Method for screening for mutants in a biological population by applying a mixed-splitting method

Abstract

This invention relates to a method for screening mutants within a biological population using a hybrid fission method. Specifically, in conventional plant breeding methods, chemical mutations can be used to randomly introduce nucleotide substitutions into the plant genome, meaning it is impossible to control the sites of nucleotide changes. Due to the complexity of the genome, the statistical probability of finding a predetermined nucleotide substitution is extremely low. However, this invention demonstrates how to develop a novel alternative use of digital polymerase chain reaction (dPCR) preferred droplet dPCR (ddPCR) for discovering specific nucleotide substitutions in mutant genes. The entire platform includes a screening method with a mutant organism library, a digital PCR-based system, and apparatus for propagating and analyzing identified mutant organisms.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780041101.5, filed on June 23, 2017, entitled "Method for screening mutants within a biological population by applying a hybrid division method". Invention Field

[0002] This invention provides highly accelerated methods and processes that can be scaled up and processed in practice for the preparation, selection, and / or propagation of organisms having specific, predetermined mutations at one or more nucleotides of interest (NOI) sites. For example, the methods of this invention can be used to improve the speed and ability to prepare plants having specific, predetermined mutations at one or more NOI sites. Background Technology

[0003] Genetic methods for generating genetically modified (GM) organisms (GMOs) are widely available. However, for many purposes, particularly in the food and beverage industry, the application of GMOs is often less than ideal. Therefore, there remains a long-standing need for improved and more precise methods for conventional crop breeding, including cereals such as barley, to easily obtain better, customized raw materials for developing and manufacturing new products. Similar limitations exist in other organisms, including microorganisms and animals. Unfortunately, conventional breeding methods lack a way to address the discovery of predetermined nucleotide mutations in the genome, thus limiting the progress of raw material development.

[0004] Methods exist that allow for genome editing and the introduction of double-strand DNA breaks at targeted sites within an organism's genome, such as through CRISPR-Cas9 technology (Jiang et al., 2016). Two key related issues exist with CRISPR-Cas9 technology:

[0005] First, CRISPR-Cas9 technology can be considered based on GM. Therefore, there is a lack of fundamental legislative information on how authorities intend to regulate new genetic tools for genome editing, including the introduction of double-strand DNA breaks at target sites in the genome;

[0006] - Secondly, there are major issues related to off-target cutting in CRISPR-Cas9 technology.

[0007] Germplasm mutations in the natural environment, such as those leading to phenotypic consequences in organisms, represent both the primary cause of variation in hereditary traits and the ultimate source of evolutionary changes with beneficial or destructive effects at the molecular level. Mutations are caused by a variety of factors, including:

[0008] - Copy error;

[0009] -DNA damage, either not repaired correctly or not repaired at all;

[0010] DNA damage caused by external factors, such as chemicals, ultraviolet radiation, and ionizing radiation;

[0011] - Endogenous factors, such as reactive oxygen species, aldehydes, or mitotic errors;

[0012] - Enzymes involved in DNA repair or genome editing;

[0013] - Viruses with inserted DNA fragments and endogenous retrotransposons.

[0014] Furthermore, current understanding suggests that exposure to mutagens may induce a large number of somatic gene mutations, most of which do not offer a clear selective advantage, but some may alter key cellular functions. Additionally, some genome-related traits may be non-mutant, such as epigenetic changes and alterations to the cellular microenvironment.

[0015] Traditional crop breeding involves random mutagenesis followed by screening for desired traits. Unfortunately, effective screening methods are lacking to identify predetermined genetic changes—specific nucleotide substitutions in genes or regulatory elements of mutant organisms—e.g., cereals that do not employ GM-based methods. In fact, GM plants have enjoyed a competitive advantage of over 10 years in terms of directional approaches to trait development itself and in broader scientific applications. However, as mentioned above, GM-based methods are not preferred.

[0016] Recent advancements in genome sequencing technologies have revolutionized our understanding of the genetic structure of traits (and the corresponding changes in response to mutagenesis). For example, it is widely expected that whole-genome sequencing and new computational methods, aided by large-scale, high-throughput gDNA sequencing, will help answer several fundamental biological questions and accelerate mutant characterization, for instance, using monocotyledonous cereal barley.

[0017] In eukaryotes, some mutational processes appear to show considerable variation in genome distribution (Pleasance et al., 2010). (Lehner and Lehner, 2012). The number of point mutations varies along the genome and is generally higher in sequence regions with low gene expression levels, repressed chromatin, and late replication times. Some of these variations can be achieved by reducing access to closed chromatin regions by mismatch repair mechanisms.

[0018] Given the background information provided above, those skilled in the art of cereal research will understand that these analytical advances have made barley and cereal research a new frontier, particularly in the R&D phase between a deeper understanding based on molecular biology and the industrial application of plants with novel characteristics. That is, despite advances in genome sequencing technologies for mutant identification, these methods remain time-consuming and logically challenging, and offer little use for identifying specific, predetermined mutations.

[0019] Two other attributes remain of interest, not only in identifying plants with predetermined mutations, but also in utilizing improvements:

[0020] - Define the range of cereal lineages and somatic mutation rates.

[0021] Current methods focus on narrowing the search for gDNA fragment mutants based on background-independent gDNA sequence analysis [e.g., the Tilling method, which is limited to screening a maximum of 5,000 to 10,000 mutants, as well as melting point differences between mutant and wild-type derived gDNA fragments (Botticella et al., 2011)].

[0022] Until now, finding predetermined and complex mutations using non-GM methods has been considered impractical. Similarly, along this line, there has been no indication of what sample size would be reliable for identifying specific nucleotide substitutions of interest. Now, this can be achieved through the guidance provided in this disclosure. Summary of the Invention

[0023] This invention discloses a method for discovering a single, conventionally developed organism with a predetermined nucleotide mutation of interest in one or more target sequences. The mutation may be a gene mutation that confers a specific, useful trait.

[0024] In particular, based on surprising and novel insights into mutant structures, this invention provides a striking yet relatively simple new genome-wide approach for mutant discovery applications. This invention provides a method for identifying organisms with predetermined mutations in one or more NOIs. The predetermined mutation can be any desired mutation. Therefore, this invention provides a non-GM method capable of identifying organisms with specific mutations. This is made possible by the ingenious use of PCR technology, particularly digital PCR. Therefore, this invention discloses a novel method for locating the mutant DNA background for screening, combined with a logically innovative system capable of analyzing a vast ensemble of mutant organisms.

[0025] A novel, time-efficient, and minimally processed procedure is also disclosed, accomplishing the challenging task of finding rare mutations, such as those predicted to be located in the aforementioned closed chromatin regions of the grain nucleus, including those in barley. Primers and / or probes for the method are designed using access to the whole genome sequence. Therefore, the whole barley genome sequence (Barley Genome Collection in Ensembl Plants, version 082214v1) can be used to design primers and probes for specific genes, making it ideal for increasing the rate of discovery of plants with predetermined mutations.

[0026] In one embodiment, the present invention provides a method for searching for predetermined mutations using conventional or routine breeding methods, said predetermined mutations being, for example, mutations leading to nucleotide substitutions in genes associated with cereal traits. Using the disclosed method, approaches are proposed that help elucidate potential molecular mechanisms associated with endogenous and induced mutagenesis.

[0027] The present invention is defined in the appended claims.

[0028] This invention provides a method for identifying an organism of a predetermined species, wherein the organism carries one or more predetermined mutations in the NOI of a target sequence, the method comprising the following steps:

[0029] a) Provide a pool representing an organism of the species or its reproductive portion representing multiple genotypes;

[0030] b) Divide the pool into one or more sub-pools containing organisms or their reproductive parts;

[0031] c) Prepare gDNA samples, each sample containing gDNA from each genotype in the sub-pool, while maintaining the proliferation potential of each genotype organism in the sub-pool;

[0032] d) Perform multiple PCR amplifications, each PCR amplification containing a gDNA sample from a sub-pool, wherein each PCR amplification includes multiple compartmentalized PCR amplifications, each compartmentalized PCR amplification containing a portion of the gDNA sample, a set of primers located flanking the target sequence, and PCR reagents, thereby amplifying the target sequence;

[0033] e) Detect PCR amplification products containing one or more target sequences, said target sequences containing mutations of the NOI of interest, thereby identifying subpools containing said mutations;

[0034] f) Divide the organisms or their reproductive parts in the identified sub-pools into secondary sub-pools;

[0035] g) Prepare gDNA samples, each sample including gDNA from each genotype in the secondary pool, while maintaining the proliferation potential of each genotype organism in the secondary pool;

[0036] h) Perform multiple PCR amplifications, each of which includes a gDNA sample from a secondary pool, a set of primers located flanking the target sequence, and PCR reagents to amplify the target sequence.

[0037] i) Detect PCR amplification products containing a target sequence containing a predetermined mutation of NOI, thereby identifying the secondary pool containing the mutation in step h).

[0038] j) Identify the organisms or their reproductive portions within the secondary pool carrying the mutation.

[0039] The present invention also provides organisms identified by the methods of the present invention. For example, the present invention provides a barley plant carrying a mutation in the HvGS1-3 gene encoding glutamine synthase, wherein the mutant gene encodes a mutant HvGS1-3 protein with reduced activity.

[0040] This invention is further illustrated in five workflows (WS) that sequentially identify specific, predetermined nucleotide mutations in a ensemble of mutant cereal grains: WS1 discloses the process for constructing conventionally induced cereal ensembles; WS2 details how to construct complex grain samples; WS3 highlights how to identify grain samples containing mutants; the analysis associated with WS4 describes how to search for individual mutant cereal grains; and WS5 focuses on how the combined use of two different digital PCR instruments can facilitate the screening of ultra-high-quantity gDNA samples from cereal grains. Attached Figure Description

[0041] Figure 1 provides an example of the overall experimental concept of this application.

[0042] Figure 2 illustrates an example of the overall experimental concept of this application using the plant species barley as an example. A involves the propagation of mutant barley grains (WS1 as explained in the detailed description of the invention); B shows various aspects of preparing an ordered library of mutant grains (WS2 as explained in the detailed description of the invention); C provides an overview of how to determine whether a grain portion contains a single grain of interest (WS3 as explained in the detailed description of the invention); D focuses on how to identify grains with specific mutations in a sample containing a mixture of grains (WS4 as explained in the detailed description of the invention); E provides an illustration of the workflow involved in detecting mutant DNA in a combined gDNA sample (WS5 as explained in the detailed description of the invention).

[0043] Detailed description of the invention

[0044] definition

[0045] The term "allele" refers to a specific form or state of a gene. As used herein, the term "mutant allele" refers to a gene carrying one or more predetermined mutations in the NOI. A mutant allele can also be an allele lacking the entire gene, even when the mutation is a deletion.

[0046] The term “approximately” as used in this document with respect to numbers means ±10%, preferably ±5%, for example ±1%.

[0047] As used in this article, the term "blocking probe" refers to an oligonucleotide that cannot be extended at the 3′ end by DNA polymerase. Blocking probes are typically oligonucleotides that are identical to or complementary to the target sequence, including a reference NOI linked to a blocking agent, which inhibits DNA polymerase extension of the blocking probe.

[0048] As used herein, the term "genotype" refers to an organism that contains a specific set of genes. Therefore, two organisms containing the same genome have the same genotype. The genotype of an organism associated with a particular gene is determined by the alleles carried by that organism. In diploid organisms, the genotype of a given gene can be AA (homozygous, dominant), Aa (heterozygous), or aa (homozygous, recessive).

[0049] As used in this article, the term "mutation detection probe" refers to an oligonucleotide optionally linked to a detectable means, wherein the oligonucleotide is identical to or complementary to the target sequence, including a predetermined mutation of NOI.

[0050] As used herein, the term "PCR" refers to Polymerase Chain Reaction. PCR is a reaction used to amplify nucleic acids. This method relies on thermal cycling and involves cycles of repeated heating and cooling to achieve sequential unwinding and enzymatic replication of the DNA. In the first step, the two strands forming the DNA double helix are physically separated at high temperature; this process is also known as DNA unwinding. In the second step, the temperature is lowered to allow enzymatic replication of the DNA. PCR may also include incubation at additional temperatures to enhance primer annealing and / or optimize the replication temperature. In PCR, the temperature is typically cycled multiple times between various temperatures.

[0051] As used herein, the term "PCR reagent" refers to reagents added to PCR in addition to the sample and a set of primers. PCR reagents contain at least nucleotides and nucleic acid polymerases. Additionally, PCR reagents may contain other compounds such as salts and buffers.

[0052] The term "ddPCR" refers to droplet digital polymerase chain reaction. In ddPCR, one or more PCR amplifications are performed, in which each reaction is separated into multiple water-oil emulsion droplets, allowing PCR amplification of the target sequence to occur in each individual droplet.

