Determination And Application Of Molecular Barcode For Diatom DNA Identification

The morphological classification of diatoms is mainly based on their siliceous shells.However,when the environmental conditions change,there may be some changes in the siliceous shells or some deformation due to other reasons.This is a huge challenge for taxonomists.Molecular classification is a method of identifying unknown individuals by using special DNA sequences of standardized genes or non-coding regions.Its advantage is that it can quickly and accurately identify diatoms without being affected by diatom deformation and microscope equipment.Diatoms have an important ecological position in the ocean,and accurate and rapid classification of diatoms is the basis for other work.We studied here the molecular sequence-based identification method,and selected a new DNA barcode.The 18S rDNA primer sequence was:downstream primer 5?GATGGATCATTCAAGTTTCTGCCC-3?;downstream primer 5?ATCATTACCTCGGTGCGCAAACC-3?.The rbcL primer sequence is:upstream primer 5?-CAACCAGGTGTAGATCCAGTAG-3?;downstream primer:5?GAAACGTTCTCTCCAACGCATG-3?.By using optical and scanning electron microscopy(LM and SEM),nearly 15 diatom species have been identified,and their 18S rDNA and rbcL sequences have been obtained.A specific DNA identification molecular database has been established for these diatoms.These diatoms are related to the following genera:Thalassiosira;Skeletonema;Coscinodiscus;Ditylum;Chaetoceros;Falcula;Melosira;Licmophora;After analyzing the molecular sequences of these diatoms in multigene phylogeny,we found that the molecular data comparison results and the morphological identification results were almost identical.It is proved that the DNA barcode selected in this study can be used in the molecular identification of diatoms and provides a reference for diatom identification.Then,the designed primers and the selected DNA barcode were used to identify 21 unknown diatoms.Combining with the morphological observation,it can provide a more favorable reference for the diatom breeding.At the same time,molecular sequencing of four morphologically-designed diatoms was performed,and it was found that the molecular sequence analysis results and morphological results were almost the same.The results showed that the two gene sequences selected in this study can be used as DNA barcodes to provide a reference for the classification and identification of diatoms.Finally,the 18S rDNA sequences,rbcL and psbC sequences measured in this paper were compared horizontally from various aspects such as base composition and accuracy of sequence identification results.The results prove that the three sequences selected in this study are relatively compared to the 18S rDNA sequence and psbC sequences,rbcL sequences are more suitable molecular markers for diatom identification.