Abstract
As the sister clade of seed plants, ferns are significant materials for plant phylogeny research. However, the genomic DNA extraction protocol for fern samples like modified CTAB method still lacks robustness. Here, we found that the amount and condition of the pinnae samples are critical for gDNA extraction in fern, Adiantum capillus-veneris L. In 500 μl CTAB solution, the recommended amount of pinnae is about 10-20 mg (2-3 pieces). The condition of the pinnae must be instantly-picked from a plant cultivated in a suitable environment. With these factors under control, it is highly reproducible to get the high-quality gDNA with low degradation rate
Keywords: Fern, DNA extraction, Adiantum capillus-veneris L., CTAB method, Fern pinnae
Background
The CTAB method has been applied to gDNA extraction from Adiantum capillus-veneris L. (Han et al., 2012; Li et al., 2017). However, the protocol still shows instability and high degradation rate in our experiment. The pinnae of ferns accumulate large quantities of secondary metabolites, such as polysaccharides and polyphenols, which is adverse to DNA extraction (Ponnusamy et al., 2015).To increase the robustness of gDNA extraction, we optimized the protocol basically from two aspects: 1) Reduce the amount of material and use only 2-3 pinnae (about 10-20 mg). 2) The pinnae must be freshly picked from a plant cultured in suitable cultivation environment (temperature: 25 °C, humidity: 65%, 16-h white light/8-h dark treatment) and instantly used for DNA extraction. Compared with the classical method, we can get gDNA of higher quality and lower degradation rate from ferns with this optimized protocol.
Materials and Reagents
Equipment
Procedure
Data analysis
The result of gel electrophoresis is shown in Figure 3. When the amount of pinnae is excessive, all the gDNA samples show high degradation rate (Figure 3A). When we decreased the amount of material, the degradation rate is dramatically reduced (Figure 3B). However, if we draw the material from a fern cultured in an unsuitable environment, the result still shows erratic degradation rate. And picking the material from a plant in a suitable environment can solve this problem.
Recipes
Acknowledgments
This protocol was optimized from procedures published in Han et al., (2012). We thank the Institute of Vegetables and Flowers, CAAS for providing a greenhouse for Adiantum cultivation. The authors declare that there are no conflicts of interest or competing interests.
References
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