Computational protocol: Isolation and characterization of microsatellite loci in Fimbristylis sericea (Cyperaceae)1

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[…] According to the method reported by , we constructed a genomic library enriched for (CT)n repeat sequences. Genomic DNA was isolated from leaves using a Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, Wisconsin, USA) and digested with MboI. Approximately 1.5 μg of the digested DNA was electrophoresed on 1.0% agarose gels. Fragments with sizes ranging from 300 to 1000 bp were excised from the gel and purified using a QIAquick Gel Extraction Kit (QIAGEN, Valencia, California, USA). The DNA fragments were resolved in 12 μL of dH2O. Two oligonucleotides, “F” (5′-TTGCTTACGCGTGGA-3′) and “R” (5′-GATCGAGTCCACGCGTAAGCAA-3′), were used to prepare a linker by denaturation at 95°C for 5 min and cooling at room temperature (). The linker was ligated to the MboI-digested DNA fragments in a total volume of 21 μL, which contained 10 μL Ligation High (Toyobo, Osaka, Japan), 2 μM linker, and 6 μL of the DNA fragments (the DNA concentration was not measured). The ligated DNA fragments were amplified by PCR in a total reaction volume of 25 μL containing 2.5 unit TaKaRa Ex Taq DNA polymerase (TaKaRa Bio Inc., Otsu, Shiga, Japan), 10× Ex Taq Buffer, 200 μM dNTP mixture, 500 nM primer F, and 3 μL of the DNA fragments. The PCR cycling conditions were 94°C for 5 min; 30 cycles at 94°C for 15 s, 52°C for 15 s, 72°C for 40 s; and a final extension step at 72°C for 7 min. The fragments were precipitated with ethanol and resolved in 48 μL dH2O. The DNA fragments were hybridized with 20 pmol of 5′-biotinylated (CT)15 oligonucleotide at 70°C overnight in a 100-μL volume containing 10× saline sodium citrate (SSC) and 5% sodium dodecyl sulfate (SDS). The hybrid fragments were captured with 300 ng of streptavidin-coated magnetic beads (Dynabeads M-280; Dynal, Oslo, Norway) at 43°C for 4.5 h. The fragments on the magnetic beads were denatured at 95°C for 10 min in 50 μL of TE buffer. PCR was conducted to restore double-stranded DNA in a total reaction volume of 50 μL, which contained 2.5 unit Ex Taq, 10× Ex Taq Buffer, 200 μM dNTP mixture, 500 nM primer F, and 3 μL single-stranded DNA fragments. Thermal conditions of the PCR were 94°C for 5 min; 30 cycles at 94°C for 15 s, 52°C for 15 s, 72°C for 40 s; and a final extension step at 72°C for 7 min.We used the technique reported by to construct a DNA library enriched with a compound microsatellite motif. The total DNA was digested with the blunt-end restriction enzyme EcoRV. The digested fragments were ligated with specific blunt adapters (a 48-mer 5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3′ and an 8-mer 5′-ACCAGCCC-NH2-3′) using Ligation High (Toyobo). Subsequently, fragments flanked by a microsatellite at one end were amplified from the EcoRV library using each compound microsatellite primer [(AC)6(AG)5, (TC)6(AC)5, (AG)6(TG)5, or (CT)6(CA)5] and an adapter primer AP2 (5′-CTATAGGGCACGCGTGGT-3′).The DNA fragments containing (CT)n or the compound microsatellite motif were cloned into the pGEM-T Easy Vector (Promega Corporation) and transformed into DH5α-competent cells (Toyobo). Next, the clones were sequenced using an ABI 3730xL DNA sequencer (Applied Biosystems, Foster City, California, USA). Out of 96 (CT)n clones and 162 compound microsatellite clones sequenced, 50 (CT)n clones and 48 compound microsatellite clones were useful for designing primers. For the (CT)n clones, the primers were designed using Primer3 (). For the compound microsatellite clones, Oligo Calculator () was used to calculate the melting temperature (Tm). PCR amplification was performed in a total volume of 10 μL, which contained 0.125 unit Blend Taq-Plus Polymerase (Toyobo), 10× buffer for Blend Taq, 200 μM of each dNTP, 500 nM of each of the primers, and approximately 10 ng of total DNA. The thermal conditions were as follows: 94°C for 5 min; 30 cycles at 94°C for 20 s, 52°C for 20 s, 72°C for 40 s; and a final extension step at 72°C for 7 min. PCR with 12 primer pairs amplified fragments with the expected sizes (). The fragment sizes were measured using a CEQ8800 system (Beckman Coulter, Fullerton, California, USA) after PCR using the forward primer labeled with a fluorescent dye (i.e., D2, D3, or D4).We assessed the polymorphisms in 64 individuals from eight populations of F. sericea. Eight individuals were collected from each of the following populations in Japan: Arahama, Niigata Prefecture (37°24′58.3″N, 138°35′05.4″E); Hamazume, Kyoto Prefecture (35°39′24.9″N, 134°57′14.4″E); Hakoishi, Kyoto Prefecture (35°39′02.8″N, 134°56′23.9″E); Ajigaura, Ibaraki Prefecture (36°24′04.6″N, 140°36′35.2″E); Fukude, Shizuoka Prefecture (34°39′49.4″N, 137°52′54.3″E); Shiratsuka, Mie Prefecture (34°46′10.9″N, 136°32′29.4″E); Irino, Kochi Prefecture (33°01′42.8″N, 133°01′22.8″E); and Ryujin, Oita Prefecture (33°30′41.6″N, 131°43′51.6″E). Voucher specimens are unavailable because only a small amount of samples were collected to limit negative impact of sampling on maintenance of the local populations. We used GenAlEx 6.5 (, ) to estimate the genetic diversity. The number of alleles per locus in the total sample and within populations was 2–5 and 1–3, with mean values of 3.5 and 1.4, respectively (). The total expected heterozygosity per locus (Ht) was 0.069–0.645, with a mean value of 0.336. The average observed heterozygosity within a population per locus (Ho) was 0.000–0.016. The average expected heterozygosity within a population per locus (He) was 0.051–0.230. We determined the deviation from Hardy–Weinberg equilibrium (HWE) and the linkage disequilibrium (LD) between all pairs of polymorphic loci using GENEPOP 4.2 (). Exact tests of the deviation from HWE detected significant excesses for homozygotes at 12 loci (P < 0.05), suggesting a high level of inbreeding. Significant LD was found for two pairs of polymorphic loci: Fse47 and Fse4-5 from Hamazume and Fse2-22 and Fse4-25 from Fukude (P < 0.05). […]

Pipeline specifications

Software tools Primer3, GenAlEx, Genepop
Applications Population genetic analysis, qPCR