论文标题:云南省主要推广核桃品种的分子标记鉴别及遗传分析
Molecular Identification and Genetic Analysis of Juglans Varieties Planted in Yunnan Province
论文作者
论文导师 陈少瑜,论文学位 硕士,论文专业 林木遗传育种
论文单位 西南林学院,点击次数 94,论文页数 73页File Size6026K
2006-04-01论文网 http://www.lw23.com/lunwen_261551757/
Juglans sigillata Dode; Molecular markers; RAPD; ISSR; Variety identification; Genetic analysis
漾濞核桃(Juglans sigillata Dode)为云南省重要的经济林树种之一，随着云南省核桃产业的发展，对于核桃遗传背景方面知识的需求和应用科学手段鉴别核桃品种，维持核桃市场秩序的需求日益增加。本研究利用RAPD(Random Amplified Polymorphic DNAs，即随机扩增多态性DNA)分子标记和ISSR(Inter-simple Sequence Repeats简单序列重复间区)分子标记技术对云南省11个漾濞核桃品种进行分子鉴别和遗传分析。为品种鉴别提供分子水平上的参考，为核桃的育种提供理论依据。参试的11个核桃品种包括4个云南乡土核桃品种：漾濞泡核桃、大姚三台核桃、夹绵核桃和铁核桃和7个优良杂交品种：云新高原核桃(云新7914号)、云新云林核桃(包括：云新7926号、云新8034号、云新8064号)、云新90301号、云新90303号和云新90306号。研究的内容及结果如下： 1．DNA提取：以漾濞泡核桃不同生长时期的叶片为材料，采取了高盐低PH法和CTAB沉淀法提取基因组DNA，并通过琼脂糖凝胶电泳、紫外分光光度计和RAPD扩增3种方法对所提取的DNA样品进行检测。比较所得DNA的产量、质量，认为不同发育时期叶片得DNA提取效果不同，其中以冬芽和新叶效果较好，老叶最差；两种提取方法得到得的DNA产量和质量有所不同，高盐低PH法提取的DNA纯度高，但是产量较低，而CTAB法则相反，产量高，纯度低。 2．建立稳定的反应体系以漾濞核桃中的大姚三台核桃为实验材料，通过对影响RAPD扩增结果的主要因子：Tag酶、Mg~(+2)、引物、模板DNA、dNTPs等不同的浓度组合和扩增程序的试验研究，确定了漾濞核桃的最适RAPD反应体系和扩增程序。即在25uL反应体系中包括0.75u.L~(-1)Taq DNA聚合酶，3.0mmol.L~(-1)Mg~(+2)，0.3mmol.L~(-1)引物，含1.6mg.L~(-1)模板，2.5ul 10×Buffer，dNTPs各0.2mmol.L~(-1)。扩增程序为：94℃预变性300s，94℃变性40s，36℃退火60s，72℃链延伸120s，45次循环后，72℃延伸600s。 ISSR反应体系和扩增程序为总的20uL反应体系中包含：模板DNA 1.0uL(50ng)，10×Buffer2.0uL，10pmol.uL~(-1)引物1.5uL，2.5mM dNTPs 1.6uL，25mM Mg~(+2)1.8uL，5U TaqDNA聚合酶0.12uL，ddH_2O 11.98uL；热循环体系为：95℃预变性600s，然后94℃变性30s，50℃退火60s，72℃链延伸120s，共35个循环最后72℃延伸420s。 3．引物筛选通过对RAPD和ISSR引物的筛选，分别筛选出8个能够扩增出清晰、稳定并能扩增出多态性位点的RAPD引物和ISSR引物。RAPD引物为：SBS A07、SBS A08、SBS B06、SBS E11、SBS F10、SBS S03、SBS S06和SBS S10；ISSR引物及其退火温
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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