[0053] As used herein, the term "reproduction" refers to both sexual and asexual reproduction. Therefore, reproduction can be the clonal propagation of an organism (also known as "asexual reproduction"). Reproduction can also produce offspring of an organism, wherein the offspring contain alleles from the parent organism. Therefore, reproduction of an organism containing mutant alleles can refer to the production of offspring of said organism, wherein the offspring contain mutant alleles. Preferably, the mutant allele carries one or more mutations in NOI.

[0054] As used herein, the term "reproductive part of an organism" refers to any part of an organism that, under suitable conditions, can grow into the whole organism. For example, in embodiments of the invention where the species is a plant, the reproductive part of the organism may be, for example, the seed, grain, or embryo of the plant. In embodiments of the invention where the species is a single-celled organism, the reproductive part is the whole organism, i.e., a single cell.

[0055] As used herein, the term "primer set flanking the target sequence" refers to a set of two primers located flanking the target sequence. Therefore, one primer contains the same sequence at the 5' end as the target sequence (also known as the "forward primer"), and the other primer contains a sequence complementary to the 3' end of the target sequence (also known as the "reverse primer"). When added to PCR along with nucleic acid containing the target sequence and PCR reagents, under conditions allowing amplification of the target sequence, the "primer set" is capable of amplifying the target sequence.

[0056] As used herein, the term "target sequence" refers to any nucleic acid sequence in which a mutation is to be generated or identified. Furthermore, the target sequence is preferably a nucleic acid sequence that can be amplified using PCR technology using primers flanking the target sequence. Additionally, the target sequence typically contains one or more NOIs. This invention provides methods for generating and / or identifying organisms carrying mutations in said NOIs. For example, the target sequence may be a nucleic acid sequence associated with a specific trait.

[0057] As used in this article, the term "reference detection probe" refers to an oligonucleotide optionally linked to a detectable mechanism, wherein the oligonucleotide is identical to or complementary to the target sequence, including a reference NOI. Typically, the target sequence including the reference NOI corresponds to the unmutated target sequence. A "reference detection probe" may also be referred to as a "wild-type probe."

[0058] As used in this article, the term "workflow" (WS) refers to a series of one or more steps in a method.

[0059] Methods for identifying organisms

[0060] This invention relates to a method for identifying an organism of a predetermined species—for example, any species described in the “Species” section below, which carries a mutation of NOI in a target sequence [e.g., any mutation described in the “Nucleotide of Interest” section below]—and the method comprises the following steps:

[0061] a. Provide a pool of organisms of a specified predetermined species or their reproductive parts, representing multiple genotypes, such as any pool described in the “Organic Pools” section below;

[0062] b. Divide the pool into one or more sub-pools of organisms or their reproductive parts, as described in the "Dividing the Pool into Sub-pools" section below;

[0063] c. Prepare gDNA samples, each sample containing gDNA from each genotype in the sub-pool while maintaining the proliferation potential of the organisms of each genotype in the sub-pool, as described, for example, in the “Preparation of DNA Samples” section below;

[0064] d. Perform multiple PCR amplifications, each PCR amplification containing a gDNA sample from a sub-pool, wherein each PCR amplification contains multiple compartmentalized PCR amplifications, each compartmentalized PCR amplification containing a portion of the gDNA sample, a set of primers flanking the target sequence, and PCR reagents, thereby amplifying the target sequence, as described, for example, in the section “PCR amplifications containing multiple compartmentalized PCR amplifications” below;

[0065] e. Detecting PCR amplification products containing target sequences with NOI mutations to identify subpools containing said mutations, as described, for example, in the “Detection of PCR Amplification Products” section below;

[0066] f. Divide the organisms or their reproductive parts of the identified sub-pools into secondary sub-pools, for example, as described in the "Dividing Sub-pools into Secondary Sub-pools" section below;

[0067] g. Prepare gDNA samples, each sample containing genomic DNA from each genotype in the secondary pool, while maintaining the proliferation potential of the organisms of each genotype in the secondary pool, as described, for example, in the “Preparation of DNA Samples” section;

[0068] h. Perform multiple PCR amplifications, each PCR amplification comprising a gDNA sample from a secondary pool, a set of primers flanking the target sequence, and PCR reagents, thereby amplifying the target sequence, as described, for example, in the “Identification of Secondary Pools” section.

[0069] i. Detect PCR amplifications containing a target sequence containing a mutation of NOI, thereby identifying secondary pools containing the mutation, as described, for example, in the “Identification of Secondary Pools” section;

[0070] j. Identify the organisms in the secondary pool carrying the mutation, as described in, for example, the “Identifying Organisms” section.

[0071] Step a. may include, for example, providing a biological pool prepared as described in WS1 of this document.

[0072] Step b can be performed as described in WS2 of this document.

[0073] Steps c., d., and e. can be performed as described in WS3 of this document.

[0074] Steps f., g., h., i., and j. can be performed as described in WS4 of this document.

[0075] Typically, PCR reagents contain at least nucleotides and nucleic acid polymerases. Furthermore, PCR reagents may preferably include one or more detection probes, such as the mutation detection probes and / or reference detection probes described in the "Detection of PCR Products" section above.

[0076] In addition to the steps outlined above, the method of the present invention may include one or more additional steps. For example, the method may include the step of preparing the organism pool, for example by mutagenesis. Methods for preparing the organism pool are described in the “Organo Pool” section below.

[0077] The method may also include the step of propagating one or more of the organism or its reproductive parts within a pool or sub-pool of an organism or its reproductive portion. In the case of simple organisms, such as asexually reproducing single-celled organisms, the propagation step may include one or more cell divisions. In the case of more complex organisms, such as sexually reproducing organisms, the step may include cultivating the organism or its reproductive portion through one or more cycles.

[0078] In the following text, reference will be made only to “organism”. However, the same considerations apply to methods using the reproductive portion of an organism. Each step in culturing an organism can result in offspring that differ from the original organism. For example, after random mutagenesis of a polyploid organism, most organisms carry any random mutation in only one allele and are therefore genetically heterozygous for that mutation. Typically, offspring organisms include organisms without the mutation, offspring that are genetically heterozygous for the mutation, and offspring that are genetically homozygous for the mutation. Thus, the offspring pool of the original pool or organism or its reproductive portion differs from the original pool but generally represents at least any mutation of NOI present in the original pool—i.e., heterozygous and / or homozygous organisms. Therefore, at least some of the said offspring contain mutant alleles. Similarly, the offspring of a sub-pool, super-pool, or secondary sub-pool may differ from the original sub-pool, super-pool, or secondary sub-pool but generally represent at least any mutation of NOI present in the original sub-pool, super-pool, or secondary sub-pool, in heterozygous or homozygous form.

[0079] Therefore, step b) of dividing the pool into another sub-pool may include the step of propagating organisms or their reproductive portions. For example, it may be performed simultaneously with dividing the pool into sub-pools. Alternatively, it may be completed after dividing the pool into sub-pools. Thus, the method of the present invention may include the step of propagating organisms or their reproductive portions within the sub-pools after step b).

[0080] Similarly, the method of the present invention may include, after step f), a step of propagating the organisms in the secondary pool or a propagating portion thereof. However, in some embodiments of the invention, particularly in embodiments where the species is a plant such as a cereal, the method preferably does not include the step of propagating the organisms in the secondary pool or a propagating portion thereof between steps f) and g).

[0081] In some embodiments of the invention, the method includes the step of propagating organisms or propagated portions thereof contained in a secondary pool, the secondary pool containing mutations of NOI. This step can be performed at any useful time, for example, between steps i) and j) above.

[0082] The method may also include the step of identifying a set of sub-pools containing mutations of NOI. This step can be performed at any useful time, but is often performed after step c) of the method described above, and may be performed as described in the “Superpools” section below.

[0083] In one embodiment, the method of the present invention can be summarized into 4 to 5 separate workflows, for example, WS1 to WS5, each divided into several separate “steps”, as described below. While a method comprising or even consisting of WS1 to WS5 represents a preferred embodiment of the invention, the invention is not limited to methods comprising these WS. For example, the method of the present invention may include only WS2 to WS5, omitting WS1. The method is illustrated, for example, in Figure 1. Figure 2 further illustrates an example of a method including WS1 to WS5, divided into 5 parts, wherein:

[0084] - Figure 2A A specific example relating to WS1 of the embodiments of the present invention is provided, wherein the species is barley. The figure illustrates the reproduction of mutant barley grains, as detailed in WS1 herein.

[0085] - Figure 2B Examples of preparing ordered mutant grain libraries are shown, as detailed in WS2 of this paper (see Examples 1 and 2).

[0086] - Figure 2C An overview example of how to determine whether the “total grains” section includes grains characterized by mutations in NOI is provided, as detailed in WS3 of this document (see Examples 3 through 7).

[0087] - Figure 2D An example from WS4 is highlighted, namely the procedure for determining which grains(s) in the “Total Grains” section contain the NOI mutation—this section was previously shown to include grains containing the NOI mutation; see [link to WS4 documentation]. Figure 2C - As detailed in step 4 of this document (see Examples 8 to 15).

[0088] - Figure 2E An example of a process overview in WS5 is given, in which compartmentalized PCR technology (e.g., ddPCR) is used to determine the “superpool” of gDNA, i.e., a combination of aliquots of gDNA samples (Example 17) containing mutations corresponding to NOI mutations.

[0089] Nucleotide of Interest (NOI)

[0090] This invention relates to a method for identifying organisms carrying one or more mutations in one or more nucleotides of interest. Specifically, this method allows for the identification of organisms carrying one or more predetermined NOI mutations. Therefore, this method allows for the identification of organisms carrying specific mutations while still relying on non-GM methods.

[0091] The mutation can be any mutation, wherein one or more NOIs of interest differ from the corresponding NOI in the reference sequence. Typically, the reference sequence is a wild-type sequence. However, the reference sequence can also be any other sequence.

[0092] Mutations can be any kind of mutation, such as deletions, insertions, substitutions, or mixtures as described above.

[0093] NOI can be a single nucleotide or several nucleotides, so NOI can consist of at least one, such as 1, such as 2, such as 3, such as 4, such as 5, such as 6, such as 7, such as 8, such as 9, such as 10, such as 10 to 20, such as 20 to 50, such as more than 50 nucleotides.

[0094] In a preferred embodiment of the invention, NOI consists of a single nucleotide – in this case, the mutation can be, for example, a substitution of a single nucleotide. Such a mutation is also called a point mutation.

[0095] In other embodiments of the invention, the mutation may be a deletion of the NOI. In other embodiments, the mutation may be the insertion of one or more nucleotides between the two nucleotides of interest.

[0096] In one implementation, the reference sequence is the wild-type sequence, i.e., the most common naturally occurring sequence, and therefore the mutation is a mutation compared to the wild-type sequence.

[0097] In one embodiment, the mutation may be associated with a desired trait within the species. Depending on the type of species, the desired trait may be selected from a variety of different traits. In embodiments of the invention, where the species is a domesticated plant, the trait may be, for example, enhanced vigor, enhanced resistance to various environmental factors, increased growth, or higher yield. In embodiments of the invention, where the species is a plant for food, feed, or beverage production, the trait may also relate to enhanced nutritional value, enhanced flavor characteristics, enhanced storage properties, or enhanced usefulness for producing said food, feed, or beverage.

[0098] NOIs can be located in any target sequence of any nucleic acid. Typically, the target sequence is part of the gDNA sequence. More preferably, the target sequence is part of the gDNA sequence. Therefore, a mutation can be a mutation in the gDNA of the organism. NOIs can be located in any part of the gDNA, both coding and non-coding regions. Typically, NOIs can be located in any part of a gene, such as within a coding region (e.g., within an exon), within an intron, or in a regulatory region of the gene, such as within a promoter, terminator, and / or intron.

[0099] Species

[0100] The method of the present invention includes identifying an organism of a predetermined species. The species can be any species, including multicellular and unicellular organisms. For example, the species can be a species included in Open Tree of Life, such as Open Tree of Life Reference Classification Version 2.9 Draft 12, generated on October 12, 2015.

[0101] This species can be a prokaryote, such as bacteria. Examples of bacteria include those used in food production, such as those selected from the genera *Acetobacter*, *Arthrobacter*, *Alactobacillus*, *Bacillus*, *Bifidobacterium*, *Brachybacterium*, *Brevibacterium*, *Carnobacterium*, *Corynebacterium*; *Enterococcus*, *Gluconacetobacter*, *Hafnia*, *Halomonas*, *Kocuria*, and *Lactobacillus*. The group consists of the genera *Cillus*, *Lactococcus*, *Leuconostoc*, *Macrococcus*, *Microbacterium*, *Micrococcus*, *Pediococcus*, *Propionibacterium*, *Proteus*, *Pseudimonas*, *Psychrobacter*, *Staphylococcus*, *Streptomyces*, *Tetragenococcus*, *Weissella*, and *Zymomonas*.

[0102] This species can also be a eukaryote, such as fungi, algae, plants, and animals.

