Sequence Analysis Of MatK Gene And ITS Region And Phylogeny On Ginkgo Biloba Var. Epiphylla Mak

Ginkgo biloba L. is a type of the relict spermatophyte found in China that has survived since the 4th Ice Age. 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. 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 chloproplast matK gene and ITS sequence were analyzed for 2 Ginkgo biloba trees and 27 Ginkgo biloba var. epiphylla trees which from China and Japan, and the phylogenetic tree was constructed based on matK gene in this study. The results obtained as follows:(1) Genomic DNA of the Ginkgo biloba var. epiphylla were extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method.The results identified by agarose gel electrophoresis showed that the total DNA was about 23 kb long, the bands were neat and distinct and did not have obvious tail, which demonstrated that the modified CTAB method could obtain high purity DNA.(2) The length of the matK genes obtained through clone for 27 Ginkgo biloba var. epiphylla individuals were 1539bp. The results of sequence analysis showed that the matK sequence had the highest T nucleotide content, and among the 27 individuals'NS2'was the highest while the'DK'was the lowest; the average contents of T, C, A, G were 33.84%, 18.75%, 28.7% and 18.73% respectively; the matK gene sequences were rich in A and T.The three codon positions of matK gene were all had the highest nucleotide T content, the average of which were 30.17%, 31.37% and 39.97% respectively; among the three codon positions the first had the highest G and C content, and the third had the lowest G and C content due to its high T nucleotide content.The ratio of variable sites in three codon positions of matK gene was 1.24:1.07:1. The variable sites in three codon positions were not strongly biased and most of them distributed at the 5' end . 512 amiod acids were encoded by the matK gene, the variable sites of the amiod acids were also mainly distributed at the 5' end. There was a linear correlation between the G and C content of the matK gene and the three codon positions for each sample of Ginkgo biloba and Ginkgo biloba var. epiphylla and slopes were closed to zero, implying that the three codon positions were subjected to the same mutation presure.The Kimura-2-parameter corrected distance based on matK gene among samples of Ginkgo biloba and Ginkgo biloba var. epiphylla varied from 0 to 0.0078. The length of the phylogenetic tree constructed based on matK gene was 1064 steps, with a CI of 0.9803 and a RI of 0.7640. All the samples that from different regions of Ginkgo biloba var. epiphylla were clustered in a clade, which showed that they had the same origin. The Ginkgo biloba var. epiphylla appeared in the basal position of the tree compared to Ginkgo biloba, reflecting that the Ginkgo biloba var. epiphylla was more originative than Ginkgo biloba.(3) The length of the ITS sequences for 27 Ginkgo biloba var. epiphylla individuals were 1224bp. The results of sequence analysis showed that the ITS sequence had the highest G nucleotide content, and among the 27 individuals'DK'was the highest while the'YZ6'was the lowest; the average contents of T, C, A, G were 18.6%,33.7%,13.6% and 34.2% respectively; the ITS sequences were rich in G and C.The ITS sequence of Ginkgo biloba var. epiphylla contained three regions, i. e. ITS1, 5.8S rDNA and ITS2, the length of which were 822bp, 161bp and 241bp respectively. The ITS1 sequence had the highest G nucleotide content, and among the 27 individuals'NS1'was the highest while the'YZ7'was the lowest; the ITS2 sequence had the highest C nucleotide content, and among the 27 individuals'NS2'was the highest while the'YZ6'and'GC3'were the lowest. There were obvious differences in base composition between ITS1 sequence and ITS2 sequence.The variable sites of ITS sequences were mainly distributed at the ITS1 sequence, the 5.8S rDNA and ITS2 sequence were more conserved.The Kimura-2-parameter corrected distance based on ITS sequence among samples of Ginkgo biloba and Ginkgo biloba var. epiphylla varied from 0 to 0.0099. The ITS sequence of Ginkgo biloba and Ginkgo biloba var. epiphylla inclued 57 variable sites, but only 2 informative sites. The informative sites were so little that it could not provide enough information to construct phylogenetic tree.The taxonomic position, phylogeny and occurrence mechanism 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.