Download An improved technique for isolating codominant compound

Springer-VerlagTokyohttp://www.springer.de102650918-94401618-0860Journal
of Plant ResearchJ
Plant ResLife Sciences27410.1007/s10265-006-0274-2
J Plant Res (2006) 119:415–417
Digital Object Identifier (DOI) 10.1007/s10265-006-0274-2
© The Botanical Society of Japan and Springer-Verlag Tokyo 2006
TECHNICAL NOTE
Chunlan Lian • Md. Abdul Wadud • Qifang Geng •
Kenichiro Shimatani • Taizo Hogetsu
An improved technique for isolating codominant compound microsatellite
markers
Received: January 4, 2006 / Accepted: February 14, 2006 / Published online: April 25, 2006
The Botanical Society of Japan and Springer-Verlag
2006
Abstract An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in
this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the
constructed DNA library using compound SSR primer
(AC)6(AG)5 or (TC)6(AC)5 and an adaptor primer for the
suppression-PCR. A locus-specific primer was designed
from the sequence flanking the compound SSR. The primer
pairs of the locus-specific and compound SSR primers were
used as a compound SSR marker. Because only one locusspecific primer was needed for design of each marker and
only a common compound SSR primer was needed as the
fluorescence-labeled primer for analyzing all the compound
SSR markers, this approach substantially reduced the cost
of developing codominant markers and analyzing their
polymorphism. We have demonstrated this technique for
Dendropanax trifidus and easily developed 11 codominant
markers with high polymorphism for D. trifidus. Use of the
technique for successful isolation of codominant compound
SSR markers for several other plant species is currently in
progress.
Key words Development · DNA marker · Microsatellites ·
Polymorphism · Simple sequence repeat
C. Lian (*) · Q. Geng
Asian Natural Environmental Science Center, The University of
Tokyo, 1-1-8 Midori-cho, Nishitokyo, Tokyo 188-0002, Japan
Fax +81-424-655601
e-mail: [email protected]
M.A. Wadud · T. Hogetsu
Graduate School of Agricultural and Life Sciences, The University of
Tokyo, Tokyo, Japan
K. Shimatani
The Institute of Statistical Mathematics, Tokyo, Japan
Introduction
Hypervariable codominant molecular markers, for example microsatellite (SSR) markers, have proven to be
extremely valuable tools for population genetic studies,
genome mapping, and marker-assisted breeding (Valdes
et al. 1993; Akkaya et al. 1995; Schuler et al. 1996). A variety of methods for SSR isolation have been developed in
recent years and the efforts required to obtain sufficient
working SSR primer pairs have been comprehensively
reviewed by Zane et al. (2002) and Squirrell et al. (2003).
Unfortunately, the conventional strategies used to develop
SSR markers are usually labor-intensive, time-consuming,
and expensive, which restricts a widespread application of
SSR markers.
To improve the efficiency of SSR marker development,
we developed a method, named the dual-suppression-PCR
technique, that did not require enrichment and screening
procedures (Lian et al. 2001; Lian and Hogetsu 2002). By
applying this technique our research group developed SSR
markers for more than 30 species, including plants, ectomycorrhizal fungi, toxic dinoflagellates, and the crown-ofthorns starfish (most of these research results have been
published in Molecular Ecology Notes). This approach
entails sequencing twice and preparation of three primers
for developing one SSR locus, however.
Hayden et al. (2004) recently prepared primers flanking
a compound SSR sequence from the bread wheat genomic
sequence database, and reported that PCR amplification
with a prepared primer and a compound SSR primer
resulted in codominant electrophoretic bands and successfully showed high polymorphism of wheat lineages. In this
method the costs of developing codominant polymorphic
markers are reduced, because a common compound SSR
primer can be used for different markers anchored by the
same type of compound repeat sequence and only one
locus-specific primer need be prepared for each marker.
Applications of this approach are limited to species whose
genomic sequence has already been registered in the database (Hayden et al. 2004), however. No technique has yet
416
been reported for development of such markers for species
for which the genomic sequence is not available.
As the first step in our dual-suppression-PCR method, a
variety of fragments flanked by an SSR sequence at one end
from each investigated species can be amplified from the
constructed restriction DNA libraries by an SSR primer and
the adaptor primer (Lian et al. 2001; Lian and Hogetsu
2002). This encouraged use of the technique to determine
sequences flanking compound SSRs. The application may
also be an improvement of the dual-suppression-PCR
method, because it omits the second step used to determine
the other sequence flanking each SSR by a “walking”
method (Siebert et al. 1995).
In this paper we describe a new technique based on this
idea.
