Methodological Systematization For The Rapid And Accurate Identification Of Nano-marine Microalgae

Microalgae are microscopic and unicellular. Either solitarily or connectively,they inhabit water environments, playing a vital role in both primary production ofaquatic environments and global atmospheric carbon dioxide acquisition. In recentyears, marine microalgal research has focus on nano-marine microalgae (2-20μm indiameter). The commercial value of nano-marine microalgae is increasingly obvious,they are rich, for example, in nutrition and important for aquacultures. Recently,nano-marine microalgae promise for biofuel production. Identification of nano-marinemicroalgae species is fundamental to both biological and practical researches.Traditional approaches are mainly morphology-based and pigment analysisdependent. Morphology-based methods are applicable for those microalgae withdistinct characteristics. However such an applicability is not always there,environments and the knowledge of identifiers influence these methods significantly.In addition, these methods are time-consuming, making their applicability in fields fora large number of samples impossible. Similarly, biochemical approaches are not alsoeffective everywhere, pigments are not typical always and physiologic andbiochemical indexes are not stable always. Therefore, traditional identificationapproaches should be supplemented with new methods like variation of carefullyselected genes.Systematics of marker genes is extremely applicable for the identification ofthose species with small sizes and remarkable geographic and cultural differences.DNA barcoding technology is experience-independent, time-saving and constantlyeffective for cells at different growing stages and from different environments. Universal DNA barcodes are not available for microalgae yet, however a group ofgenes are putatively applicable for microalgae. A widely adopted strategy at present isto amplify all possible marker genes from nano-marine microalgae, compare themwith the related genes in databank and identify them according to their similarity andphylogenetic relationship with the known species. Rich and desirable identificationshave been achieved in red alga, brown alga, diatom and dinoflagellate, unfortunatelysuccessful identifications are rare in other microalgae.In this study, a Nannochloropsis strain was identified as N. oceanica LAMB0002with morphological, biochemical and molecular approaches in combination.Morphological characteristics included those observed under light microscopy andtransmission electron microscope, biochemical properties included the compositionsof fatty acids and pigments and molecular approaches included the phylogeneticrelationships retrieved from18S rRNA gene and rbcL sequences. Identifying fourNannochloropsis strains from lab with the method, and the results are consistent withthe original information, which fully verify the identification method can meetaccurate identification for Nannochloropsis strains.Morphological and biochemical approaches in this study cannot identify theinterspecific differences in Nannochloropsis, so we modified the established methodby wiping off these approaches, using light microscope observation for primaryidentification and then dominating approaches with phylogenetic analysis. As wasexpected, we identified two unknown strains into N. oceanic and N.gaditana, whichfurther proved that the method is perfectly appropriate for identification ofNannochloropsis strains. We identified two Nannochloropsis strains into species withonly18S rRNA gene sequence, so we applied this simplified method to identifynano-marine microalgae. Using light microscope observation for primaryidentification and then dominating approaches with phylogenetic analysis, wesuccessifully identified9nano-marine microalgae strains,5strains in familypavlovaceae,3in group chromulinals and1in family prorocentraceae.In this study, we got an accurate identification method starting fromNannochloropsis, then the method was modified and applied on Nannochloropsis strains from outdoor sampling. At last, we identify nano-marine microalgae strainsrapid and accurate with the method, results indicated this method works in somenano-marine microalgae strains.