【作者】 李芳芳；

【导师】 李永华； 柳新红；

【作者基本信息】 河南农业大学 ， 园林植物与观赏园艺， 2015， 硕士

【摘要】 枫香(Liquidambar formosana Hance.)系金缕梅科(Hamamelidaceae)枫香属落叶乔木,分布于秦岭淮河以南地区,跨北热带和南、中、北亚热带等4个气候带,是我国重要的乡土阔叶速生树种,集观赏、药用和材用等多种价值于一身。近年来对枫香的研究主要集中在无性繁殖技术、人工林和混交林技术、叶片的叶色分析、种子生物学特性、枫香树林的生理生化研究及园林应用等方面,而对其分子水平的遗传多样性研究较少,本研究将SRAP这种新的DNA分子标记运用到枫香的遗传多样性研究上,以期为枫香不同居群的遗传多样性分析、种质鉴定、遗传连锁图谱构建和分子育种等领域研究提供参考本试验以浙江省7个枫香群体(杭州市淳安县、杭州市桐庐县、丽水市莲都区、温州市文成县、衢州市开化县、台州市天台县、舟山市定海区)的叶片为材料,通过对不同的DNA提取方法对比,探讨最优的枫香基因组DNA提取方法;同时对枫香基因组SRAP技术体系进行优化,并将该体系应用于SRAP扩增分析中,研究各群体间的遗传多样性。本试验具体研究结论如下:1.枫香基因组DNA提取方法优化本试验采用改良CTAB法和试剂盒法(Bioteke公司研发的)两种方法对枫香叶片DNA提取结果进行对比,发现两种方法提取的枫香嫩叶DNA在含量和纯度上相差不大,均能满足后续PCR扩增要求。而对于老叶,改良CTAB法提取老叶基因组DNA的产率和纯度均较试剂盒法高。后者不仅含量较低,且含有其他杂质,DNA降解较严重,将对下游PCR反应和其他DNA分子操作产生不利影响。使用改良CTAB提取法有效地解决了枫香树基因组DNA提取中的褐变和多酚、多糖和蛋白质等次生物质干扰等问题。从检测所得纯度可知该DNA模板完全可以用于SRAP分析;另外,该方法还具有成本低、操作简单、省时等特点。2.枫香基因组DNA的SRAP-PCR扩增体系优化采用L16(45)正交实验设计,对影响SRAP-PCR反应体系的Mg2+、d NTPs、引物浓度及Taq DNA聚合酶和模板DNA用量等5个因素进行了优化,确立枫香树SRAP-PCR最佳反应体系为:总体积20ul,含1×PCR Buffer、1.5 mmol·L-1 Mg2+、0.16 mmol·L-1 dNTPs、0.5umol·L-1引物、0.9 U Taq DNA聚合酶和40 ng模板DNA。SRAP-PCR扩增程序为:95℃预变性5 min,94℃变性30 sec,35℃退火1 min,72℃延伸2 min,5次循环;94℃变性30 sec,50℃退火30 sec,72℃延伸1 min,30次循环;72℃延伸10 min,4℃保存。结果显示随机选择的引物对随机选择的基因组DNA均可扩增出多态性丰富且条带清晰的DNA片段。说明该SRAP-PCR反应体系稳定可靠,适用于枫香树基因组DNA的SRAP-PCR扩增反应。3.枫香树群体的遗传多样性应用SRAP分子标记的16对引物组合对采自浙江省7个枫香树群体的120个个体样品进行SRAP-PCR分析,共扩增902个条带,其中多态性条带897个,枫香树物种水平上的多态性条带百分率为82.18%,shannon表型多样性指数为0.4935。在群体水平上,多态性条带百分率变化范围为54.29%~74.29%,其均值为66.74%;shannon表型多样性指数的变化范围为0.3199~0.3928,其均值为0.3674。枫香天然群体内和群体间都存在着显著的遗传变异(P<0.001),群体间的遗传变异占总变异的15.04%,群体内的遗传变异占总变异的84.96%。4.枫香树天然群体的遗传距离由Nei’指数计算枫香树群体的遗传相似度和遗传距离,结果表明群体的遗传相似度在0.8999~0.9685之间变化。遗传距离在0.1055~0.0320。其中淳安群体与桐庐群体的遗传距离最小为0.0320,而淳安群体和文成群体的遗传距离最大为0.1055。5枫香树天然群体的聚类图根据枫香树种群间的遗传距离,利用UPGMA聚类方法对7个枫香群体进行聚类分析,得出反映各群体间亲缘关系的树状图。可将7个种群分为两大类:第一类中淳安群体和桐庐群体的遗传距离最近,首先聚在一起,再与台州群体聚在一起,最后与生长环境不同的舟山群体聚类;另一类为丽水和文成先聚在一起,再与距离相对较远的开化群体聚在一起。发现枫香树群体的聚类结果与其地理分布格局大致相吻合。更多还原

