柑橘变异材料的遗传鉴定与品质分析

The Genetic Identification and Fruit Quality Analysis of the Variations in Citrus

作者: 专业:果树学 导师:伊华林 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

late maturity Jincheng Orange, fresh edible Valencia Sweet Orange cultivars, mutant, AFLP

        柑橘是我国第一大水果,现阶段,我国柑橘产业结构仍需要进行改进,突出的问题主要表现在晚熟加工品种稀缺,鲜食品种供应期集中。本研究鉴定的两个变异材料能够填补目前市场上的空白,为我国柑橘品种的改良起到重要作用。本研究以在兴山发现的晚熟锦橙[Citrus sinensis (L.) Osbeck]变异和在秭归发现的适合鲜食的伏令夏橙[Citrus sinensis (L.) Osbeck]变异为材料,使用AFLP分子标记技术对变异材料及其母本进行遗传鉴定,鉴定出变异与母本的差异。对果实的常规品质进行分析的结果显示:晚熟锦橙变异的果实品质与母本变化不大,其加工品质无变化,伏令夏橙变异果实表现为无籽,固酸比高等性状,具有作为鲜食品种推广的潜力。具体研究结果如下:1.使用流式细胞仪对伏令夏橙变异和母本进行倍性鉴定,结果表明变异和母本都是二倍体。使用了120组AFLP引物组合在晚熟锦橙变异及其母本中鉴定出16条多态性条带;在伏令夏橙变异及其母本中鉴定出2条多态性条带。将鉴定出的多态性条带测序,根据序列比对的结果设计引物,设计的部分引物在两组变异和母本材料中能够扩增出来,说明变异材料是不同于母本的芽变新种质,能够作为品种资源加以利用。2.晚熟锦橙果实的常规品质和母本变化不大,主要的加工品质中,出汁率为59.74%,可溶性固形物(TSS)含量为11.27%,可滴定酸(TA)含量为1.19%,Vc含量为52.55mg/100g,满足加工品种的品质要求。气相色谱(GC)分析果实中可溶性糖酸含量也无较大差异。伏令夏橙变异果实大小(纵径8.82cm,横径8.65cm)较母本大,同时表现无籽,固酸比高(14.84)等特性。GC分析显示伏令夏橙变异果实苹果酸(0.71mg/g)和蔗糖含量(43.11mg/g)显著高于母本,柠檬酸含量(3.02mg/g)显著低于母本,其他种类可溶性糖酸无显著差异,从而构成变异单株果实的风味特点,有可能成为鲜食品种加以推广。
    Citrus is the most important fruit crops in China. At present, there is still lack of late mature cultivars for processing, the supply of fresh edible fruit is concentrated. In this research, we find two mutation to fill in the blanks of the market, provide contributions to improve the citrus varieties in China.In this study, we used AFLP to perform genetic identification with the mutation of late mature Jincheng Orange [Citrus.sinensis.(L.)Osbeck] found in XingShan, and the fresh edible variation of Valencia Sweet Orange [Citrus. sinensis.(L.)Osbeck] cultivars foung in ZiGui. Polymorphisms between the mutation and the original were found out. Quality analysis were also performed,there is little difference in fruit quality between the mutation and the original in Jincheng Orange,the processing quality shows no change; at the same time, the mutation of Valencia Sweet Orange cultivars has potential to be introduced as edible cultivars which shows better edible characters such as seedless, high solid-acid ratio.The main research results are as follows:1. Ploidy identification of the Valencia Sweet Orange cultivars was performed by flow cytometry, and the result shows the mutation was diploid, the same as the original.120 pairs of AFLP primers were used, and there are 16 polymorphism bands we found between the mutation and the original in Jincheng Orange,2 between the variation and the original in Valencia Sweet Orange cultivars. All of the polymorphism bands sequences were measured, and from which the primers designed to amplified in the mutations and the originals. Some primers were able to get amplified successfully which shows the truth of the polymorphism between the mutations and the originals. As the proof of mutant.2. There shows little differences in common quality between the variation of Jincheng Orange. For main processing quality,the juice yield was 38%, the TSS was 11.27%, TA was 1.19%, and the amount of Vc was 52.55mg/100g,which meet the requirements of processing. Analysis of sugar to acid by GC shows little differences between the mutation and the original neither. The mutation of Valencia Sweet Orange cultivars shows larger firut (length8.82cm, & width 8.65cm), seedless, high sugar-acid ratio (14.84). The malic acid (0.71mg/g) and the sucroce (43.71mg/g) of the mutation were higher than the original. The citrus acid (3.02mg/g) was less than the original, which constitute the flavor of the mutation fruit.It has potential to be introduced as fresh edible cultivars.
        