[0103] In one implementation, the species is selected from fungi. Therefore, the species can be a unicellular or multicellular organism. For example, the species can be selected from genera such as *Aspergillus*, *Candida*, *Cystofilobasidium*, *Cyberlindnera*, *Debaryomyces*, *Fusarium*, *Geotrichum*, *Issatchenkia*, *Kazachstania*, *Kloeckera*, *Klyveromyces*, *Mucor*, and *Neurospora*. Fungi belonging to the genera *Penicillium*, *Pichia*, *Rhiozopus*, *Rhodosporidium*, *Rhodotorula*, *Saccharomyces*, *Torulaspora*, *Torulopsis*, *Thrichosporon*, *Verticillium*, *Yarrowia*, and *Zygotorulaspora*.

[0104] Specifically, this species can be a yeast, such as the group consisting of *Saccharomyces cerevisiae*, *Saccharomyces pastorianus*, *Saccharomyces bayanus*, and *Saccharomyces uvarum*. Other yeasts of interest include the genus *Brettanomyces*.

[0105] In a preferred embodiment of the invention, the species is a plant. The plant can be a green plant, such as those selected from the group consisting of flowering plants, conifers, gymnosperms, ferns, lycophytes, hornworts, mosses, bryophytes, and green algae. The plant can be, for example, a monocotyledonous or dicotyledonous plant.

[0106] In particular, the plant can be a domesticated plant. The domesticated plant can be any plant cultivated by humans, for example, as a source of food, feed, or raw material for the production of goods or for aesthetic purposes.

[0107] In a preferred embodiment of the invention, the species is a cereal. As defined herein, "cereal" refers to members of the Poaceae family of plants cultivated primarily for their starchy seeds or grains. Cereals include, but are not limited to, barley (Hordeum), wheat (Triticum), rice (Oryza), maize (Zea), rye (Secale), oats (Avena), sorghum (Sorghum), and wheat-rye hybrids such as black wheat.

[0108] The plant could also be other domesticated plants, including tomatoes.

[0109] As mentioned above, the species can also be an animal, such as a domesticated animal, such as a cow, chicken, pig, sheep, goat, camel, horse, turkey, duck and rabbit.

[0110] biological pond

[0111] The method of the present invention includes providing a pool of organisms representing a given species with multiple genotypes. For example, the species can be any species described in the "Species" section above.

[0112] Therefore, the organism pool comprises multiple organisms belonging to the same species but representing different genotypes of said species. The pool may contain more than one type of organism for each genotype. However, the pool must contain multiple organisms with different genotypes. Preferably, the pool contains at least 100, more preferably at least 1000, even more preferably at least 5000, even more preferably at least 10,000, even more preferably at least 50,000, even more preferably at least 100,000, for example at least 500,000, for example at least 1,000,000 organisms, or their reproductive portions, with different genotypes.

[0113] Preferably, the organism pool contains a sufficient number of organisms or their reproductive portions with different genotypes, such that the pool can theoretically include all possible mutations in all genes of the organisms. For example, in an embodiment of the invention, where the species is barley, a pool containing ~500,000 randomly mutated barley grains is believed to theoretically include all possible mutations in all genes of the barley (based on the assumption that ~10,000 single-base mutations are induced in a grain with 1 mM NaN3). To improve the efficiency of the method of the invention, the pool may contain at least 2×, for example, at least 3×, of organisms or their reproductive portions theoretically expected to contain all possible mutations, having different genotypes. Thus, the organism pool can include at least 500,000, for example, at least 1,000,000, for example, at least 1,500,000 organisms or their reproductive portions with different genotypes. For example, this could be the case in an embodiment where the species under discussion is barley.

[0114] In some embodiments, the pool may contain at least 30,000 organisms or their reproductive parts with different genotypes, for example, in the range of 30,000 to 500,000.

[0115] In some embodiments, the organism pool may contain at least 5,000,000, for example, from 1,000,000 to 100,000,000 organisms or their reproductive portions having different genotypes. For example, this may be the case in embodiments of the invention, where the organisms are small organisms, such as single-celled organisms.

[0116] In other embodiments, the pool may contain 100,000 to 500,000 organisms or their reproductive portions having different genotypes. For example, this could be the case in embodiments of the invention where the organisms are single-celled organisms.

[0117] One advantage of the method of the present invention is that it allows screening of a large number of organisms with different genotypes. Therefore, the organism pool can contain an extremely large number of organisms with different genotypes. Furthermore, the method of the present invention can allow the identification of organisms with a predetermined mutation in any NOI. To be able to identify organisms with any NOI mutation, this requires the organism pool to contain a large number of different genotypes, such as the number of different genotypes mentioned above.

[0118] You can obtain a pool of organisms in any useful way.

[0119] In one implementation, an organism pool is obtained by collecting individual organisms. This can be achieved, for example, from a seed bank (assuming the species is a plant), from a cell collection (assuming the species is a single-celled organism), or from other organism collections. It can also be achieved by collecting individual organisms or samples of said organisms in any other manner.

[0120] In a preferred embodiment of the invention, a pool of organisms is prepared by mutagenesis, particularly by random mutagenesis. Therefore, multiple organisms can be mutagenized to obtain a pool of organisms. The mutagenesis can be, in particular, random mutagenesis, which can be carried out, for example, as described below.

[0121] In an embodiment of the invention, the organism is a single-celled organism, and then multiple complete organisms are typically subjected to random mutagenesis.

[0122] In embodiments of the invention, where the species is a multicellular organism, it may be sufficient to subject the reproductive portion of the multicellular organism to the random mutagenesis. In such embodiments, random mutagenesis is performed on multiple organisms or multiple reproductive portions of organisms or mixtures thereof.

[0123] In an embodiment of the invention, the species is a plant, and the pool may contain multiple seeds representing multiple genotypes. Therefore, the pool may contain multiple seeds that have undergone mutagenesis. However, the pool may also contain offspring of seeds that have undergone mutagenesis.

[0124] Here, seeds that have undergone mutagenesis (e.g., grains of cereal) can be referred to as Generation M0. Seeds, such as grains of cereal, that can be sown and allowed to develop into mature plants are considered Generation M1. Generation M1 seeds can be sown and allowed to grow into mature plants; these seeds are considered Generation M2, and so on. The principle is as follows: Figure 2A As shown.

[0125] The organism pool may include seeds, such as cereal grains from any of the aforementioned generations. Therefore, the pool does not necessarily contain seeds that have previously undergone direct mutagenesis. The organism pool may also contain seeds from generations M1, M2, or M3, such as cereal grains.

[0126] The random mutagenesis can be carried out in any useful manner, for example, by radiation or chemical treatment. Radiation can be ultraviolet irradiation, X-ray irradiation, or radioactive irradiation. Chemical mutagenesis can be performed with any mutagenic chemical, such as compounds selected from the group consisting of: sodium azide (NaN3), alkylating agents such as N-ethyl-N-nitrosourea (ENU), methylnitrosoguanidine (MNNG), and ethyl methanesulfonate (EMS), or alkylating agents mentioned below. NaN3, ENU, and EMS are commonly used to generate random mutants. MNNG and EMS are frequently used to prepare yeast cultures for random mutagenesis.

[0127] To induce random mutations in the gDNA of plants, grains or regenerative plant tissues can be treated with mutagens or mixtures of mutagens, including but not limited to alkylating agents such as sulfonates (e.g., ethyl methanesulfonate (EMS), diethyl sulfonate (DES)); sulfur mustard, such as ethyl-2-chloroethyl sulfide; nitrogen mustard, such as 2-chloroethyl-dimethylamine; and epoxides, such as ethylene oxide. Others include ethyleneimine, hydroxylamine (NH₂OH), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), NaN₃, and diazomethane. The treated tissues or grains can then be propagated to produce progeny organisms. This random mutagenesis can be used on plants, such as crops, including cereals, but not limited to wheat, corn, rice, sorghum, and millet, and dicotyledonous crops, including but not limited to rapeseed, cotton, soybean, and sugar beets.

[0128] The method of this invention is not limited to any particular type of mutagenesis. Therefore, any type of mutagenesis can be used, such as any form of random mutagenesis.

[0129] In one embodiment of the invention, the organism pool can then be prepared as described in the "WS1" section of the following embodiments. In this embodiment, where the species is barley, the organism pool can be prepared as described in the "WS1" section of the following embodiments concerning barley. Those skilled in the art can modify the method described in WS1 to apply it to other flowering plants, including other cereals. In another embodiment, where the species is yeast, the organism pool can be prepared as described in the "WS1" section of the following embodiments concerning yeast. Those skilled in the art can modify the method described in WS1 to apply it to other single-celled organisms.

[0130] The biological pond is divided into sub-ponds.

[0131] The method of the present invention includes the step of dividing a pool of organisms into one or more sub-pools, wherein each sub-pool includes a plurality of organisms or their reproductive portions. Preferably, each sub-pool includes a plurality of organisms or their reproductive portions representing multiple genotypes.

[0132] Typically, the pool is divided into multiple sub-pools, preferably into at least 5 sub-pools, more preferably into at least 10 sub-pools, even more preferably into at least 30 sub-pools, more preferably into at least 50 sub-pools, even more preferably into at least 70 sub-pools, and even more preferably into at least 90 sub-pools.

[0133] In some embodiments, the pool is divided into at least 500, for example, at least 1000, or at least 1500 sub-pools. In particular, this can be the case in embodiments of the invention, where the pools contain a large number of organisms with different genotypes.

[0134] In principle, there is no upper limit to the number of sub-pools. However, pools are typically divided into a maximum of 50,000, or a maximum of 25,000, or a maximum of 10,000 sub-pools.

[0135] In some implementations, the pool is divided into 90 to 500 sub-pools.

[0136] Each subpool preferably contains multiple organisms or their reproductive portions representing multiple genotypes. Preferably, each subpool includes at least 10, more preferably at least 100, even more preferably at least 500, even more preferably at least 1000, even more preferably at least 5000, even more preferably at least 10,000, such as at least 50,000, such as at least 100,000 organisms or their reproductive portions with different genotypes. In some embodiments, each subpool contains 2000 to 10,000, such as 3000 to 5000 organisms or their reproductive portions with different genotypes.

[0137] In some implementations, each sub-pool contains 1,000 to 2,000 organisms or their reproductive portions representing different genotypes.

[0138] Subpools can be ordered in any way desired. If a pool contains multiple organisms of the same genotype, it is preferable that most or even all organisms of the same genotype are included in the same subpool. This can be ensured in various ways, depending on the species, for example, as described below. In embodiments of the invention, where a pool of organisms or its reproductive portions are prepared by random mutagenesis, the pool is preferably divided in such a way that all offspring of an organism or its reproductive portion from the pool are included in one subpool.

[0139] For example, the pool can be divided into sub-pools, and a proliferation step can be performed on the sub-pools. The proliferation step may also be performed simultaneously with the step of dividing the pool into sub-pools, so that the offspring of a single organism or its reproductive part eventually enter the same sub-pool.

[0140] It is also preferable that each sub-pool contains more than one organism or its reproductive portion for each genotype. This can be ensured in different ways. For example, the sub-pool can undergo a proliferation step, allowing each organism or its reproductive portion in the sub-pool to produce offspring. In particular, it is preferable that each sub-pool contains a sufficient number of organisms for each genotype to randomly divide the sub-pool into 2, 3, or 4 portions such that each portion theoretically contains an organism (or its reproductive portion) representing each genotype of the sub-pool. Thus, it is preferable that each sub-pool contains at least 5, preferably at least 10, and even more preferably at least 15 organisms representing each genotype. In particular, this can be the case in embodiments of the invention where the species is a plant, such as a cereal. In embodiments of the invention where the species is a single-celled organism, it is preferable that each sub-pool contains even more organisms for each genotype, such as at least 100, at least 1000, or at least 10,000.

[0141] Figure 1A The document provides examples of methods for dividing a pool into sub-pools.

[0142] In embodiments of the present invention, the species is a single-celled organism, and sub-pools can be prepared, for example, in the following manner by mutagenesis of multiple single-celled organisms:

[0143] - Following mutagenesis, each single-celled organism is allowed to reproduce independently, allowing each single-celled organism to develop into a clonal culture. This can be achieved by culturing colonies of each clone on a solid medium or by culturing each clone in a separate space with a liquid medium, such as in tubes or wells. Multiple clones of single-celled organisms can be combined to form sub-pools.

[0144] - Immediately after mutagenesis, the single-celled organism is divided into sub-pools, and each sub-pool is allowed to proliferate.

[0145] Therefore, the method may include the following steps:

[0146] - Provides multiple single-celled organisms, such as yeast;

[0147] - Randomly induced mutagenesis in the organism;

[0148] - Divide the mutated organisms into sub-pools;

[0149] - Perform a proliferation step in each sub-pool.

[0150] The proliferation step may include incubating each sub-pool in a culture medium under conditions that allow the organism to grow. For example, this step may include incubating the sub-pools in a culture medium for 1 to 5 days at a temperature that allows the organism to grow.

