DNA Barcode Data Collection And Phylogenetic Relationship Reconstruction In Shenzhen

DNA bar code(barcoding DNA)technology is a kind of automatic identification technology,which is used for rapid and accurate identification of species with a section(or several sections)standard,with enough variation,easy to be amplified and relatively short DNA sequences.Its technical principle is similar to the bar code technology in our daily contact,and it uses A,T,C and G to identify the species of the four bases in the gene sequence.The new identification technology has been developed rapidly in recent years,which provides a new solution to solve the problems of traditional taxonomy.Rich plant resources,Shenzhen City,Guangdong Province,the distribution of wild ferns have 41 families,86 genera 188 species,contain six advantages Selaginellaceae families(families),Pteridaceae(a),Athyrium ferns Athyriaceae,Thelypteridaceae Thelypteridaceae,angle iron ferns(Aspleniaceae),scales of Dryopteris,Dryopteridaceae,Polypodiaceae Polypodiaceae and some precious endangered ferns such as the double leaf fern Dipteris chinensis,Osmunda mildei Osmunda mildei,Fujian Avalokitesvara lotus Angiopteris fokiensis etc.,with development,protection and research of multiple values.DNA bar code technology through the identification of mitochondrial CO1 gene animals,can be efficient and accurate identification of species.In the study of plant bar code,people found that the plant mitochondrial gene evolution rate is slow,can not be used as a standard bar code.In the next work to find a common bar code will become a plant DNA bar code of a research focus.After a large number of studies,CBOL Consortium the for the Barcode of Life and were used to determine the use of chloroplast rbc L and mat K as plant universal bar code in the Third International Barcode conference in 2009.But the combination is not universal in all plant groups,such as mat K gene because of its universal primer,the problem is difficult to use as a standard bar code.It is still an important research content to find the DNA bar code which is universal in the whole fern group.In this study,the ycf1,rps4,rbc L and ITS genes were selected,and the data were collected and analyzed by using the DNA bar code data in Shenzhen city.The time span of the field collection is about one year,all the collection sites are within the urban area of Shenzhen.A total of 384 species of wild fern were obtained,including 146 different species.In previous experiments,it was found that the ycf1 fragment of chloroplast and nuclear gene ITS was low in general,and the amplification efficiency was difficult to use,so the selection of chloroplast trn L-F and trn H-psb A fragment was replaced,and the subsequent experiment was carried out.Research results show that the selected fragments have different features.The chloroplast fragments ycf1 gene although for amplification and sequencing of the effect is not ideal is to abandon the use,but the quality of the data has been obtained better and identification in all segments of the greatest variation,there is a 100% success rate.It is suggested that the following research can start from the angle of primer development,and study the possibility of ycf1 as a universal bar code.trn H-psb A had the highest amplification rate(81.96%)and second high sequencing success rate(71.70%),and the identification success rate was second high(98%),suitable for use as a generic DNA bar code for fern.This fragment also has the advantage of shorter length than the other segments in the identification of damaged material or the amount of material obtained is less than the other segments.The disadvantage is that the trn H-psb A fragment contains a lot of insertions and deletions,which caused great difficulties to the data analysis.Chloroplast rbc L gene is used in most of the fern DNA barcoding fragment,the amplification success rate(95%),sequencing success rate is low(63.55%),levels were significantly lower than those of previous similar studies,identification of success rate to 92.15%.Suitable for use as a fern DNA bar code.The other two fragments trn L-F and rps4 showed poor performance in both extraction and species identification,and could be used as candidate fragments.Finally through the establishment of the system of phylogenetic tree to discuss the phylogenetic relationship that Pyrrosia genus should not be independent division,agree with the Adiantum into the view of generalized Pteridaceae.