Research And Development Of Molecular Barcode Of Kareniaceae And Its Application In The Coastal Waters Of China

Kareniaceae species are common toxic and harmful microalgae that are the main causative species of dinoflagellate harmful algal blooms(HABs).Due to their small size,fragile algae cell,and lack of obvious morphological characteristics,it is difficult to accurately identify Kareniaceae species with the traditional microscopic classification method.In recent years,studies have shown that more and more HABs are caused by multiple Kareniaceae species,while the algae cell density of Kareniaceae species in water during non-bloom period is rather low,making them easily to be ignored during routine sampling.In order to quickly and accurately identify the causative species of Kareniaceae HABs,as well as to understand the diversity of Kareniaceae species in the environment,a specific molecular barcoding detection method was established in this study,make it possible to effectively carry out early warning and monitoring of Kareniaceae HABs.The main research contents and the results are as following:(1)By comparing the interspecific genetic distances of the ribosomal small subunit DNA sequence(18S rDNA),the large subunit DNA sequence(28S rDNA)and internal transcribed spacer(ITS)sequence of Kareniaceae species,and phylogenetic analysis of ITS sequences of Kareniaceae species,it was found that compared with 18 S rDNA and28 S rDNA,ITS had a suitable mutation rate,which could better distinguish the species of three genera of Kareniaceae and found the differences between different geographical strains of the same species.Therefore,the ITS region was determined as the target region for the design of molecular barcodes of Kareniaceae.As a result,two pairs of molecular barcodes(Kare and KarlTa)were designed based on the alignment results of ITS sequences of Kareniaceae species,of which,the Kare barcodes were used for Karenia species,and the KarlTa barcodes were used for the detection of Karlodinium and Takayama species.The PCR reaction system and the optimal working temperature for the two pairs of molecular barcodes were determined by PCR experiments,and two molecular barcodes could detect the minimum target alga genomic DNA of 3.2 pg,corresponding to 0.17-0.60 target alga cells.Based on online assessment tools and specificity experiments,two pairs of molecular barcodes could be used for the detection of multiple target algae,while there was no positive amplification for species other than Kareniaceae.Collectively,the established molecular barcodes showed good specificity and sensitivity,and will be effective to detect Kareniaceae species.(2)In order to study the species diversity and geographical distribution characteristics of Kareniaceae in China coastal waters,88 environmental samples were collected along the coastline of China,and the detection was carried out based on the established method.The results showed that 50 samples obtained target bands by amplification using Kare molecular barcodes,and the detected samples were collected from the Yellow Sea,Bohai Sea,East China Sea and central area of South China Sea.While only 28 samples were successfully amplified by KarlTa barcodes,and the corresponding samples were mainly from the East China Sea and central area of South China Sea.The results implied that the Kareniaceae species were widely distributed in the coastal waters of China,and their distribution showed regional characteristics,which laid a good foundation for the diversity analysis of Kareniaceae species.(3)The above 78 PCR products of environmental samples with target band were used to construct high-throughput self-built libraries,and the sequencing data found that the number of homologous sequences of ITS gene of Kareniaceae species in most sequencing libraries accounted for more than 80% of the total effective sequences,and the PCR products of the two pairs of molecular barcodes had good correspondence with the species of different genus of Kareniaceae,which further proved the specificity of this detection method.A total of 24603 OTUs were obtained by clustering these homologous sequences of ITS gene of Kareniaceae species,of which,the OTUs with similarity greater than 90% accounted for 92.6% of the total OTUs,indicating that the species of known genera of Kareniaceae in the coastal waters of China had rich genetic diversity.According to the statistics on representative species of OTU with similarity greater than 98% among92.6% OTUs,16 known Kareniaceae species were found in the natural environment of China coastal waters,besides common 10 species(Karenia mikimotoi,Karenia papilionacea,Karenia selliformis,Karenia longicanalis,Karenia brevis,Karlodinium veneficum,Karlodinium digitatum,Takayama acrotrocha,Takayama tasmanica and Takayama xiamenensis),6 species were discovered for the first time in the coastal waters of China,including Karenia bicuneiformis,Karenia brevisulcata,Karlodinium ballantinum,Karlodinium azanzae,Karlodinium armiger and Karlodinium sp.,and the monotype species of Brachidinium,Brachidinium capitatum.Among them,the main dominant species were Karenia mikimotoi and Karenia papilionacea in Karenia,and Karlodinium veneficum in Karlodinium.Meantime,it was also found that the diversity of Kareniaceae species in different coaltline of China was different,with the highest diversity in the central area of the South China Sea and the lowest diversity in the yangtze river estuary.Related studies have preliminarily revealed the species type,main species and geographical distribution characteristics of Kareniaceae in the coastal waters of China.In conclusion,this study developed two pairs of molecular barcodes for Kareniaceae species,with high sensitivity and specificity,which can be used for rapid identification and detection of low-density Kareniaceae species in the natural environment samples.By using the high-throughput sequencing technology based on the established molecular barcodes,it can further determine the Kareniaceae species and contribute to the diversity analysis of Kareniaceae.The establishment of this detection system will provide important technical support for the effective early warning and monitoring of Kareniaceae HABs.