【作者】 桂腾琴；

【导师】 乔爱民； 孙敏； 王心燕；

【作者基本信息】 西南大学 ， 植物学， 2008， 硕士

【摘要】 果梅（Prunus mume Sieb.et Zucc.）隶属蔷薇科（Rosaceae）李亚科（Prunoideae）李属（Prunus L）,原产于中国。果梅是我国南方重要外销型创汇果树,外销出口的比例很大,广东主产区近年来产量的一半以上销往境外。我国果梅栽培历史悠久,种质资源丰富,长期天然杂交和传统生产上的实生繁殖,使现存种质资源和人工栽培品种的遗传背景十分复杂,同物异名、同名异物现象严重。为了更好地保存和利用这些种质资源,我们利用简单重复序列间区（ISSR）分子标记技术对果梅遗传多样性进行分析,研究结果如下:1果梅基因组DNA提取的探讨为从顽拗植物果梅叶片中获得可供后续分子生物学操作的基因组DNA,针对其组织细胞中富含多糖、多酚、有机酸等物质的特点,采用改良CTAB法提取果梅基因组DNA。在研磨前加入适量PVP和2%β-巯基乙醇,以防止酚氧化成醌,避免褐变;在核裂解之前先破碎细胞,将细胞质中的次生物质去除后再裂解细胞核;用高浓度的CTAB/NaCl溶液与氯仿/异戊醇共沉淀有效去除多糖等措施,经A₂₆₀和A₂₈₀值的测定,以及电泳检测和PCR扩增,结果表明改良CTAB法所提取的DNA无论在纯度上、完整性上都比改良的SDS法和传统的CTAB法好。2果梅ISSR反应体系的建立对影响ISSR扩增反应的主要因子采用两种方法进行优化:一为正交设计法;二为单因素逐项优化方法。建立了果梅最佳ISSR反应体系,即在20μL反应体系中含2μL 10×buffer、2.5 mmol/L MgCl₂、0.2 mmol/L dNTPs、0.32μmol/L引物、20-80 ng模板DNA、1U Taq DNA聚合酶。最佳反应程序:94℃预变性5 min;94℃变性45 s,47.1℃-54.3℃（退火温度随引物不同而定）45 s,72℃延伸2min,循环35次;72℃最后延伸7min,4℃保存。3果梅遗传多样性分析（1）多态性分析从51条ISSR引物中筛选出扩增条带清晰,稳定性好的10条引物共扩增出120个位点,平均每个引物扩增出12个位点,DNA片段大小分布在0.2-3.0kb之间。不同引物扩增的多态性也存在较大的差异,扩增的多态性条带数的范围为8-12条,每个引物检测到的多态位点平均为9.8个。（2）聚类分析39个果梅品种的遗传相似性分析（Jaccard系数）表明,各基因型间的Jaccard相似系数在0.5263-0.9910之间。通过非加权算术平均数聚类（UPGMA）法,绘制了39个果梅品种遗传关系树状图。以0.65为阈值将39份材料分为3类,第3类（Ⅲ）的34个品种又分成5个亚类,这与传统分类学上按照成熟果皮颜色的划分结果基本一致,聚类结果与地域无明显相关。ISSR分析结果显示,果梅遗传多样性丰富,可以利用ISSR分子标记对果梅进行分子水平的鉴定和遗传多样性的分析。更多还原

【Abstract】 Japanese apricot （Prunus mume Sieb. et Zucc.） which belongs to the genus of Prunus L. in Rosaceae was originated in southern China and has high economic value. Japanese apricots was an important fruit tree which have been exported abroad over half the total output the main producing areas in Guangdong in recent years. Japanese apricots are rich in genetic resources and cultivated with a long history, but their morphological characters are very similar. It is a very serious phenomenon that different cultivars have the same name in production. There is much confusion and difficulty in identifying the cultivars. In order to preserve and utilize the germplasm better, it is necessary to study their genetic diversity by ISSR molecular markers.1 Study on genomic DNA extraction from recalcitrant Prunus mume Sieb. et Zucc.The modified CTAB method has been developed to extract the high quality genomic DNA from the recalcitrant fruit tree Japanese apricot （Prunus mume Sieb. et Zucc.） which contains abundant polysaccharide, polyphenol, organic acid and some other secondary metabolites. Some measures were taken to eliminate them including a proper amount of PVP and 2 %β-mercaptoethanol were added during grinding to prevent oxidation of phenolic compounds; using high concentrate of CTAB/ NaCl solution to separate polysaccharides. The results of ratio of A₂₆₀ and A₂₈₀ , electrophoresis and amplification suggested that the modified CTAB method could obtain higher quality genomic DNA with better purity and integrity compared with modified SDS while the conventional CTAB method is not suitable for the DNA extraction from Japanese apricot.2 Establishment of an ISSR reaction system in Japanese apricot （Prunus mume Sieb. et Zucc.）Based on the high quality genomic DNA extracted by the modified CTAB method, five essential factors that might affect the results of ISSR were compared by orthogonal design and single factor test. Comprehensive results of the two methods, a suitable ISSR-PCR reaction system （20 uL） was established, i. e. 2 uL 10×buffer, 2.5 mmol/L Mg²⁺, 0.2 mmol/L dNTPs, 0.32μmol/L primer, 20-80 ng template DNA and 1 unit of DNA polymerase. The parameters were: 5 min at 94℃for initial activation step, followed by 35 cycles of 45 s at 94℃, 45s at annealing temperature 47.1℃-54.3℃and a 2 min extension at 72℃. At the end, amplified products were elongated for 7 min at 72℃and preserved at 4℃.3 The analysis of genetic diversity of Japanese apricot（1） The polymorphism analysis of accessions The genetic diversity of Japanese apricot were studied by ISSR. 10 primers were screened from 51 arbitrary ISSR primers. A total of 120 DNA fragments ranging from 0.2-3.0kb were amplified, among which 98 （81.67 %） were polymorphic, using these 10 primers. The average number of DNA band produced by each prime was 12. The polymorphic band difference was very significant among different primers. The average number of DNA polymorphic band produced by each prime was 9.8.（2） The cluster analysis of accessions According to the result of PCR, the genetic similarity analysis for 39 cultivars was calculated by the software of NTSYS pc - 2.1e. The result of genetic similarity analysis showed that the Jaccard coefficient ranged from 0.5263-0.9910. A DNA molecular dendrogram was established for 39 cultivars based on UPGMA cluster analysis. When the similarity coefficient value was 0.65, the 39 Japanese apricot cultivars were divided into 3 groups, and 34 cultivars of the third group （III） were further divided into 5 subgroups, as was fundamentally accorded with the traditional classification base on fruit-ripening color. There was no obvious difference in geographic relationship among the clustering results. The study indicated that ISSR produced high polymorphism on Japanese apricot. ISSR analysis can be used for the molecular evaluation and studies of genetic diversity of Japanese apricot germplasm.更多还原