Somatic Cell Cytoplasmic Inheritance And Chloroplast Simple Sequence Repeat (SSR) Primer Development In Citrus

In the present study, cytoplasmic inheritance of Citrus somatic hybrids and cybrids from 38 intergeneric and interspecific fusion combinations was analyzed by restriction fragment length polymorphisms (RFLPs), cleaved amplified polymorphic sequence (CAPS) and chloroplast simple sequence repeat (cpSSR) markers. The experimental procedures were modified and optimized, cpSSR, a novel marker method was developed in Citrus and other tropical fruit crops. Meanwhile, organelle genetic variation of 23 Citrus calli was analyzed. The main results were as follows:1. A simple and efficient method for genomic DNA extraction from woody fruit crops containing high level of polysaccharides was developed. This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using water saturated ether at the presence of 1.25-1.30 M NaCl. The quality of DNA samples extracted by the present method was suitable for PCR and RFLPs analysis and for long-term storage. In addition, this procedure was successfully applied in DNA isolation from frost withered old leaves of Citrus and more than 20 kinds of tropical and subtropical fruit crops.2. Five pairs of chloroplast DNA (cpDNA) universal primer and 3 pairs of mitochondrial DNA (mtDNA) universal primers generated monomorphic fragments among hybrids of 4 intergeneric and 3 interspefic fusion combinations. After being digested by restriction endonuclease, polymorphic mitochondrial CAPS markers were displayed in the 4 intergeneric combinations, while polymorphic chloroplast CAPS markers were found in 3 intergeneric and 2 interspecific fusion combinations. The results showed that the specific bands of mitochondria among all the hybrids were identical to embryogenic suspension parents, while the chloroplast were identical either to the mesophyll parents or to the embryogenic suspension parents.3. Detailed RFLPs analysis of mitochondrial genome among the hybrids of 38 fusion combinations were performed by using 5 restriction endonuclease to digest the genomic DNA and hybridize with 12 mitochondrial probes, the results revealed that the mitochondrial genome of Citrus somatic hybrids &cybrids is derived biasly from the embryogenic suspension parent, and rearrangement occurred commonly. The characterization of mitochondria! genome recombination was influenced by nuclear background of the hybrids, more complex mtDNA composition usually existed in allotetraploid somatic hybrids than those in diploid cybrids. The common mtDNA fragments in the fusion parents could be usually detected in the offspring. Mitochondrial inheritance ability is related with the genotype of the fusion parents. The ancient ancestor had stronger mitochondrial transfer ability than their offspring. When both fusion parents are evolutionarily latter than C. reticulata, there had the possibilities of C. reticulata like mitochondrial RFLP banding pattern displayed in their hybrids. When modern genotype acts as embryogenic suspension parent and ancient genotype as mesophyll parent, the mitochondria in the fusion hybrids may derived biasly from the mesophyll parent.4. Leaves of 'Valencia' orange + 'Meiwa' kumquat somatic hybrid were harvested monthly for a whole year, and RFLPs analysis were performed. Combined with the field observation, the results revealed that the loss of the mtDNA fragments was correlated with the plant died back, as suggested that the genetic instability of mitochondrial genome is one possible reasons for the growth abnormality of the somatic hybrids.5. Combined the data of CAPS with RFLPs in chloroplast genome analysis, 14 out of the 38 fusion combinations showed some degree of polymorphisms, chloroplasts were randomly segregated among these combinations. To further study the origin of chloroplast genome among the other combinations, new marker need to be developed.6. To study the organelle characterization at the expression level, RT-PCR was carried out, and Northern hybridization was employed in mitochondria as well. The results showed th...