New microsatellite markers for Tricyrtis macrantha (Convallariaceae) and cross-amplification in closely related species1

• Premise of the study: Microsatellite markers were characterized in Tricyrtis macrantha (sect. Brachycyrtis, Convallariaceae), a vulnerable species endemic to Japan, to investigate its genetic diversity and population structure. • Methods and Results: Eleven microsatellite markers were developed. The number of alleles per locus ranged from one to seven, and genetic diversity per locus ranged from 0.142 to 0.812. Four markers were successfully amplified in other species of sect. Brachycyrtis (T. ishiiana, T. ishiiana var. surugensis, and T. macranthopsis). • Conclusions: The microsatellite markers can be used to investigate the genetic diversity and population structure of the vulnerable and endangered species of Tricyrtis sect. Brachycyrtis, to aid in the development of conservation strategies for each species.

; Ministry of the Environment of Japan, 2000 ).
Nine microsatellite markers were previously developed for T. ishiiana ( Setoguchi et al., 2011 ); however, only four markers could be amplifi ed in all species of sect. Brachycyrtis . Development of additional microsatellite markers would contribute not only to the elucidation of the genetic structure of T. macrantha populations but will also allow us to compare the genetic diversity among the four taxa of Tricyrtis sect. Brachycyrtis using the same markers. Here, we report the development and characterization of microsatellite markers for T. macrantha and the transferability of T. macrantha markers to the other species of sect. Brachycyrtis .

METHODS AND RESULTS
Genomic DNA was extracted from fresh leaves of one T. macrantha individual from the Kyoto Prefectural Botanical Garden, originally collected from a wild population at Mt. Yokokura (Ochi-cho, Takaoka-gun), in Kochi Prefecture, Japan (33 ° 32 ′ N, 133 ° 12 ′ E). The voucher specimen ( H. Setoguchi JP12005 ) was deposited at the herbarium of Kyoto University (KYO).
Fresh leaf material was frozen in liquid nitrogen and then ground into a fi ne powder. The leaf powder was suspended in 2-[4-(2-hydroxyethyl)-1piperazinyl] ethanesulfonic acid (HEPES) buffer (pH 8.0) and centrifuged (10,000 rpm at 20 ° C for 5 min) to remove polysaccharides ( Setoguchi and Ohba, 1995 ). Total DNA was then isolated from each pellet using cetyltrimethylammonium bromide (CTAB; Doyle and Doyle, 1990 ). The extracted DNA was dissolved in 100 μ L of TE buffer. Microsatellite loci were isolated using an improved technique for isolating codominant compound microsatellite markers ( Lian and Hogetsu, 2002 ;Lian et al., 2006 ). A total volume of 100 μ L containing ~2.5 μ g genomic DNA was digested separately with the blunt-end restriction enzymes Ssp I, Eco RV, and Alu I. The restricted fragments were ligated with a specifi c blunt adapter (consisting of the 48-mer 5 ′ -GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGG-C TGGT-3 ′ and an 8-mer with the 3 ′ -end capped with an amino residue: 5 ′ -ACCAGCCC-NH 2 -3 ′ ) using a DNA ligation kit (TaKaRa Biotechnology Co., Ohtsu, Shiga, Japan). Fragments were amplifi ed from Ssp I, Eco RV, and Alu I DNA libraries using one of the compound simple sequence repeat (SSR) primers (AC) 6 (AG) 5 or (TC) 6 (AC) 5 and an adapter primer (5 ′ -CTA-TAGGGCACGCGTGGT-3 ′ ). PCR was performed in a total reaction volume of 50 μ L containing 38.25 μ L sterilized water, 0.2 mM dNTP mixture, 0.125 U AmpliTaq Gold (Applied Biosystems, Foster City, California, USA), 1.5 mM reaction buffer with MgCl 2 (Applied Biosystems), 0.5 μ M of each primer, and 0.5 μ L of template DNA (10 ng/ μ L). The amplifi cation profi les included initial denaturation at 94 ° C for 9 min; followed by 20 cycles of 30 s at 94 ° C, 30 s at 66-60 ° C (reduced 0.3 ° C/cycle), and 1 min at 72 ° C; 20 cycles of 30 s at 94 ° C, 30 s at 60 ° C, and 1 min at 72 ° C; and a fi nal extension at 72 ° C for 6 min. After electrophoresis on a 1.5% agarose gel, the fragments (400-800 bp) were purifi ed using the GENECLEAN II kit (QBioGene, Solon, Ohio, USA) and cloned using the QIAGEN PCR Cloning plus Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions. Recombinant clones were identifi ed using blue/white screening on Luria-Bertani agar plates containing ampicillin, X-gal, and isopropyl-β -D -1-thiogalactopyranoside (IPTG ).

