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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. References Akkaya MS, Shoemaker RC, Specht JE, Bhagwat AA, Cregan PB (1995) Integration of simple sequence repeat and markers into a soybean linkage map. Crop Sci 35:1439–1445 Hayden MJ, Stephenson P, Logojan AM, Khatkar D, Rogers C, Koebner RMD, Snape JW, Sharp PJ (2004) A new approach to extending the wheat marker pool by anchored PCR amplification of compound SSRs. Theor Appl Genet 108:733–742 Lian C, Hogetsu T (2002) Development of microsatellite markers in black locust (Robinia pseudoacacia) using a dual-suppression-PCR technique. Mol Ecol Notes 2:211–213 Lian CL, Zhou ZH, Hogetsu T (2001) A simple method for developing microsatellite markers using amplified fragments of inter-simple sequence repeat (ISSR). 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