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<p>The study aimed to develop and standardize a reliable, cost-effective, and reproducible protocol for isolating high-quality total genomic DNA suitable for chloroplast marker amplification from two <i>Dendrobium</i> hybrids—<i>Dendrobium ‘</i>Sonia Earsakul<i>’</i> and <i>Dendrobium</i> ‘Singapore White’—and to assess its suitability for downstream molecular applications, such as PCR amplification using selected chloroplast markers. Leaf tissues were collected from healthy plants and subjected to three different DNA extraction methods: (i) a modified cetyltrimethylammonium bromide (CTAB) method, (ii) a modified sodium dodecyl sulfate (SDS) method, and (iii) the DNeasy Plant Mini Kit. The optimized CTAB protocol incorporated polyvinylpyrrolidone (PVP) and β-mercaptoethanol to counteract secondary metabolites present in orchid tissues. DNA yield and purity were measured using a Colibri + Microvolume UV-Vis spectrophotometer, while integrity was assessed by agarose gel electrophoresis. The extracted total genomic DNA was further validated through PCR amplification using three widely applied chloroplast markers: matK, trnL-F, and trnH-psbA. The standardized CTAB method produced the highest yield and purity, with DNA concentrations of 145–150 ng/µL, A260/A280 ratios of 1.88–1.92, and A260/A230 ratios of 2.03–2.11. In contrast, the SDS method and DNeasy kit produced lower yields and showed signs of contamination. Agarose gel electrophoresis confirmed intact, high-molecular-weight genomic DNA suitable for downstream plastid marker amplification. Successful amplification of all three chloroplast markers confirmed the suitability of the isolated DNA for downstream molecular studies. The optimized CTAB-based protocol provides a reliable and cost-effective method for genomic DNA extraction from <i>Dendrobium</i> hybrids. Its higher DNA yield and purity values observed in this study compared with the SDS method and the commercial kit suggest its potential utility in DNA barcoding, phylogenetics, population genetic analysis, and species authentication, which are essential tools for conservation genetics, monitoring illegal orchid trade, and biodiversity conservation of <i>Dendrobium</i> species. It is important to note that the protocol yields total genomic DNA; however, the quality obtained was sufficient for robust amplification of chloroplast markers without the need for chloroplast enrichment procedures.</p>

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An optimized CTAB-based protocol for high-quality genomic DNA extraction suitable for chloroplast marker amplification in Dendrobium hybrids

  • 期刊论文

  • 发表日期：

    2026年4月6日

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  • Nisruti Anuja Behura,
  • Naga Jogayya Kothakota

摘要

The study aimed to develop and standardize a reliable, cost-effective, and reproducible protocol for isolating high-quality total genomic DNA suitable for chloroplast marker amplification from two Dendrobium hybrids—Dendrobium ‘Sonia Earsakul’ and Dendrobium ‘Singapore White’—and to assess its suitability for downstream molecular applications, such as PCR amplification using selected chloroplast markers. Leaf tissues were collected from healthy plants and subjected to three different DNA extraction methods: (i) a modified cetyltrimethylammonium bromide (CTAB) method, (ii) a modified sodium dodecyl sulfate (SDS) method, and (iii) the DNeasy Plant Mini Kit. The optimized CTAB protocol incorporated polyvinylpyrrolidone (PVP) and β-mercaptoethanol to counteract secondary metabolites present in orchid tissues. DNA yield and purity were measured using a Colibri + Microvolume UV-Vis spectrophotometer, while integrity was assessed by agarose gel electrophoresis. The extracted total genomic DNA was further validated through PCR amplification using three widely applied chloroplast markers: matK, trnL-F, and trnH-psbA. The standardized CTAB method produced the highest yield and purity, with DNA concentrations of 145–150 ng/µL, A260/A280 ratios of 1.88–1.92, and A260/A230 ratios of 2.03–2.11. In contrast, the SDS method and DNeasy kit produced lower yields and showed signs of contamination. Agarose gel electrophoresis confirmed intact, high-molecular-weight genomic DNA suitable for downstream plastid marker amplification. Successful amplification of all three chloroplast markers confirmed the suitability of the isolated DNA for downstream molecular studies. The optimized CTAB-based protocol provides a reliable and cost-effective method for genomic DNA extraction from Dendrobium hybrids. Its higher DNA yield and purity values observed in this study compared with the SDS method and the commercial kit suggest its potential utility in DNA barcoding, phylogenetics, population genetic analysis, and species authentication, which are essential tools for conservation genetics, monitoring illegal orchid trade, and biodiversity conservation of Dendrobium species. It is important to note that the protocol yields total genomic DNA; however, the quality obtained was sufficient for robust amplification of chloroplast markers without the need for chloroplast enrichment procedures.