CONCLUSIONS
The microsatellite markers presented here will be useful for investigating the genetic diversity and population structure of the species of Tricyrtis sect. Brachycyrtis . The markers that amplifi ed in all species of Tricyrtis sect. Brachycyrtis , developed by Setoguchi et al. (2011) and this study, are now being used to Two hundred and fi fty insert-positive clones were amplifi ed using M13 forward primer (5 ′ -GTAAAACGACGGCCAGT-3 ′ ) and M13 reverse primer (5 ′ -AACAGCTATGACCATG-3 ′ ). The amplifi cation profi les included initial denaturation at 95 ° C for 9 min; followed by 40 cycles of 30 s at 94 ° C, 1.5 min at 54 ° C, and 1 min at 72 ° C; and a fi nal extension at 72 ° C for 4 min. PCR was performed in a total reaction volume of 10 μ L containing 6.75 μ L sterilized water, 0.08 mM dNTP mixture, 0.25 U TaKaRa Ex Taq (TaKaRa Biotechnology Co.), 1.0 mM reaction buffer (TaKaRa Ex Taq ), 0.2 μ M of each primer, and 1.0 μ L of template DNA (10 ng/ μ L). The PCR products in a total reaction volume of 5 μ L, using M13 forward primer, were sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). For 48 fragments containing the (AC) 6 (AG) n or (TC) 6 (AC) n compound SSR sequence at one end, primers were designed using Primer3 version 0.4.0 ( Rozen and Skaletsky, 2000 ). Each primer was designed to have <50% of GC content with optimal annealing temperature at 58 ° C, and total size of approximately 20-25 bp. PCR amplifi cations were performed following the standard protocol of the QIAGEN Multiplex PCR Kit (QIAGEN) in a fi nal volume of 6 μ L. Compound SSR primers [(AC) 6 (AG) 5 or (TC) 6 (AC) 5 ] were labeled with the fl uorochromes 6-FAM or HEX (Applied Biosystems). The amplifi cation profi les included initial denaturation at 95 ° C for 15 min; followed by 35 cycles of 30 s at 94 ° C, 1.5 min at 58 ° C, and 1 min at 72 ° C; and a fi nal extension at 60 ° C for 30 min. The size of the PCR products was measured using the ABI PRISM 3100 Genetic Analyzer with GeneScan 350 ROX Size Standard (Applied Biosystems) and GeneMapper analysis software (Applied Biosystems).
Eleven microsatellite loci that showed a clear and strong band on the electrophoretic gel were selected in the preliminary screening ( Table 1 ) . Each reverse primer was designed to contain six repeats of AC followed by fi ve repeats of AG [(AC) 6 (AG) 5 ] or six repeats of TC followed by fi ve repeats of AC [(TC) 6 (AC) 5 ]. For further characterization, 20 individuals of T. macrantha were genotyped using the procedure described above. These plant samples were collected from a population at Mt. Yokokura in Kochi Prefecture, Japan. Three loci were monomorphic and the number of alleles per locus varied from four to seven in polymorphic loci ( Table 2 ) . The observed heterozygosity and genetic diversity per locus ( H S ; Nei, 1987 ) ranged from 0.050 to 0.851 and 0.142 to 0.812, respectively.
Tests for deviations from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium were conducted using FSTAT version 2.9.3.2 ( Goudet, 1995 ). Signifi cant deviations ( P < 0.05) from HWE were detected in all polymorphic loci except TM_11. No signifi cant linkage disequilibrium was found in the pairwise comparisons of the polymorphic loci. Of the 11 microsatellite markers