Method for inducing mutations in algae and method for evaluating neutron irradiation conditions

Abstract

To provide a method for inducing mutations in algae and a method for evaluating neutron irradiation conditions.SOLUTION: A method for inducing mutations in algae includes irradiating the algae present in water with neutron beams, where the absorbed dose of the neutron beams by the algae is within the range of 0.1 to 100 Gy.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to methods for inducing mutations in algae and methods for evaluating neutron irradiation conditions. [Background technology]

[0002] To date, several methods have been used to introduce mutations into laboratory organisms and organisms related to agriculture and fisheries.

[0003] To create mutants using recombinant gene technology and genome editing technology, a huge amount of genomic information on the target organism is required, and it is necessary to identify the genes that are directly linked to the desired phenotypic change. In addition, to modify or edit genes, it is necessary to introduce a vector (nucleic acid) or a DNA cleavage enzyme (nucleic acid and / or protein) that acts as a transmitter of foreign genes from outside the cell, so there is a limitation that it cannot be immediately applied to all organisms (Non-Patent Document 1). In particular, the availability of genome information and gene modification technology is limited for algae.

[0004] The method of inducing mutations using light energy or electromagnetic energy of electromagnetic waves including ultraviolet light is limited in its effect of causing ionization and excitation of biomolecules, so the scope of mutation induction is limited. For example, in the case of mutation induction by ultraviolet light irradiation, the double bond between the thymine and cytosine bases in DNA is used to absorb ultraviolet light, and when the adjacent bases are thymine or cytosine, a covalent bond is formed between the two bases to induce a mutation. Therefore, the applicable base sequences and applications are limited (Non-Patent Document 2).

[0005] Mutation induction by the introduction of chemical substances such as ethyl methanesulfonate (EMS) induces mutations by replacing the bonds of DNA base pairs, so it depends on the gene sequence and does not necessarily induce the desired mutation in all gene sequences (Non-Patent Document 3). In addition, there are problems associated with the handling of carcinogenic chemical substances.

[0006] The method of irradiating low LET rays such as gamma rays or X-rays causes mutations through ionization or excitation of water molecules, so that applicable cells and irradiation conditions are limited. On the other hand, when using high LET rays such as proton beams and heavy particle beams, the particles directly act on biomolecules to cause ionization or excitation, but in the case of subjects (e.g., microalgae) that require mutagenesis while being cultured in liquid culture medium or water, the particles have low permeability to the culture medium or water, making it difficult to irradiate uniformly to the inside of the target organism (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jinek M, et al:A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337:816-821, 2012 [Non-Patent Document 2] Ikehara and Ono, The mechanisms of UV mutagenesis. Journal of radiation research, 2011;52(2):115-25. [Non-Patent Document 3] Sega, A review of the genetic effects of ethyl methanesulfonate. Mutation Research, 1984;134(2-3):113-42. [Non-Patent Document 4] Sage and Shikazono. Radiation-induced clustered DNA lesions: Repair and mutagenesis. Free radical biology and medicine. 2017;107:125-135. Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for mutagenesis methods that can generate algae with useful traits.

[0009] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for inducing mutations in algae and a method for evaluating neutron beam irradiation conditions. [Means for solving the problem]

[0010] A method for inducing mutation in algae in one embodiment of the present disclosure includes irradiating the algae present in water with neutron beams, wherein the absorbed dose of the neutron beams by the algae is within a range of 0.1 to 100 Gy.

[0011] In another aspect of the present disclosure, a method for evaluating neutron beam irradiation conditions for inducing mutation in algae using neutron beams includes: (a1) dividing algae in water into a plurality of irradiation condition groups and irradiating them with neutron beams; (a2) culturing each of the plurality of irradiation condition groups after the irradiation or their progeny as a plurality of single individual cultures isolated into individual individuals; and (a3) ​​comparing the coefficient of variation of cell proliferation amount obtained from the plurality of single individual cultures between the plurality of irradiation condition groups; or includes: (b1) dividing algae in water into a plurality of irradiation condition groups and irradiating them with neutron beams; (b2) culturing the plurality of irradiation condition groups after the irradiation or their progeny on a solid medium containing uracil and 5-fluoroorotic acid; and (b3) comparing the number of colonies that appear between the plurality of irradiation condition groups. Effect of the Invention

[0012] According to the present disclosure, it is possible to provide a method for inducing mutations in algae and a method for evaluating neutron beam irradiation conditions. [Brief description of the drawings]

[0013] [Figure 1]This shows the relationship between the absorbed dose (Gy) when the red alga Schizon was irradiated with high-energy neutron rays and the number of colonies that appeared on an agar medium containing 5-FOA and uracil. [Diagram 2] This shows the relationship between the absorbed dose (Gy) when the red alga Schizon was irradiated with thermal neutrons and the number of colonies that appeared on an agar medium containing 5-FOA and uracil. [Diagram 3] 1 shows the positions of mutations in the URA gene after high-energy neutron irradiation of the red alga Schizon. [Figure 4] This shows the mutation pattern in the URA gene of the red alga Schizon after high-energy neutron irradiation. [Figure 5A] Growth (OD860) curves from individual cells of Euglena irradiated with neutron beams are shown. The Euglena cultures corresponding to the boxed points were used for the analyses in Figures 5B and 5C. [Figure 5B] The coefficient of variation of OD860 of Euglena clonal lines irradiated with high-energy neutrons (black) or thermal neutrons (gray) is plotted against the radiation dose (Gy). [Figure 5C] The average OD860 values ​​of Euglena clonal lines irradiated with high-energy neutrons (black) or thermal neutrons (gray) are plotted against the radiation dose (Gy). [Figure 6A] This figure shows the absorbed dose (Gy) and proliferation (relative value of OD750 with no irradiation set as 1) of red alga Schizon irradiated with high-energy neutron beams for each current value of the particle accelerator. ▲ indicates irradiation at 10 μA, ■ indicates 65 μA, and ● indicates 120 μA. [Figure 6B] This figure shows the absorbed dose (Gy) and growth (relative OD750 value, with no irradiation taken as 1) of the red alga Schizon irradiated with high-energy neutron rays, across all current value conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0015] <Method for inducing mutations in algae>

[0016] Although neutron beams are highly penetrating, little research has been done on what kind of mutations can be induced under what conditions, including the dose, duration, and cycle of irradiation, etc. In particular, if it were possible to evaluate and clarify the neutron beam irradiation conditions that can induce useful algal traits by irradiating algal cells present in a culture solution with neutron beams, and if it were possible to efficiently introduce mutations into algae using such conditions, it could potentially open up a new field in the creation of algae with useful traits, which is still largely unexplored.

