II. MATERIALS AND METHODS Leaf samples from individual trees of 1 Rhizophora apiculata, 1 Rhizophora mucronata and 1 putatively natural mangrove hybrid were collected from the Sirindhorn International Environmental Park, Phetchaburi, Thailand. Leaf samples were stored in silica gel before further analyses. DNA extraction, PCR amplification, and sequencing. Genomic DNA was extracted from approximately 20 mg of silica gel-dried leaf material using the DNeasy Plant Mini Kit (QIAGEN). One chloroplast DNA (cpDNA) region: atpB–rbcL intergenic spacer; and 3 partial nuclear gene (nDNA) r e g i o n s : DLDH ( d i h y d r o l i p o ami d e dehydrogenase), SBE2 (starch branching enzyme) and FMRrm11 were amplified from the genomic DNA of R. apiculata (Figure 3) R. mucronata (Figure 4) and putatively natural mangrove hybrid (Figure 5 and 6). Genomic DNA was extracted from approximately 20 mg of dried leaf material using the DNeasy Plant Mini Kit (QIAGEN). Three nuclear regions: DLDH, SBE2 and FMRrm11 and One cpDNA region: atpB-rbcL intergenic spacer were PCR-amplified in all sampled individuals. The primers used for PCR are listed in Table 1. Primers for loci atp-BrbcL, DLDH, and SBE2 were previously described by Inomata et. al. (2009), while the primers for the FMRrm11 locus were described by Cerón- Souza et. al. (2010). PCR amplifications were performed in 20 μl reaction mixtures, each containing 10–50 ng of genomic DNA, 1×Ex-Taq buffer (2 mM of Mg2+), dNTP mixture (0.2 mM of each dNTP, 0.2 μM of each primer, and 1.0 U of Ex-Taq DNA polymerase. The Fig.3 Rhizophora apiculata Fig.4 Rhizophora mucronata 54 Proceedings of the International Conference on Climate Change, Biodiversity and Ecosystem Services for the Sustainable Development Goals (SDGs): Policy and Practice 27-29 June 2016, Cha-am, Phetchaburi, Thailand
Proceedings of International Conference on Climate Change, Biodiversity and Ecosystem Services for the Sustainable Development Goals : Policy and Practice 27-29 June 2016 at the Sirindhorn International Environmental Park, Cha-am, Phetchaburi, Thailand
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