A simple protocol for isolating genomic DNA from chestnut rose (<Emphasis Type="Italic">Rosa roxburghii</Emphasis> tratt) for RFLP and PCR analyses |
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Authors: | Qiang?Xu Xiaopeng?Wen Email author" target="_blank">Xiuxin?DengEmail author |
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Institution: | (1) National Key Laboratory of Crop Genetic Improvement, Huazhong Agriculture University, 430070 Wuhan, PR China;(2) Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou University, Guiyang, PR China |
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Abstract: | Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols,
polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol
for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified
hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a
washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of
polyvinylpyrrolidone (2% w/v]), CTAB (3% w/v]), and β-mercaptoethanol (3% v/v]) in the high-salt-concentration extraction
buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins
and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This
protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream
results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction
(PCR) analyses. |
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Keywords: | chestnut rose (Rosa roxburghii Tratt) DNA isolation PCR RFLP polysaccharides |
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