| Retrotransposons are the most abundant and widespread class of transposable elements (TEs) in plants. LTR retrotransposons are further subdivided into the Tyl-copia and the Ty3-gypsy groups. They transpose through an RNA intermediate by a "copy and paste" mechanism, and thus retrotransposons generated stable mutation and had great affect on the genome size, genomic structure, gene function and the evolution of gene and genome in plants.Japanese apricot, stone tree of Rosaceae Prunus L., originated in China, and has a long cultural history. The fruit are nutritious and healthy. Although many researches have been done on this fruit tree, however, there are no reports on the existence of TEs in the Japanese apricot genome. The researches on the amounts, heterogeneities, distribution and transcription activity of retrotransposons contribute much to our understanding on the genome constitue, genomic structure and genome evolution, The genome characterization with reference to the content, heterogeneity, activity and overall distribution of retrotransposons might contribute to our understanding about Japanese apricot genome organization and its evolution.Japanese apricot was used to isolate two major classes LTR retrotransposons: Tyl-copia group and Ty3-gypsy group and analysis the heterogeneity, transcriptional activity, copy number of retrotransposons. Tyl-copia LTR sequences were also isolated from Japanese apricot, and SSAP based on the Tyl-copia LTR sequences are used to analysis the genetic diversity of P. mume.1 At present, the materials to extract DNA from P. mume mostly were young leaves. Although the leaves had the advantages of higher DNA yield and easy to gather, they could be influenced by the seasons and long distance transportation. The phloem layers of dormant branch used to isolation genomic DNA not only overcome the problem of the DNA degradation causing by the long distance transportation, but extended the gathering time of materials and elongated the gathering ranges of materials.In this study, the genomic DNA of P. mume were isolated from the phloem layers of dormant branch of 5 cultivars by improved CTAB method, and the effects of DNA extraction were contrasted with young leaves. The results showed:each of the genomic DNA extracted from phloem and cambial layers of 5 cultivars by this method was pure, integral, the values of D260nm/D280nm were 1.80 to 2.0, degradation was free nearly, the RNA was eliminated completely, and suitable for digestion by restriction endonucleases. SSAP analysis indicated that the primer (LTR-1/EcoR-ACC, LTR-2/EcoR-ACC) produced clear polymorphic patterns, which were completely the same to the corresponding young leaves. Therefore, this method could be used for extracting total DNA from the phloem layers of dormant branch of P. mume and suitable for SSAP.2 The conserved domains of RT genes of roughly 260 bp for Tyl-copia and 430 bp for Ty3-gypsy groups of LTR retrotransposons were amplified from the Japanese apricot genome using degenerate oligonucleotide primers, whose sequence analyses showed that 32.3% of Ty1-copia and 27.5% of Ty3-gypsy RT sequences possessed stop codons and/or frameshifts, and all sequences were AT-rich. The significantly high heterogeneity among RT sequences corresponding to both Ty1-copia and Ty3-gypsy group retrotransposons was observed, but Ty3-gypsy elements were less heterogeneity than Tyl-copia group elements, with the difference of 45.3% and 40.7%, respectively. Southern dot blot hybridization result revealed that both group retrotransposons were present with high copy number in the genome of Japanese apricot, about 7.9×103 (18.4% of P. mume genome) and 1.0×104 (33.3% of P. mume genome) copies for Ty1-copia and Ty3-gypsy, respectively. Phylogenetic analysis illustrated that some of the clones were more closely related to the representative elements present in other plant species than to other clones of Japanese apricot. The ratios of dN/dS of the open reading frames among members of each subgroup of both group retrotransposons were less than 1, suggested that most of the Japanese apricot retrotransposons might be active. However, RT-PCR amplification from total RNA, which was extracted from young leaves of Japanese apricot treated with UV light and 2,4-D either individually or in both combinations, did not yield either of RT fragments.3 Based on the principle of Pearce’s method, we have successfully developed an effective method to isolate the LTR sequences of Ty1-copia group retrotranspsons from the genome of Japanese apricot. We have successfully isolated 11 RNase-LTR sequences from Japanese apricot. There were some differences among the length, PPT and IR of the 11 sequences of Japanese apricot, and the PPTs of 5 of continuous purines with a pyrimidine insertion. Sequence PmLTR6 contained recombination mutations. Based on the homogeneity of amino acid sequence of RNaseH,11 sequences were divided into three groups. The promoter analysis of LTR sequences showed that there were "CAAT" box and "TATA" box in 10 and 9 sequences, while the motif regulated by light, heat, drought and plant hormone (abscisic acid, gibberellin) were also observed. There were also motifs which were special expressed in meristem and endosperm in sequence PmLTR10.4 Based on the isolated 3’-LTR sequences from the genome of Japanese apricot, the SSAP molecular system was established. The results showed that adding a base in the 3’of LTR primer could improve the definition of bands and polymorphism, and different annealing temperature had no effective affects on the PCR products. The estabilished SSAP molecular system was used on the genetic diversity of 60 cultivars of Japanese of apricot. According to the different countries, two populations were classified and genetic diversity analyzed by SSAP showed that the diversity of Chinese population was higher than Japanese population. According to the origin place,4 populations were classified. Among the 4 populations, ZhejiangJiangsu local species group with special differentiation in the population was the most variable. The phylogenetic relationship of the 4 populations showed that YunnanSichuanHunan local species group was found to be the most similar one among the 4 to Japanese population with the highest genetic identity of 0.9669. That showed us that the genetic differentiation among the 2 populations (Japanese population and YunnanSichuanHunan local species group) was small and YunnanSichuanHunan local species group could be the origin of Japanese population. The phylogenetic relationship among the 60 cultivars showed that all species could be divided into three groups which showed no relationship with the classification of Green mei, Red mei and White mei. The genetic structure released cultivar population was conferred. |
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