【Abstract】 Liquidambar formosana Hance. is Hamame lidaceae deciduous trees. Distributed in the Qinling Mountains area to the south of Huaihe, Cross the North tropical and south, in the tropical north, 4 climate zones, is a fast-growing tree species in our country important indigenous broadleaf, ornamental, medicinal and timber and other values in a body. In this experiment, The leaves of Liquidambar for materials, through the comparison of different DNA extraction methods, Extraction of genomic DNA of optimal Liquidambar formosana, At the same time, Liquidambar genomic SRAP system were optimized, and the system is applied to the 7 groups of Liquidambar materials were amplified by SRAP analysis. The results are as follows:1.In this experiment, by using the modified CTAB method and kit method(developed by Bioteke) the two methods were compared for t Liquidambar formosana leaves DNA extraction results, Found that the two kinds of extraction methods of Liquidambar leaves DNA content and purity are not significant, which can meet the requirements of the subsequent PCR amplification. For old leaves, The yield and purity of the modified CTAB method to extract genomic DNA of old leaves were relatively high kit method. The latter not only content is low, and contain other impurities, degradation of DNA is more serious, will adversely affect the response to the downstream of PCR and other DNA molecules. Using the modified CTAB extraction method effectively solves the problem of Liquidambar genomic DNA extraction of browning and polyphenol, polysaccharide and protein secondary metabolites interference and other issues. From the detection of the purity of the DNA template can be used for SRAP analysis; in addition, the method also has the advantages of low cost, simple operation, time-saving features.2.By the orthogonal experiment design Ll6(45), five factors including Mg2+ concentration, d NTPs concentration, primer concentration, Taq DNA polymerase and template DNA amount in SRAP-PCR reaction system were optimized, he optimized SRAP-PCR reaction system was as follow: total volume 20 μL, containing 1×PCR Buffer, 1.5 mmol?L-1 Mg2+, 0.16 mmol?L-1 d NTPs, 0.9 U Taq DNA polymerase, 0.5 μmol?L-1 primer, 40 ng template DNA. SRAP-PCR amplification procedure: pre-denaturation at 95℃for 5 min, 94 ℃ degeneration for 30 sec, annealed at 35 ℃ for 1 min, 72 ℃extension of 2 min, 5 cycles; 94 ℃ degeneration for 30 sec, annealed at 50 ℃ for 30 sec, 72 ℃extension of 1 min,30 cycles; 72 ℃extension of 10 min; kept at 4℃. The results showed that the primers were selected on random selection of genomic DNA could be amplified polymorphic and clear bands of DNA fragments. The description of the SRAP-PCR reaction system is stable and reliable, suitable for Liquidambar genomic DNA for SRAP-PCR amplification reaction.3. Genetic diversity of different populations of Liquidambar formosanaThe application of a molecular markers by 16 primer combinations in seven collected from zhe jiang province maple tree group(hang zhou, lis hui, wen zhou, qu zhou, tai zhou, zhou shan) SRAP- PCR analysis of samples of 120 individuals, total of 902 amplified bands, of which 897 bands were polymorphic.Percentage of polymorphic bands(PPB)and Shannon ’ s phenotypic diversity index(Ho)were respectively 82.18%and 0.4935 at the species level, at the population level, Percentage of polymorphic bands(PPB) excursion is 54.29~74.29. Shannon’s phenotypic diversity index(Ho) excursion is 0.3199~0.3928.In natural populations of Liquidambar formosana within and between groups had significant genetic variation(P<0.001). The genetic variation among populations accounted for 15.04% of the total variance, Genetic variation within the groups account for 84.96% of the total variance.4. The genetic distance of the natural populations of Liquidambar formosanaThe genetic similarity and genetic distance calculated Liquidambar group by Nei ’index, The results showed that the group genetic similarity between the changes in the 0.8999~0.9685, The mean genetic similarity of 0.9396. The genetic distance of 0.1055~0.0320, Mean genetic distance was 0.0624. Chun An and Tong Lu the smallest genetic distance is 0.0320, While Cun An and Wen Chen into the largest genetic distance was 0.1055.5. The dendrogram of the natural population of Liquidambar formosanaAccording to the genetic distance of Liquidambar formosana species groups, Cluster analysis was performed on 7 groups of Liquidambar formosana Hance by UPGMA clustering method, The dendrogram of genetic relationship of reflection between the various groups. The clustering results of Liquidambar formosana populations found their geographic distribution pattern of roughly coincide.更多还原