Plant Mol Biol Rep (2009) 27:86–93

DOI 10.1007/s11105-008-0060-5

Start Codon Targeted (SCoT) Polymorphism: A Simple,

Novel DNA Marker Technique for Generating
Gene-Targeted Markers in Plants
Bertrand C. Y. Collard & David J. Mackill

Published online: 16 September 2008

# Springer-Verlag 2008

Abstract Random amplified polymorphic DNA (RAPD) Introduction

markers have been used for numerous applications in plant
molecular genetics research despite having disadvantages of DNA markers have numerous applications in plant molec-
poor reproducibility and not generally being associated with ular genetic research (Gupta et al. 1999; Semagn et al.
gene regions. A novel method for generating plant DNA 2006; Winter and Kahl 1995). Two of the most common
markers was developed based on the short conserved region uses of DNA markers have been the assessment of genetic
flanking the ATG start codon in plant genes. This method uses diversity within crop germplasm and the construction of
single 18-mer primers in single primer polymerase chain linkage maps for mapping genes or quantitative trait loci
reaction (PCR) and an annealing temperature of 50°C. PCR (QTL) controlling agronomically important traits (Collard
amplicons are resolved using standard agarose gel electro- et al. 2005; Farooq and Azam 2002). The use of random
phoresis. This method was validated in rice using a genetically amplified polymorphic DNA (RAPD) markers (Welsh and
diverse set of genotypes and a backcross population. McClelland 1990; Williams et al. 1990), inter simple
Reproducibility was evaluated by using duplicate samples sequence repeat (ISSR) markers (Blair et al. 1999), and
and conducting PCR on different days. Start codon targeted amplified fragment length polymorphism (AFLP) markers
(SCoT) markers were generally reproducible but exceptions (Vos et al. 1995) have been very popular because these
indicated that primer length and annealing temperature are not marker techniques may generate relatively high numbers of
the sole factors determining reproducibility. SCoT marker DNA markers per sample and are technically simple. These
PCR amplification profiles indicated dominant marker like methods have been used extensively in genetic analysis of
RAPD markers. We propose that this method could be used in prokaryotes and eukaryotes. In crops, RAPD, ISSR, and
conjunction with these markers for applications such as AFLP markers have been used extensively for genetic
genetic analysis, bulked segregant analysis, and quantitative diversity analysis and QTL mapping (Botha and Venter
trait loci mapping, especially in laboratories with a preference 2000; Dziechciarkova et al. 2004; Gostimsky et al. 2005;
for agarose gel electrophoresis. Gupta et al. 1999; Kelly and Miklas 1998; Mueller and
Wolfenbarger 1999; Rao et al. 2002). Each marker system
Keywords Gene-targeted markers . Start codon . Genetic has certain advantages and disadvantages compared to each
diversity . QTL mapping other. In many research labs, the selection of marker systems
is largely influenced by crop species, technical expertise,
B. C. Y. Collard (*) : D. J. Mackill available equipment, and available research funding.
Plant Breeding, Genetics and Biotechnology Division,
In recent years, many new alternative and promising marker
International Rice Research Institute (IRRI),
DAPO Box 7777, Metro Manila, Philippines techniques have emerged. These techniques include inter
e-mail: [email protected] retrotransposon amplified polymorphism, retrotransposon
microsatellite amplified polymorphism (Kalendar et al.
Present address:
1999), sequence-related amplified polymorphism (Li and
B. C. Y. Collard
Department of Primary Industries, Quiros 2001), and target region amplified polymorphism
Bundoora, Victoria 3083, Australia (TRAP; Hu and Vick 2003). Coupled with the rapid growth
Plant Mol Biol Rep (2009) 27:86–93 87