[0151] In an embodiment of the invention, where the species is a plant, the subpool can be prepared in such a way that all the seeds of a particular plant are contained in a single subpool. Therefore, in one embodiment of the invention, the method includes the following steps:

[0152] - Provides multiple seeds of a plant, such as grains;

[0153] - Mutagenesis of the seeds to obtain M0 generation seeds;

[0154] -Grow the M0 generation seeds into mature plants and obtain seeds from the mature plants, wherein the seeds are M1 generation seeds;

[0155] -Optionally repeat the previous step X times to obtain a plant containing M(1+X) generation seeds;

[0156] - Obtain seeds of generation M1 or M(1+X) from the mature plant to obtain a seed pool (e.g., a grain pool);

[0157] - Divide the pool into sub-pools, in which all seeds (e.g., grains) from a given mature plant are placed in the same sub-pool.

[0158] Figure 2A and 2B These steps are illustrated, using barley as an example. The method may include... Figure 2A and 2B The steps are shown in the figure. Figure 2A and 2B The steps shown can be performed using any flowering plant, and are therefore not limited to barley. Furthermore, Figure 2A and 2BThe steps shown can be implemented using any number of mutant plants (the numbers provided in the figure are just one example).

[0159] Therefore, the method may include the following steps:

[0160] - Provides multiple plant seeds, such as grains;

[0161] - Mutagenesis of the seeds to obtain M0 generation seeds;

[0162] - Optionally, the M0 generation seeds are grown into mature plants, and seeds are obtained from the mature plants, wherein the seeds are M1 generation seeds;

[0163] -Optionally repeat the previous step X times to obtain a plant containing M(1+X) generation seeds;

[0164] - M0, M1 or M(1+X) generation seeds are cultivated in different fields and grown into mature plants, wherein the seeds of the mature plants constitute a seed pool;

[0165] - Divide the area into plots;

[0166] - Harvest all the seeds of all the plants in a small plot of land to obtain a seed pool (e.g., a grain grain pool), which can be called the "total grains" (see See also: Total Grain Quantity). Figure 2B ).

[0167] Besides cultivating seeds in fields, these seeds can be cultivated in any useful way to divide the plants into subgroups, thus obtaining seed pools. For example, seeds can be grown in separate containers, each containing one or more plants. Seeds can also be grown in greenhouses.

[0168] An example of a method for dividing a pool into sub-pools is described below in WS2.

[0169] Identification of sub-pools

[0170] The method of the present invention includes the step of dividing a pool of an organism or its reproductive portion into sub-pools. The next step is the identification of the sub-pools, which contain organisms or their reproductive portions with NOI mutations.

[0171] The identification of the sub-pool may include the following steps:

[0172] a) Prepare gDNA samples, each sample containing gDNA from each genotype within a subpool, which may be performed, for example, as described in the “Preparation of DNA Samples” section below;

[0173] b) Perform multiple PCR amplifications, each of which includes multiple compartmentalized PCR amplifications, for example, as described in the "PCR amplifications comprising multiple compartmentalized PCR amplifications" section below;

[0174] c) Detect PCR amplification products containing mutations of interest in the NOI to identify the subpool, for example, as described in the “Detection of PCR Amplification Products” section below.

[0175] For example, sub-pools can be directly identified as long as the PCR amplification product containing the NOI mutation can be detected directly after or during PCR amplification. This can be done, for example, if the PCR amplification product contains a detection mechanism that generates one or more detectable signals, provided the PCR amplification product contains a target sequence containing the NOI mutation. Such a detection mechanism, described in more detail in the "Detection of PCR Amplification Products" section below, can be, for example, a mutation detection probe.

[0176] Figure 1B The document illustrates an example of a method for identifying subpools containing one or more specific mutations, highlighting the following steps:

[0177] - Provides sub-pools;

[0178] - Prepare a sample from a portion of the sub-pool;

[0179] - Prepare gDNA samples from the samples;

[0180] - Perform PCR amplification using all single gDNA samples from all sub-pools, for example, in individual wells of a plate, such as a microtiter plate;

[0181] - Select samples that contain NOI mutations.

[0182] Figure 2C A more specific example of the method used to identify subpools is shown. In this example, the species belongs to the Cereals section. Figure 2C The specific figures provided are merely examples, and those skilled in the art will understand that this method can be implemented with another number of grains. For example, the identification of subpools containing mutations of NOI can be implemented as outlined in WS3 below.

[0183] DNA sample preparation

[0184] The method of the present invention includes one or more steps of preparing a DNA sample, particularly a gDNA sample. Specifically, the method may include a step of preparing a gDNA sample from a sub-pool and a step of preparing a gDNA sample from a secondary sub-pool. The method may also include a step of preparing a gDNA sample from a superpool.

[0185] Typically, the gDNA sample is prepared in such a way that the theoretical gDNA sample contains gDNA from each genotype in the sub-pool, secondary sub-pool, or super-pool, while maintaining the proliferation potential of the organisms of each genotype in the sub-pool, secondary sub-pool, or super-pool.

[0186] This can be ensured in different ways. For example, it is preferable that each sub-pool and super-pool contains more than one single organism or its reproductive portion for each genotype. In particular, it is preferable that each sub-pool or super-pool contains a sufficient number of organisms for each genotype so that the sub-pool or super-pool can be randomly divided into 2, 3, or 4 portions, such that each portion theoretically includes an organism or its reproductive portion representing each genotype of the sub-pool or super-pool.

[0187] Thus, a portion of the organisms or their reproductive portions from the sub-pools or super-pools, for example, 10 to 90%, preferably 10 to 50%, or for example 25 to 50% of the organisms or their reproductive portions from each sub-pool or super-pool, can be used to prepare gDNA samples. The organisms or their reproductive portions are also referred to herein as “organism samples.” The remaining portions of the organisms from the sub-pools can be stored while preserving the proliferative potential of the organisms or their reproductive portions. In embodiments where the organism is a plant, storing the seeds of the plant may be sufficient. Seeds, such as grains, can often be stored in any dry and dark place. In embodiments of the invention where the organism is a single-celled organism, freezing the organism is preferable, for example, in the presence of a cryoprotectant such as glycerol.

[0188] Similarly, each of the secondary pools may contain more than one individual organism or its reproductive portion for each genotype. Therefore, a portion of the organisms or their reproductive portions in each secondary pool, such as 10 to 90%, preferably 40 to 60%, or for example 25 to 50%, of each secondary pool, can be used to prepare gDNA samples. However, in some embodiments, particularly when the species are large organisms, it is preferred that the gDNA samples of the secondary pools be prepared from a portion of the sample from each organism or its reproductive portion, as described below.

[0189] The invention also includes the possibility that the gDNA sample can be prepared from a sample from each organism or its reproductive portion in a sub-pool, secondary sub-pool, or super-pool. For example, the invention includes the possibility that each sub-pool, super-pool, and secondary sub-pool may contain only one or more organisms or their reproductive portions for each genotype. In the described embodiments, the gDNA sample can be prepared from a sample from each organism or its reproductive portion. Typically, in embodiments of the invention, samples are only obtainable from each organism of sufficient size to obtain the sample. This may be particularly relevant to embodiments of the invention where the species is an animal or plant, such as a flowering plant.

[0190] While sub-pools and super-pools preferably comprise several individual organisms or their reproductive portions for each genotype as described elsewhere herein, secondary sub-pools may typically contain only a few, and sometimes even only a single organism or its reproductive portion, for each genotype. Therefore, in embodiments of the invention where the species is a plant (e.g., cereal), it is preferable to prepare gDNA samples from secondary sub-pools by obtaining samples of each individual organism and preparing gDNA samples from said samples.

[0191] When obtaining samples from each organism or its reproductive parts, it is preferable to obtain the samples in a manner that does not significantly impair the reproductive potential of said organism or its reproductive parts. Therefore, preferably, the samples comprise, or consist of, portions of an organism or its reproductive parts that are not essential for reproduction. Depending on the species, samples can be obtained in any useful manner, for example, by biopsy, cutting, drilling, grinding, tearing, or using a syringe equipped with a needle.

[0192] For example, in embodiments where the species is cereal and the sub-pools, super-pools, or secondary sub-pools contain cereal grains, the sample preferably contains portions of the cereal grains that are not essential for reproduction. The sample can be obtained in various ways, such as by cutting a portion of the grain using a sharp instrument like a knife, scalpel, or scissors, or by drilling a hole in the grain. In the latter case, the sample could be flour obtained after drilling.

[0193] Once a biological sample is obtained, or a sample is obtained from an organism or a portion thereof (collectively referred to herein as the “sample”), a gDNA sample can be prepared from said sample by any useful method. If the sample contains a large structure, such as a whole seed, the first step in preparing a gDNA sample typically involves breaking the contents of the sample into smaller fractions, for example by physical methods such as crushing or grinding. Methods for preparing a gDNA sample typically involve steps that disrupt cells and / or tissues, such as using detergents, enzymes (e.g., lysins), ultrasound, or combinations thereof, to produce a crude lysate. The lysate can be separated from any residual fragments by any useful means. The crude lysate may constitute a gDNA sample. Alternatively, the gDNA can be further purified, for example by separating the gDNA from the remainder of the lysate, such as by binding to a selective matrix, centrifugation, gradient centrifugation, and / or precipitation (e.g., using a precipitating agent, such as salt, alcohol, or magnetic beads). Prior to separation, other components of the lysate—including proteins and / or nucleoproteins—can be denatured or destroyed, for example by using enzymes and / or denaturing agents. Other RNA-containing molecules can be removed, for example, with the aid of enzymes. Useful methods for preparing gDNA samples are described, for example, in Sambrook et al., Molecular Cloning-Laboratory Manual, ISBN 978-1-936113-42-2.

[0194] PCR amplification including multi-compartment PCR amplification

[0195] The method of the present invention includes the steps of performing multiple PCR amplifications, each PCR amplification comprising a gDNA sample (e.g., prepared as described above) from a sub-pool to amplify a target sequence. Each PCR amplification may include multiple compartmentalized PCR amplifications, each compartmentalized PCR amplification comprising a portion of the gDNA sample, one or more sets of primers and PCR reagents located flanking the target sequence.

[0196] The entire PCR reaction comprising multiple compartmentalized PCR amplifications can be prepared in a variety of different ways. In one embodiment, the PCR amplification comprising multiple compartmentalized PCR amplifications can be performed as digital PCR (dPCR) amplification. Any dPCR amplification known to those skilled in the art can be used with this invention. Typically, at least one dPCR amplification comprising multiple compartmentalized PCR amplifications is prepared for each type of gDNA sample prepared. Therefore, at least one dPCR amplification comprising multiple compartmentalized dPCR amplifications will be prepared for each sub-pool.

[0197] Typically, compartmentalized dPCR amplification is prepared using a method that includes the following steps:

[0198] -Preparation of dPCR amplification, which includes a gDNA sample, a set of primers located flanking the target sequence, and PCR reagents;

[0199] - The dPCR amplification is partitioned so that nucleic acid molecules in the sample are located and concentrated in multiple spatially separated compartments;

[0200] - Perform dPCR amplification;

[0201] - Detection of dPCR-based amplification products.

[0202] The separated compartment can be any individual compartment capable of PCR amplification. For example, it can be a well of a plate, such as a microplate or microtiter plate, a microfluidic compartment, a capillary, a dispersed phase of an emulsion, or a droplet or miniaturized compartment of a microchamber array. The separated compartment can also be discrete points on a solid support, such as discrete nucleic acid binding surfaces.

[0203] Typically, it is preferred to randomly allocate gDNA samples into the sample for compartmentalized dPCR amplification. It is also preferred that each compartmentalized dPCR amplification includes only a small number of nucleic acids containing the target sequence. Due to the random distribution nature, the number of nucleic acid molecules included in each compartmentalized dPCR amplification may vary. In one embodiment, each compartmentalized dPCR amplification contains an average of up to 10, for example, up to 5, nucleic acid molecules containing the target sequence.

[0204] In a preferred embodiment of the invention, the dPCR amplification comprising multiple compartmentalized PCR amplifications is droplet digital polymerase chain reaction (ddPCR). ddPCR is a method for dPCR based on water-oil emulsion droplet technology. PCR amplification is fractionated into multiple droplets, and PCR amplification of the target sequence occurs in each individual droplet. Typically, ddPCR technology employs PCR reagents and workflows similar to those used in conventional PCR. Therefore, the compartmentalization of PCR amplification is a key aspect of ddPCR technology.

[0205] Therefore, compartmentalized ddPCR amplification can be contained in droplets, which may, for example, comprise an emulsion composition or a mixture of two or more immiscible fluids (e.g., as described in U.S. Patent No. 7,622,280 or as illustrated in the examples below). Droplets can be generated using the apparatus described in WO / 2010 / 036352. In particular, droplets can be prepared using a droplet generator, such as the QX200 DropletGenerator available from Bio-Rad Laboratories, USA (hereinafter referred to as Bio-Rad). As used herein, the term emulsion can refer to a mixture of immiscible fluids (e.g., oil and water). For example, an emulsion can be a water-in-oil droplet, as described in [Hindson, BJ et al. (2011), High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83: 8604-8610]. Thus, an emulsion can comprise aqueous droplets situated in a continuous oil phase. Emulsions can also be oil-in-water emulsions, where the droplets are oil droplets located within a continuous aqueous phase. The droplets used in this paper are typically designed to prevent mixing between compartments, protecting not only the contents of a single compartment from evaporation but also preventing aggregation of the contents of other compartments. Therefore, each droplet can be considered a spatially separated compartment.

