论文标题:猕猴桃属植物的遗传多样性及种质超低温保存研究
Studies on Genetic Diversity and Germplasm Cryopreservation of Genus Actinidia
论文作者 徐小彪
论文导师 张秋明,论文学位 博士,论文专业 果树学
论文单位 湖南农业大学,点击次数 272,论文页数 119页File Size9236k
2004-10-30论文网 http://www.lw23.com/lunwen_2958162/ 猕猴桃属;植物;遗传多样性;AFLP;超低温保存
Genus Actinidia; Plant; Genetic Diversity; AFLP; Cryopreservation
中国是猕猴桃属植物的起源和分布中心，种质资源极为丰富，可为猕猴桃遗传改良和资源利用提供广泛的背景材料。长期以来，对猕猴桃属植物的遗传多样性尤其是DNA水平上的遗传多样性及种质的超低温保存缺乏系统研究。鉴于此，作者以重要分布区之一的江西猕猴桃属植物为对象，在查清其种类、分布及若干性状评价的基础上，进行AFLP分析与种质超低温保存的研究。主要结果如下： 1 江西猕猴桃属植物的分布及若干性状评价 (1) 通过野外实地调查与查阅标本，对江西猕猴桃属(Actinidia)植物的分布状况进行系统研究，基本查清了其分布种类及规律。结果表明，江西境内分布有猕猴桃属植物20种11变种，其中葛枣猕猴桃(Actinidia．polygamya(Sieb．et Zucc．)Maxim．)、灰毛猕猴桃(A．cinerascens C．F．Liang)、楔叶猕猴桃(A．fasciculoides var cuneata C．F．Liang)和簇花猕猴桃(A．fasciculoides C．F．Liang)为江西首次发现。依据其种类与变种的多样性形态特征，编制了江西猕猴桃属植物分类检索表。 (2) 江西猕猴桃属植物的地理分布区域较广，中华猕猴桃在全省各地均有分布，各种类与变种之间对气候及土壤等生态条件要求的多样性导致其地理分布的广泛性。其区系特征主要表现为：种类丰富，特有现象较明显，江西分布国有特产种17种，占国产该属特有种的30.4％，其中有2种为江西特有：种间、种内分化较强烈，江西猕猴桃属植物的种与变种在该属4个组均有代表：与邻近地区猕猴桃属植物的关系密切，其中与福建关系最为密切。 (3) 通过对主要种与变种果实性状观测、营养成分和氨基酸含量测定，表明江西猕猴桃属植物在果实形状、毛被状况、果皮色泽、果肉色泽、果实风味、果实Vc含量及氨基酸含量方面存在丰富的多样性。 (4) 对江西猕猴桃属植物中的矮型(赣猕5号)和耐贮(97-3)中华猕猴桃特异种质资源进行了初步研究，为其保存与利用奠定基础。 2 猕猴桃属植物的AFLP分析 (1) 4种经优化的猕猴桃基因组DNA提取方法即CTAB区室法、SDS区室法、CTAB常规法、SDS常规法均适于提取猕猴桃嫩叶基因组DNA。经综合对比分析，认为CTAB区室法为猕猴桃属植物AFLP分析的最佳方法，其步骤简单、快捷，抽提的基因组DNA降解少、杂质含量少，能获得高质量的DNA模板。通过对所得到的DNA模板进行AFLP分析，获得了清晰的DNA指纹图谱。 (2)从AFLP试剂盒所提供的64对引物组合中筛选出扩增带信号强度一致性好、条带分布均匀、多态性高的4对引物组合即E一从C+M--CAC、E一从G+MweCTG、E一AAC十M--以G和E一从C+M一CTA对31份称猴桃种质进行了DNA指纹分析。4对引物共得到扩增位点190个，其中多态性位点179个，多态性比例为94.2%，区分率达100%。该4对引物适用于称猴桃属植物的AFLP分析。各种质的多态性带数存在差异，多态性带数最多的是黑蕊称猴桃(108条)，多态性比例为60.3%;多态性带数最少的是早鲜称猴桃(54条)，其多态性比例为30.2%。结果进一步显示，称猴桃属植物在DNA分子水平上存在丰富的遗传多样性。 (3)首次运用AFLP技术构建了31份称猴桃种质的DNA指纹图谱，通过对扩增结果进行UPGMA聚类分析，其相似系数为0.50一0.85，表明种质之间遗传关系较远。依相似系数0.56的水平，将供试的31份称猴桃种质分为4个类群:净果组和斑果组为一类群，糙毛组为一类群，星毛组为一类群，中华称猴桃和美味称猴桃为一类群，该结果与传统的果面毛被特征和皮孔状况的形态分类系统中组的划分大体一致，并有按地理分布聚类的趋势。AFLP的分子分类可弥补传统形态分类系统的不足，其分类范围更细，可为称猴桃属植物的分类提供依据。 (4)聚类分析表明，称猴桃属植物矮型种质“赣称5号”独立于中华称猴桃种，基于AFLP指纹，从分子水平上分析，可提升为称猴桃属中华称猴桃种内的一个变型。3称猴桃种质超低温保存研究 (1)以“赣称5号”为试材，筛选了其离体培养的激素组合。MS+B凡.0+NAAo.，与MS+BAI.砰NAAo.l较适于称猴桃外植体的诱导及增殖。无菌小芽在MS+I BA10培养基上出根率较高，根系生长正常。 (2)称猴桃的组培苗每4周继代一次，在增殖培养基上连续继代3一5次后转入到低浓度生长调节剂的培养基上继代一次，然后继续增殖。生根试管苗经炼苗移栽后可达到91%的成活率。 (3)首次采用玻璃化法保存了称猴桃种质离体茎尖，其适宜的程序为:继代20d的1。m长嫩梢，接种到含蔗糖3%的MS培养基上，5℃低温下锻炼6周。切取1.5砚.51nlll的茎尖，于5%DMSO+5%蔗糖+MS基本培养基上预培养3d，在室温下用Zm。比Gly+0.4m。比蔗糖预处理30而n，再于O℃下用PvS之脱水处理4伪nin，换一次新鲜的PVSZ溶液后，迅速投入到LN中。保存24h后，在40℃水浴中化冻，用3%蔗糖+MS基本培养基洗涤两次后接种到恢复培养基上，可获得56.7%的成活率和51石%的再生率。超低温保存后的称猴桃无菌小苗在形态上与对照一致。 (4)应用流式细胞仪和TEM对超低温保存后的材料进行倍性检测以及超低温保存过程中细胞的超微结构变化进行观察。结果表明，其染色体倍性未发生变异。在含3%蔗糖的MS培养基上，5℃?