of genomics research, there has been a trend away from following the method by Zheng et al. (1995) with minor
random DNA markers towards gene-targeted markers modifications described by Collard et al. (2007).
(Andersen and Lubberstedt 2003; Gupta and Rustgi
2004). Genome sequence data offers enormous potential Primer Design
for the development of new markers in diverse plant species
(Holland et al. 2001). Primers were designed from consensus sequences derived
In this study, we describe a novel marker system called from the studies by Joshi et al. (1997) and Sawant et al.
start codon targeted polymorphism that is based on the (1999). For primer design, the ATG codon (+1, +2, and +3),
short conserved region in plant genes surrounding the ATG ‘G’ at position +4, and A, C, and C at positions +7, +8, and
translation start (or initiation) codon that has been well +9, respectively, were fixed (Table 2).
characterized in previous studies (Joshi et al. 1997; Sawant All primers were 18-mer and ranged in GC content
et al. 1999). DNA markers are produced by polymerase between 50% and 72% (Table 3). There were no degener-
chain reaction (PCR) using single primers that are designed acies. Primers were checked for dimers and hairpin loops
from the short conserved region flanking the ATG start using the program ‘FAST PCR’ (Kalendar 2007).
codon that is conserved for all genes. Therefore, in
principle, this technique is similar to RAPD or ISSR or PCR and Electrophoresis
single primer amplification reaction because a single primer
is used as the forward and the reverse primer (Gupta et al. PCR was optimized for testing the SCoT method. The final
1994; Williams et al. 1990). Markers are visualized by optimized protocol is reported here. All PCR reactions were
standard gel electrophoresis with agarose gels and staining performed within a total volume of 10 μl in 96-well plates
making this technique suitable for the vast majority of plant using a PTC-100 Thermocycler (MJ Research Model
research labs with standard equipment. We have validated PTC100). PCR reaction mixtures contained PCR buffer
this technique using the important crop and model species (Promega; 20 mM Tris-HCl (pH 8.4), 50 mM KCl),
rice (Oryza sativa). 1.5 mM MgCl2, 0.24 mM of each deoxyribonucleotide
triphosphates, 0.5 U of Taq polymerase (Promega), and
0.8 μm of primer. Each reaction contained 25 ng of
Materials and Methods template DNA. A standard PCR cycle was used: an initial
denaturation step at 94°C for 3 min, followed by 35 cycles
Plant Material of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min;
the final extension at 72°C was held for 5 min. All PCR
A representative set of ten rice genotypes was used for testing amplification products were separated on 1.2% agarose
the level of polymorphism. These genotypes include popular gels in Tris-borate buffer stained with ethidium bromide
‘mega-varieties’, common rice ‘reference’ genotypes, and and visualized under UV light.
important donor parents currently used in International Rice
Research Institute’s breeding program (Table 1). A random Data Analysis
set of 14 BC1 lines were obtained from a larger BC1
population derived from a IR64 (recurrent parent)×FL478 PCR-amplified SCoT fragments detected on gels were
cross. DNA was extracted from a 2-week-old seedling tissue scored as absent (0) or present (1). Only clear, reproducible

Table 1 List of rice genotypes selected for SCoT genotyping

Genotype Subspecies Information

IR64 indica Popular and widely grown rice ‘mega variety’a

FL478 indica Salt tolerance donor breeding line derived from Pokkali
Swarna indica Popular rice mega variety grown in India
Samba Mahsuri indica Popular rice mega variety grown in India
BR28 indica Popular rice mega variety grown in Bangladesh
BR11 indica Popular rice mega variety grown in Bangladesh
IR40931 indica Submergence tolerant donor breeding line derived from FR13
IR36 indica Popular and widely grown rice ‘mega variety’
Nipponbare japonica Reference genotype (genome sequence available)
Azucena japonica Drought tolerant
a
The term ‘mega variety’ refers to extremely popular rice varieties that are widely grown by farmers.
88 Plant Mol Biol Rep (2009) 27:86–93

A/C
15
bands were scored. Pairwise comparisons between acces-

N
–
sions, based on the proportion of shared bands produced by

C/T
the primers, were calculated using the Dice similarity
14

A
–
13
coefficients (expressed as percentages) using the program
G FreeTree (Pavlicek et al. 1999). A dendrogram showing the
A
N
genetic relationships between accessions, based on the
12

N
A unweighted pair-group method with arithmetic averages,
C

was constructed using TreeView (Page 1996).

