In the present study,cytoplasmic inheritance of Citrus somatic hybrids and cybrids from 38 intergeneric and interspecific fusion combinations was analyzed with restriction fragment length polymorphisms (RFLPs),cleaved amplified polymorphic sequence (CAPS) and chloroplast simple sequence repeat (cpSSR) markers.The experimental procedures were modified and optimized.A novel marker,cpSSR,was developed in Citrus and other tropical fruit crops.Meanwhile,genetic variations of organelle from 23 Citrus calli were 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 mol/L NaCl.The quality of DNA samples extracted with this method was suitable for PCR and RFLPs analysis and for long-term storage.In addition,this procedure was successfully applied in DNA isolation from the freezed or withered or senile leaves of Citrus and more than 20 kinds of tropical and subtropical fruit crops.2.Five universal pairs of chloroplast DNA (cpDNA) primer and 3 universal pairs of mitochondrial DNA (mtDNA) primers amplified monomorphic fragments among 4 intergeneric hybrids and 3 interspefic fusion combinations.After 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 with embryogenic suspension parents,while the chloroplast were identical either with the mesophyll parents or with the embryogenic suspension parents.3.Detailed RFLPs analysis of mitochondrial genome among the hybrids of 38 fusion combinations were performed through digesting the genomic DNA with 5 restriction endonucleases and then hybridizing with 12 mitochondrial probes,results revealed that the mitochondrial genome of Citrus somatic hybrids and cybrids was biasly derived from the embryogenic suspension parent with commonly occurred rearrangements.The characterization of mitochondrial genome recombination was influenced by nuclear background of the hybrids,and mtDNA composition usually existed in allotetraploid somatic hybrids was more complicated than that in diploid cybrids.The common mtDNA fragments in the fusion parents could be usually detected in its offsprings.Mitochondrial inheritability was correlated with the genotype of the fusion parents.The ancient ancestor had stronger mitochondrial transferability than its offspring had.When both fusion parents are evolutionarily later 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 from the somatic hybrid of ‘Valencia’ orange and ‘Meiwa’ kumquat were harvested monthly for a whole year and analyzed with RFLPs marker combined with the field observation.Results revealed that the loss of the mtDNA fragments was correlated with the plant died back,suggesting 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 other combinations,new markers are required to be developed.6.To characterize the organelle at the expression level,RT-PCR and Northern hybridization were used in mitochondria.Results showed that expression of mitochondrial genes in somatic hybrids was higher than that in their corresponding fusion parents,while no clear difference existed among cybrids and their parents.7.Thirty-three pairs of cpSSR primers designed according to the chloroplast genomes of pine (Pinus thunbergii),rice (Oryza sativa),tobacco (Nicotiana tabacum) and Arabidopsis thaliana were amplified with Citrus genomic DNA.After strictly screened,fourteen specific primers were obtained and named as SPCC1 to SPCC14,and subsequent studies were performed as follows:1)Thirty-four representative accessions of Citrus,Fortunella,Poncirus and some of their hybrids were selected and amplified with 14 SPCC primers.Loci among the 34 genotypes ranged from 1 to 8 with an average of 3.54,and the mean PIC value was 0.356.Results of neighbor-joining cluster based upon the cpSSR data showed that all the samples were divided into 7 clusters,namely citron (C.medica),lime (C.aurantifolia),pummelo (C.grandiss),Yichang papeda:(C.ichangensis),kumquat (Fortunella spp.),trifoliate orange (Poncirus spp) and mandarin (C.reticulata) clusters.2)Seventeen fragments of SPCC1 and 21 of SPCC11 were cloned,sequenced and aligned withsequences of the original primer motif from tobacco and Arabidopsis ,the outcomes revealed that all the cloned sequences were from the chloroplast genomes and the polymorphism was caused by mononucleotide repeat of poly (A) or poly (T).3)According to the cpSSR classification results,suitable SPCC primers were selected to analyze the chloroplast genomes among the hybrids of the 38 fusion combinations,random segregation of chloroplast in Citrus fusion hybrids was further proved,and 1∶1 segregation ratio were revealed in the fusion combinations of Kinnow tangerine+Jincheng orange and Bonanza navel orange+Goutou sour orange,respectively.4)Sixty-four DNA samples belonging to 31 kinds of tropical and subtropical fruit crops were amplifiedwith the synthesized cpSSR primers,and a set of wide adaptable universal primers were screened.For instance,SPCC1 was rich in polymorphism among intergenera or interspecies,such as persimmon (Diospyros kaki ) and dateplum persimmon (Diospyros lotus),banana (Musa spp.),guava (Psidium spp.),papaya (Carica spp.) and grape (Vitis spp.).8.Twenty-three genotypes of calli adequately representing the Citrus species and related genera were selected,and corresponding leaves were used as the control to analyze the stability of mitochondrial and chloroplast (plastome) genome.RFLPs,histological section,cpSSR and mtDNA transferring from the cytoplasm to the nuclei were analyzed.The results were as follows:1)Low frequency of mtDNA mutation existed in Citrus calli,while the mtDNA contents increased in individual mitochondria,and no such phenomena were observed in chloroplast genome.2)Analyzed with cpSSR,novel bands were detected among most of the calli.Both Southern hybridization and sequencing proved that the bands were amplified uniquely,suggesting that short sequences insertion occurred frequently in Citrus calli plastid genomes.3)Dot blotting analyses were performed by using nuclear DNA hybridized with mitochondrial probes.Results showed that mitochondrial DNA transfering from cytoplasm into nuclei occurred during long-term subculture.