[0206] Each droplet in ddPCR can have any useful volume. However, it is preferable that the droplet has a volume in the range of nL. Therefore, it is preferred that the average droplet volume is in the range of 0.1 to 10 nL.

[0207] Microfluidic methods are known for generating monodisperse or polydisperse emulsions, using microchannel cross-flow focusing or physical stirring to produce emulsion droplets. The droplets can be monodisperse. Furthermore, droplets can be generated such that their size variation does not exceed ±5% of the average droplet size. In some cases, droplets are generated such that the droplet size variation is only within 2% of the average droplet size.

[0208] Higher mechanical stability is desirable for microfluidic manipulation and fluid handling at higher shear rates (e.g., in microfluidic capillaries or through 90° turns, such as valves in fluid paths). Droplets or capsules before and after heat treatment are mechanically stable for standard pipetting operations and centrifugation.

[0209] Droplets can be formed by passing the oil phase through an aqueous sample. The aqueous phase may contain or consist of components used in PCR amplification, such as a PCR amplification comprising a gDNA sample, a set of primers flanking the target sequence, and PCR reagents, such as any PCR reagents described in the “PCR Reagents” section below.

[0210] The oil phase may contain a fluorinated base oil, which may be further stabilized by combination with a fluorinated surfactant (e.g., a perfluorinated polyether). In some cases, the base oil may be one or more of HFE 7500, FC-40, FC-43, FC-70, or other common fluorinated oils. In some cases, the anionic surfactant is an ammonium salt of Krytox (Krytox-AM), an ammonium salt of KrytoxFSH, or a morpholine derivative of Krytox-FSH.

[0211] The oil phase may further contain additives to adjust the properties of the oil, such as vapor pressure, viscosity, or surface tension. Non-limiting examples include perfluorooctanol and 1H,1H,2H,2H-perfluorodecanol. The oil phase may also be an oil that produces droplets, such as Droplet Generation Oil from Bio-Rad.

[0212] This emulsion can be formulated to produce highly monodisperse droplets with a liquid interfacial film, which can be transformed into microcapsules with a solid interfacial film by heating; such microcapsules can serve as bioreactors, retaining their contents through reaction processes such as PCR amplification. Transformation into microcapsule form can occur upon heating. For example, this transformation can occur at temperatures greater than 50, 60, 70, 80, 90, or 95°C. In some cases, the heating is performed using a thermal cycler. During heating, a fluid or mineral oil coating can be used to prevent evaporation.

[0213] In some cases, commercially available droplet generators are used to produce droplets, such as the Bio-Rad QX100. TM DropletGenerator or Bio-Rad QX200 TM Droplet Generator. ddPCR and subsequent assays can be performed using commercially available droplet readers, such as the Bio-Rad QX100 or QX200. TM Droplet Reader.

[0214] Each PCR amplification can be compartmentalized into any suitable number of compartments. However, in a preferred embodiment, each PCR amplification is compartmentalized into 1,000 to 100,000 compartments (e.g., droplets). For example, each PCR amplification can be compartmentalized into 10,000 to 50,000 compartments (e.g., droplets). For example, each PCR amplification can be compartmentalized into 15,000 to 25,000 compartments (e.g., droplets). Further, each PCR amplification can be compartmentalized into approximately 20,000 compartments (e.g., droplets).

[0215] PCR reagents

[0216] The method involves performing several PCR amplifications, at least some of which may include multiple compartmentalized PCR amplifications.

[0217] Regardless of whether the PCR involves compartmentalized PCR amplification, PCR amplification typically includes a gDNA sample, a set of primers flanking the target sequence, and PCR reagents. The PCR reagents can be any of the PCR reagents described in this section.

[0218] Typically, PCR reagents contain at least nucleotides and a nucleic acid polymerase. The nucleotides can be deoxyribonucleotide triphosphate molecules, and preferably, PCR reagents contain at least dATP, dCTP, dGTP, and dTTP. In some cases, PCR reagents also contain dUTP.

[0219] Nucleic acid polymerases can be any enzyme capable of catalyzing template-dependent polymerization, i.e., replication, of nucleotides. The nucleic acid polymerase should be resistant to the temperatures of PCR amplification and should retain catalytic activity at extension temperatures. Several thermostable nucleic acid polymerases are known to those skilled in the art.

[0220] In some embodiments of the present invention, the nucleic acid polymerase has 5'-3' nuclease activity and is therefore suitable for amplification reactions with TaqMan probes.

[0221] The nucleic acid polymerase can be *E. coli* DNA polymerase I. It can also be Taq DNA polymerase, which has a DNA synthesis-dependent strand that substitutes for 5'-3' exonuclease activity. Other polymerases with 5'-3' exonuclease activity include, but are not limited to, rTth DNA polymerase. Taq DNA polymerase, for example, obtained from New England Biolabs, may include Crimon... Taq DNA polymerase, Crimson Taq DNA polymerase, Hemo KlenTaq TM or Taq.

[0222] In some cases, the nucleic acid polymerase can be, for example, E. coli DNA polymerase, the Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, T4 DNA polymerase, Taq polymerase, Pfu DNA polymerase, Vent DNA polymerase, phage 29, or REDTaq. TM Genomic DNA polymerase or sequencing enzyme. DNA polymerases, for example, are described in U.S. Patent Application Publication No. 20120258501.

[0223] In addition, PCR reagents may include salts, buffers, and detection methods. Buffers can be any useful buffer, such as TRIS. Salts can be any useful salt, such as potassium chloride, magnesium chloride, magnesium acetate, or magnesium sulfate.

[0224] PCR reagents may contain nonspecific blocking agents, such as BSA, gelatin from bovine hide, β-lactoglobulin, casein, dried milk, salmon sperm DNA, or other common blocking agents.

[0225] PCR reagents may also contain biological preservatives (e.g., NaN3), PCR enhancers (e.g., betaine, trehalose, etc.), and inhibitors (e.g., RNase inhibitors). Other additives may include dimethyl sulfoxide (DMSO), glycerol, betaine (mono)hydrate, trehalose, 7-deaza-2′-deoxyguanosine triphosphate (7-deaza-2′-dGTP), bovine serum albumin (BSA), formamide, tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives [e.g., tetraethylammonium chloride (TEA-Cl)]; tetrapropylammonium chloride (TPrA-Cl) or nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40 (NP-40), or PREXCEL-Q.

[0226] Furthermore, the PCR reagent may also contain one or more means for detecting PCR amplification products containing NOI mutations. These means can be any detectable means, and they can be added as a single compound or bound to one of the primers, or even covalently linked. Detectable means include, but are not limited to, dyes, radioactive compounds, bioluminescent and fluorescent compounds. In a preferred embodiment, the detection means is one or more probes. Therefore, it is preferred that the PCR reagent contains one or more detection probes, such as any probe described in the "Detection of PCR Amplification Products" section below.

[0227] Primers located on the flanking side of the target sequence

[0228] The method of this invention involves using one or more primer sets flanking a target sequence. A discrete "primer set" flanking the target sequence includes a primer containing a sequence identical to the 5' end of the target sequence (also called a "forward primer") and a primer containing a sequence complementary to the 3' end of the target sequence (also called a "reverse primer"). When the primer set is added to PCR along with nucleic acid containing the target sequence and PCR reagents, under conditions allowing amplification of the target sequence, the primer set is capable of amplifying the target sequence. The same primer set can be used for all PCR amplifications of the method of this invention, and even different primer sets may be used for PCR amplifications in different steps of the invention.

[0229] In addition to the sequence identical to the 5' end of the target sequence, the forward primer may contain additional sequences. Similarly, in addition to the sequence complementary to the 3' end of the target sequence, the reverse primer may contain additional sequences. For example, the primer may contain an additional nucleic acid sequence at the 5' end that does not hybridize with the target nucleic acid but is helpful in processing the primer or PCR amplification product, such as detecting the product.

[0230] The lengths of the forward and reverse primers can be determined based on the target sequence. For example, the primer lengths can be adjusted to obtain the desired melting temperature (Tm). Therefore, the lengths of the forward and reverse primers can be in the range of 10 to 100 nucleotides, for example, 10 to 50 nucleotides, for example, 15 to 20 nucleotides, for example, 15 to 25 nucleotides, for example, 15 to 30 nucleotides, for example, 15 to 40 nucleotides, for example, 15 to 45 nucleotides, for example, and for example, 15 to 50 nucleotides. Typically, the Tm of the forward and reverse primers is adjusted to the range of 40 to 70 °C.

[0231] The primer concentration in the aqueous phase for PCR amplification can be, for example, in the range of 0.05 to 2.0 μM, for example, in the range of 0.1 to 1.0 μM, for example, in the range of 0.2 to 1.0 μM, for example, in the range of 0.3 to 1.0 μM, for example, in the range of 0.4 to 1.0 μM, or in the range of 0.5 to 1.0 μM.

[0232] Typically, forward and reverse primers contain or are even composed of oligonucleotides. However, in some cases, primers may contain nucleotide analogs. Many nucleotide analogs are known to those skilled in the art and include derivatives in which sugars are modified, such as 2′-O-methyl, 2′-deoxy-2′-fluorine, and 2′,3′-dideoxynucleotide derivatives; nucleic acids based on other sugar backbones, such as threose, locked nucleic acids (LNA), LNA derivatives, peptide nucleic acids (PNA), glycol nucleic acids (GNA), threonine nucleic acids (TNA), bicyclic sugars or hexoses, glycerol and glycol sugars; nucleic acid analogs based on nonionic backbones; or nucleic acids and analogs with nonlinear topology, such as dendritic macromolecules, comb structures, and nanostructures.

[0233] Primers can also be linked to various tags (such as fluorescent tags, functionalized tags, or binding tags), which can optionally bind to their ends, sugars, or nucleobases.

[0234] Primers can be prepared by a variety of methods, including but not limited to cloning suitable sequences using methods well known in the art and direct chemical synthesis [Narang et al., Methods Enzymol. 68:90 (1979); Brown et al., Methods Enzymol. 68:109 (1979)]. Primers are also available from commercial sources.

[0235] The forward and reverse primers can have the same or similar melting temperatures, for example, ±5°C. The primer length can be extended or shortened at the 5′ and / or 3′ ends to produce a primer pair with the desired melting temperature. Therefore, one primer in a primer pair can be longer than the other.

[0236] Primers can be designed based on melting temperature. The equation for determining the melting temperature of primers smaller than 25 bp is known as Wallace's Rule [T...]. d = 2×(A+T)+4×(G+C)]. Here, T d It is the temperature at a specific salt concentration where 50% of the oligonucleotides and their perfectly filtered binding complements are in a double-stranded conformation. Typically, T... d Determined in 0.9 M NaCl. However, other considerations are also important when designing primers, such as their predicted secondary structure. Several computer programs and online services are available for primer design.

[0237] In one implementation, primer sets can be designed such that the primers specifically amplify target sequences containing mutated NOIs, but not target sequences containing reference NOIs. For example, this can be achieved by designing forward primers to include the same sequence as the mutated NOI, and / or by designing reverse primers to include one or more sequences complementary to the mutated NOI.

[0238] In other embodiments, the primer set can be designed in a manner that enables primer-specific amplification of target sequences containing a reference NOI, but not of target sequences containing mutated NOIs. This can be achieved, for example, by designing forward primers to contain the same sequence as the reference NOI, and / or by designing reverse primers to contain a sequence complementary to the reference NOI.

[0239] However, in a preferred embodiment of the invention, the primer set is capable of amplifying a target sequence containing a mutation of NOI and a target sequence containing a reference NOI.

[0240] It is worth noting that the method of the present invention includes PCR amplification with more than one set of primers, such as two sets of primers, three sets of primers, or two to ten sets of primers. Therefore, each PCR amplification may contain several sets of primers flanking different target sequences. This allows for the detection of more than one different mutation during a single PCR amplification.

[0241] Detection of PCR products

[0242] The method of this invention includes at least two steps of detecting PCR amplification products containing a target sequence, said target sequence containing a mutation of the NOI of interest. The PCR amplification products can be detected by any useful means.

[0243] As mentioned above, the mutation can be a substitution, deletion, and / or insertion, involving only one or a few nucleotides to a large number of nucleotides. Therefore, detection can be adapted to the specific mutations specified by the NOI.

[0244] In some embodiments, primer sets are designed such that the primers specifically amplify target sequences containing mutated NOI, but not target sequences containing a reference NOI. In other embodiments, primer sets can be designed such that these primers specifically amplify target sequences containing a reference NOI, but not target sequences containing mutated NOI. In such embodiments, detection can be performed simply based on the presence or absence of the PCR amplification product. In particular, this can be the case in embodiments of the invention where the mutation is a large number of NOI mutations.