China is the center of origin and distribution of genus Actinidia and there are various genotypes, which provides broad breeding materials for creating new cultivars. It has not systematically studied for genetic diversity especially for the evaluation on the DNA level and cryopreservation of genus Actinidia in past. Therefore, we have formulated this study to investigate the distribution and main traits, and to conduct AFLP analysis and cryopreservation of genus Actinidia in Jiangxi province which is one of the important distribution region. The major results are summarized and reported in this paper.1. Distribution and major Traits of Genus Actinidia in Jiangxi Province(1)The geographical distribution and floristic characters of genus Actinidia from Jiangxi province were studied by means of field surveys and examining herbarium specimens. The results indicate that there are 20 species and 11 varieties of Actinidia distributed in Jiangxi province, among them Actinidia. polygamya (Sieb.et Zucc.)Maxim., A. cinerascens C.F.Liang, A. fasciculoides var cuneata C.F.Liang, A. fasciculoides CF.Liang are newly recorded in Jiangxi Province. The distribution of genus Actinidia was tabulated according to its morphological various characters.(2)The geographical distribution is broad for genus Actinidia in Jiangxi province. It is distributed for Actinidia Chinensis Planch.all over this region. The universality of distribution for genus Actinidia is the result of diversity such ecological conditions as climate and soil. Jiangxi province is rich in endemic species of the genus, and 17 species endemic to China are found in whole province counting up 30.4% of total species. Interspecific and intraspecific differentiation of Actinidia was significantly high. The geographical impacts on the species are complex. The species endemic to China are widely distributed in the province. Species of Actinidia in Jiangxi province is closely related to that distributed in bordering regions of Fujian province.(3)The fruit characters of genus Actinidia in Jiangxi were determined and the nutritional constituents were analyzed. The results showed that the genus Actinidia in Jiangxi contained abundant genetic diversity in morphological characters and the nutritional constituents.(4)Dwarf genotype(Actinidia Chinensis Planch.cv.Ganmi No.5) and long-storage life genotype(Actinidia Chinensis Planch.line97-3) distributed in Jiangxi province were studied, and its utilization approach and preservation strategy were proposed.2. AFLP Analysis of Genus Actinidia(1)The results of four modified extraction methods for genomic DNA such as CTAB subarea method , SDS subarea method , CTAB regular method, and SDS regular method were suitable to kiwifruit. It has showed that CTAB subarea method was the best for AFLP analysis by comprehensive comparison. Its simple and quick procedure led to high-quality genomic DNA, which had lower elimination and impurity. By employing this method, very clear AFLP fingerprinting pattern was obtained.(2)Four pairs of primers: E-AAC+M-CAC; E-AAG+M-CTG; E-AAC + M-CAG ; E-AAC+M-CTAwere screened out from 64 pair of primers. Their signal intensity of amplified pattern was consistent, and distributed uniformly. A total of 190 sites were amplified, among which 179(94.2%) sites were polymorphic. The identification rates among 31 germplasms of genus Actinidia were up to 100%. Therefore, the results indicated that four pairs of primers are suitable for the AFLP analysis on genus Actinidia plants. The polymorphic band numbers for the germplasms showed some differences. The largest number for polymorphic band was in Actinidia melanandra French with polymorphic rate 60.3%, whole the smallest was in Actinidia Chinensis Planch.cv.Zaoxian with polymorphic rate 30.2%.The results indicated that the genetic diversity for genus Actinidia was abundant on the molecular level.(3)DNA fingerprint map of 31 kiwifruit accessions were constructed by AFLP technique for the first time. UPGMA analysis baseded on AFLP markers indicated that the similarity coefficient o
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