柑橘变异材料的遗传鉴定与品质分析

摘要6-7
Abstract7-8
缩略词表9-10
1 前言10-17
    1.1 课题的提出10-11
    1.2 文献综述11-16
        1.2.1 柑橘的生物学特性11-12
        1.2.2 柑橘的育种途径12-13
            1.2.2.1 杂交育种12
            1.2.2.2 芽变和实生选种12
            1.2.2.3 生物技术育种12-13
        1.2.3 芽变选种在柑橘育种中的应用13-14
        1.2.4 柑橘芽变的鉴定手段14-16
            1.2.4.1 形态学鉴定14
            1.2.4.2 同工酶检测14-15
            1.2.4.3 DNA分子标记鉴定15-16
    1.3 本研究的目的和内容16-17
2 材料和方法17-25
    2.1 研究材料17
    2.2 研究方法17-25
        2.2.1 倍性分析17
        2.2.2 AFLP分子标记鉴定17-21
            2.2.2.1 基因组DNA提取17-18
            2.2.2.2 酶切基因组DNA18-19
            2.2.2.3 连接反应19-20
            2.2.2.4 预扩增反应20
            2.2.2.5 选择性扩增反应20
            2.2.2.6 聚丙烯酰胺凝胶电泳20-21
        2.2.3 差异片段的回收,克隆和测序21
        2.2.4 差异条带的生物信息学分析并设计特异引物21
        2.2.5 果实常规品质测定21-24
            2.2.5.1 果实大小21-22
            2.2.5.2 单果重22
            2.2.5.3 果皮,果肉色差值22
            2.2.5.4 出汁率22
            2.2.5.5 可溶性固形物含量22
            2.2.5.6 可滴定酸含量22-23
            2.2.5.7 VC含量23-24
        2.2.6 GC检测果肉中可溶性糖和有机酸含量24-25
            2.2.6.1 可溶性糖和有机酸的提取24
            2.2.6.2 提取物的两步衍生化24
            2.2.6.3 GC检测条件24-25
            2.2.6.4 糖酸成分的定性25
            2.2.6.5 标准曲线的绘制25
3 结果与分析25-34
    3.1 遗传鉴定结果25-30
        3.1.1 倍性分析结果25-26
        3.1.2 AFLP鉴定结果26-28
            3.1.2.1 晚熟锦橙的鉴定结果26-27
            3.1.2.2 伏令夏橙变异的鉴定结果27-28
        3.1.3 差异条带的测序及生物信息学分析28
        3.1.4 根据序列比对设计引物在基因组DNA中特异性扩增28-30
    3.2 品质分析结果30-34
        3.2.1 果实常规品质测定结果分析30-32
            3.2.1.1 晚熟锦橙及对照果实常规品质测定30-31
            3.2.1.2 伏令夏橙变异及对照果实常规品质测定31-32
        3.2.2 GC测定果实糖酸结果分析32-34
            3.2.2.1 晚熟锦橙及母本果实糖酸分析32
            3.2.2.2 伏令夏橙变异及母本果实糖酸分析32-34
4 讨论34-36
    4.1 AFLP分子标记在芽变鉴定中的应用34
    4.2 差异条带的分析和应用价值34-35
    4.3 果实品质分析及未来应用前景35-36
参考文献36-41
图版41-43
致谢43
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