11

N
C
C
A/G
T/A
10

Results and Discussion

C/G

G
C
9

Rationale and Concept

N
C
C
8

Gene-targeted markers are preferable for numerous appli-

A/G

cations in plant molecular genetics especially QTL mapping

N
N
7

since recombination levels between marker and gene/QTL

are generally lower compared with ‘indirect random
G

G
T
6

markers’ such as RAPDs, ISSRs, or SSRs (Andersen and

N
C
C
5

Lubberstedt 2003). The SCoT technique is based on the

single primer amplified region principle since it uses a
G
G
G
4

single primer as a forward and reverse primer, like the RAPD

Table 2 Consensus sequences flanking the ATG start codon from Joshi et al. (1997) and Sawant et al. (1999)

G
G
G
3

or ISSR technique. However, PCR amplification using SCoT

primers targets gene regions surrounding the ATG initiation
T
T
T
2

codon on both DNA strands as shown in Fig. 1. It is possible

A
A
A
1

that some SCoT markers would be codominant due to

insertion–deletion mutations; these would be the minority
−1

A
N
C

like codominant RAPDs (Davis et al. 1995).

Due to the basis of SCoT primer design, we expect
C/A

Dataset II was based on lowly expressed genes described in Sawant et al. (1999).
Dataset I was based on highly expressed genes described in Sawant et al. (1999).
−2

A
C

SCoT markers to be distributed within gene regions that

contain genes on both plus and minus DNA strands. It is
A/G

G/A

also possible that pseudogenes and (genes within) trans-

−3

posable elements may be used as primer binding sites by

A/C

SCoT polymorphism technique. An important factor is

−4

A
N

the distance in base pairs between primer binding sites of

the template. Therefore, a relatively long extension time
A/C
−5

N
C

of the thermal cycle is important and we recommend at

least 2 min.
−6

N
T

Primer Design and PCR Protocol

Sawant et al. (1999)
Sawant et al. (1999)
Joshi et al. (1997)

There are many important parameters that influence the

reliability and reproducibility of PCR-amplified markers
Reference

(Tyler et al. 1997). SCoT primers in this study were

designed based on a consensus sequence for the flanking
region around the ATG start codon derived from the
Consensus sequence

previous studies by Joshi et al. (1997) and Sawant et al.

Monocot consensus
Nucleotide position

(1999). Specific nucleotides in the primer sequence were

fixed: the ATG codon (at positions +1, +2, and +3), ‘G’ at
Dataset IIb

position +4, ‘C’ at position +5, and A, C, and C at positions

Dataset Ia

+7, +8, and +9, respectively. Most primers differed from

each other by at least one nucleotide with an emphasis
b
a
Plant Mol Biol Rep (2009) 27:86–93 89

Table 3 SCoT primer sequences on variations at the 3′ end, which has been shown to
SCoT primer Sequence (5′-3′) %GC critical for primer-template specificity (Kwok et al. 1990;
Sommer and Tautz 1989). Nonconserved nucleotide posi-
1 CAACAATGGCTACCACCA 50 tions (i.e., ‘N’) were exploited, by designing primers such
2 CAACAATGGCTACCACCC 56 that these nonconserved nucleotides typically occurred
3 CAACAATGGCTACCACCG 56
within the last three or four nucleotides at the 3′ end.
4 CAACAATGGCTACCACCT 50
Previous research studies have indicated that primer
5 CAACAATGGCTACCACGA 50
6 CAACAATGGCTACCACGC 56 length and annealing temperature are important factors for
7 CAACAATGGCTACCACGG 56 the reproducibility of PCR-based DNA markers. Longer
8 CAACAATGGCTACCACGT 50 primers have been shown to improve reproducibility
9 CAACAATGGCTACCAGCA 50 (Debener and Mattiesch 1998; Tanaka and Taniguchi
10 CAACAATGGCTACCAGCC 56 2002; Ye et al. 1996). The study by Gillings and Holley
11 AAGCAATGGCTACCACCA 50 (1997) suggest that primers between 18 to 24 nucleotides
12 ACGACATGGCGACCAACG 61
are preferable for producing reproducible markers. SCoT
13 ACGACATGGCGACCATCG 61
14 ACGACATGGCGACCACGC 67
primer design was constrained by the number of highly
15 ACGACATGGCGACCGCGA 67 conserved nucleotides within the conversed ATG region,
16 ACCATGGCTACCACCGAC 56 and 18-mer primers were considered to be the maximum
17 ACCATGGCTACCACCGAG 61 length. An initial comparison of 12-mer versus 18-mer
18 ACCATGGCTACCACCGCC 67 primers indicated that the latter primer length was superior
19 ACCATGGCTACCACCGGC 67 in terms of reproducibility (data not shown). The optimal
20 ACCATGGCTACCACCGCG 67 primer length in the TRAP technique was also found to be
21 ACGACATGGCGACCCACA 61
18 nucleotides (Hu and Vick 2003).
22 AACCATGGCTACCACCAC 56
23 CACCATGGCTACCACCAG 61 Higher annealing temperatures also improve marker
24 CACCATGGCTACCACCAT 56 reproducibility (Atienzar et al. 2000; Johnson and Clabots
25 ACCATGGCTACCACCGGG 67 2000). A range of annealing temperatures was tested (data
26 ACCATGGCTACCACCGTC 61 not shown). There was a noticeable decrease in the number
27 ACCATGGCTACCACCGTG 61 of scorable markers when annealing temperatures exceeded
28 CCATGGCTACCACCGCCA 67 55°C, and, therefore, 50°C was selected due to the number
29 CCATGGCTACCACCGGCC 72
of reproducible scorable bands when this annealing
30 CCATGGCTACCACCGGCG 72
temperature was used.
31 CCATGGCTACCACCGCCT 67
32 CCATGGCTACCACCGCAC 67 Magnesium chloride, an important cofactor for Taq DNA
33 CCATGGCTACCACCGCAG 67 Polymerase and for primer-template binding, is another
34 ACCATGGCTACCACCGCA 61 important factor that influences reproducibility (Perry et al.
35 CATGGCTACCACCGGCCC 72 2003). It was reasoned that a standard, relatively low, fixed
36 GCAACAATGGCTACCACC 56 concentration of 1.5 mM would minimize the likelihood of