[0017] In one embodiment, there is provided a method for inducing mutation in algae, comprising irradiating algae present in water with neutron beams, wherein the absorbed dose of the neutron beams by the algae is within a range of 0.1 to 100 Gy. The absorbed dose referred to in this disclosure does not refer to the amount of Gy received by an individual algae, but rather to the amount of Gy received by the algae present in water, i.e., an aqueous suspension of algae.

[0018] The type of algae in the present disclosure is not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae. The algae may be, for example, any of the following classifications: Aurantiochytrium, Chlamydomonas, Chlorella, Schizosaccharomyces, Spirulina, Botryococcus, Euglena, Cyanobacteria, Diatoms, Xanthophytes, Dinoflagellates, and Seaweeds. Schizosaccharomyces and Euglena are particularly suitable examples. The algae to be irradiated with neutron rays may be a single species of algae or a population containing multiple species of algae. A genetically homogeneous population of algae (pure line) of the same species may be used, or a population of algae having genetic variation may be used. As the algae, a line isolated from the wild may be used, or a mutant strain generated by mutation from such a line may be used for further mutation introduction. A line created by genome editing, transduction, transformation, or the like may also be used.

[0019] In the embodiment, the term "red algae Schizon" includes at least algae belonging to the genus Cyanidioschyzon. Examples of algae belonging to Cyanidioschyzon include Cyanidioschyzon merolae.

[0020] In the embodiment, "Euglena" includes at least algae belonging to the genus Euglena. Examples of Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, and Euglena viridis.

[0021] Mutation refers to a qualitative or quantitative change in the genetic material of a target organism (including, for example, a change in base sequence and a change in the number of chromosomes), but it is preferable that the induced mutation is a change that occurs in DNA. The DNA in which the change occurs is not limited, but may be, for example, genomic DNA. The type of change induced in genomic DNA by mutation is not particularly limited, but may be a single base deletion, insertion, or substitution, a two-base deletion, or a substitution from three bases to one base, etc. of genomic DNA. A trait change caused by a deletion, insertion, or substitution of a single base is particularly preferable because it is a trait change that minimizes genome modification. Alternatively, the induced mutation may be a mutation or rearrangement of a larger genome sequence than these, for example, a deletion, insertion, or substitution of several or more bases, or a chromosome rearrangement. The chromosome rearrangement may be a deletion, inversion, duplication, translocation, aneuploidization, or polyploidization. The mutation of genomic DNA includes any mutation and rearrangement pattern known by a person skilled in the art, in addition to those specifically exemplified above.

[0022] The mutagenesis method of the present disclosure induces mutations in the target algae. Although the mutations induced by such a method may occur only temporarily and may be subsequently repaired by an intracellular repair mechanism, the mutations are preferably maintained in the individual in an unrepaired state, and more preferably, at least a portion of the mutations are genetically inherited to the next generation.

[0023] The algae of the embodiment are present in water. Here, "in water" may mean in pure water, tap water, river water, lake water, or seawater, or in an aqueous medium for culturing the algae. In other words, the term "in water" broadly refers to a suspension of algae in water, and does not exclude the possibility that other substances in addition to the algae are dissolved or suspended in the water. The aqueous medium may be a medium for culturing algae known by those skilled in the art, for example, a medium containing nutrients, carbon sources, rare metals, etc. Specific examples of the aqueous medium include Koren-Hutner (KH) medium, Cramer-Myers (CM) medium, and mixtures thereof.

[0024] The composition of KH medium is as shown in the table below. The remainder is water. When preparing the medium, the pH is usually adjusted to 3.5 with an aqueous NaOH solution, and the medium can be sterilized by autoclaving. [Table 1]

[0025] The composition of CM medium is as shown in the table below. The remainder is water. When preparing the medium, the pH is usually adjusted to 3.5 with sulfuric acid, and the medium can be sterilized by autoclaving. [Table 2]

[0026] In embodiments, the algae present in the water may be, for example, algae suspended in water or a medium. The algae present in the water may be stirred continuously, periodically, or irregularly to maintain uniformity.

[0027] The type of neutron beam in the embodiment is not limited, but preferably, thermal neutron beams can be used. As will be understood by those skilled in the art, thermal neutron beams refer to neutron beams that contain, as a majority component (i.e., more than half) neutrons that are in thermal equilibrium with a substance in the environment or have low energy close to the energy of neutrons in such thermal equilibrium, and typically contain, as a majority component, neutrons with energies of 4 to 100 millielectron volts. Thermal neutron beams are preferred because they are considered to be suitable for steadily obtaining target mutations while avoiding the induction of excessive mutations.

[0028] Alternatively, as the neutron beam in the embodiment, a high-energy neutron beam may be preferably used. As will be understood by those skilled in the art, a high-energy neutron beam may also be called a fast neutron beam, and is a neutron beam that typically contains a large number of neutrons with an energy of 0.1 megaelectron volts or more. It has been observed that the efficiency of high-energy neutron beams in generating mutations is higher than that of thermal neutron beams. Due to the difference in energy of neutron beams, such as thermal neutron beams and high-energy neutron beams, the probability of electrons or protons being released from the collided water molecules may differ, and therefore the biological aspects of mutation generation may differ even with the same absorbed dose value.

[0029] The method of generating neutron beams in the embodiments is not limited, but as known to those skilled in the art, neutron beams can be generated by, for example, irradiating charged particles accelerated by a particle accelerator onto a neutron generating target, such as beryllium, tungsten, lithium, lead, or mercury.

[0030] In an embodiment, the neutron irradiation conditions can be adjusted in different parts of the irradiation device. For example, the neutron beam intensity can be changed by changing the current value of the accelerator. For example, the neutron beam intensity can be adjusted by setting the current value to 10 μA, 65 μA, 100 μA, or 120 μA.

[0031] Alternatively, the energy of the neutron beam can be adjusted by using a moderator between the neutron generation target and the algae culture solution to be irradiated. For example, by changing the type and thickness of the moderator, it is possible to generate a neutron beam mainly composed of high-energy neutrons or thermal neutrons. Examples of moderators that can be used include water, heavy water, polyethylene, and methane.

[0032] Alternatively, the absorbed dose can be adjusted by varying the distance between the neutron generating target and the algal culture to be irradiated.

[0033] It will be understood by those skilled in the art that by modifying or combining the various irradiation conditions described above, it is possible to generate neutron beam types having the required characteristic values ​​such as energy and intensity.

