An effective protocol to solve the problem in genomic DNA isolation of tung tree
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An effective protocol to solve the problem in genomic DNA isolation of tung tree Lingling Zhang & Lijing Dai & Junbo Gou & Junhua Peng
Received: 9 August 2012 / Accepted: 1 December 2012 # Society for Plant Biochemistry and Biotechnology 2013
Abstract Tung tree (Vernicia fordii) is a native and oilproducing woody plant in China. The oil is industrially important and promising biodiesel raw material. However, until recently the lack of effective protocols for the extraction of genomic DNA had made DNA-based molecular studies of tung tree difficult. Here, four conventional protocols and one novel protocol were compared for their capacity in isolating DNA from tung tree leaves of different age. Our results showed that all the four conventional protocols could isolate DNA from old leaves, two from matured leaves, but none from young leaves. However, the detectable DNA samples contained many contaminations, leading to overestimation of DNA concentration measured by ultraviolet spectrophotometer, also interfering with the downstream PCR reaction. The novel protocol could produce high-yield and good-quality DNA from tung tree leaves regardless of leaf age. Its key steps were that a single leaf tissue sample could be recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations
L. Zhang : J. Gou : J. Peng Wuhan Botanical Garden, Chinese Academy of Science, Wuhan, Hubei 430074, China L. Zhang e-mail: [email protected] L. Zhang : L. Dai : J. Gou : J. Peng (*) Key lab of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei 430074, China e-mail: [email protected] J. Peng e-mail: [email protected] L. Dai e-mail: [email protected] L. Zhang : L. Dai Graduate University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
(termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. All the four DNA samples of a single tissue were good template for PCR reaction. The novel protocol is an effective method for genomic DNA isolation of tung tree. Keywords DNA isolation . Leaf age . Plant . Tung tree . Protocol . Vernicia fordii Abbreviations EDTA Ethylene Diamine Tetraacetic Acid PVP Polyvinyl Pyrrolidone CTAB Cetyltrimethyl Ammonium Bromide SDS Sodium Dodecyl Sulfate
Tung tree, Vernicia fordii, belonging to Vernicia Lour, Euphorbiaceae, is a native and oil-producing woody plant in China, whose oil extracted from the fruit is a drying oil used in paints, varnishes and polymerization (Chen et al. 2012). Currently, tung oil is expected to be converted to biodiesel to cope with worldwide energy crisis and increasingly serious environmental problems (Park et al. 2008; Shang et al. 2010; Chen et al. 2012). DNA extraction is essential for molecular genetic analyses and marker-assisted improvement of this oil-producing species. Leaf of tung tree contains high level of secondary metabolites such as alkaloids, flavanoids, polyphenols, and polysaccharides. The second
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