Fig. 1 Diagram showing GENOTYPE #1

principle of SCoT PCR
amplification ,
Gene A 3
,
5

SCoT primer SCoT primer

, ,
3 Gene B 5

PRESENT
GENOTYPE #2

, ,
5 Gene A 3

SCoT primer

, ,
3 5

ABSENT
90 Plant Mol Biol Rep (2009) 27:86–93

SCoT 1 SCoT 9
nonspecific binding, even though higher number of markers 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
may be obtained using higher concentrations.
1500

1000 1500
Generation of Polymorphic DNA Markers 1000
250

Preliminary evaluation using a representative set of rice 250

genotypes has indicated that SCoT primers generate DNA SCoT 12 SCoT 13
fingerprints similar to those generated by using RAPD 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

markers (Fig. 2). The size range was typically between 1500 1500

200–1,500 bp. Using IR64 and Nipponbare as reference 1000 1000

genotypes, between two and six markers were usually

250 250
scored, the exception was using SCoT 9 which produced
only a single marker for most genotypes. This is compa-
rable to the number of markers typically generated by SCoT 18 SCoT 19
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

RAPD and ISSR techniques in rice (Kaushik et al. 2003;

1500 1500
Mackill 1995; Parsons et al. 1997).
1000 1000
The results also indicated the effect of altering a
single nucleotide within the last three nucleotides at the 250 250

3′ end (Fig. 2). For example, SCoT primers 29, 30, and
31 differ only in the last nucleotide at the 3′ end yet SCoT 20 SCoT 22
produced different profiles. SCoT primers 18 and 20 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

differed only in the second last nucleotide and SCoT 1500 1500
primers 12 and 13 differed only at the third last nucleotide 1000 1000
yet also produced very different DNA marker profiles
(Fig. 2). Two primers differed by a single nucleotide at the 250 250

5′ end (SCoT 1 and SCoT 11), which also generated

different DNA marker profiles (data not shown). Further SCoT 29 SCoT 30
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

experiments are needed to determine the full effects of

nucleotide substitution on the generation of polymorphism 1500 1500

(Table 3). 1000 1000

Reproducibility 250 250

SCoT 31 SCoT 34
RAPD markers have been notorious for having problems
1 2 3 4 5 6 7 8 9 10 11
with reproducibility, especially between laboratories (Hallden 1 2 3 4 5 6 7 8 9 10 11

et al. 1996; Jones et al. 1997; Penner 1996). Since the SCoT 1500 1500

technique was based on the same principle of single primer 1000

1000

PCR, reproducibility was thoroughly tested and consisted

250
of two stages: (1) comparing PCR amplification results 250

between duplicate samples of selected genotypes and (2)

