Development Of Applicable Methodology For Of The Identification Of Marine Microalgae

Marine microalgae are a group of primitive plants responsible for the primaryproduction in marine ecosystems. These important marine organisms survive everyparts of ocean. Marine microalgae are diverse in species and tremendous in biomassand speed of propagation. Many species of microalgae are highly valuable ineconomics and important for matter and energy cycles.The precise identification of marine microalgae is fundamental to variousstudies. Since the19th century, the identification of microalgae has experienced theevolvement from the morphology based approaches to physiological and biochemicalproperties dependent apprioaches and currently molecular biological approaches,each have limitations. Classification of marine microalgae has not yet reached thelevel in higher plants and animals, such as reproductive isolation and reproductionpatterns have not been applied in this area.This study investigated the molecular biological and morphological methods formicroalgal taxonom, then explored the application of reproduction pattern formicroalgal classification:1. In this study, molecular and morphological information of preserved ofmarine microalgae samples were linked together through single cells. Single-cellPCR was adopted to retrieve18S rRNA gene sequence from the genomes of40microplanktons (>20micrometers in length) collected in South China Sea. Thesesequences were linked with the morphological characteristics of species. It was foundthat the isolated microplankton (mainly microalgae) can be identified to genus with amolecular biological method without culturing.2. In this study, Nannochloropsis oceanica S. Suda et Miyashita wasinvetigated as a model for raising identification to species level with molecular biological methods. Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae. The species of Nannochloropsis are finerotifer feed and rich in eicosapentaenoic acid (EPA). The species in this genus areusually2-5μm in size and are morphologically similar each other, making theiridentification difficult. The18S rRNA gene, rbcL gene and the ITS region ofribosomal RNA transcription unit were used to the identification of Nannochloropsis.A monoclone of Nannochloropsis was obtained with its DNA extracted. The18Sribosomal RNA gene, ITS region of ribosomal RNA transcription unit and rbcL genewere amplified from the DNA extracted. The phylogenetic analysis was carried out byconstructing the neighbor-joining trees with Tamura-Nei distances. It was found thatthe monoclone is N. oceanica.18S rRNA gene, rbcL gene and ITS region sequence incombination can successfully identify Nannocloropsis to species.3. The traditional classification of microalgae regards reproduction little.However reproduction which affects the stability of species is an important stage inthe microalgal life history, therefore being the important basis for classification. Inthis study, the genome of N. oceanica was sequenced with the next-generationIllumina GA sequencing technologies, reveling N. oceanica a premeiotic or ameioticalga. The genome was~30Mb in size, which contained11,129protein-encodinggenes. Less frequent polymorphic nucleotides (one in22.06kb) and the obviousdeviation from1:1(major: minor, minor≥10) expectation indicated the nuclearmonoploidy of N. oceanica. The lack of the majority of meiosis-specific proteinsimplied the asexual reproduction of this alga.4. Sexual reproduction patterns of diatoms are their evolutionary characteristics,which can serve as the extra morphological characterisrtics for their identification. Inorder to achieve a perfect identification of marine microalgae, the sexual reproductionpattern of Phaeodactylum tricornutum was observed in this study.P. tricornutum has been intensively studied in past a few decades; however, itsnuclear transition has never been described, and accordingly, its sexual reproductionremained a puzzle. In this study, the nuclear movement among P. tricornutum cellswas observed. It was found that two cells can conjugate sometime with the nucleus of one cell moved into the other. The conjugation started when two cells jointed at theirapices and followed by immediate swinging and aligning. The cell pairs conjugatedfor nuclear movement were different from the cell pairs formed during mitosis inhypovalve thickness and cellular arrangement.Our observation matched the second pattern proposed by Hustedt, in whichone cell was released from two attached cells; paired cells can only be distinguishedby behavior. Our findings indicated the existence of sexual reproduction in P.tricornutum. This finding is conducive to understand the life cycle of P. tricornutumand its evolutionary position.