Study On Molecular Phylogeny Of Angiopteris

DNA is an important biological information molecule, as well as an important material of phylogenetic research on plant. The extraction of genomic DNA is the most elementary and important step to the molecular study. Due to losing water in long-term conservation, dried specimens of plant had resulting in a number of secondary products, and DNA degradation occurred in varying degrees. So compared to fresh plant material, their DNA extraction is more difficult. In this study, six kinds of dried specimens of plants which had conversed for nine years are three kinds of dicotyledonous plants (Thalictrum aquilegifolium L., Cotinus coggyria Scop., Syneilesis aconitifolia (Bunge) Maxim.), monocotyledon (Paris polyphylla Smith), gymnosperm (Ginkgo biloba L) and pteridophyte (Polystichum shensiense Christ). They were soaked in three kinds of solution to pre-: TE buffer (pH8.0), eliminating polyphenolic compounds buffer and Tris-HCl buffer. The DNA extraction methods were CTAB method, SDS method and High-salt low-pH method. The DNA qualities were tested with the UV spectrophotometry and the PCR amplification of chloroplast DNA trnL-F. The results showed that: The pretreatment method of TE buffer got the best effect to the dry specimen Polystichum shensiense Christ, and to the other five dry specimens the Tris-HCl buffer got the best effect. Among the DNA extraction methods, high-salt low-pH method was the optimum method for the dicotyledonous plant ( Thalictrum aquilegifolium L., Cotinus coggyria Scop and Syneilesis aconitifolia (Bunge) Maxim), CTAB method was the optimum method for the monocotyledon (Paris polyphylla Smith), high-salt low-pH method was the optimum method for the gymnosperm (Ginkgo biloba L) and pteridophyte (Polystichum shensiense Christ). It is suggested choosing the different method of DNA extraction according to the different plant materials. Most of the Angiopteridaceae plants (especially Angiopteris) are large fleshy ferns.Their specimen collection and pressing are difficult. Its classification has long been one of the difficulties in the classification of ferns. And so far the researches about Angiopteridaceae plants are mainly focused on the morphological classification, few on molecular phylogeny. In this study, the CTAB method was used for the total genomic DNA extraction of nine kinds of plant specimens——Angiopteris six, Archangiopteris one and the other two outgroup plants which were stored at low-temperature for 3 months. Chloroplast DNA trnL -F and part of atpB of all plants were done PCR amplification, then cloned and sequenced. The corresponding gene sequence of four plants(including outgroups relatives of plants and ingroups) were downloaded from the Genebank. CLUSTAL X2 software was used for sequence alignment of target DNA. The result of sequence alignment was used to construct the phylogenetic tree with the neighbor-joining, NJ of MEGA4.1. It was used Kimura 2-parameter algorithm in neighbor joining analysis of genetic distance. Through the analysis of phylogenetic trees constructed with trnL-F sequences and atpB sequences, it is showed that: Danaea were separated earlier than Angiopteridaceae and Marattia , and Marattia were separated earlier than Angiopteridaceae ; it did not agree with the idea——Archangiopteris is evolved from Angiopteris; in the study on molecular phylogeny of ferns, trnL-F sequence can be used to the classification inter-genera, and atpB sequence can be used to the classification of not only families, but also under-genus; Angiopteris and Archangiopteris were experienced rapid evolutionary radiation process. It was difficult to separate them. So it is needed to more data of molecular phylogeny to determine the classification of their relationship.