| Juglans sigillata Dode is one of the most important economic forestry plants in Yunnan Province. With the development of walnut industry, the demand of making clear the walnut's genetic background and identifying varieties using a scientific method is increasing. In the research Random Amplified polymorphic DNA (RAPD) markers and inter-simple sequence repeat (ISSR) markers were applied to identify 11 cultivars of Yangbi walnut (Juglans sigillata Dode) and their genetic variation was detected. The purpose of the research is to provide molecular evidence for variety identification and to lay scientific foundation for breeding. The varieties involved in the research are 4 Yunnan local varieties(Yangbi pao walnut, Santai walnut, Jiamian walnut and tie walnut) and 7 walnut hybrids varieties(Yunxin 7914,Yunxin 7926, Yunxin 8034, Yunxin 8064, Yunxin 90301, Yunxin 90303 and Yunxin 90306)The methods and results are as follows:1 .Genome DNA extraction: Taking different development stage of Juglans Silillata leaves: dormant bud, young leaf, mature leaf and old leaf as samples, two methods of CTAB precipitation and low PH extraction medium with high salt were used to extract the genomic DNA. The DNA obtained by the above methods were tested by agarose gel electrophoresis, ultraviolet spectrophotometer and RAPD. The results show that the quality and quantity of DNA from different samples are different. Among them, the quality and quantity of DNA extracted from dormant bud and young leaf are better than that from the old leaf. As for the two methods, the method of low PH extraction medium with high salt can obtain better quality but lower yield of DNA. On the contrary, the method of CTAB Precipitation can obtain higher yield but worse quality DNA.2. Stable PCR-reaction procedure establishment: Taking the leaves of Juglans silillata as DNA extraction samples, factors of Taq DNA polymerase, Mg+2, template DNA, primers and dNTPs which will influence the results of RAPD were studied in the experiment. Moreover five PCR-reaction programs were tested. An optimal reaction system and the PCR reaction program have been established, that is, the reaction system25ul amplification reaction solution consists of 0.75u.L-1 Taq DNA polymerase, 0.3mmol.L-1 Mg+2, 0.3mmol.L-1 primer, 1.6 mg.L-1 template DNA, 2.5ul 10×Buffer and 0.2mmol.L-1 dNTPs. The PCR amplification program is that predenaturing at 94℃ for 300s, followed by denaturing at 94℃ 60s, anneling at 36℃ for 60s, extension at 72 ℃ for 120s, cycling 45 times, last extension at 72 ℃ for 600s.As for ISSR, reaction volumes of 20 μL, contains 1.0 μL of genomic DNA (50 ng), 2.0... |
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