Phylogenetic Analysis Of Zygophyllaceae Based On Nuclear ITS DNA And Chloroplast TrnL-F Sequences

This paper describes the optimized method for the total DNA isolation of Zygophyllaceae plant.A DNA extraction method for the PCR of Zygophyllaceae plant was found and ITS was used for the first time in the identification of whole Zygophyllaceae plant.We established and optimized ITS and trnL-F's PCR procedure to fit the Zygophyllaceae plant.Meanwhile,we sequenced ITS and trnL-F DNA fragment of 31 species Zygophyllaceae plant,Using Bioedit to complete blast,using Clustwal to complete compositor,using Paup to construct Phylogenetic tree.Result are follows:1. The optimized CTAB method was seccessfully used to extract the polysaccharide-rich genomic DNA of N.tanutorum. Bobr.and N.sibirica.Pall.This method can obtain satisfactory purity and quantity of the extraction.The extracted DNA is suitable for ITS and trnL-F's PCR.2. The optimized ITS and trnL-F's PCR system of Zygophyllaceae plant was:25μL reaction system contained 50ng template,1μM primer,2.0mM Mg2+,0.2mM dNTP,11.1μL的ddH2O and 2U Taq polymerase.The PCR parameter was: 94℃pre-denature for 7min;94℃denature for 30sec,52℃annealing for 45sec,72℃extended for 2min,45cycles;72℃extended for 7min,the product of PCR was kept at 4℃.By using the optimized conditions,we could obtain repeatable results and legible electrophoretic patterns.3. Use MP and UPGMA respectively to construct Phylogenetic tree,sameness is:Z.macropterum C.A.Mey and var. microphyllum Boriss. gather for a category;Subsp.dolichocarpum Popov. and Z. iliense Popov gather for a category;Z. fabago L.and Subsp.orientale Boriss. gather for a category; Z.macropodum Boriss. and Z.fabago L.,Subsp.orientale Boriss gather for a category;Z.jaxarticum Popov and Z.rosovil Bunge gather for a category;N.tangutorum Bobr. and N.roborowskii Kom. gather for a category ;S.kaschgaricum Bunge and S. xanthoxylon (Boriss.) Y.X.Liou gather for a category;dissimilarity is:Z.pterocarpum Bunge and Z. brachypterum Kar.et Kir. gather for a category in MP,Z.pterocarpum Bunge and Z.loczyi Kanitz gather for a category in UPGMA;Tribulus L.,Tetraena Maxim.'s clade gather with Peganum L.,Nitraria L.'s clade as a category,then gather with Sarcozygium Bunge in MP;Tribulus L.,Tetraena Maxim. and Zygophyllum L.,Sarcozygium Bunge's clade gather for a category in UPGMA ;Zygophyllum L. Plant's differentiation is great in MP,while Zygophyllum L. Plant all gather with each other.4. Use UPGMA,NJ and ML respectively to construct trnL-F Phylogenetic tree,result as follows:in UPGMA,Tribulus L.,Tetrana Maxim. and Zygophyllum L.,Sarcozygium Bunge's clade has far relationship with Peganum L. and Nitraria L.;In NJ,Tribulus L.,Tetrana Maxim.,Peganum L. Nitraria L.and Sarcozygium Bunge's clade has far relationship with Zygophyllum L.;the result caused by ML is as same as NJ's.