American Journal of Plant Sciences, 2012, 3, 1322-1327 Published Online September 2012 (
Effective Procedure for Development of EST-SSR Markers
Using cDNA Library
Kyung A Kim1, Hee-Cheon Park1, Jae-Keun Sohn2, Kyung-Min Kim2*
1Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, Korea (South); 2School of Applied
Biosciences, College of Agriculture and Life Science, Kyungpook National University, Daegu, Korea (South).
Email: Kyung-AKim,, Hee-Cheon Park,, Jae-Keun Sohn,,
*Kyung-Min Kim,
Received August 8th, 2012; revised September 5th, 2012; accepted September 13th, 2012
The present study was conducted to develop EST-SSR markers using the cDNA library from rice plant. Total RNA ex-
tracted from the leaves of brown plant hopper resistance gene originated from a rice cultivar “Cheongcheong” and sen-
sitive rice cultivar “Nakdong” were used to synthesize a cDNA library. As a result of analyzing the cDNA library, the
17 EST-SSR primer sets were developed. This study enables to provide effective marker assisted selection (MAS)
methods on the selection of white-backed planthopper resistance gene originated from a rice plant more simply, quickly
and precisely. Furthermore, using this marker’s advantage of deriving from cDNA, it is possible to identify the
white-backed planthopper resistance gene. In addition, this study introduces a technique for construction of a cDNA
library safely without using radioactivity.
Keywords: cDNA; EST-SSR Markers; Radioactivity; Rice
1. Introduction
Several protocols for cDNA library construction have
been investigated to date; however, these are each com-
plicated and difficult to control [1-6]. Moreover, the
procedure for size fraction selection requires the use of
radioactivity. Therefore, this study was conducted to de-
velop expressed sequence tag-simple sequence repeat
(EST-SSR) markers of the brown plant hopper resistance
gene for the “Cheongcheong” and the sensitive rice culti-
var, “Nakdong”. Furthermore, we introduce a safe and
easy method for construction of a cDNA library without
the use of radioactivity. The white-backed planthopper is
one of the most harmful insects to crop growth [7]. Most
farmers are currently cultivating a japonica sensitive rice
cultivar in Korea; however, it is very difficult to examine
brown plant hopper resistance in rice [8,9]. Marker as-
sisted selection (MAS) is able to select brown plant hop-
per resistant rice cultivars based on DNA markers [10-
13]. Using MAS, it is possible to overcome the complex-
ity and imprecision associated with traditional methods
of selecting brown plant hopper resistant rice cultivars
[14]. So, we carried out the development of EST-SSR
markers for molecular breeding in rice.
2. Materials and Methods
2.1. Extraction of Total RNA and Genomic DNA
Total RNA was extracted using the RNeasy Mini Kit
according to the manufacturer’s instructions (Qiagen
GmbH, Hilden, Germany).Briefly, RLT Buffer contain-
ing β-mercaptoethanol (β-ME) was added to fresh leaves
of “Cheongcheong” and “Nakdong” and thoroughly
mixed. The mixture was then transferred to a supplied
spin column and centrifuged for 5 minutes. Next, the
supernatant from the spin column was carefully trans-
ferred to a new 1.5 ml tube, to which an equal volume of
70% ethanol was added and mixed by pipetting. The
sample was then immediately transferred to an RNeasy
spin column and centrifuged for 1 minute at 13,000 rpm,
after which the flow-through was discarded. Next, 700 μl
of buffer RW1 was added to the spin column and the
sample was centrifuged for 1 minute at 13,000 rpm to
wash the column membrane. The flow-through was then
discarded, after which 500 μl of buffer RPE was added to
the RNeasy spin column, which was then centrifuged for
1 minute at 13,000 rpm to wash the membrane. The
flow-through was again discarded and this process was
repeated. Next, the RNeasy spin column was placed in a
new 1.5 ml collection tube and 30 µl of DEPC water was
added directly to membrane. After incubating for 10
*Corresponding author.
Copyright © 2012 SciRes. AJPS
Effective Procedure for Development of EST-SSR Markers Using cDNA Library 1323
minutes, the column was centrifuged for 1 minute at
13,000 rpm to elute the RNA and the extracted RNA was
then stored at –80˚C. The genomic DNA was extracted
using a DNeasy Mini Kit according to the manufacturer’s
instructions (Qiagen GmbH, Hilden, Germany).