[0245] In a preferred embodiment, the PCR amplification product is detected using a detection probe. This is particularly true when the mutation is a small number of NOI mutations (e.g., one or more point mutations).

[0246] Detection probes can be oligonucleotides comprising the same sequence as the NOI containing the mutation. Additionally, the probe typically also contains a sequence identical to the target sequence region flanking the NOI. Similarly, detection probes can be oligonucleotides comprising a sequence complementary to the NOI containing the mutation, and further, the probe may contain a sequence complementary to the target sequence region flanking the NOI. The regions flanking the NOI can be the 5′ and / or 3′ sequences of the NOI. Such probes are preferably annealed to PCR amplification products containing the NOI mutation, but not to PCR amplification products containing a reference NOI. Such detection probes are also referred to herein as “mutant detection probes.” Mutation detection probes typically contain 10 to 30 nucleotides. In particular, mutation detection probes may comprise a continuous sequence of 10 to 30 nucleotides that is identical to or complementary to the target sequence containing the NOI mutation.

[0247] The detection probe can also be an oligonucleotide containing the same sequence as the reference NOI. Additionally, the probe typically also contains a sequence identical to the target sequence region flanking the NOI. Similarly, the detection probe can be an oligonucleotide containing a sequence complementary to the reference NOI, and the probe may also contain a sequence complementary to the target sequence region flanking the NOI. Such probes are preferably annealed to PCR amplification products containing the reference NOI, but not to amplification products containing NOI mutations. Such detection probes are also referred to herein as "reference detection probes". Reference detection probes typically contain 10 to 30 nucleotides. Specifically, reference detection probes may comprise a continuous sequence of 10 to 30 nucleotides that is identical to or complementary to the target sequence containing the reference NOI.

[0248] In some implementations, the PCR reagent comprises a set of probes consisting of a mutation detection probe and a reference detection probe. When added to PCR amplification along with a nucleic acid containing the target sequence, the PCR reagent, and a primer set, under conditions allowing amplification of the target sequence, the "probe set" preferably competes for the binding site at the NOI. As mentioned above, the detection probes are typically oligonucleotides. In particular, they can be short nucleotide fragments of single-stranded DNA. Detection probes can bind to or even covalently link to detectable means, including but not limited to reporter molecules, dyes, radioactive compounds, bioluminescent compounds, fluorescent compounds, and fluorophore / quencher pairs.

[0249] In some implementations, the last 5' nucleotide of the detection probe is not G. Therefore, the mutation detection probe can contain an oligonucleotide in which the last 5' nucleotide is not G. Similarly, the reference detection probe can contain an oligonucleotide in which the last 5' nucleotide is not G.

[0250] The method of the present invention may include the use of mutation detection probes and reference detection probes. In such cases, preferably, the probes are differentially labeled, for example, such that one probe is bound or covalently linked to a detectable means, while the other is not. Both probes may be bound or covalently linked to different detectable means.

[0251] In one embodiment, the reference detection probe is labeled with a fluorophore at its 5' end and with a quencher at its 3' end. The fluorophore and quencher can be, for example, HEX and Black-Hole Quencher, respectively. In one embodiment, the mutation detection probe is labeled with a fluorophore at its 5' end and with a quencher at its 3' end. The fluorophore and quencher can be, for example, FAM and Black-Hole Quencher, respectively.

[0252] The PCR reaction may contain similar amounts of mutation detection probes and wild-type detection probes. However, in some embodiments, it is preferable to use an excess of mutation detection probes, especially when detecting PCR products after superpool PCR amplification. In such cases, the PCR reaction may include at least a 2-fold excess, more preferably at least a 4-fold excess, of the mutation detection probe compared to a reference detection probe.

[0253] In some implementations, the detection probe is Probes (Heid et al., 1996) utilize the 5' exonuclease activity of nucleic acid polymerases. This is why PCR reagents preferably include nucleic acid polymerases with 5' exonuclease activity (e.g., Taq polymerase). Typically, The probe can be a mutation detection probe as described above, or a reference detection probe as described above, covalently linked to a fluorophore / quencher pair. Therefore, the probe may contain a fluorophore, typically located at or near the 5' base. Furthermore, The probe may contain a quencher, which may be located at or near the 3' base and capable of quenching the fluorescence of the fluorophore [see Tyagi et al., Nature Biotechnology 16: 49-53 (1998)]. When irradiated, the excited fluorophore transfers energy to the nearby quencher instead of fluorescing [Forste or fluorescence resonance energy transfer (FRET)]. Therefore, the close proximity of the fluorophore and the quencher prevents any fluorescence emission while the probe remains intact. However, when The probe anneals to the internal region of the target sequence, and the polymerase replicates the binding on it. When the probe is templated, its 5′ exonuclease activity can cleave the probe. This series of events eliminates the quenching function, i.e., without FRET, the fluorophore begins to emit fluorescence, which can be measured by any useful means.

[0254] It is worth noting that, as emphasized above, the present invention also includes the ability of these methods to identify more than one different mutation. This can be achieved by using multiple sets of primers as described above. However, this can also be achieved by using several different detection probes. Therefore, in one embodiment, the method can be used to identify more than one different mutation of NOI in a target sequence. In this embodiment, the PCR reagent may comprise a reference detection probe and several mutation detection probes, wherein each mutation detection probe comprises an oligonucleotide of the same or complementary sequence to a mutation. Preferably, the reference detection probe and the mutation detection probes are linked to different detection methods. All mutation detection probes may be linked to the same detection method, similar detection methods, or different detection methods.

[0255] In some cases, the detection probe is a molecular beacon (MB), which is a probe containing a complementary sequence (also called a "stem") capable of self-hybridization, creating a "hairpin loop" structure. The loop of the MB may contain a sequence complementary to or identical to the NOI (or a mutant or reference NOI). Furthermore, the MB typically includes a fluorophore and a quencher at either end of the MB, such that they are close to each other when the probe hybridizes with itself. The MB can be a mutation detection probe or a reference detection probe as described above, covalently linked to the fluorophore / quencher pair and the stem sequence providing the complementary region of the MB. Further details regarding standard methods for preparing and using MBs have been given in the literature, and MBs are available from many commercial reagent sources.

[0256] In some cases, primers / probes are used; in others, primers / probes are Scorpions. TM The probe can provide a FRET-based stem-loop detection mechanism similar to MB, but with the difference that it also has a connecting segment that can be used as a forward or reverse primer (see Whitcombe et al., Nature Biotechnol. 1999, Aug 17(8): 804-7; U.S. Patent No. 6,326,145). Scorpions TM The probe can maintain its stem-loop configuration in the unhybridized state, and the fluorophore is quenched. Scorpions TM The probe can have a longer, multi-component structure, such as a 5' fluorophore, followed by a target-specific stem-loop moiety, then a quencher, then a blocking agent [e.g., hexanediol (HEG)], and finally a 3' primer sequence. The blocking agent prevents the product from extending backwards onto the probe.

[0257] In some cases, primers / probes are Sunrise. TM The probe comprises primers attached to a hairpin probe that extends during amplification. This setup separates the internal quencher from the 5' fluorophore (Nazarenko et al., Nucl. Acids Res. 1997, 25: 2516-2521).

[0258] Detection probes can be of any useful length, such as 10 to 60 nucleotides. Oligonucleotide probes can also be in the range of 10 to 30 nucleotides in length. The precise sequence and length of an oligonucleotide probe can depend in part on the nature of the target polynucleotide it binds to. The binding site and length can be modified to achieve appropriate annealing and unwinding properties under specific conditions. For example, detection probes can be designed to have the same unwinding temperature as the forward and / or reverse primers, such as within ±10°C, or even within ±5°C.

[0259] The 3' terminal nucleotide of the detection probe can be blocked or prevented from extending by a nucleic acid polymerase. This blocking can be conveniently achieved by attaching a detectable element to the terminal 3' base of the oligonucleotide probe via a ligation site, using a fluorophore or quencher.

[0260] The literature contains numerous practical guidelines describing useful fluorophore-quencher pairs, including methods for selecting fluorophore-quencher pairs, as follows:

[0261] -Clegg, Meth. Enzymol., 211:353-388 (1992); Wo et al., Anal. Biochem., 218:113 (1994); Pesce et al., eds., Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence A analysis: A practical approach (Marcel Dekker, New York, 1970); etc.

[0262] The literature also includes comprehensive lists of fluorescent and chromophore molecules, as well as references for selecting report molecule-quencher pairs and their relevant optical properties, such as Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd edition (Academic Press, New York, 1971); Griffiths, Color and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland, Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); and so on.

[0263] - In addition, there are extensive guidelines in the literature for deriving reporter molecules and quencher molecules through covalent linkage via common reactive groups that can be added to oligonucleotides, as shown in the following references: Haugland (cited above); Ullman et al., U.S. Patent No. 3,996,345; Khanna et al., U.S. Patent No. 4,351,760; etc.

[0264] The fluorophore and quencher can be selected, for example, from fluorescein and rhodamine dyes. These dyes, combined with appropriate ligation methods for linking to oligonucleotides, are described in numerous references, e.g., Khanna et al. (as described above); Marshall, Histochemical J., 7:299-303 (1975); Menchen et al., U.S. Patent No. 5,188,934; Menchen et al., European Patent Application 87310256.0; and Bergot et al., International Application PCT / US90 / 05565. The latter four are incorporated herein by reference.

[0265] The fluorophore / quencher pair can, for example, use a fluorophore such as EDANS or fluorescein at the 5′ end and a quencher such as Dabcyl at the 3′ end.

[0266] Detection of PCR amplification products may include a step of comparing the signal obtained from PCR amplification containing a given gDNA sample with the signal obtained from control PCR amplification. This signal is typically associated with a detectable means, such as a mutation detection probe and / or a wild-type detection probe. Therefore, if the probe is attached to a fluorophore, the signal may be fluorescence. Control PCR amplification may be PCR amplification performed under the same conditions but without a gDNA sample; that is, the control PCR amplification may lack a gDNA template, or it may only include control DNA containing only a reference target sequence, such as wild-type gDNA. In some embodiments, any signal stronger than the signal from control PCR amplification may be considered a positive signal, i.e., a signal indicating the presence of a target sequence containing one or more mutations of NOI.

[0267] In one implementation, any signal exceeding a given threshold can be considered a positive signal. The threshold can be determined by any useful means, typically using suitable software such as the Biorad QX200. TM DropletReader and Quantasoft TM software.

[0268] In an embodiment of the invention using a mutation detection reference probe, the fractional abundance between the signal obtained by the mutation detection probe and the signal of the reference probe can be determined and used to assess the presence of PCR products. The fractional abundance can be determined as: [(signal of the mutation detection probe)] divided by [(signal of the reference probe + signal of the mutation detection probe)].

[0269] For example, it can be assumed that the sample contains target DNA with a mutation including NOI, provided that it has the following characteristics compared to the control PCR amplification:

[0270] 1) Increased fractional abundance, and / or;

[0271] 2) Increased concentration of mutant droplets, and / or;

[0272] 3) The number of mutant events increased, 50% or more higher than the average.

[0273] Mutant droplets can be droplets that generate positive signals from mutation detection probes. In this paper, a "mutant event" is equal to the number of mutant droplets.

[0274] Divide the sub-pool into secondary sub-pools.

[0275] Once a subpool containing an organism carrying the NOI mutation or its reproductive portion has been identified, the method of the present invention includes the step of dividing the subpool into a plurality of secondary subpools.

[0276] As described above, a portion of the sub-pool has already been used to prepare gDNA samples. Therefore, only the remaining portion of the sub-pool can be used to divide it into secondary sub-pools. The present invention also includes dividing the sub-pool into several parts, with only one part used for preparing secondary sub-pools.

[0277] Each sub-pool may contain only one organism or its reproductive portion. Alternatively, each sub-pool may include multiple organisms or their reproductive portions. For example, each sub-pool may include multiple organisms or their reproductive portions representing multiple genotypes.

[0278] Typically, a subpool or a portion thereof is divided into multiple secondary subpools, preferably at least 5, more preferably at least 10, even more preferably at least 30, more preferably at least 50, even more preferably at least 70, and even more preferably at least 90. In principle, there is no upper limit to the number of secondary subpools. However, the pool is typically divided into a maximum of 50,000, for example, a maximum of 25,000, or even a maximum of 10,000 secondary subpools.

[0279] Each sub-pool may include one or more organisms or their reproductive portions representing multiple genotypes. Preferably, each sub-pool contains 1 to 100, more preferably 1 to 50, and even more preferably 1 to 20 organisms or their reproductive portions with different genotypes.

[0280] Secondary pools can be sorted in any desired manner. In some implementations, secondary pools contain only a limited number of organisms or their reproductive portions.