[0034] In an embodiment, the algae can be irradiated with neutrons after the algae have been cultured to the logarithmic growth phase and then transferred to a container such as a centrifuge tube. The material of the container such as a centrifuge tube can be, for example, glass, polypropylene, polycarbonate, polyethylene, polystyrene, polyethylene terephthalate, or a combination thereof, but is not limited thereto.

[0035] The absorbed dose of the irradiated neutron beam may be, for example, within the range of 0.1 Gy to 100 Gy (0.1 Gy or more and 100 Gy or less), but is preferably within the range of 0.2 Gy to 30 Gy (0.2 Gy or more and 30 Gy or less), and more preferably 20 Gy or less, for example, within the range of 0.2 Gy to 20 Gy (0.2 Gy or more and 20 Gy or less). It will be understood by those skilled in the art that the absorbed dose of the irradiated neutron beam can be adjusted to a desired value in both thermal neutron beams and high-energy neutron beams by adjusting one or more of the irradiation time, the distance between the neutron generation target and the algae culture solution, and the beam current value of the accelerator.

[0036] Irradiating algae with neutrons at an absorbed dose within this range enables efficient production of algae strains with useful traits (e.g., high lipid accumulation strains). Irradiating algae with neutrons at these preferred absorbed doses sufficiently ensures the occurrence of targeted mutations in the algae while avoiding unnecessary or excessive mutations, and thus avoids impairing the survival and health of the target algae, and therefore prevents, for example, a decrease in the growth rate of the algae even after mutation.

[0037] <How to create algae strains>

[0038] In one aspect, a method is provided for producing an algal strain with increased lipid accumulation, comprising mutagenizing a population of algae using the methods described in the Methods for Mutagenizing Algae section, isolating an algal strain from the mutagenized population of algae, measuring lipid accumulation of the algal strain, and identifying an algal strain with increased lipid accumulation based on the measured lipid accumulation.

[0039] In the embodiment methods of producing algal strains, populations of algae are mutagenized using the methods described in the section Methods of Mutagenizing Algae, and therefore the description of each element provided with respect to the embodiment methods of mutagenizing algae may also be applied to the embodiment methods of producing algal strains, and vice versa.

[0040] In the embodiment of the method for producing an algal strain, isolating an algal strain from a population of mutagenized algae can be performed by isolating a single algae individual after irradiating the algae with neutron radiation. For small algae, such as unicellular algae, isolating the single individual can be performed using a cell sorter. The isolated individual can be stored, for example, in a well of a cell culture plate containing medium and used for further experiments.

[0041] In the method for producing an algal strain according to the embodiment, the amount of lipid accumulation in the algal strain can be measured by using a dye that stains lipids, which is known to those skilled in the art. The dye may be a fluorescent dye. An example of the fluorescent dye is a BODIPY dye, more specifically, a BODIPY dye. 505 / 515 The lipid content of algal cells can be measured by measuring the fluorescence value of the algal cells stained with a fluorescent dye using a cell sorter. In other words, isolating an algal strain and measuring the lipid accumulation amount of the algal strain can be performed in different orders or in parallel.

[0042] In the method for producing an algae strain according to the embodiment, the lipid accumulation of the algae strain can be measured by extracting lipids from a culture of the isolated algae strain. The algae can be cultured using a medium for algae known by those skilled in the art. The lipids can also be extracted by a method known by those skilled in the art. For example, the lipids can be extracted using an organic solvent such as hexane after crushing the algae, or the lipids can be extracted using a lipid extraction method by two-phase partitioning such as the Bligh & Dyer method. The amount of extracted lipids can be quantified by evaporating the solvent and then measuring the weight. Alternatively, the amount of lipids can be quantified by a method known by those skilled in the art, such as gas chromatography, liquid chromatography, mass spectrometry, or a combination of these methods.

[0043] In embodiments, algae strains that have increased lipid accumulation are identified based on the measured lipid accumulation, for example, algae strains that have increased lipid accumulation compared to before mutagenesis can be identified.

[0044] In the embodiment of the method for producing an algal strain, measuring the lipid accumulation amount of the algal strain may include measuring the lipid accumulation amount after subjecting the algal strain to anaerobically treatment. Algae such as Euglena stop growing under nitrogen-deficient conditions and accumulate polysaccharides in the form of paramylon. Furthermore, when there is a shortage of oxygen in the surroundings, oxygen-sensitive pyruvate dehydrogenase (PNO) becomes active and metabolizes pyruvic acid that is metabolized anaerobically and accumulates, which is then synthesized into lipids in the form of wax esters via fatty acid synthesis. Therefore, by measuring the lipid accumulation amount after subjecting the algal strain to anaerobically treatment, it may be possible to appropriately evaluate the lipid production ability of the algal strain.

[0045] The anaerobic treatment of algae can be carried out by nitrogen substitution or the like using an anaerobic culture method for algae known to those skilled in the art.

[0046] In the measurement of lipid accumulation amount of an algae strain in the method for producing an algae strain according to the embodiment, the measured lipid accumulation amount can be corrected by multiplying it by the ratio of the cell weight after the anaerobic treatment to the cell weight before the anaerobic treatment (i.e., (cell weight after anaerobic treatment) / (cell weight before anaerobic treatment)). The metabolic pathway of wax ester synthesis is a reaction in which a small amount of carbon dioxide is released during the process, and it is known that amino acids and organic acids are also released outside the cells. Therefore, the weight of each cell decreases with lipid accumulation. For this reason, if the lipid content as weight is calculated by calculating the amount of lipid relative to the measured dry algae powder, the content value after anaerobic treatment may be unduly high when simply comparing before and after anaerobic treatment. For this reason, it is easy to understand in terms of practical productivity to correct and evaluate the lipid content based on the cell weight before anaerobic treatment. For this reason, for example, the cell concentration (g / L) before and after anaerobic treatment is measured, and the calculation is performed using the following formula. Fat content (corrected) = Fat content (uncorrected) × correction factor Where: Correction factor = cell weight after anaerobic treatment / cell weight before anaerobic treatment The cell weight may be a dry weight, and more preferably, the cell weight may be a dry weight obtained by freeze-drying or drying in an oven heated to 100° C. or higher.

