Polymorphism Of MtDNA And CpDNA In Diospyros Kaki Thunb. And Some Closely Related Species

Japanese persimmon(Diospyros kaki Thunb.) belongs to the Diospyros genus, Ebenaceae Family. Because of maternal inheritance of cytoplasm, mitochondrial and chloroplast genomes were employed to evaluate the genetic polymorphism and phylogenetic relationships of Diospyros spp. in the present study. The materials included seven species: D. kaki Thunb.; D. lotus L.; D. glaucifolia Metc; D. oleifera Cheng; Jinzaoshi; D.rhombifolia Herosl.; D. Virginiana L.. The cytoplasm polymorphisms were investigated in order to identify the genetic relationships and provide the evidence for the scientific and efficient utilization of the persimmon germplasm. The results were as follows:1. Universal primers were used to amplify mtDNA non-coding regions in Diospyros spp. including 6 related species and 20 genotypes of Diospyros kaki Thunb.. The results showed: 1) Thirty two universal primers successfully amplified either introns or intergenic regions in Diospyros spp. A total of 110 bands were polymorphic. 2) Twenty three universal primers were used to analysis genetic diversity at the level of intra-specific, which revealed that the mitochondrial genomes had abundant variation during recombination. Japanese persimmon of PCNA genotypes from Japan and China were separated distinctly from each other by clustering analysis, which suggested that they had different origins; 3) Two Chinese PCNA genotypes of Japanese persimmon, Baogai-tianshi and Eshi No.1, have part of unique bands to other materials, which showed they would have derived from the same female parent according to the maternal inheritance of mitochondrial genome.2. Ten universal primer pairs of chloroplast genome were used to amplify chloroplast DNA(cpDNA) non-coding regions in 7 Diospyros species including 29 genotypes. Approximately 20.4kb, 12.7% of the chloroplast genome was analysed. The products were digested by seven restriction enzymes. The results showed abundant interspecific cpDNA variations within the Diospyros spp.. However, no visually variation was detected among 22 genotypes in D. kaki, excepting of Male strain No.9 by trnS-trnfM/Rsal. The species D. kaki had closely relationship with D. lotus and D. glaucifolia, but distant from D.virginiana and D.rhombifolia in diagram based on principal coordinates analysis by NTSYS 2.10e program and unrooted phylogenetic trees based on PHYLIP 3.62 Wagner parsimony method. Investigated the digesting pattern discrepancy in cpDNA, the genotype Jinzaoshi was distinct with the remaining Diospyros spp., which suggested that Jinzaoshi could be considered as a new species.3. A universal primer pair was used to amplify the trnS-trnfM region in chloroplast genome of 9 persimmon cultivars from D. kaki and 1 genotype of related species D. lotus. The nucleotide sequence of 1155～1160 bp were surveyed from four coding regions, trnS, psbZ, trnG and trnfM, and three intergenic spacer regions. A total of 17 mutations including 11 base substitutions, 3 insertions and 3 deletions were identified used Clustal X software. Among persimmon cultivars from China, only 2 base substitutions were found, whereas 14 from Japan, which revealed the distinct discrepancy. The sequences were conserved and homology among centuries-old genotypes, which showed relatively little divergence from their furcation points. However, Youhou, new cross-breed cultivars had the most mutations and were same change with female parent Fuyuu. Neighbor-joining trees were constructed from site mutations data were indicated persimmon cultivars from China and Japan were formed different group and suggested both of which maybe have different origin. Chloroplast DNA homology and phylogenetic analysis revealed an immediate common progenitor for Baogai-tianshi and Eshi No.1 was closely relationship with PCA persimmon Mopanshi.4. The ORJF25 pimers were used to amplify the mtDNA, then the products were cloned and sequenced. The genetic diversity and amplified specificity were identified. Comparative sequence revealed some base mutations were occured in this region and a second copy maybe presented in mtDNA. Phylogentic analysis indicated the divergence time between Zenjimaru(PVNA) and Hiratanenashi(PVA) should not be long. The persimmon genotypes, which had different astringent types and ploidy, were employed to evaluate potential utilization of twelve universal cpSSR primers. Six cpSSR primers designed according to the chloroplast genomes of Citrus(ARCP4, ARCP 5, ARCP9) and Nicotiana tabacum(ccmp3, NTCP9, NTCP40) were amplified with Diospyros genomic DNA. The results showed this set primer pairs had high consensus abilities and potentiality applied in phylogeny research of Diospyros spp..5. Intra-specific cross compatibilities of Japanese persimmon(D. kaki Thunb.) Mopanshi(PCA) with Luotian-tianshi(PCNA) was investigated using a combination of UV fluorescence microscopy. The genetic characterizations were analyzed among the parent and the progenies obtained by embryo rescue using RAPD and SSR molecular markers and the relationships were surveyed with other PCNA persimmon germplasm from China. Although viable pollens of Luotian-tianshi loading on the stigma could germinate, the pollen tube showed very a few compatible growths along the stylar tissue after pollination. A range of abnormalities in both pollen tube growth and callose deposition were detected. It appeared that the fertilization barriers characterized by the poor performance of pollen tube in style was responsible mainly for the low seed set of Mopanshi. The results of this study revealed Mopanshi had predominantly parthenogenesis with a few sexual reproductions. Among 186 hand-pollinated treatments, 82 fruits and 13 seeds were obtained by hybriding Mopanshi with Luotian-tianshi. Evidence from RAPD and SSR markers confirmed that the seedlings obtained by embryo rescue were F₁ progenies of Mopanshi×Luotian-tianshi, part of the which were identical patterns with pollinated constant non-astringent persimmons native to China, Baogai-tianshi and Eshi No. 1.In conclusion, a large amount of universal primers were used to amplified the cytoplasm genome of persimmon, including mtDNA and cpDNA, then the amplified products were detected by agarose gel, digested by restriction enzymes or sequenced directly in this study,. The genetic relationship were analysed using UPGMA, Wagner parsimony method and PCoA. The results were mostly agreed to the conclusion based on molecular markers of total genomes, which revealed that: 1) the mitochondrial genomes of Japanese persimmon had abundant variation; 2) cpDNA PCR-RFLP markers were efficiency to phylogeny analysis at interspecies level of Diospyros spp.; 3) Polyploidy species, D.kaki, D.virginiana and D.rhombifolia have different evolution process; 4) Jinzaoshi could be considered as a new species of Diospyros L.; 5) The characters of parthenogenesis with sexual reproductions in Mopanshi and staminate flowers in Luotian-tianshi provide the opportunity for them as parents of PCNA persimmon hybridization breeding.