[0281] In other implementations, the secondary pool contains multiple organisms with the same genotype. This can be ensured by subjecting the secondary pool to a reproductive step. The reproductive step may also occur simultaneously with the step of dividing the pool into secondary pools, in a way that allows the offspring of a single organism or its reproductive portion to eventually enter the same secondary pool.

[0282] The method of the present invention includes preparing one or more gDNA samples from a secondary pool. In embodiments of the invention where the secondary pool contains only one or more organisms or their reproductive portions for each genotype, the gDNA samples are typically prepared from samples of each organism or its reproductive portions, as described in the "Preparation of DNA Samples" section above.

[0283] In embodiments where a secondary pool contains multiple organisms of a single genotype or their reproductive portions, gDNA samples can be prepared from a portion of the secondary pool.

[0284] Figure 1C The diagram provides an example of a method for dividing a sub-pool into secondary sub-pools, where the first step of the diagram shows the division of the sub-pools and the acquisition of gDNA samples.

[0285] In an embodiment of the invention where the species is a single-celled organism, the secondary pool can be prepared as follows:

[0286] - After identifying sub-pools containing NOI mutations, each single-celled organism in the sub-pool is allowed to proliferate individually, resulting in clonal cultures from each single-celled organism. This can be achieved by culturing colonies of each clone on a solid medium or by culturing each clone in a space separate from the liquid medium, such as in the wells of a microtube or microtiter plate. Secondary sub-pools can be formed by combining single-celled organisms of multiple clones;

[0287] - After identifying the sub-pool of interest, the single-celled organism can be immediately divided into secondary sub-pools, and each secondary sub-pool can be allowed to proliferate.

[0288] In one implementation, the secondary pool is prepared through the following steps:

[0289] i) Provide a sub-pool containing NOI mutations;

[0290] ii) to proliferate some or all of the organisms in the sub-pool in a clonal manner to obtain clonal cultures;

[0291] iii) Combining a subset of organisms from multiple said clonal cultures to obtain a secondary pool.

[0292] As described above, gDNA samples can be prepared from a portion of the organisms in the sub-pool while the remaining organisms in the sub-pool are stored. When the organisms are single-celled, they can be cryopreserved. In the described embodiment, step i) above may include incubating the organisms in a culture medium at a temperature sufficient to grow them to revive them. Preferably, the revival step involves only minimal reproduction.

[0293] Step ii) above can be performed, for example, by spreading the organisms on a solid culture medium at a sufficiently low titer to spatially separate the organisms from one another in the culture medium.

[0294] Step iii) above may include combining 10 to 1000, preferably 10 to 500, more preferably 10 to 100, for example 30 to 70 independent clonal culture fractions. Preferably, two backups are obtained for each sub-pool. This can be done by combining the fractions as described above and then dividing each sub-pool into at least two parts. Alternatively, two backups for each sub-pool can be prepared by combining fractions of the same clonal culture from two different containers from the beginning. Typically, one part of the sub-pool is used to prepare gDNA samples, while the other part is stored (e.g., frozen under a cryoprotectant). Before preparing gDNA samples and / or before storage, the sub-pools may undergo a proliferation step, for example, incubation in a culture medium at temperatures suitable for organism growth.

[0295] In an embodiment of the invention where the species is a plant, a secondary sub-pool can be prepared by obtaining a sample of each seed within a sub-pool and combining samples from a predetermined number of seeds. In this embodiment, it is important to sort the secondary sub-pools so that the seeds of a sub-pool and the samples of that sub-pool can be identified. Therefore, in one embodiment of the invention, the method includes the following steps:

[0296] - Provide a sub-pool containing multiple seeds of a plant, such as grains, wherein the sub-pool contains a mutation of NOI.

[0297] - Divide the seeds in the sub-pool into secondary sub-pools, each secondary sub-pool containing at least one seed, for example, in the range of 1 to 100 seeds.

[0298] - Take samples from each seed in the secondary pool in such a way that they are fully developed into plants, and combine all samples from all seeds in each secondary pool.

[0299] - Prepare gDNA samples from the combined samples.

[0300] The steps outlined above are in Figure 2D The upper part is shown, using grains as an example. Therefore, the method can include... Figure 2D The steps shown. Furthermore, Figure 2D The steps shown can be performed using any flowering plant, and are therefore not limited to grains. Furthermore, any number of mutant plants can be used to perform them. Figure 2D The steps shown ( Figure 2D The figures provided are just one example.

[0301] An example of a method for dividing a subpool into secondary subpools is described below in WS4.

[0302] Identify secondary sub-pools

[0303] The method of the present invention includes the following steps: dividing a sub-pool of an organism containing one or more mutations of NOI or its reproductive portion into a secondary sub-pool, and then identifying the secondary sub-pool, wherein the secondary sub-pool contains an organism containing a mutation specified by NOI or its reproductive portion.

[0304] The identification of the secondary sub-pool may include the following steps:

[0305] a) Prepare gDNA samples, each sample containing gDNA from each genotype in a sub-pool, as described in the “Preparation of DNA Samples” section above;

[0306] b) Perform multiple PCR amplifications to amplify the target sequence;

[0307] c) Detecting PCR amplification products containing NOI mutations to identify the secondary pool, as described, for example, in the “PCR amplification product detection” section below.

[0308] The PCR amplification described above can be any PCR amplification of the target sequence. Therefore, the PCR amplification can be conventional PCR amplification or PCR amplification that includes the above-described multiple compartmentalized PCR amplifications.

[0309] Typically, each PCR amplification includes a gDNA sample from a secondary pool, a set of primers flanking the target sequence, and PCR reagents. The primers can be any of the primers described in the "Primers Flanking the Target Sequence" section above. The PCR reagents can be any PCR reagents described in the "PCR Reagents" section above. Specifically, the PCR reagents contain nucleotides and a nucleic acid polymerase. Preferably, the PCR reagents also contain one or more detection probes, such as mutation detection probes and / or reference detection probes, as described in the "Detection of PCR Products" section above.

[0310] For example, secondary pools can be directly identified, provided that PCR amplification products containing the NOI mutation can be detected directly after or during the PCR amplification process. This can be done, for example, in cases where the PCR amplification includes a detection method that generates a detectable signal, provided that the PCR amplification product includes a target sequence containing the NOI mutation. Such detection methods are described in more detail in the "Detection of PCR Amplification Products" section above and may include, for example, probes for mutation detection.

[0311] exist Figure 1C The lower part shows an example of a method for identifying secondary pools containing the aforementioned mutations, illustrating the following steps:

[0312] - Provides secondary sub-pools;

[0313] - Prepare a sample from a portion of the secondary sub-pool;

[0314] - Prepare gDNA samples from the samples;

[0315] - Prepare PCR amplification using all individual gDNA samples from all sub-pools, for example, in individual wells of a plate, such as in a microtiter plate;

[0316] - Detect the presence of target sequences containing NOI mutations.

[0317] Figure 2D A more specific example of a method for identifying secondary pools is shown. In this example, the species under discussion is a cereal plant. Figure 2D The specific figures provided are merely examples, and those skilled in the art will understand that the method can be implemented with an additional number of grains.

[0318] It is worth noting that, as outlined in WS4 below, secondary pools containing NOI-specific mutations can be identified.

[0319] Identifying organisms

[0320] Once a secondary pool containing an organism or its reproductive portion is identified, the method includes identifying organisms containing mutations of NOI.

[0321] It can be accomplished in a variety of ways, depending on the species and the sub-pools.

[0322] In an embodiment of the invention where the secondary sub-pool contains only one organism or its reproductive portion, the organism or its reproductive portion is identified once the relevant secondary sub-pool is identified.

[0323] In embodiments of the invention where the secondary pool contains more than one organism or its reproductive portion, the method typically includes an organism identification step. Prior to this, a proliferation step may be performed on the secondary pool containing the NOI mutation, either simultaneously with or after the identification of the organism or its reproductive portion.

[0324] In embodiments of the invention where the species is a single-celled organism, the proliferation step can be clonal expansion of the organism, for example, cloning each organism in a secondary pool in a spatially segregated manner to achieve clonal expansion of the organism. Therefore, the method may include the following steps:

[0325] i) Provide a secondary pool containing mutations of NOI;

[0326] ii) to proliferate some or all of the organisms in the sub-pool in a clonal manner;

[0327] iiii) Identify which clones include organisms with NOI mutations.

[0328] Step ii) above can be performed, for example, by spreading the organisms on a solid culture medium at a sufficiently low titer to spatially separate the organisms from one another in the culture medium.

[0329] Step iii) above may involve preparing a gDNA sample from a portion of each clone and performing PCR amplification, for example, PCR amplification as basically described in the “Identification of Secondary Sub-pools” section above.

[0330] In an embodiment of the invention where the species is a plant and the secondary pool contains seeds of the plant, the method may include the following steps:

[0331] A. Identify secondary pools containing organisms with NOI mutations or their reproductive portions;

[0332] B. Cultivate all seeds in the secondary seed pool to allow germination and optionally grow plants from each seed;

[0333] C. Take a sample from each germinated seed;

[0334] D. Detecting the presence of the NOI mutation in the sample, wherein the detection can be performed by any method, such as by preparing a gDNA sample from the sample and performing PCR amplification as described above.

[0335] This led to the identification of a plant carrying a NOI mutation.

[0336] In step B above, the seed may, for example, germinate and be allowed to develop into a small plantlet containing roots, stems, and leaves. In the embodiment described, the sample obtained in step C may be, for example, a leaf, root, or part thereof of the small plantlet, such as a portion of the first leaves. After obtaining the sample, the germinated seed or small plantlet may grow to maturity. The invention also includes the plant growing to maturity, in which case the sample obtained in step C may be, for example, a leaf, flower, root, seed, stem, or part thereof.

[0337] After identifying a single organism or its reproductive portion containing one or more NOI mutations, the organism or its reproductive portion typically undergoes one or more proliferation steps to obtain multiple organisms containing the mutations.

[0338] In an embodiment of the invention where the identified organism is heterozygous relative to the mutation in question, the method may include the step of cultivating the organism to be homozygous relative to the mutation.

[0339] After identifying an organism containing the NOI mutation, the method of the present invention may include a step of further breeding the organism to retain the NOI mutation. The purpose of this breeding is to combine the trait conferred by the NOI mutation with traits from other organisms of the same species.

[0340] This method may also include additional steps to verify the presence of mutations in the NOI. For example, the target sequence or the gene containing the target sequence may be sequenced in its entirety.

[0341] Super Pool

[0342] In one embodiment of the invention, the method includes the step of identifying a set of sub-pools containing mutations in NOI, also referred to herein as superpools. In this way, a very large number of potential organisms or their reproductive portions can be screened to determine the presence of NOI mutations. This step is preferably performed after the pool has been divided into sub-pools and gDNA samples have been prepared from the sub-pools, for example, after step c) of the method of the invention.

[0343] Therefore, the method includes performing the following steps after step c):

[0344] - Prepare a portion of each gDNA sample from each sub-pool;

[0345] - Combine multiple sub-pools into a super-pool to obtain a super-pool gDNA sample, which contains gDNA from multiple sub-pools;

[0346] - Perform multiple PCR amplifications, each PCR amplification containing a gDNA sample from a superpool, wherein each PCR amplification includes multiple compartmentalized PCR amplifications, each compartmentalized PCR amplification containing a portion of the gDNA sample, a set of primers located flanking the target sequence, and PCR reagents to amplify the target sequence, wherein the PCR is performed, for example, as described in the "PCR amplification in a superpool" section below.

[0347] - Detect PCR amplification products containing target sequences with NOI mutations to identify superpools containing said mutations, wherein said detection steps are performed, for example, as described in the "Detection of PCR Products" section above.

[0348] In some implementations, it is preferable to enrich the gDNA sample from the superpool. This can facilitate the specific detection of target sequences containing NOI mutations. The enrichment step may include the following steps:

[0349] - Provide gDNA samples from the superpool, which contain gDNA from the multiple subpools mentioned above;

[0350] - Perform PCR amplification, each PCR amplification containing a superpool of gDNA sample, wherein each PCR amplification includes a set of primers flanking the target sequence, blocking probes, and PCR reagents.

[0351] The PCR amplification performed during the enrichment step is typically routine PCR amplification and can be performed as described in the “PCR” section below. Typically, this PCR involves a relatively small number of cycles, such as 10 to 30, or even 15 to 25 PCR cycles.

[0352] Blocking probes are typically designed to inhibit the amplification of a target sequence containing a reference NOI. Therefore, blocking probes can be, for example, oligonucleotides, which:

[0353] • Cannot be extended at the 3' end by DNA polymerase;

[0354] • Prioritize binding to target sequences containing a reference NOI or its complementary sequence, rather than target sequences containing a predetermined NOI mutation.

[0355] Blocking probes typically contain 10 to 30 nucleotides. Specifically, blocking probes may comprise a continuous sequence of 10 to 30 nucleotides that is identical to or complementary to the target sequence containing the reference NOI. Furthermore, blocking probes are typically linked to an inhibitor that inhibits probe elongation via DNA polymerase.