[0047] <Method of evaluating neutron irradiation conditions>

[0048] In one aspect, a method for evaluating neutron irradiation conditions for neutron-beam-based mutagenesis of algae is provided, the method comprising: (a1) dividing underwater algae into a plurality of irradiation condition groups and irradiating them with neutron beams; (a2) culturing each of the plurality of irradiation condition groups after the irradiation or their progeny as a plurality of single-individual cultures isolated into individual individuals; and (a3) ​​comparing the coefficient of variation of the cell proliferation amount obtained from the plurality of single-individual cultures between the plurality of irradiation condition groups, or The method includes (b1) dividing underwater algae into a plurality of irradiation condition groups and irradiating them with neutron beams, (b2) culturing the plurality of irradiation condition groups after the irradiation or their progeny on a solid medium containing uracil and 5-fluoroorotic acid, and (b3) comparing the number of colonies that appear among the plurality of irradiation condition groups.

[0049] In the method for evaluating neutron irradiation conditions of the embodiment, the methodology described in the section <Method for inducing mutation in algae> may be used to divide algae in water into a plurality of groups of irradiation conditions, irradiate them with neutrons, and culture the algae. Therefore, the description of each element provided for the embodiment of the method for inducing mutation in algae may also be applied to the embodiment of the method for evaluating neutron irradiation conditions, and vice versa. For example, the plurality of groups of irradiation conditions may be groups of irradiation conditions within an absorbed dose range of 0.1 to 100 Gy, but are not necessarily limited thereto, and the evaluation method may also be performed with different absorbed dose ranges or a wider absorbed dose range.

[0050] In the method for evaluating neutron beam irradiation conditions according to the embodiment, neutron beams are irradiated to algae in water under a plurality of irradiation condition groups, where the plurality of irradiation conditions may include neutron beam energy, intensity, absorbed dose, and combinations thereof.

[0051] The method for evaluating neutron irradiation conditions of the embodiment may include culturing each of the plurality of irradiation condition groups or their progeny as a plurality of single-individual cultures isolated into individual individuals. Here, the single-individual cultures isolated into individual individuals refer to cultures prepared by isolating a plurality of individuals from algae irradiated by the plurality of irradiation condition groups and starting a culture from each individual. Isolation of the unicellular algae individuals may be performed by flow cytometry or the method described in the section <Method of producing an algae strain>, or more simply, by sucking up the individuals using a pipette or the like under a microscope. The number of the plurality of single-individual cultures may be, for example, 10 or more, 20 or more, 30 or more, or more for each irradiation condition group.

[0052] The method for evaluating neutron beam irradiation conditions according to the embodiment may include comparing the coefficient of variation of the cell proliferation amount obtained from the plurality of single-individual cultures among the plurality of irradiation condition groups. The coefficient of variation of the cell proliferation amount can be calculated by calculating the average value and variance of the cell proliferation amount, which can be calculated from the absorbance (e.g., OD860) according to the type of algae, from the plurality of single-individual cultures irradiated under specific irradiation conditions, and applying the following formula: Coefficient of variation = standard deviation / mean

[0053] The coefficient of variation is a value obtained by standardizing the standard deviation in each irradiation condition group by the average value, and is therefore considered to be a suitable value for comparing the variation in cell (progeny population) proliferation rate specific to each irradiation condition group at the time of isolation while minimizing the random effects of each irradiation experiment. It was found that algal populations mutated under different neutron irradiation conditions may lack visible differences in the short-term average proliferation rate of the entire population, even if the mutation rates of individual genes differ from population to population. In other words, the average proliferation rate of the entire population may be a poor indicator for evaluating the neutron irradiation conditions. In contrast, the method of the present embodiment allows for highly sensitive evaluation of the specific effects of different neutron irradiation conditions on algal proliferation.

[0054] Alternatively, a method for evaluating neutron beam irradiation conditions of an embodiment may include culturing a plurality of irradiation condition groups or their progeny after irradiation on a solid medium containing uracil and 5-fluoroorotic acid (5-FOA) and comparing the number of colonies that appear among the plurality of irradiation condition groups.

[0055] Orotidine-5'-phosphate decarboxylase, which is found in wild-type algae, is an enzyme that produces uracil nucleotides from orotidine 5'-phosphate, and is referred to as URA in this disclosure. When 5-FOA is used as a substrate, URA produces the cytotoxic 5-fluorouracil (5-FU), which causes the death of wild-type strains. On the other hand, algae that have become uracil-requiring due to a mutation in URA can grow by culturing them in a medium containing uracil, and do not produce cytotoxicity even in the presence of 5-FOA. Therefore, algae that produce colonies on a solid medium (e.g., an agar medium) containing 5-FOA and uracil can be said to be a strain in which a mutation has been caused in the URA gene. Therefore, by comparing the number of colonies that appear among the multiple irradiation condition groups, the frequency of occurrence of mutations can be compared. The method of this embodiment can be used not only to assay the mutation frequency for the URA gene, but also to estimate the rate at which mutations occur in any target gene in the genome. In addition, by sequencing the URA genes of the algae that have formed colonies, it will be possible to investigate the pattern of mutations that occur for each group of irradiation conditions (for example, whether single-base mutations or two or more base mutations are more likely to occur, or whether deletions, insertions, or substitutions are more likely to occur).

[0056] In the embodiment, the solid medium containing uracil and 5-fluoroorotic acid is not particularly limited as long as it is a solid medium suitable for growing algae and contains added uracil and 5-fluoroorotic acid, and can be prepared using a liquid medium for algae known to those skilled in the art and agar, etc.

[0057] The number of colonies appearing on a solid medium can be counted by methods known to those skilled in the art, for example, by visual inspection.

[0058] According to the method for evaluating neutron beam irradiation conditions of the embodiment, the suitable neutron beam type and absorbed dose when irradiating algae with neutron beams to induce mutations are derived from the relationship between the absorbed dose and the algae growth or colony number. Since the relationship is absorbed dose = intensity x time, it is possible to produce a similar effect by increasing the intensity and shortening the irradiation time, or by irradiating algae with low intensity for a long time. EXAMPLES

[0059] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0060] (Example 1) Mutation induction in red alga Schizon by high-energy neutron radiation

[0061] An experiment was conducted to provide a technique for producing algae with useful characteristics by inducing genetic mutations in algae by irradiating them with neutron beams. Neutron-irradiated red algae Schizon algae cells (approximately 1x10^8 cells) were spread on an agar medium containing 5-FOA and uracil, and the number of colonies that appeared was detected. When wild-type orotidine-5'-phosphate decarboxylase (URA) catalyzes a reaction using 5-FOA as a substrate, it produces a cytotoxin and kills the host cell. Therefore, the algae that appeared on the agar medium containing 5-FOA and uracil can be said to be a strain in which a mutation has been induced in the URA gene. In Example 1, high-energy neutron beams were used, and in Example 2 described below, thermal neutron beams were used, and the results were compared by conducting experiments within the same absorbed dose range. In the case of high-energy neutron beams in Example 1, the absorbed dose absorbed by the algae culture solution was changed by changing the accelerator beam current value, irradiation time, and distance from the neutron generation target.

