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Method for determining size of single-cell eukaryotic algae genome

A measurement method and genome technology, applied in the field of microalgae species, can solve the problems of reducing the fluorescence intensity of PI and affecting the accuracy of genome measurement by flow cytometry, achieving high operability and accuracy, and improving the effect of staining

Active Publication Date: 2019-02-01
JINAN UNIVERSITY
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Problems solved by technology

At present, there are few studies on the genome evaluation of unicellular eukaryotic microalgae species, because their cells are small and usually contain thicker cell walls, coupled with the presence of a large number of pigments and secondary metabolites in the cells, which will reduce the fluorescence of PI strength
In addition, due to the large evolutionary scale between different algae species, the genome size of species in the same family may vary greatly. Therefore, the selection of internal reference species seriously affects the accuracy of genome size determination by flow cytometry. We need to Further clarification of methods suitable for genome determination of unicellular eukaryotic algae

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  • Method for determining size of single-cell eukaryotic algae genome
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  • Method for determining size of single-cell eukaryotic algae genome

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Embodiment 1

[0046] Taking two unicellular eukaryotic microalgae with unknown genomes, E.cf.polyphem and V.stellata as examples, and Chlamydomonas reinhardtii (C.reinhardtii) as examples The first internal reference, N.oculata and M.salina were the second internal reference, and the genomes of the three known species were mutually calibrated. 5 microalgae cultures (about 50mL, 1×10 5 cells / mL) after centrifugation, add 50mL methanol, magnetically stir, 1-2h each time, centrifuge at 3000rpm to remove the supernatant, repeat the extraction 3-5 times until the cells become white; add 5mL Lysisbuffer LB01 to re- Suspend the cells, process and lyse the nuclei for 0.5h, obtain the cell nucleus suspension, and keep it on ice for later use; take 1mL of the cell nucleus suspension, add 50μL PI (1mg / mL), 50μL RNase (1mg / mL) and 5μL β-mercaptoethanol, mix well Stain in the dark for 0.5 h at 4°C; take 300 μL of the stained cell suspension, add it to a flow-type special round-bottom test tube (Fluores...

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Abstract

The invention discloses a method for determining size of single-cell eukaryotic algae genome. The method is based on the characteristics of single-cell eukaryotic algae, optimizes an extraction and staining method of the nucleus, and obtains the more accurate flow cytometry analysis result, combined with a high-throughput second-generation illumina sequencing technology, the genome size of algae cells is comprehensively evaluated, and the characteristics of the genome can be clarified. The technical method optimizes the flow cytometry analysis of traditional plants, removes the interference ofalgal cytochrome and cell wall by using methanol, greatly improves the dyeing effect of PI, and improves the accuracy of flow cytometry analysis, and combined with a current leading high-flux sequencing technology, the genome structure characteristics of the to-be-tested species are comprehensively analyzed, which can provide a theoretical basis for a genetic structure research of subsequent species and an assembly strategy of whole genome sequencing.

Description

technical field [0001] The invention relates to a method for measuring the genome size of single-cell eukaryotic algae, which is used to jointly analyze the genome size of the algae by flow cytometry analysis and next-generation sequencing technology, and particularly relates to microalgae species. Background technique [0002] In order to determine the complexity of an unknown species, it is first necessary to understand the characteristics of the genome from the genome level, and quickly obtain the genome size of the unknown species. Usually, flow cytometry (FCM) is used, and the external standard method or internal standard method is used to perform fluorescence staining of the nuclei with propidium iodide (PI), and then the flow cytometer is used for determination. FCM is currently one of the most commonly used methods for estimating the genome size of species. Compared with earlier methods such as pulsed field gel electrophoresis, it has the advantages of simple operati...

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122
Inventor 张成武黄罗冬高保燕
Owner JINAN UNIVERSITY
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