄄
cation efficiency). In this study, the optimum an鄄
nealing temperature for the primer combinations
GYM_5. 8SF1 / ITS4 and GYM_5. 8SF1 / S3R was 48 益
and 50 益, respectively. The remaining five primer
combinations showed the same optimum annealing
temperature of 52 益 (Table 1).
Universality of the candidate primer combinations
In the universality assessment at the family鄄lev鄄
el on 14 samples, primer pair 5. 8SR / ITS4 yielded
strong PCR bands for seven samples (50% ), weak
PCR bands for five samples (35. 7%), and no bands
for two species (Ginkgo biloba and Sciadopitys verti鄄
cillata). Though the primer combinations of 5. 8SRa /
ITS4 and GYM_5. 8SF2 / S3R produced bands for all
14 samples (100% ), the PCR products for some
species showed double bands on the agarose gel.
Because of the low universality and the amplification
of more than one band, these three primer pair were
excluded from further assessments. The remaining
four ITS2 primer combinations ( GYM _ 5. 8SF1 /
ITS4, GYM_5. 8SF1 / S3R, GYM_5. 8SF2 / ITS4,
and S2F / S3R) showed 100% PCR success (Table
1), though the primer combinations GYM_5. 8SF1 /
ITS4 and GYM_5. 8SF1 / S3R produced a weak PCR
band for Ginkgo biloba (Table 1). The primer com鄄
bination GYM_5. 8SF2 / ITS4 yielded a strong PCR
657摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
band and S2F / S3R a medium bright PCR band for
Ginkgo biloba (G4), respectively (Figure 1). The
remaining sampled species showed a single strong
PCR band on the family鄄level assessment for the four
primer combinations (Figure 1, Table 1).
The universality of the four ITS2 candidate primer
combinations performed uniformly well on the remai鄄
ning 42 sampled species with 100% PCR success and a
single strong PCR band per reaction except for Cun鄄
ninghamia lanceolata (G22) for which consistently
medium bright PCR band were obtained (Figure 1, Ta鄄
ble 1 ). Thus, the overall universality assessment
across all 56 species for the four candidate primer com鄄
binations was 100% PCR success. Considering the
band intensities, primer combination GYM_5. 8SF2 /
ITS4 performed best, followed by the pair S2F / S3R.
Fig. 1摇 PCR amplification profiles of ITS2 with primer combinations GYM_5. 8SF1 / S3R, GYM_5. 8SF2 / ITS4, and S2F / S3R, on all 56
species sampled for this study. Sample code as in Table 1. C means negative control for PCR, M means DNA ladder (100 bp)
7576 期摇 摇 摇 摇 摇 摇 摇 LI and GAO: Highly Universal DNA Barcoding Primers of ITS2 for Gymnosperms摇 摇 摇 摇 摇 摇 摇 摇
Sequencing success and sequence quality
The assessment of the sequencing universality
and the quality of the generated sequence traces for
the two best PCR performing ITS2 primer combina鄄
tions, GYM_5. 8SF2 / ITS4 and S2F / S3R, demon鄄
strated 100% sequencing success with the seven
species tested. All the 28 generated sequence traces
of the seven tested species were of high base call
quality, with few low鄄quality bases needing trimming
out. The complete amplicon length for the two primer
combinations ranged from 380 bp (GYM_5. 8SF2 /
ITS4) to 510 bp (S2F / S3R), including primer sites,
for Cycas guizhouensis. When using the default set鄄
tings for sequence trimming in Sequencher 91. 2 -
99. 4% and 95. 5-99. 5% of the bases (QV>30)
remained with primer combination GYM_5. 8SF2 /
ITS4 and S2F / S3R, respectively. Thus, the se鄄
quence quality was very high and similar for the two
primer combinations. The assembled contigs showed
>70% overlap in the alignment of the forward and
reverse reads without low鄄quality base calls for any
of the generated ITS2 sequences.