[0062] Materials and Methods

[0063] Unicellular red algae were cultured to logarithmic growth phase (40°C, 2% CO2 (v / v), approximately 50 μmol m -2 s -1), transferred to a 50 ml centrifuge tube, and irradiated with high-energy neutrons. More specifically, the accelerator beam current was set to 120 μA, the culture medium was placed 62 cm away from the neutron generation target, and irradiation was performed for approximately 15 minutes per 10 Gy to prepare irradiated algal cells with absorbed doses ranging from 10 Gy to 80 Gy. Note that after every 15 minutes of irradiation, the centrifuge tube was gently shaken up and down 2-3 times to maintain the homogeneity of the culture medium. Non-irradiated cells were cells that were irradiated under the same conditions, but without irradiation. The irradiated or non-irradiated cells were transferred to a vented-capped tissue culture flask (600 ml capacity) and stored in a room set at 25°C (approximately 5 umolμmol m -2 s -1 ) for 2 to 4 weeks, after which the number of cells per ml of culture medium was calculated using a hemocytometer.

[0064] MA2 solid medium (Bio-protocol 9(4): e3172) containing 5-FOA (final concentration 0.9 mg / ml) and uracil (final concentration 0.6 mg / ml) was prepared, and 1 x 10^8 cells were plated on each plate by the top starch method (see the same reference). Then, one AnaeroPouch (registered trademark) CO2 carbon dioxide culture pouch agent A-63 and two plates were placed in a pouch bag (for AnaeroPack (registered trademark)) W zipper small A-65, and incubated at 40°C (approximately 50 umol μmol m -2 s -1 ) for approximately one month, and the number of colonies formed was counted.

[0065] result

[0066] The relationship between absorbed dose and the number of colonies that appeared is shown in Figure 1. The number of colonies was high at approximately 10 Gy to approximately 30 Gy, making it clear that mutations were efficiently introduced under these conditions.

[0067] (Example 2) Mutation induction in red alga Schizon by thermal neutron radiation

[0068] The relationship between the radiation dose and the number of colonies that appeared was investigated using thermal neutrons. In the experiments using thermal neutrons, the distance from the neutron generation target and the accelerator beam current were kept constant, and the absorbed dose was changed by changing the radiation time.

[0069] Materials and Methods

[0070] Unicellular red algae were cultured to logarithmic growth phase (40°C, 2% CO2 (v / v), approximately 50 μmol m -2 s -1 ), then transferred to a 50 ml centrifuge tube and the algae were irradiated with thermal neutrons. More specifically, the accelerator beam current was set to 100 μA, the culture medium was placed 62.1 cm away from the neutron generation target, and irradiation was performed for 5 hours per 13 Gy to prepare irradiated algal cells with absorbed doses of 13 Gy or 26 Gy. During irradiation, the rotor with the algae attached was rotated at 2 rpm. During this time, the sample was not exposed to light, so non-irradiated cells were also rotated in the same manner in the dark. Irradiated or non-irradiated cells were transferred to a vented-capped tissue culture flask (600 ml capacity) and placed in an incubator set at 28°C (approximately 5 umolμmol m -2 s -1 The subsequent procedures were the same as in Example 1.

[0071] result

[0072] The relationship between the absorbed dose and the number of colonies that appeared is shown in Figure 2. The number of colonies was high with 13 Gy irradiation, making it clear that mutations were efficiently introduced under these conditions.

[0073] (Example 3) Identification of the mutation site and mutation pattern of red alga Schizon gracilis induced by high-energy neutron irradiation

[0074] The URA gene region of the 5-FOA-resistant strain obtained by high-energy neutron irradiation was amplified by PCR, and the nucleic acid sequence was confirmed by Sanger sequencing.

[0075] Materials and Methods

[0076] 1.5 ml of MA2 medium containing 5-FOA and uracil (final concentration of both: 0.5 mg / ml) was placed in each well of a 24-well culture plate, and the 5-FOA-resistant colonies obtained in the experiment in Fig. 1 were transferred to each well and cultured in liquid. The 24-well plate was enclosed in a pouch bag together with one pouch agent A-63 for carbon dioxide culture, and cultured at 40°C with approximately 50 umolμmol m -2 s -1 The cells were cultured at 4°C for 1 h. Genomic DNA was purified from the grown cells by phenol extraction and used as a template for PCR. If the A of the translation initiation codon ATG of the URA gene (called the URA5.3 gene in the case of Schizon) is set to +1 (+1 to +1392 is the ORF of URA5.3), the DNA fragment was amplified by PCR in a thermal cycler using PCR primers designed to amplify the fragment from +540 to +1685. After confirming by agarose electrophoresis that the DNA containing the URA5.3 gene sequence had been amplified as a single band, the PCR product was purified.

[0077] The sequence of the URA5.3 gene was obtained by Sanger sequencing using the purified PCR product as a template, and was compared with the wild-type URA5.3 gene sequence registered in the Schizon gene database (http: / / czon.jp / ) to analyze whether mutations had been introduced and, if any, the type of mutation.

[0078] result

[0079] Mutations were confirmed throughout the URA gene region (Fig. 3), indicating that the introduction of mutations by neutron irradiation is essentially random and does not depend on the base sequence. The most common mutation patterns were single-base deletions, insertions, and substitutions, with single-base substitutions accounting for the majority of mutations, and two-base deletions and three-base to one-base substitutions were also observed. It became clear that the majority of mutations were caused by base sequence changes of a small number of base units, rather than large-scale base changes in the genome sequence (Fig. 4).

[0080] (Example 4) Evaluation of neutron irradiation conditions for Euglena

[0081] The effects of neutron irradiation on Euglena were investigated. For each irradiation condition group, the variation in growth (coefficient of variation) between clone lines was examined, and a comparison was also made between high-energy neutrons and thermal neutrons.