2.2. Construction of cDNA Library
A cDNA library was produced using a cDNA Synthesis
Kit (TaKaRa, Shiga, Japan) and a cDNA PCR Library
Kit (TaKaRa, Shiga, Japan). Briefly, template RNA was
added to a mixture composed of 2 μl 5× first strand syn-
thesis buffer, 0.5 μl dNTPs mixture (10 mM), 0.5 μl
RNase inhibitor (20 unit·μl–1), 1 μl oligodT RA primer (1
μμl–1) and 0.5 μl of M-MLV reverse transcriptase.
Upon adding the template RNA, the volume was adjusted
to 10 μl. The mixture was then reacted for 10 minutes at
room temperature, after which it was incubated at 42˚C
for 1 hour, then heated to 80˚C for 5 minutes to produce
the first strand of cDNA. Next, 10 μl of the first-strand
cDNA synthesis mixture was mixed with 15 μl of 5×
second strand synthesis buffer and 1.5 μl of dNTP mix-
ture (10 mM), after which the volume was adjusted to 71
μl with DEPC water. In the next step, 1 μl of E. coli
DNA polymerase I (20 unit·μl–1) and 1 μl of E. coli-
RNase H/E. coli DNA ligase mixture was added, mixed
well and reacted at 16˚C for 2 hours. The mixture was
then heated to 72˚C, after which 2 μl of T4 DNA poly-
merase (1 unit·μl–1) was added and thoroughly mixed.
The mixture was then allowed to react at 37˚C for 10
minutes, at which time 6 μl of stop solution (0.2M EDTA,
2 mg·ml–1 glycogen, pH 8.0) was added. An equal vo-
lume of PCI (phenol: chloroform: isoamyl alcohol =
25:24:1) was then added, after which the mixture was
stirred for 1 minute and centrifuged at 15,000 rpm for 5
minutes to generate pure second-strand cDNA. The su-
pernatant was subsequently transferred to a new 1.5 ml
tube, after which an equal volume of CI (chloroform:
isoamylalcohol 24:1) was added and the mixture was
stirred for 1 minute and then centrifuged at 15,000 rpm
for 5 minutes. The supernatant was subsequently trans-
ferred to a new 1.5 ml tube and equal volumes of 4 M
ammonium acetate and isopropanol were added. Follow-
ing incubation at –80˚C for 1 hour, the mixture was cen-
trifuged at 15,000 rpm for 5 minutes.
2.3. Construction of Second-Strand cDNA
without Radioactivity
The supernatant was then removed and 1 ml of 70%
ethanol was added to wash the precipitate. After the pre-
cipitate dried, it was dissolved in 5 μl of RNase-free wa-
ter and ExTaq polymerase (TaKaRa, Shiga, Japan) was
used to synthesize an adaptor sequence with a restriction
enzyme site at each terminal of the second-strand cDNA.
To accomplish this, 30 μl of second strand cDNA, 5 μl of
10× ExTaq buffer, 4 μl of dNTP mixture (2.5 mM), 1 μl
of CA primer (20 p mole·μl–1, 5’-CGTGGTACCATG-
GTCTAGAGT-3’), 1 μl of RA primer (20 pmole·μl–1,
RaExTaq polymerase (5 unit·μl–1) (TaKaRa, Shiga, Japan)
and 8.5 μl nuclear free water were mixed to give a final
volume of 50 μl, after which PCR was performed by sub-
jecting the samples tothe following conditions: 2 minutes
at 94˚C followed, by 35 cycles of 30 seconds at 94˚C, 30
seconds at 60˚C and 3 minutes at 4˚C and then final ex-
tension for 10 minutes at 72˚C. To select 500 bp to 1 kb
for the cDNA library, the cDNA PCR amplicons were
electrophoresed in 1.0% agarose gel, after which selected
bands were cut out and extracted from the gel (Qiagen-
Co., Germany). After the gel extraction procedure, PCR
was performed (TaKaRa, Shiga, Japan) by subjecting the
samples to the same conditions as described above.