Discussion
The nuclear ribosomal DNA (nrDNA) internal
transcribed spacer ( ITS) region is one of the most
used DNA region for molecular systematics and evo鄄
lution (魣lvarez and Wendel, 2003; Kress et al.,
2005; Nieto et al., 2008). It can provide signifi鄄
cant sequence variability at the species level or lower
(Yao et al., 2010; Liu et al., 2011). Because of
the greater discriminatory power of nrITS over plastid
regions at low taxonomic levels among plant species,
it has been proposed as a DNA barcode in several
studies (Roy et al., 2010; Li et al., 2011b; Muell鄄
ner et al., 2011; Simon et al., 2012). The remark鄄
able length variation of ITS in gymnosperms (Liston
et al., 1996, 1999; Gernandt and Liston 1999) is
an obvious obstacle for a DNA barcode (Liu et al.,
2011). The “universal冶 ITS primer pair for gymno鄄
sperms has revealed low level of PCR and sequen鄄
cing success (Li et al., 2011b). It limited the use
of ITS as a barcode in gymnosperms even though it
was proposed as a core barcode for seed plants (Li et
al., 2011b). However, ITS2, the second spacer of
the ITS region, has been suggested as an effective
DNA barcode for plants (Chen et al., 2010; Pang
et al., 2010; Yao et al., 2010; Garc侏a鄄Robledo et
al., 2013). If ITS in its entirety is difficult to am鄄
plify and directly sequence, ITS2 could be an alter鄄
native for gymnosperms because it is shorter and eas鄄
ier to sequence than the entire ITS region (Coleman
et al., 2003). Therefore, ITS2 can represent a use鄄
ful back鄄up barcode of ITS (Li et al., 2011b).
The ITS2 primer pair used for gymnosperms
showed relative low PCR and / or sequencing success
in previous study (Chen et al., 2010; Pang et al.,
2012). It is therefore the lack of a highly universal
primer pair for ITS2 that prevents its wider use in
gymnosperms. Thus, primer universality is one of
the first to fulfilling criteria when screening for a
DNA barcode ( Chase et al., 2007; Kress and
Erickson, 2007; Ford et al., 2009; Hollingsworth et
al., 2009). In this study, we designed three new
primers based on sequences of the 5. 8S gene greatly
conserved across 55 genera of gymnosperms. When
the polymorphic site exists within the primers re鄄
gion, we used the combined base in the prime (Ta鄄
ble 2). Four of the seven candidate ITS2 primer
combinations here performed very well with 100%
PCR success for all the tested 56 species covering all
the families of the eight orders of gymnosperms. On鄄
ly Ginkgo biloba and Cunninghamia lanceolata yiel鄄
ded weak or medium strong PCR bands for the four
primer combinations. Ginkgo biloba is the only living
representative of the family Ginkgoaceae with an iso鄄
lated systematic position in the gymnosperms. This
distant genetic relationship of Ginkgo biloba with
other groups of gymnosperms may have resulted in
changes in the annealing sites for most combinations
(except for GYM_5. 8SF2 / ITS4) and may explain
the weak or medium PCR band of the candidate ITS2
primers for this species ( c. f. M觟ller, 2000). The
case for Cunninghamia lanceolata ( Cupressaceae)
857摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
is likely different, because all other sampled species
of Cupressaceae yielded strong PCR bands. A rela鄄
tively poor quality of template DNA used here or the
presence of inhibitors might be responsible for the
consistently weak PCR bands of Cunninghamia lan鄄
ceolata.
Based on the PCR success and yield of the PCR
products of the four ITS2 primer combinations fully
tested, two combinations GYM_5. 8SF2 / ITS4 and
S2F / S3R performed slightly better. Of these two
GYM_5. 8SF2 / ITS4 performed slightly better than
the benchmark primer pair S2F / S3R, and yielded
strong PCR band for most sampled species here.
Both primer combinations, GYM_5. 8SF2 / ITS4 and
S2F / S3R, performed equally well in their universal鄄
ity of sequencing. The generated sequences showed
high levels of sequence quality and coverage as well,
which is also an important criterion for DNA barcode
screening. Based on our overall assessments of prim鄄
er universality and sequence quality and coverage,
we propose GYM_5. 8SF2 / ITS4 as the most “univer鄄
sal冶 primer pair for ITS2 for barcoding in gymno鄄
sperms.
Acknowledgements: We are grateful to QL Zhang, JB
Yang, J Yang, HT Li and LJ Yan for their help in lab work.
We also thank Michael M觟ller from Royal Botanic Garden Ed鄄
inburgh for improving English of the manuscript. Laboratory
work was performed at the Laboratory of Molecular Biology at
the Germplasm Bank of Wild Species, Kunming Institute of
Botany, Chinese Academy of Sciences.
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