Microsatellite markers were isolated for Daphne kiusiana var. kiusiana (Thymelaeaceae), an evergreen broad-leaved shrub endemic to Korea and Japan. Because its populations in Jeju Island are morphologically controversial, and consistently threatened by anthropogenic pressures, taxonomic delimitation...
Microsatellite markers were isolated for Daphne kiusiana var. kiusiana (Thymelaeaceae), an evergreen broad-leaved shrub endemic to Korea and Japan. Because its populations in Jeju Island are morphologically controversial, and consistently threatened by anthropogenic pressures, taxonomic delimitation and conservation effort are required at the genetic level. We developed 21 polymorphic microsatellite loci from Next Generation Sequencing data. The primer set included di-, tri-, and tetra-nucleotide repeats. Variability in the markers was tested for 80 individuals of D. kiusiana from three natural populations in Jeju Island and Japan. Among the 21 loci, three were unavailable for population JKJU of Japan. The Neighbor-Joining tree based on microsatellite markers described here classified the three populations into two groups according to geographical or morphological traits. These will be a powerful genetics tool for determining the taxonomic boundary and establishing suitable conservation strategies for D. kiusiana in Jeju Island.
Microsatellite markers were isolated for Daphne kiusiana var. kiusiana (Thymelaeaceae), an evergreen broad-leaved shrub endemic to Korea and Japan. Because its populations in Jeju Island are morphologically controversial, and consistently threatened by anthropogenic pressures, taxonomic delimitation and conservation effort are required at the genetic level. We developed 21 polymorphic microsatellite loci from Next Generation Sequencing data. The primer set included di-, tri-, and tetra-nucleotide repeats. Variability in the markers was tested for 80 individuals of D. kiusiana from three natural populations in Jeju Island and Japan. Among the 21 loci, three were unavailable for population JKJU of Japan. The Neighbor-Joining tree based on microsatellite markers described here classified the three populations into two groups according to geographical or morphological traits. These will be a powerful genetics tool for determining the taxonomic boundary and establishing suitable conservation strategies for D. kiusiana in Jeju Island.
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제안 방법
The DNA was extracted as described above, and PCR was conducted with a GeneAmp® PCR System 2700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA), using a final volume of 10 μL that comprised 15 to 20 ng of extracted DNA, 5 µL of SolgTM 2× Taq PCR Smart-Mix I (Solgent, Daejeon, Korea), 0.01 µM forward primer, 0.2 µM reverse primer, and 0.1 µM of the M13 primer (fluorescently labeled).
The authors thank two colleagues Choi IS and Jin DP at the Plant Systematics Laboratory of Inha University for help in assisting with statistical analysis. This work was supported by the National Research Foundation of Korea (NRF) (No.
, 2013). The parameters were set for detection of di-, tri-, or tetra-nucleotide motifs with flanking regions larger than 50 bp and having at least 10, 6, or 4 repeats, respectively. From this screening, we acquired 28,495 sequences that were then re-filtered in an attempt to reference-map all of the reads to each microsatellite that contained a singleton.
Table 1. Voucher and location information for Daphne kiusiana samples used in this study. One specimen per population was deposited in the herbarium at the Korea Institute of Oriental Medicine (KIOM).
대상 데이터
To test the effectiveness of the developed microsatellite loci, we collected 80 individuals of D. kiusiana from three natural populations in Korea and Japan (Table 1). To minimize the damage to this study species, only one plant per population was taken to prepare a voucher specimen that was then deposited in the herbarium at the Korea Institute of Oriental Medicine (KIOM).
이론/모형
The pair-wise genetic distance (Dps) was generated with MSA software (Dieringer and Schlöterer, 2003) using a bootstrap analysis of 1,000 replicates. From these distance matrices, we constructed 50% consensus trees by the Neighbor-Joining (NJ) method, as implemented in PHYLIP ver. 3.68 (Felsenstein, 2004).
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