Materials and methods
DNA extraction
Genomic DNA was extracted from fresh leaves of Dendropanax trifidus using a modified CTAB method. The fresh
leaves were directly homogenized in 2× CTAB solution (2%
cetyltrimethylammonium bromide, 0.1 mol L−1 Tris–HCl
(pH 8.0), 20 mmol L−1 EDTA (pH 8.0), 1.4 mol L−1 NaCl,
and 0.5% 2-mercaptoethanol) and then incubated at 60°C
for 30 min. After chloroform–isoamyl alcohol (24:1) extraction, isopropanol precipitation, and 70% ethanol wash,
DNA was dissolved in 100 µL sterilized water and ribonuclease solution (RNase; 10 mg mL−1, 1 µL; Nippon Gene,
Tokyo, Japan) was added. After 30 min incubation at 37°C,
DNA solution was extracted with an equal volume of
chloroform–isoamyl alcohol (24:1) and stored at −30°C until
use.
DNA library construction
An adaptor-ligated DNA library was constructed by using
the method reported previously (Lian et al. 2001). Briefly,
DNA was digested with blunt-end restriction enzyme,
EcoRV, and the restricted fragments were then ligated
with a specific blunt adaptor (consisting of a 48-mer: 5′GTAATACGACTCACTATAGGGCACGCGTGGTCGA
CGGCCCGGGCTGGT-3′ and an 8-mer with the 3′-end
capped by an amino residue: 5′-ACCAGCCC-NH2-3′) by
use of a DNA ligation kit (Takara Shuzo, Japan).
Primer designing of a compound SSR marker
Fragments were amplified from the EcoRV DNA library
using compound SSR primer (AC)6(AG)5 or (TC)6(AC)5
and an adaptor primer AP2 (5′-CTATAGGGCACGCGT
GGT-3′). The amplified fragments were integrated into the
plasmids using pT7 Blue Perfectly Blunt Cloning Kit
(Novagen) and the plasmids were transferred into Escherichia coli according to the manufacturer’s instructions. The
cloned fragments were amplified from the plasmid DNA of
positive clones using the U19 and M13 reverse primers, and
inserted fragment lengths were checked by 1.5% agarose
gel electrophoresis. Amplified fragments were sequenced
directly using Thermo Sequence Pre-mixed Cycle Sequencing Kit (Amersham Biosciences, USA) with a Texas Redlabeled T7 primer (Sigma-Aldrich, Japan) in an SQ-5500E
sequencer (Hitachi). For each fragment containing
(AC)6(AG)n or (TC)6(AC)n compound SSR sequences at
one end, a specific primer (IP1) was designed from the
sequence flanking the compound SSR. The primer pairs of
specific primer (IP1) and compound SSR primer were used
as a compound SSR marker.
Polymorphic analysis of designed primer pairs
To examine the effectiveness of primer pairs designed as
compound SSR markers, 22 D. trifidus individuals were collected from Tsushima Island, southern Japan (34°09′N,
129°13′E). Template DNA was extracted from silica geldried leaves. PCR amplification was performed with a PCR
thermal cycler (TP3000; Takara Shuzo). Five microliters of
the reaction mixture contained 0.5 µL template DNA,
0.2 mmol L−1 of each dNTP, 1× PCR buffer (Mg2+ free,
Applied Biosystems, USA), 2.5 mmol L−1 Mg2+, 0.125 U of
Ampli Taq Gold (Applied Biosystems), and 0.5 µmol L−1 of
each IP1 and a Texas Red-labeled compound SSR primer
((AC)6(AG)5 or (TC)6(AC)5). The PCR cycling conditions
were 9 min at 94°C, then 40 cycles of 30 s at 94°C, 30 s at
the annealing temperature for each designed specific
primer, and 1 min at 72°C, with a 5 min extension of 72°C
treatment in the final cycle. The reaction products were
electrophoresed on a 6% Long Ranger sequencing gel
(FMC BioProducts, ME, USA) using an SQ-5500E
sequencer. Electrophoretic patterns were analyzed by Fraglys version 3 software (Hitachi).
Cervus version 2.0 (Marshall et al. 1998) was used to
calculate observed and expected heterozygosities. Genepop
version 3.4 on the web (Raymond and Rousset 1995) was
used to test the Hardy–Weinberg equilibrium (HWE) and
linkage disequilibria between the loci.
