| Ginkgo biloba L. is a type of the relict spermatophyte found in China that has survived since the 4th Ice Age which has long been called living fossil.There were obvious morphological variaton occured in seeds and leaves of Ginkgo biloba along the long evolutionary process. In 1891, Shirai discovered the first Ginkgo biloba var. epiphylla Mak. tree of the wolrd in Japan and Dong Chunyao Guo discovered the first in Yiyuan,Shandong province of China,Guo Shanji reported it at 1984. The Ginkgo biloba var. epiphylla which is a special germplasm of Ginkgo family has great ornamental and scientific values. However, mostly of the studies on Ginkgo biloba var. epiphylla are focus on simple observation and description of the morphology at present. A convincing concept for the taxonomic position and phylogeny of Ginkgo biloba var. epiphylla is still laking. Thus, it is in urgent need to explore the variation mechanism of Ginkgo biloba var. epiphylla using molecular biotechnology.The study was used 2 Ginkgo biloba trees and 26 Ginkgo biloba var. epiphylla trees which from China and 1 fromJapan.Using primers trnS-GF(5′-GCCGCTTTA- GTCCACTCAGC-3′) and trnS-GR(5′-GAACGAATCACACTTTTACCAC-3′) reported in current literature,the auther amplified a portion of trnS-trnG from the total genomic DNA of the Ginkgo biloba trees and Ginkgo biloba var. epiphylla trees accessions used in the present study. The PCR products were first purified and then ligated to the plasmid vector, which were introduced intoDH5a competent cell of E. coli. The positive clones were screened on the LB solid culture medium containing ampicilin and IPTG/X-gal according to'blue and white reaction'. The positive clones (in white color) were cultured in LB liquid culture medium at 37℃for 12 hours. The selected plasmid DNAs were sequenced by Shenggong. The sequences were aligned with ClustalX computer program. The gene trees were constructed based on the aligned sequences.The length of the trnS-trnG genes obtained through clone for 27 Ginkgo biloba var. epiphylla individuals most were 1005bp,the most shoted is YZ4 which from YIYUAN only 1002bp The results of sequence analysis showed that the trnS-trnG sequence had the highest T nucleotide content. The average contents of T, C, A, G were 31.28%,19.43%,31.04% and 18.24% respectively. Between the sequence have low differences. the trnS-trnG gene sequences were rich in A and T.The sequences of all samples were 1008bp after aligned,and includ 34 variable sites that all changed by substitutions, but only 3 informative sites,each in 34bp,37bp and 359bp codon positions.The Kimura-2-parameter corrected distance based on trnS-trnG gene among samples of Ginkgo biloba and Ginkgo biloba var. epiphylla varied from 0 to 0.008,the average is 0.0027.The genetic distance is minimal and on the range of variation of intraspecific. The length of the phylogenetic tree constructed based on trnS-trnG gene was 684 steps, with a CI of 0.9853 and a RI of 0.8361.It is strict consistent tree's consistency index is more higer and on the range of variation of intraspecific,which showed that the variation of frequency was very low in trnS-trnG gene of Ginkgo biloba and Ginkgo biloba var. epiphylla. All the samples that from different regions of Ginkgo biloba var. epiphylla were clustered in a clade in most parsimonious, which showed that they had the same origin; The samples that from different regions of Ginkgo biloba var. epiphylla were constituted a branch in phylogenetic tree,which indicated that genesis mechanism of Ginkgo biloba var. epiphylla were independent of geography position. Ginkgo biloba var. epiphylla had a near relationship with Cycas of outgroup.The sequence variation characteristics and phylogeny of Ginkgo biloba var. epiphylla were discussed in this paper. This research is of great importance to understand the phylogeny and molecular evolution of Ginkgo biloba var. epiphylla and gymnosperm and to the preservation, utilization and exploitation of this ornamental resource. |
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