Taxonomical study of Citrus and its related genera by mitochondrial DNA analysis

柑橘属及其近缘属的分类仍存在争议。细胞质中的线粒体DNA(MtDNA)是系统发育研究的标志物之一。本研究为柑橘属及其近缘属的分类研究建立了线粒体DNA的提取和纯化方法。叶片中的苯酚化合物和精油等次生代谢物抑制线粒体DNA的提取。采用继代培养的愈伤组织作为mtDNA提取材料。除了开发从叶片和愈伤组织中提取线粒体DNA的程序外，还利用温州蜜柑开发了染色体DNA(CpDNA)的提取程序。温州蜜柑叶片和愈伤组织的mtDNA产量分别为0.46ug/gFW和0.34ug/gFW。温州蜜柑叶片cpDNA的得率为1.05 ug/g FW。从叶片中提取mtDNA，用限制性内切酶EcoRI进行限制性内切酶消化，但限制性片段长度多态性较差。琼脂和Gellite等胶凝剂可抑制线粒体DNA的消化。对柑橘亚科植物7属19种进行了同工酶分析。小柑橘和小柑橘具有相同的子房室基因。通过同工酶分析鉴定了柑橘属、海棠属和金桔属植物。分析了‘金鸡儿’、‘金鸡儿’和温州蜜柑不同组织发生层发育的组织器官以及3个无性系品种的珠心幼苗的同工酶图谱。经证实，‘Kinkoji-unshu’确实是一个倾角周围嵌合体。

The taxonomy of Citrus and its related genera is still controversial. Mitochondrial DNA (mtDNA) in a cytoplasm is one of the markers for phylogenetic study. In this profection, the procedures to extract and purify mitochondrial DNA were developed for the taxonomical study of Citrus and its related genera.1. Second metabolites such as phenol compounds and essential oils in leaves inhibited the extraction of mtDNA. Subcultured calli were used for the material of mtDNA extraction.2. In addition to the development of the procedures for the extraction of mtDNA from leaves and calli, the procedures of chroloplast DNA (cpDNA) were developed using satsuma. The yield of mtDNA from satsuma leaves and calli was 0.46 ug and 0.34 ug/g FW, respectively. The yield of cpDNA from satsuma leaves was 1.05 ug/g FW.3. MtDNA was extracted from leaves and digested by restriction enzyme EcoRI, but the RFLP was smeary. Gelling agent, e. g. agar and Gellite inhibited the digestion of mtDNA.4. Isozyme analysis of seven genera and 19 species of orange subfamily plants was carried out. Microcitrus and Eremocitrus have same gene loculus. Species of Citropsis, Atalantia and Fortunella were identified by Isozyme analysis.5. The Isozyme pattern of tissues and organs developed from the different histogenic layers of the cultivars 'Kinkoji-unshu', 'Kinkoji' and satsuma, as well as the three nucellar seedlings derived from the three clonal cultivars were analyzed. It is confirmed that 'Kinkoji-unshu' is indeed a periclinal chimera.