[0082] Materials and Methods

[0083] 4-1. Preparation of Euglena cells for neutron irradiation

[0084] The eu029 strain was used as a wild-type Euglena gracilis strain. This strain was a strain that was independently subcultured and adapted from the IAM E-6 strain obtained from the Institute of Applied Microbiology (IAM) culture collection at the University of Tokyo, and is of the same origin as NIES-48, which is currently available at the National Institute for Environmental Studies. After pre-culturing the eu029 strain using KH medium, it was cultured for 4 days in 50 mL of CM medium in each of three 100 mL test tubes. At the time, the culture was performed under constant light conditions with a culture starting concentration of 0.25 (measured at λ = 860 nm using an absorption photometer, hereinafter referred to as OD860), a temperature of 26 to 29 °C, an aeration volume of 50 mL / min (5% CO2), and light irradiation PPFD of 100 μmol / m2s. The culture medium reached a concentration of about 2 (OD860) and was diluted with CM medium, 45 mL of which was sealed in a 50 mL centrifuge tube, and sent to a neutron irradiation facility.

[0085] 4-2.Irradiation

[0086] Neutron irradiation was carried out in the same manner as that used for the red alga Schizon in Example 1.

[0087] 4-3. Cell isolation from each population after irradiation and culture test

[0088] The samples were opened on the day they were returned after irradiation, or the next day after they were left at room temperature (approximately 26°C) overnight, and cells were isolated using a cell sorter (Beckman Coulter, MofloXDP). The conditions were as described in Scientific Reports 6.1 (2016): 1-8. The cell sorter was equipped with a 100 μm nozzle, and 180 μL of a 4:1 mixture of CM medium and KH medium was dispensed into each well of a 96-well flat-bottom cell culture plate for sorting, and then sorting was performed. From each irradiation condition group, one cell was randomly sorted into 30 wells, without any selection based on fluorescence value, etc. The plate from which the cells were sorted was placed on a culture shelf maintained at 26-29°C, and cultured under constant light conditions with a light irradiation PPFD of 100 μmol / m2s. After about one week, the absorbance (OD860) was measured every few days using an absorbance plate reader.

[0089] 4-4. Evaluation of the effect of each irradiation condition on the proliferation ability of individual cells

[0090] To evaluate the variation in growth rate affected by neutron irradiation within each irradiation condition group, the coefficient of variation of the culture medium concentration (OD860) formed by multiple single individual cultures isolated from each group on the 13th day of culture was calculated and a scatter plot was created. Data from wells in which no cell growth was observed on the 13th day was discarded, and the average OD860 value of wells in which growth was observed was calculated. Similarly, the standard deviation of OD860 of wells in which growth was observed was calculated. The coefficient of variation was calculated using the following formula. Coefficient of variation = standard deviation / mean

[0091] A scatter plot was created based on the coefficient of variation (vertical axis) for each irradiation condition group and the neutron beam exposure (horizontal axis).

[0092] The above coefficient of variation is a value obtained by normalizing the standard deviation in each irradiation condition group by the average value, and is therefore considered to be a suitable value for comparing the variation in cell (progeny population) proliferation rate specific to each irradiation condition group while minimizing the random effects of each irradiation experiment. In a situation where the evaluation criteria for the degree of phenotypic change in response to the introduction of mutations in Euglena are almost limited to lethality or a decrease / increase in proliferation rate, the above coefficient of variation is effective for evaluating the latter aspect.

[0093] 4-5. Confirmation of proliferation ability as a group After irradiation, 40 mL of KH medium was added to 10 mL of the culture medium (CM medium) of each group, and recovery culture was performed using a sterilized Erlenmeyer flask with a capacity of 100 mL. At that time, the culture was performed for 3 days under the conditions of room temperature 26-29 ° C, rotary shaking at 100 rpm on a rotary shaker, and constant light irradiation at 50 μmol / m2s. The concentration (OD860) of the obtained culture medium was then measured with an absorption photometer, diluted with KH medium so that the OD860 was about 0.1, and 1 mL of each irradiation condition group was dispensed into 3 wells of a 24-well flat-bottom plate. This well plate was placed on a culture shelf maintained at 26-29 ° C, and culture was performed under constant light conditions of light irradiation PPFD 100 μmol / m2s. The absorbance (OD860) was measured each day with an absorption plate reader.

[0094] result

[0095] The change in OD860 over time for the control group is shown in Figure 5A. It can be seen that the control group maintained genetic uniformity, and thus the variation in growth rate was relatively small. Figure 5B shows the results of evaluation based on the coefficient of variation of OD860 on the 13th day (boxed in Figure 5A). High-energy neutron irradiation (black) generally had a greater effect on growth than thermal neutron irradiation (gray) at the same Gy (Figure 5B). On the other hand, evaluation based on the average OD860 showed no significant difference between high-energy neutron irradiation and thermal neutron irradiation at the same Gy, nor was there any significant difference from the non-irradiated control group (Figure 5C). Although extremely strong irradiation that causes large-scale changes throughout the genome may result in a decrease in the average growth rate of the entire population in the short term, at irradiation levels that can induce mutations in individual genes, the short-term average growth rate was not seen to be a useful indicator.

[0096] (Example 5) Screening of Euglena mutants with high lipid content

[0097] We screened for lipid-rich mutants of Euglena. To evaluate changes in the amount of lipids accumulated in cells, we used the fluorescent dye BODIPY, which specifically stains neutral lipids, and separated cells with increased lipid accumulation based on the fluorescence intensity detected by a cell sorter.

[0098] Materials and Methods

[0099] 5-1. Euglena BODIPY 505 / 515 staining

[0100] BODIPY 505 / 515(TargetMol, T36958) was dissolved in dimethyl sulfoxide at 1 mM and stored at -20°C. The above solution was diluted with water to 10 μM immediately before use and used as a staining solution. For staining, 100 μL of staining solution was added to 100 μL of culture solution sample placed in a 1.5 mL tube, mixed well, and then left to stand in the dark for 5 minutes. After 5 minutes, the sample was centrifuged at 2,000 g for 5 seconds and the supernatant was removed. Then, 400 μL of water was added, and screening was performed using flow cytometry or a cell sorter.