2.4. cDNA Library Transformation to Vector
The cDNA librarywas purified using a PCR purification
kit (QiagenCo., Germany) and then ligated into the
pGEM-T Easy Vector (Promega Co., USA). After adding
5 μl of 2× rapid ligation buffer and 1 μl of T4 DNA li-
gase (unit·μl–1), the pGEM-T Easy Vector (50 ng·μl–1)
and cDNA PCR products were adjusted to the ratio. The
volume was then adjusted to 10 μl with nuclease-free
water, after which the sample was allowed to react at 4˚C
for 12 hours. Next, 5 μl of ligation product and 50 μl of
DH 5α competent cells were mixed and reacted on ice for
30 minutes and then incubated at 42˚C for 1 minute. The
mixture was again incubated on ice for 2 minutes, after
which 250 μl of LB broth (10 g Bactotryptone, 5 g Bacto
yeast extract and 5 g NaCl) was added and the samples
were cultured at 37˚C for 1 hour while shaking at 180
rpm. The culture was then incubated at 37˚C for 12 hours
after platingon LB agar amended with 100 ppm·l–1 ampi-
cillin, 0.5 mM IPTG and 80 µg·ml–1 X-Gal. Blue-colored
and white-colored colonies appeared after culture, with
white indicating that transformation had occurred. Ac-
cordingly, white colonieswere selected, added to 5 ml of
LB broth, and then cultured at 37˚C for 14 hours. Fol-
lowing incubation, the culture medium was centrifuged
at 3000 rpm for 10 minutes and then extracted using a
Qiaprep Spin Miniprep Kit (QiagenCo., Germany). After
removing the supernatant, 250 μl of P1 buffer was added
to dissolve the colonies, and the sample was then trans-
ferred to a 1.5 ml tube and mixed carefully with 250 μl of
P2 buffer. Next, 350 μl of N3 buffer was added and the
sample was centrifuged at 13,000 rpm for 10 minutes.
The supernatant was then transferred to a new 1.5 ml
tube, centrifuged again at 13,000 rpm for 5 minutes, and
then transferred to a new 1.5 ml tube. Next, 700 μl of
supernatant was transferred to a spin column and centri-
Copyright © 2012 SciRes. AJPS
Effective Procedure for Development of EST-SSR Markers Using cDNA Library
fuged at 13,000 rpm for 1 minute, after which the flow
through was discarded. PB buffer (5 μl) was then added
to the column, after which it was centrifuged at 13,000
rpm for 1 minute, and 750 μl of PE buffer was added to
wash the plasmid DNA. The flow through from the col-
lection tube was then discarded, after which the column
was centrifuged to remove any residual buffer. The spin
column was then added to a 1.5 ml tube containing 30 μl
of nuclease-free water to extract the plasmid DNA.
2.5. DNA Sequencing and Primer Design
The plasmid DNA sequence was analyzed by SolGent
(SolGent Co., Korea), which verified that the cDNA was
inserted into the pGEM-T Easy Vector (Promega Co.,
USA). The DNA sequencing analysis results were then
used for SSR-site analysis in the EST sequence using a
microsatellite analysis program
( [15], and the SSR
sites analyzed were used to prepare the forward and re-
verse primers.
2.6. Polymorphism Analysis Using an EST-SSR
Polymorphism analysis was conducted using the markers
of EST-SSR primer sets developed from this study.
Briefly, PCR was used to investigate genomic crane
DNA using TaKaRaExTaq polymerase (TaKaRa, Shiga,
Japan) and the EST-SSR primer set (forward and reverse
primers) developed in this study. The PCR conditions
were as follows: initial denaturation at 94˚C for 5 min-
utes, followed by 35 cycles of 94˚C for 30 seconds, 56˚C
- 58˚C for 30 seconds and 72˚C for 45 seconds and then
final extension at 72˚C for 5 minutes. QIAxcel (Qiagen-
Co., Germany) was used to verify the amplified band in
the PCR product, and the size was verified using the QX
DNA size marker (FX174/Hae). Only bands that were
verified with a 0% noise cut-off (0% - 99%) and 100%
contrast (1% - 100%) were selected; the red portion of
the band peak size (bp) and concentration (ng·μl–1) were
referenced in a data table.
3. Results
It is laborious and time-consuming to examine brown
plant hopper resistance in rice cultivars. To obtain good
quality results and reduce labor, we developed a simple
and safe method of cDNA construction for development
of EST-SSR markers. This procedure is able to select a
cDNA library of hopping size, about 500 bp to 1 kbp.
Fragments of this size can be ligated into the pGEM-
T-Easy vector efficiently, thereby enabling a high yield
of good quality ligated products. This procedure can also
be carried out safely without the need to use radioactivity
to determine size fraction. The EST-SSR markers of the
brown plant hopper resistance gene originating from the
“Cheongcheong” and the sensitive rice cultivar “Nak-
dong” were developed. Seventeen EST-SSR primer sets
were designed the expected size was shown (Table 1).
