Wood Identification Based On DNA Molecule Of Part Species In Ulmus Spp.,Zelkova Spp.and Celtis Spp.

With the development of DNA molecular identification technique,it is becoming popularly used in wood identification..Some of the woods of Ulmus,Zelkova,and Celtis in the Ulmaceae are commonly used materials.In order to accurately and efficiently identify the wood of Ulmaceae microscopically,DNA molecular marker technology and DNA barcode technology were used to identify the selected 13 wood species from 3 genera of Ulmus spp.,Zelkova spp.,and Celtis spp.This paper analyzed the ability of six DNA extraction methods to extract residual DNA from different air-dry conditions and screened ISSR primers for PCR amplification.The band polymorphism was compared by ISSR amplification with 13 tree species.13 tree species were amplified with DNA barcodes PsbA-TrnH,TrnL-F,ITS 1,ITS 2,followed by analysis of polymorphic base sites and phylogenetic trees,Finally,the following conclusions are presented:1)Different DNA extraction methods can be used for wood of Zelkova schneideriana with different air drying time.Wood with a dry air time of 1 a can use The new rapid plant genomic DNA extraction kit can be applied when the air drying time of wood is within 1a and CTAB-SDS method can be conducted for wood within 4a,while PTB method can only be used for long-term air-dry wood.Among the six DNA extraction methods,the CTAB-SDS method has the highest extraction concentration and the best quality,followed by the CTAB method,and the common plant genomic DNA extraction kit has the worst extraction effect;As the air drying time is prolonged,the DNA of the Z.schneideriana can be extracted less and less when the quality of the DNA is also reduced.However,the length of the DNA molecule extractable within 4a is at least 1 500 bp;2)27 primers with bands were screened out from 52 randomly selected ISSR primers.After comparing the band quality and quantity of 27 primers,it was found that ISSR primers818,828 and 836 could amplify the DNA fragment of beech with high abundance and fragments exceeding 1000 bp of Z.schneideriana.After ISSR-PCR amplification of 13 tree species,836 primers were most suitable.3)ISSR primer 836 amplifies 34 band loci in 13 tree species and the total polymorphism ratio is 100%.The number of band locus in the three genera of Ulmus spp.,Zelkova spp.,and Celtis spp.are respectively 23,11 and 13.In terms of polymorphism ratio,Ulmus spp.(100%)>Celtis spp.(92.3%)> Zelkova spp.(90.9%).Primer 836 is therefore suitable for non-specific amplification of these 13 species.There are 16 repeating band loci between 13 tree species of the three genera,which account for 45.7% of the total band locus.There are 19 unique band loci,accounting for 54.3% of the total band loci.Except for celtis julianae,the remaining 12 species have 1-3 unique band loci.Therefore,the 13 tree species of the three genera are both conserved and rich in genetic diversity,allowing the primer 836 to easily distinguish 13 species.4)TrnL-F sequence,ITS-1 sequence,ITS-2 sequence and PsbA-TrnH sequence have characteristic nucleotide loci among 13 species of the three genera.There are abundant mutation sites among the three genera,which can easily determine the genera of tree species.However,not all tree species have specific interspecific variation loci,so it is not possible to distinguishall tree species by only one sequence of variation loci.5)When constructing a phylogenetic tree in a single sequence,none of the four sequences can completely distinguish 13 wood species individually.For Ulmus spp.,the TrnL-F sequence can identify the most tree species-4;for Zelkova spp.,the PsbA-TrnH sequence can identify the most species-2;for Celtis spp.,the ITS 1 sequence can identify the most species-3.The identification ability of the phylogenetic tree of the combined sequence system is significantly higher than that of the single sequence phylogenetic tree.As the number of combined sequences increases,the bootstrap value increases,the clustering accuracy of the wood species increases,and the number of identified trees increases.When the number of combined sequences reaches3,the 13 wood species can be completely distinguished.The sequence combination ITS 1+ITS2+PsbA-TrnH,ITS 1+ITS 2+TrnL-F and ITS 1+ITS 2+PsbA-TrnH+TrnL-F can accurately identify the 13 wood species.When constructing the phylogenetic tree,the NJ tree is better than the ML tree.The NJ tree is more accurate for the clustering of 13 wood species,and it is only one which fully distinguishes the 13 species.