Cloning And Phylogenetic Analysis Of LEAFY Homologous Gene Of Dendrobium

Dendrobium Sw., which belongs to Epidendroideae (Orchidaceae), contains about 1,000 species in the world and mainly spread in tropical and subtropical Asia and Oceania. In China, they mainly distribute in the mountains which lies in the south of Qinling Mountains and expecially in southern part of Yunnan province. The traditional classification of Dendrobium was mainly based on morphological features, and ITS sequences were the only frequently-used molecular data in phylogenetic research. However, there were some limitations in resolving the phylogenetic relationship among Dendrobium just depending on the morphology evidence or analyzing single gene sequence. Therefore, more molecular markers are needed to reconstruct the phylogenetic relationship of Dendrobium. In the trends of studies of molecular systematic, low-copy nuclear gene has become a new tool in phylogenetic research. According to the previous studies, LFY homologous gene was a kind of low-copy gene and have been used for phylogenetic reconstruction in lower level species.In this study, the LFY homologous gene of Dendrobium moniliforme (L.) Sw. was cloned by new primers which were designed based on the conservative region of known sequences of orchid LEAFY gene. Partial LFY homologous gene was cloned by common PCR, then the complete LFY homologous gene DenLFY was obtained by Tail-PCR. The complete sequence of DenLFY gene was 3,575 bp which contained three exons and two introns. By comparison analysis among the exon of LFY homologous gene using BLAST method, it was indicted that the DenLFY gene had high identity with other orchids LFY homologous (over 80%). Homologous analysis demonstrated that the C-terminal amino acids were highly conserved. The research of exons and amino acid of DenLFY gene were a start of its functional study. The lengths of the second intron showed difference between Dendrobium moniliforme and Dendrobium officinale, which can be inferred that the second intron can be used in authentication research of two Dendrobium plants.Phylogenetic tree of 36 Dendrobium species were reconstructed based on the first intron of LFY gene, ITS and trnL-trnF sequence respectively. The results demonstrated that the first intron of LFY gene was suitable for Dendrobium phylogeny study, due to its appropriate length and many variable and parsimony sites. From the phylogenetic tree based on the first intron of LFY gene, the relationship among most species were well resolved with higher support compared with previous studies. Through the amplification of low-copy nuclear LFY gene successfully, this gene can be developed in Dendrobium phylogenetic study. From the comparison among phylogenetic trees based on single gene sequence and combined molecular sequences, the concatenation data have more variable and parsimony sites and the relationship among Dendrobium were fully resolved with higher support value.The species in our research belong to nine sections of Dendrobium. The result of phylogenetic study deduced by combined data revealed that, Dendrobium phalaenopsis and Dendrobium salaccense lied in the base of the phylogenetic tree. Dendrobium section which contains the most species was divided into two clades and the relationship among some species were in consistent with morphology evidence based on combined data. In Dendrobium section, Dendrobium officinale and Dendrobium flexicaule, Dendrobium guangxiense and Dendrobium tosaense, Dendrobium moniliforme and Dendrobium huoshanense, Dendrobium linawianum and Dendrobium nobile were sister together seperately. Dendrobium densiflorum and Dendrobium thyrsiflorum, Dendrobium lindleyi and Dendrobium jenkinsii which have the similiar inflorescence and labellum were clustered together, and Dendrobium chrysotoxum belonged to Chrysotoxae section while it had far relationship with other species in Chrysotoxae section. Dendrobium hercoglossum and Dendrobium aduncum were both in Breviflores section according to morphological classification, but they were involved into Dendroum section based on molecular evidence and this maybe due to the simialr leafy, stem between the two sections.Overall, the relationships of species among Dendrobium are complicated. Moderate molecular makers and morphological characters are the two keys to resolve the relationships among Dendrobium. The first intron of LFY gene is suitable for Dendrobium phylogenetic research due to its appropriate length and more parsimony informative sites. The analyses of combined molecular data have great significant to this research according to our study.