The definite PCR bands of the amplified DNA from rice
cultivarsusing the EST-SSR primer sets was achieved
(Figure 1(a)). For the mapping of resistant gene to
white-backed planthopper, the doubled-haploid (DB) po-
pulations was developed by anther culture of F1 plants
from a cross “Cheongcheong/Nagdong”. PCR analysis of
the DNA from the 22 “Cheongcheong/Nakdong” Dou-
bled Haploid (CNDH) populations using the CC24-2F2R
primer set showed clear bands between each populations
(Figure 1(b)). All patterns of the PCR bands using EST-
SSR primers were shown very clearly and precisely, re-
4. Discussions
The several protocols for cDNA library constructionwere
previously reported that exploit the mRNA cap structure
to enrich for full-length sequence [3], the “oligo-capping”
method [4,5], CAPture [2], CAP-trapper [1] and
SMARTTM technology of constructing full-length cDNA
library [6]. We also obtained good quality cDNA library
using safe size fraction method. Overall, the methods
presented here should reduce the labor and effort re-
quired for identification or resistant strains of rice while
increasing safety by removing the need to use radioactive
materials. Sequencing was performed on a total of 147
cDNA insertions confirmed through EcoRІ digestion.
Using the DNA STAR SeqMan expert sequence
analysis software (version 5.01), the forward and reverse
primers were selected as EST-SSR sites were found. As a
result of analyzing the EST-SSR sites of the sequence
using the Microsatellite analysis program
( [15] on the 20 se-
lected primer sets and used to develop the EST-SSR
primers (Table 1). To verify their availability as markers,
PCR was performed on each of the designed EST-SSR
primer sets and seventeen primer sets with high repro-
ducibility as EST-SSR markers were selected. Many of
ESTs are available in public databases, which offer an
opportunity to identify SSRs in ESTs by data mining.
These sequences may provide an estimate of diversity in
the expressed portion of the genome and may be useful
for comparative mapping, for tagging important traits of
interest, and for additional map-based cloning of impor-
tant genes [16]. For effective conservation and the use of
genetic resources, evaluation of the genetic variation is
crucial and could be dramatically enhanced by using mo-
lecular genotyping tools. Evaluation of germplasm with
SSRs derived from ESTs (EST-SSRs) may enhance the
role of genetic markers by assaying variation in trans-
cribed and known-function genes. Furthermore, since the
Copyright © 2012 SciRes. AJPS
Effective Procedure for Development of EST-SSR Markers Using cDNA Library
Copyright © 2012 SciRes. AJPS
Table 1. Characterization of seventeen EST-SSR primers from cDNA library in rice cultivar.
EST-SSR primer Repeat motif Primer (5'-3') Annealing
Temp. (˚C)
Expect size
(cloned sequence)
F: N-terminal of the SSR region sequence; R: C-terminal of the SSR region sequence.
Effective Procedure for Development of EST-SSR Markers Using cDNA Library
Figure 1. Analysis of PCR fragments amplified using the EST-SSR primer set for the “Cheongcheong” (C) and “Nakdong”
(N) rice cultivars. QIAxcel was used to verify the amplified band in the PCR product. (a): Amplified with each primers, 1:
CC15-2F2R primer, 2: CC24-2F2R primer, 3: CC58FR primer, 4: CC38FR primer, 5: ND17FR primer, 6: ND92-2F2R
primer. (b): Amplified with CC24-2F2R primer, 1 - 22: Cheongcheong/Nakdong Doubled Haploid populations. M: QX DNA
size marker.
EST-SSR markers represent transcribed regions of the
genome, it is also of interest to compare estimates of ge-
netic diversity calculated from both genomic and EST-
derived SSR markers [17]. Recent increase in the avail-
ability of expressed sequence tag (EST) data has facili-
tated the development of microsatellite or simple se-
quence repeat (SSR) markers in a number of species
group [18]. There are several advanced studied about
EST-SSR for wheat [17,19], cotton [16] and several
grass species [20,21] et al. EST-SSR markers from rice
cultivars were effective and easy MAS methods on the
selection of white-backed planthopper resistance gene
originnated from a rice cultivar. The patterns of the PCR
bands using EST-SSR primers were shown very clearly
and precisely. This method maybe introduce that the se-
lection of using EST-SSR markers is possible to get over
too much like hard work of traditional methods of se-
lecting brown plant hopper resistant rice cultivars.
5. Acknowledgements
This work was supported by a grant from the Next-Gene-
ration BioGreen 21 Program (No. PJ0080822011), Rural
Development Administration, Republic of Korea.This
research was supported by Kyungpook National Univer-
sity Research Fund, 2012. We are grateful to SolGent Co.
in Korea for sequencing of our all cDNA library se-
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