Cellular And Molecular Identification Of Chlorophyta Associated With Green Tide

Green tides usually refer to an ecological disaster caused by the excessive proliferation and growth or accumulation of some large marine green algae in sea water under specific environmental conditions.Since 2007,the green tides have been periodically and large-scale outbreaks in the Yellow Sea area of China from May to July each year,which have seriously affected the surrounding aquaculture and tourism industry,bringing serious ecological disasters and economic losses.Ulva prolifera is a common large marine green alga,belonging to Chlorophyta,Chlorophyceae,Ulvales,Ulva.It has been confirmed as the main dominant species of the green tide in the Yellow Sea.Its algae are bright green or dark green,and are tubular when inflated,with obvious main branches and fine branches.They grow in the gravel,rocks,tidal flats,and stony marshes of low and middle tide areas along the ocean coast.The homogeneous generation of U.prolifera and its adaptability to salinity,temperature,light,and dry-out conditions make U.prolifera the main species of green tide outbreak,and it is also one of the main niche competitors of artificially cultivated laver in the raft area.In this study,a large number of green algae samples were collected at different sites in the seaweed rafts in Jiangsu and coastal areas of Shandong.Combined with many commonly used molecular markers to identify green algal species,the green algae species at different sites were identified the appropriate molecular marker combination was selected to facilitate subsequent species identification.In addition,we also performed ploidy identification of the yellow sea tide,by combining flow cytometry detection with chromosome observation,and the steps of preparation of protoplasts and selection of lysate in flow cytometry sampling system were optimized.Finally,the propagation method of floating U.prolifera was discussed in conjunction with the extinction phototaxis experiment.Through the screening of different molecular markers,we found that ITS is highly conserved for molecular identification between species,but cannot distinguish between U.prolifera and U.linza;5S spacers can effectively distinguish U.prolifera-linza was found in the LPP complex,and the 18S method showed many deficiencies in the identification of interspecific species due to excessive conservation.for example,the construction of a phylogenetic tree after amplification and sequencing showed that it was impossible to cluster closer species into different branches.In addition,we found that the tufA gene has a good interspecies identification effect;the screening results show that the combination of the tufA gene and the 5S spacer can effectively identify species of different green algae species.At the same time,We also screened for green tide U.prolifera ISSR-PCR polymorphic bands and designed a pair of effective molecular primers based on specific bands,which can be directly identified from the bands without cutting gel recovery,which greatly saves The time for identification by molecular means after collecting the samples is given.In this study,the distribution of different species in Jiangsu,Shandong and other places was identified through the selection of molecular marker combinations.U.compressa,U.intestinalis,and U.pertusa can be collected in coastal areas of Shandong all year round,and there are U.prolifera,U.intestinalis,U.linza,U.flexuosa,and Blidingia minima that are associated with perennial rafts in Jiangsu.Through the combination of flow cytometry and chromosome observation technology to explore the ploidy of the yellow sea green tide floating U.prolifera,the results obtained by flow cytometry found that the samples collected at different sites were mainly sporophytes,and only a few were gametophytes.At the same time,the phenomenon of coexistence of two generations also exists,and the chromosomal data further confirms our conclusion:the yellow sea green tide U.prolifer a is the main advantage of sporophytes,and the detection of a small number of gametophytes may be due to meiosis of some algal bodies.The reason why multi-generation coexistence is detected at the same time may be that there is no guarantee that the sample is a complete single plant during sample processing.We also conducted a phototaxis experiment on the green tide U.prolifera of the Yellow Sea,and found that the amount of gametes released by U.prolifera is much larger than the amount of spores,indicating that sexual reproduction is a common method of propagation of the green tide.In this study,the green tide-related green algae in the Yellow Sea were studied from both cellular and molecular aspects.The former provides evidence for the ploidy study of the green tide floating U.prolifera,and the latter screening based on different molecular markers is helpful for the subsequent rapid identification of different species of Ulva.