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

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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.