[0101] 5-2. Primary screening of mutants

[0102] Mutant screening was performed according to the method described in Scientific Reports 6.1 (2016): 1-8. After irradiation, the population was cultured for recovery, and 100 μL of the culture medium was used to extract the lipid BODIPY. 505 / 515 The stained cells were sorted using a cell sorter (Beckman Coulter, MofloXDP) equipped with a 100 μm nozzle, using the FL1 (488 nm excitation, 529 ± 28 nm fluorescence) value as an index, and the cells showing high values, which were in the top 0.1%, were collected in a 1.5 mL tube containing 1 mL of KH medium. 6Approximately 1,000 cells were collected from the cells and grown in a 1.5 mL tube for one week. When the cells had grown sufficiently, they were added to 50 mL of KH medium and cultured in a 100 mL flask with shaking. The cells were cultured for one week at room temperature of 26-29°C, with a rotary shaker at 100 rpm and constant light of 50 μmol / m2s. The above series of mutant enrichment procedures were repeated again, and then a third sorting was performed using a cell sorter. In the third sorting, one cell with a high FL1 value was isolated in each well of a 96-well flat-bottom cell culture plate. Each well was pre-dispensed with 180 μL of a medium made by mixing CM medium and KH medium in a ratio of 4:1. From each group, the cells were sorted into half the wells (48 wells) of the 96-well plate. The plate from which the cells were sorted was placed on a culture shelf maintained at 26-29°C and cultured for two weeks under constant light conditions of PPFD 100 μmol / m2s.

[0103] 5-3. Secondary screening of mutants

[0104] After the cells had sufficiently expanded, 10 strains were randomly selected from each population, and 100 μL of the culture medium was used to infect BODIPY 505 / 515 The phenotype was confirmed by staining. 505 / 515 The cells were stained with , and flow cytometry was performed on the stained cells using a cell sorter (Beckman Coulter, MofloXDP) to measure the FL1 (488 nm excitation, 529 ± 28 nm fluorescence) value in each stained population. As a result, several lines that clearly showed high FL1 values ​​were selected from each irradiation condition population. The remaining culture medium of the selected lineages was added to 50 mL of KH medium, and each was cultured with shaking in a 100 mL flask. The culture was performed for one week under conditions of room temperature 26-29 °C, rotating at 100 rpm on a rotary shaker, and irradiating with constant light at 50 μmol / ms. Approximately 100 μL of the culture medium of the proliferated cells was used to incubate with BODIPY. 505 / 515The phenotype was confirmed again by staining. The same measurement was also performed on the wild-type strain (eu029) that was cultured in parallel under the same conditions. If any strains showed clearly higher FL1 values ​​than the wild-type strain, the strain showing the highest value from each irradiation condition group was established as a mutant strain and stored.

[0105] result

[0106] In screening of mutant strains, three mutant strains with the desired trait (high staining with BODIPY) were obtained under each of the high-energy neutron and thermal neutron irradiation conditions, confirming that useful mutants could be obtained with either irradiation method (Table 5 below).

[0107] (Example 6) Evaluation of the obtained lipid-rich mutant candidate strains

[0108] The lipid-rich mutant candidate strains obtained in Example 5 were evaluated based on the lipid content corrected by the ratio of the cell weight after anaerobic treatment to the cell weight before anaerobic treatment, as described herein.

[0109] Materials and Methods

[0110] 6-1.Culture operation

[0111] Euglena strains were precultured under the heterotrophic conditions shown in the table below. [Table 3]

[0112] Using the preculture medium, cells for phenotypic analysis were cultured under the conditions shown in the table below. The cell concentration (OD860) was measured each day using an absorption spectrophotometer and compared with that of the wild-type strain (eu029). [Table 4]

[0113] 6-2. Harvesting and sample preparation by anaerobic fermentation

[0114] 15 mL of each strain sample was collected in two 15 mL centrifuge tubes. The weight of the 15 mL centrifuge tube itself was measured in advance. One 15 mL centrifuge tube was centrifuged (2,000 g, 2 min), the supernatant was discarded, and the precipitated cells were frozen and stored at -20°C. The other centrifuge tube was stored stationary at 29°C for 1 day in the dark and subjected to anaerobic fermentation (anaerobic treatment). After the anaerobic treatment was completed, the tube was centrifuged (2,000 g, 2 min), the supernatant was discarded, and the precipitated cells were frozen and stored at -20°C. The frozen samples before and after anaerobic treatment were each dried overnight in a freeze dryer. The weight of each tube containing the dry cell powder was then measured, and the dry weight of the harvested cells was determined by subtracting the weight of the centrifuge tube itself.

[0115] 6-3.BODIPY 505 / 515 Phenotype confirmation by staining

[0116] After the main culture was completed, the remaining culture medium was used to culture BODIPY according to the method described in 5-1 of Example 5. 505 / 515 After staining, flow cytometry was performed using a cell sorter (Beckman Coulter, MofloXDP) to measure the FL1 (488 nm excitation, 529±28 nm fluorescence) value in each stained population.

[0117] 6-4. Quantification of lipid content

[0118] The above dry powder (about 50 mg) was weighed and placed in a glass vial. Then, 10 mL of normal hexane was added as an extraction medium, and the cells were disrupted for 1 minute using an ultrasonic disintegrator (Tommy, UD211) at a dial setting of 4. The extraction solution was then filtered through a glass filter (Whatman, GF / C). Furthermore, the residue on the glass filter was returned to the glass vial, 10 mL of normal hexane was added, and the cells were ultrasonically disintegrated under the same conditions, and the extraction solution was filtered and collected. The collected filtrate was collected in a ground eggplant flask whose dry weight had been measured, and the solvent was evaporated using a rotary evaporator. Furthermore, the lipids remaining in the ground eggplant flask were stored in an oven heated to 105 ° C for 1 hour, and the moisture was completely evaporated. The ground eggplant flask was cooled while reducing the pressure in a desiccator, and the weight was measured after it returned to near room temperature for about 1 hour. The weight of the extracted lipid was calculated from the weight difference of the ground eggplant flask before and after lipid extraction, and the lipid content was calculated by dividing the weight by the weight of the dry cell powder used. The reduction rate of the cell weight after anaerobic treatment compared to before anaerobic treatment was calculated, and the lipid content after anaerobic treatment was also calculated as follows, based on the estimated weight of the same number of cells before anaerobic treatment. Corrected lipid content = lipid content after anaerobic treatment × (cell weight after anaerobic treatment / cell weight before anaerobic treatment)

[0119] The lipid contents before anaerobic treatment and the corrected lipid contents after anaerobic treatment of the obtained mutants are shown in Table 5. It was confirmed that the mutant strains obtained by irradiation with thermal neutrons or high-energy neutrons were able to accumulate more lipids than the wild-type strain.

[0120] [Table 5]

[0121] (Reference Example) Further consideration of irradiation conditions

[0122] Finally, we investigated whether there is a difference in mutation induction between short-term irradiation with high-intensity neutron beams and long-term irradiation with low-intensity neutron beams (with the same absorbed dose), in other words, whether the cumulative absorbed dose determines the frequency of mutations. To clarify this, we analyzed the turbidity (= number of cells per unit volume) of the algae after culturing for several weeks after irradiation. This is because if mutations essential for cell growth are introduced, this will affect long-term cell accumulation.