repeating PCR reactions on different days and using Fig. 2 Examples of SCoT marker profiles. Lane 1 IR64, 2 FL478, 3
Swarna, 4 Samba Mahsuri, 5 BR28, 6 BR11, 7 IR40931, 8 IR36, 9
different thermal cyclers. Primers were initially prescreened Nipponbare, 10 Azucena, 11 no template control. Left most lane—
on samples of selected genotypes; only primers that passed 250 bp DNA ladder (Invitrogen Cat. No: 10596-013)
stage 1 (i.e., produced reproducible markers) were consid-
ered for stage 2 evaluation. In general, SCoT markers were
reproducible when using duplicate samples, although some
primers did not produce reproducible profiles suggesting like RAPD. These results may also imply that further
that primer length and annealing temperature per se do not optimization of PCR conditions is required for these
ensure reproducibility. Markers that ‘passed’ stage 1 testing primers since the primer length of 18 nucleotides and
were found to be reproducible when PCR was performed annealing temperature of 50°C do not guarantee reproduc-
on different days and using different thermal cyclers with ibility. In this study, it was observed that primers with a
the exception of SCoT 28. This emphasizes the need to higher GC content were generally more reproducible (data
carefully test all markers generated by using this technique, not shown).
Plant Mol Biol Rep (2009) 27:86–93 91

Validation of SCoT Markers: Genetic Diversity Analysis SCoT 33

and Testing in a Backcross Population

FL478
IR64
1 2 3 4 5 6 7 8 9 10 11 12 13 14

1500
SCoT primers were used to fingerprint a small diverse set
of rice genotypes. The 13 primers generated a total of 50 1000
polymorphic SCoT markers, which were scored based on at
least two replicates. Only markers that were clearly scorable
250
and detected on both gels were considered for diversity
analysis. Clustering of the rice genotypes was generally
consistent with known taxonomic and pedigree information SCoT 34
(Fig. 3). The two japonica genotypes, Nipponbare and

FL478
Azucena, clearly clustered together and were more diverse

IR64
1 2 3 4 5 6 7 8 9 10 11 12 13 14
compared to the indica genotypes. Samba Mahsuri and 1500
Swarna both have Mahsuri as a common parent and clustered
together. The Bangladeshi varieties, BR11 and BR28, 1000

clustered together which is consistent with the common

parents IR262 and Peta in their pedigrees. The abiotic stress 250
tolerant breeding lines FL478 and IR40931 also clustered
together, which is consistent with IR1561 being a common
ancestor. Genotyping of a small random subset of backcross Fig. 4 Validation of SCoT markers in 14 backcross lines
lines indicated typical segregation of a dominant marker and
high reproducibility since identical marker profiles were
generated between replications (Fig. 4). or by targeting different regions of the conserved region
flanking the ATG start codon. Since the region flanking the
ATG start codon is highly conserved in all plant species, we
Conclusion predict that the SCoT method will be useful for generating
DNA markers in diverse plant species although this has not
Although we have only tested 36 primers in this study, been experimentally validated.
many additional primers could be designed by minor We anticipate that there will be three main applications
alterations of the primer sequences described in this study of SCoT markers: QTL mapping, bulked segregant analy-
sis, and genetic diversity studies. For QTL mapping, SCoT
markers could be integrated into existing framework maps
to increase the marker density or to target specific
Azucena chromosomal regions. In another analogy with RAPD, we
propose that important SCoT markers, such as those
Nipponbare
identified to be tightly linked to a gene or QTL of interest,
IR64 could be converted into sequence characterized amplified
regions markers or sequence tagged site markers in order to
make the marker single locus and improve robustness
(Monna et al. 1994; Paran and Michelmore 1993).
Swarna In summary, a novel DNA marker technique that targets
plant gene regions by the design of primers was developed.
It is agarose-based and therefore simple and relatively
Samba Mahsuri cheap to use. Furthermore, markers should be associated
IR36
with gene regions based on their primer design. This
marker technique should provide an additional option to the
BR28 FL478 RAPD or ISSR techniques for several applications in plant
BR11 IR40931 genetic analysis.

Acknowledgements Technical assistance for gel electrophoresis by

0.1 Ms. Miladie Penarubia is gratefully acknowledged. We also thank Dr.
C. Raghavan and two anonymous reviewers for valuable comments on
Fig. 3 Radial tree of rice genotypes based on 50 SCoT markers the manuscript.
92 Plant Mol Biol Rep (2009) 27:86–93

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