Results and discussion
Among cloned fragments whose lengths were more than
150 bp, 12 fragments produced with an (AC)6(AG)5 primer
and 12 produced with a (TC)6(AC)5 primer were randomly
chosen and sequenced. Of the former 12 fragments, eight
were successfully sequenced and two pairs had the same
sequences. Of the latter 12 fragments, nine were successfully
sequenced, four had the same sequence, and one contained
a tandem connection of two different fragments. All successfully sequenced fragments were flanked by a compound
SSR sequence at one end. In total we designed 13 different
specific primers from these fragments, seven from the
(TC)6(AC)5 fragments (two from the fragment contained a
417
Table 1. Characteristics of compound microsatellite markers isolated from Dendropanax trifidus
Locus
Repeat
Primer sequence (5′–3′)
Ta (°C)
Size range
(bp)
Dt02
(AC)6(AG)5
56
100–150
Dt03
(AC)6(AG)32
56
Dt05
(TC)6(AC)11
Dt06a
(AC)6(AG)9
Dt07
(AC)6(AG)5
Dt08
(AC)6(AG)10
Dt09a
(TC)6(AC)10
Dt10
(TC)6(AC)7TT(AC)7
Dt11
(TC)6(AC)7
Dt12
(TC)6(AC)8
Dt13
(TC)6(AC)6
CATGAGTACTACAGTGAATCC
ACACACACACACAGAGAGAGAG
CCCAATAATGGATTAACATGG
ACACACACACACAGAGAGAGAG
CAGCAATAACTGAAAGCCTGT
TCTCTCTCTCTCACACACACAC
GGAAGTTGGAGAAGTCATTAA
ACACACACACACAGAGAGAGAG
GTTGGCAAAACCACCAGATCT
ACACACACACACAGAGAGAGAG
ATCCCCTTTCTATCTCTAATG
ACACACACACACAGAGAGAGAG
GTCGTGCAAGACATTCCATGT
TCTCTCTCTCTCACACACACAC
ATCGGCGATCGAAATCGATAA
TCTCTCTCTCTCACACACACAC
GCTGCTGAAAAGAATGACATT
TCTCTCTCTCTCACACACACAC
ATCTAGAGGAGGTATCTCCAA
TCTCTCTCTCTCACACACACAC
GCCCTTAGAAATTATGTTTCC
TCTCTCTCTCTCACACACACAC
No. of
alleles
HO
HE
GenBank
accession no.
6
0.864
0.726
AB250284
126–188
12
1.000
0.897
AB250285
56
93–107
6
0.818
0.757
AB250286
56
100–114
5
0.591
0.684
AB250287
56
116–124
4
0.682
0.687
AB250288
56
90–120
8
0.773
0.846
AB250289
56
90–104
4
0.182
0.716
AB250290
56
122–152
6
0.773
0.710
AB250291
56
89–101
4
0.500
0.586
AB250292
56
93–103
4
0.636
0.628
AB250293
56
73–89
4
0.455
0.569
AB250294
Ta annealing temperature, HO observed heterozygosity, HE expected heterozygosity
a
Significant deviation from Hardy–Weinberg equilibrium (P < 0.05)
tandem connection of two different fragments), and six
from the (AC)6(AG)5 fragments.
We used the individually designed primer in combination
with a compound SSR primer ((TC)6(AC)5 or (AC)6(AG)5)
to amplify each compound SSR region of 22 D. trifidus
individual trees (Table 1). Of these 13 primer pairs, 12 produced one or two bands, and one produced multiple bands.
The former 12 primer pairs contained one producing a
monomorphic band for all individuals. The other 11 primer
pairs showed polymorphism ranging from 4 to 12 alleles per
locus. The observed and expected heterozygosities ranged
from 0.182 to 1.000 and from 0.569 to 0.897, respectively.
Two loci (Dt06 and Dt09) deviated significantly from HWE,
because of the excess of homozygotes (Table 1). No significant linkage disequilibrium was found among these loci.
The above results indicate that codominant markers with
high polymorphism for D. trifidus were easily developed by
this technique. Because it requires sequencing only once in
comparison with our previous technique (Lian and Hogetsu
2002), this approach substantially reduces time and cost in
the development of codominant polymorphic markers.
Another merit is that because a common fluorescent compound SSR primer can be used in polymorphism analyses
for different loci and the fluorescent primer is rather expensive, this may further save investigation costs (Hayden et al.
2004). Finally, when codominant polymorphic markers must
be developed simultaneously for several species, a common
fluorescent compound SSR primer could also be used in
polymorphism analyses for these several different species.
This technique is, in principle, applicable to other species,
and successful isolation of codominant compound SSR
markers for several other plant species, by use of this
method, is currently in progress.
Acknowledgments This work was supported by Grants-in-Aid from
the Ministry of Education, Culture, Sports, Science, and Technology of
Japan.
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