[0123] Materials and Methods

[0124] In the high-energy neutron irradiation experiment of the red alga Schizon shown in Example 1, the beam current value of the accelerator was changed, i.e., the intensity of the neutron beam was changed, and neutral beam irradiation was performed. After several weeks of neutron beam irradiation, the OD750 of the culture solution was measured using a spectrophotometer.

[0125] result

[0126] The relative turbidity values ​​of the culture solution under each condition, with the turbidity value of unirradiated algae set at 1, are shown in Figures 6A and 6B. Figure 6A shows a plot that distinguishes data from different beam current values, while Figure 6B shows a plot that does not distinguish data from all beam current values.

[0127] At each intensity, the slope of the ratio of turbidity to neutron absorbed dose was almost the same (Fig. 6A and Fig. 6B), confirming that the integrated value of absorbed dose determines the mutation frequency.

[0128] By using clearly defined irradiation conditions, it is possible to breed algae strains that exhibit useful traits (such as strains with improved lipid accumulation) without causing unnecessary damage to the algae through excessive irradiation. The introduction of mutations and the acquisition of strains with useful traits, as exemplified in the above examples using the red algae Schizon and Euglena, are highly versatile techniques that can be applied to algae in general.

[0129] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various modifications can be made to the configuration and details of the present invention within the scope of the present disclosure.

[0130] <Additional Notes> The present disclosure includes the following embodiments. (Appendix 1) A method for inducing mutations in algae, comprising the steps of: irradiating the algae present in water with neutron beams; The absorbed dose of the neutron radiation to the algae is within a range of 0.1 to 100 Gy. Mutagenesis methods. (Appendix 2) 2. The method of claim 1, wherein the neutron radiation is thermal neutron radiation. (Appendix 3) 2. The method of claim 1, wherein the neutron radiation is a high-energy neutron radiation. (Appendix 4) The method according to any one of claims 1 to 3, wherein the alga is the red alga Schizon or Euglena. (Appendix 5) 5. The method according to any one of claims 1 to 4, wherein the absorbed dose of the neutron beam is 20 Gy or less. (Appendix 6) 1. A method for producing an algae strain with increased lipid accumulation, comprising: mutagenizing a population of algae using the method of any one of claims 1 to 5; Isolating an algal strain from the population of mutagenized algae; Measuring lipid accumulation in said algal strain; and identifying an algae strain having increased lipid accumulation based on the measured lipid accumulation. (Appendix 7) Measuring the amount of lipid accumulation Measuring lipid accumulation after anaerobically treating the algal strain; and Correcting the measured amount of lipid accumulation by multiplying it by the ratio of the cell weight after the anaerobic treatment to the cell weight before the anaerobic treatment. 7. The method of claim 6, comprising: (Appendix 8) A method for evaluating neutron irradiation conditions in the induction of mutations in algae using neutron beams, comprising: (a1) dividing underwater algae into a plurality of irradiation condition groups and irradiating the algae with neutron beams; (a2) culturing each of the plurality of irradiation condition groups or their progeny after the irradiation as a plurality of single individual cultures isolated into individual individuals; (a3) comparing the coefficients of variation of the cell proliferation amounts obtained from the plurality of single individual cultures among the plurality of irradiation condition groups; or (b1) Irradiating underwater algae with neutron beams under a plurality of irradiation condition groups; (b2) culturing the plurality of irradiation condition groups or their progeny after the irradiation on a solid medium containing uracil and 5-fluoroorotic acid; (b3) comparing the number of colonies that appear among the plurality of irradiation condition groups; A method comprising:

Claims

1. A method for inducing mutations in algae, This includes irradiating the aforementioned algae present in water with neutron beams, The absorbed dose of neutrons from the aforementioned algae is in the range of 0.1 to 100 Gy. A method for inducing mutations.

2. The method according to claim 1, wherein the neutron beam is a thermal neutron beam.

3. The method according to claim 1, wherein the neutron beam is a high-energy neutron beam.

4. The method according to claim 1, wherein the algae is the red alga Schizophyllum or Euglena.

5. The method according to claim 1, wherein the absorbed dose of the neutron beam is 20 Gy or less.

6. A method for producing algal strains with increased lipid accumulation, Inducing mutations in a population of algae using the method described in any one of claims 1 to 5, Isolating algal strains from the aforementioned population of mutated algae, To measure the amount of lipid accumulation in the aforementioned algal strain, A method comprising identifying algal strains with increased lipid accumulation based on the measured lipid accumulation amount.

7. Measuring the amount of lipid accumulation means The amount of lipid accumulation is measured after the aforementioned algal strain is subjected to anaerobic treatment, and The method according to claim 6, further comprising correcting the measured lipid accumulation by multiplying it by the ratio of the cell weight after anaerobic treatment to the cell weight before anaerobic treatment.

8. A method for evaluating neutron irradiation conditions in the induction of algal mutations using neutron beams, (a1) Irradiating aquatic algae with neutron beams under multiple irradiation condition groups, (a2) Culturing each of the multiple irradiation condition groups after irradiation, or their offspring, as multiple single-individual cultures isolated from individual organisms, (a3) Comparing the coefficient of variation of cell proliferation obtained from the plurality of single-organism cultures among the plurality of irradiation condition groups, or (b1) Irradiating aquatic algae with neutron beams under multiple irradiation condition groups, (b2) The plurality of irradiation condition groups or their offspring after irradiation are cultured on a solid medium containing uracil and 5-fluoroorotic acid, (b3) A method comprising comparing the number of colonies that appear among the plurality of irradiation condition groups.

9. A red alga, Schizon, having a mutation in the URA gene and requiring uracil.

10. The red alga Schizon according to claim 9, wherein the mutation is one of a single base deletion, a single base insertion, a single base substitution, a two-base deletion, or a three-base to one-base substitution.

11. Euglena in which the lipid content before anaerobic treatment and the lipid content after anaerobic treatment, corrected by multiplying by the ratio of cell weight after anaerobic treatment to cell weight before anaerobic treatment, are both higher than the corresponding lipid content of Euglena gracilis eu029 strain.

12. The lipid content before anaerobic treatment is 3.9% or more, The Euglena according to claim 11, wherein the lipid content after anaerobic treatment, corrected by multiplying by the ratio of the cell weight after anaerobic treatment to the cell weight before anaerobic treatment, is 15.1% or more.

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