[0356] The blocking agent can be, for example, the portion covalently linked to the 3'most nucleotide of the blocking probe. In fact, most 3' modifications will block the extension. For example, the blocking agent can be selected from the group consisting of a 2',3'-dideoxy C spacer, a 3' dd C, and a 3' reverse d T. The blocking agent can also be a modification of the terminal 3' hydroxyl group, for example, having an amino group (e.g., 3' amino) or an alkyl group, such as a 3' C3 spacer. The blocking agent can also be a phosphorylation of the 3' nucleotide.

[0357] Once a superpool containing mutations of one or more NOIs is identified, step d) and subsequent steps can be performed. However, step d) may be limited to preparing PCR amplification using gDNA samples from subpools contained in the superpool.

[0358] After preparing the sub-pool in step b), the identification of the super-pool can also be performed directly. In this case, the method may include the following steps after step b):

[0359] - If the subpool contains only a few of each genotype, such as only one organism or its reproductive portion, then the subpool can subsequently undergo a proliferation step;

[0360] - Prepare a portion of each subpool, which represents each genotype within the subpool, while the remainder of the subpool also represents each genotype within the subpool;

[0361] - Combine multiple parts into a super pool, thereby obtaining a super pool containing an organism or its reproductive parts from multiple sub-pools, wherein the organism or its reproductive parts exist in only one super pool;

[0362] - Prepare gDNA samples, each sample containing gDNA from each genotype within the superpool, which may be performed, for example, as described in the "Preparation of DNA Samples" section above;

[0363] - Perform multiple PCR amplifications, each PCR amplification containing a superpool of gDNA sample, wherein each PCR amplification includes multiple compartmentalized PCR amplifications, each compartmentalized PCR amplification containing a portion of the gDNA sample, a set of primers located flanking the target sequence, and PCR reagents to amplify the target sequence, wherein the PCR is performed, for example, as described in the "PCR amplification of superpool" section below.

[0364] - Detect PCR amplification products containing a target sequence that includes a mutation in NOI, thereby identifying superpools containing said mutation.

[0365] In the described embodiment, it is not important that the gDNA samples of the superpool are prepared in a manner that preserves the reproductive potential of organisms of each genotype within the superpool. This is generally the expected result, since each superpool contains DNA from multiple subpools, and each subpool contains an organism (or its reproductive portion) representing each genotype within the subpool.

[0366] Once a superpool containing the NOI mutation is identified, step c) and subsequent steps can be performed. However, step c) may be limited to preparing gDNA samples from the subpools included in the superpool.

[0367] Any desired number of superpools can be prepared, for example, in the range of 5 to 100, or in the range of 5 to 50, or in the range of 5 to 20.

[0368] Figure 1D An example of a method used to identify superpools is shown. Another example, where the species is a grain, such as... Figure 2E As shown. It should be understood that, Figure 2E The numbers provided are merely examples, and the method can be performed with other numbers of grains, etc.

[0369] Non-limiting examples of the preparation and identification of supercells are also described below in WS5.

[0370] PCR amplification in superpool

[0371] The method of the present invention includes the step of performing multiple PCR amplifications, each PCR amplification comprising a gDNA sample from a superpool, such as that prepared in the above section, wherein each PCR amplification comprises multiple compartmentalized PCR amplifications, each compartmentalized PCR amplification comprising a portion of the gDNA sample, a set of primers located flanking the target sequence, and PCR reagents, thereby amplifying the target sequence.

[0372] Typically, the PCR can be performed as described in the "PCR amplification containing multiple compartmentalized PCR amplification" section above, but preferably, with the exceptions described below:

[0373] As described above, compartmentalized PCR amplification can be contained within droplets. High sensitivity is crucial for superpool PCR amplification, while lower sensitivity is required for subpool PCR amplification. Although high-sensitivity PCR amplification typically requires a large amount of reagent, lower-sensitivity PCR amplification requires a smaller amount. Therefore, superpool PCR amplification preferably has the sensitivity to detect at least 200,000, more preferably at least 250,000 reference NOIs, including one or more mutant NOIs.

[0374] For PCR amplification in a superpool, it may be preferable that each droplet has a very small volume, for example, in the pL range. Therefore, it is preferable that the average volume of the droplets is 0.1 to 10 μL.

[0375] Each PCR amplification can be compartmentalized into any suitable number of compartments. However, in a preferred embodiment, each PCR amplification is compartmentalized into 200,000 to 100,000,000 compartments (e.g., droplets). For example, each PCR amplification can be compartmentalized into 500,000 to 50,000,000 compartments (e.g., droplets), or, for example, into 1,000,000 to 10,000,000 compartments (e.g., droplets). Preferably, each PCR amplification is compartmentalized into at least 1,000,000, more preferably at least 3,000,000, and even more preferably at least 5,000,000, such as at least 7,000,000 compartments (e.g., droplets).

[0376] - In some cases, droplets are generated using commercially available droplet generators, such as Raindance Technologies' RainDrop Digital PCR System, which can also be used to detect PCR amplification products.

[0377] Once the compartmentalized PCR reaction is prepared, it can be amplified as described in the “PCR” section below.

[0378] The presence of a target sequence containing the mutant NOI can be detected as described in the "Detection of PCR Products" section above.

[0379] In one embodiment, detection is preferably performed using a reference detection probe and a mutation detection probe as described in the "Detection of PCR Products" section, wherein an excess of the mutation detection probe is used. Preferably, the PCR reaction contains at least 2 times, more preferably at least 4 times, an excess of the mutation detection probe compared to the reference detection probe. Alternatively, the mutation detection probe may be used without the reference detection probe.

[0380] PCR

[0381] The methods described herein include multiple steps for performing PCR amplification, including compartmentalized PCR amplification, such as the PCR amplification described in the "PCR amplification comprising multiple compartmentalized PCR amplification" section or the "PCR amplification in a superpool" section.

[0382] Here, PCR amplification typically includes the following steps:

[0383] 1) Preparation of PCR amplification, which includes a gDNA sample, a set of primers located on the flanking side of the target sequence, and PCR reagents;

[0384] 2) Perform PCR amplification, which typically includes the following steps:

[0385] - Incubate PCR amplification at denaturing temperatures, such as 85 to 100°C, or 90 to 98°C, for a time sufficient to denature the double-stranded DNA, such as 15 to 30 minutes, or 30 seconds to 5 minutes.

[0386] - Implement multiple loops, such as 10 to 60 loops, or 20 to 40 loops, including the following steps:

[0387] - Incubate at the annealing temperature to allow annealing between the primer and target DNA, for example, 15 seconds to 2 minutes, or 30 seconds to 1 minute. Typically, the annealing temperature is similar to or lower than the primer's melting temperature, for example, 45 to 75°C. Incubation at the annealing temperature can also elongate the primer;

[0388] -Optionally, incubate at an extension temperature where the nucleic acid polymerase is active, such as 55 to 75°C for 15 seconds to 2 minutes, such as 30 seconds to 1 minute;

[0389] - Incubate at denaturing temperatures, such as 85 to 100°C or 90 to 98°C, for 15 seconds to 2 minutes, such as 30 seconds to 1 minute;

[0390] - Incubate at the extended temperature of DNA polymerase, for example, 55 to 98°C, for example, 15 to 20 seconds, or for example, 1 to 15 minutes.

[0391] Before the amplification step, such as before step 2) in the previous list, the PCR can be divided into multiple spatially separated compartments to obtain compartmentalized PCR amplification. This can be achieved, for example, by generating droplets, such as by adding oil to generate droplets and preparing the droplets in a droplet generator.

[0392] Barley plants carrying mutations in the GS1-3 gene

[0393] In one embodiment, the present invention relates to barley plants carrying mutations in a gene encoding glutamine synthase 1, particularly isotype 3 (GS1-3). The gene may also be referred to as the HvGS1-3 gene, the protein-coding sequence of which is shown in SEQ ID NO: 1 herein, and the amino acid sequence encoding the HvGS1-3 enzyme as shown in SEQ ID NO: 2.

[0394] Preferably, the mutation refers to a mutation within the gene that confers a sequence encoding a mutant HvGS1-3 enzyme with reduced activity. The activity of HvGS1-3 is one or more of the following:

[0395] i. Catalytic condensation of ammonium;

[0396] ii. Catalyzes the condensation of ammonia and glutamic acid into glutamine;

[0397] iii. Catalyzes the synthesis of glutamate, hydroxylamine, and ATP into gamma-hydroxyoxime.

[0398] Specifically, the activity of HvGS1-3 can be as described in iii. Here, the activity can be determined, for example, according to Example 19 in the "In Vitro Activity Assay of Recombinant HvGS1-3" section below.

[0399] The mutant HvGS1-3 protein preferably exhibits activity lower than that of the wild-type protein. However, the enzyme should preferably retain at least some activity. Specifically, when detected as described in Example 19, the k... cat (min-1) is the wild-type HvGS1-3 protein k cat 1 to 20% of (min-1), for example, 2 to 10%.

[0400] The wild-type HvGS1-3 enzyme is considered to be a decameric consisting of two rings, each composed of five subunits. In one embodiment, the mutant HvGS1-3 protein contains a mutation at the amino acid residues located at the interface between the two rings. Specifically, the mutation may be a mutation of amino acid residues selected from SEQ ID NO: 2, specifically amino acid residues 141, 235, 285, 287, and 290.

[0401] Therefore, barley plants can contain mutations in the gene encoding HvGS1-3, which encodes a mutated HvGS1-3 protein carrying a mutation in one of the aforementioned amino acid residues.

[0402] Specifically, the mutant HvGS1-3 protein can be a protein with a mutation at amino acid residue 287 of SEQ ID NO: 2, for example, a mutation from Gly to any other amino acid residue, such as any other naturally occurring amino acid residue. The mutation can also be a mutation at amino acid residue 287 of SEQ ID NO: 2, i.e., a mutation from Gly to a polar or charged amino acid residue, for example, an amino acid residue selected from the group consisting of Arg, Lys, Asp, Glu, Gln, Asn, His, Ser, Thr, Tyr, Cys, Met, and Trp, or an amino acid residue selected from the group consisting of Arg, Lys, Asp, Glu, Tyr, Phe, Met, and Trp. The mutation can also be a mutation at amino acid residue 287 of SEQ ID NO: 2 from Gly to a charged residue, for example, an amino acid residue selected from the group consisting of Arg, Lys, Asp, and Glu. Specifically, the mutation can also be a mutation at amino acid residue 287 of SEQ ID NO: 2 from Gly to Asp.

[0403] Therefore, a mutation in the HvGS1-3 gene can be any mutation that results in the HvGS1-3 gene encoding one or more of the above-described mutations. In one embodiment, the gene mutation can be one or more mutations in nucleotides 859, 860, and 861, thereby obtaining a codon encoding one of the above-described amino acid residues. In one example, the mutation is a gene mutation in nucleotide 860 from G to A.

[0404] In one embodiment, the mutated HvGS1-3 protein can be a protein carrying a mutated 235th amino acid residue of SEQ ID NO: 2, for example, a mutation from Asp to any other amino acid residue. The mutation can also be a mutation of the 235th amino acid residue of SEQ ID NO: 2 from Asp to a positively charged residue, for example, an amino acid residue selected from Arg and Lys. Therefore, the mutation in the HvGS1-3 gene can be any mutation in the HvGS1-3 gene that results in a HvGS1-3 protein encoding any of the above-described mutations.

[0405] In one embodiment, the mutated HvGS1-3 protein can be a protein carrying a mutation at amino acid residue 285 of SEQ ID NO: 2, for example, a mutation from Gly to any other amino acid, such as any other naturally occurring amino acid. The mutation can also be a mutation at amino acid residue 285 of SEQ ID NO: 2 from Gly to a polar or charged amino acid, such as an amino acid residue selected from the group consisting of Arg, Lys, Asp, Glu, Gln, Asn, His, Ser, Thr, Tyr, Cys, Met, and Trp. Therefore, the mutation in the HvGS13 gene can be any mutation in the HvGS1-3 gene that results in encoding the aforementioned mutated HvGS1-3 protein.

[0406] In one embodiment, the mutant HvGS1-3 protein can be a protein carrying a mutation at amino acid residue 290 of SEQ ID NO: 2, for example, a mutation from Arg to any other amino acid residue. The mutation can also be a mutation at amino acid residue 290 of SEQ ID NO: 2, from Arg to a negatively charged amino acid residue, such as an amino acid residue selected from Asp and Glu. Therefore, the mutation in the HvGS13 gene can be any mutation in the HvGS1-3 gene that results in encoding the mutant HvGS1-3 protein described above.

[0407] In one embodiment, the mutant HvGS1-3 protein can be a protein carrying a mutation at amino acid residue 141 of SEQ ID NO: 2, for example, a mutation from Trp to any other amino acid residue. The mutation can also be a mutation at amino acid residue 141 of SEQ ID NO: 2 from Trp to any other naturally occurring amino acid, other than Tyr or Trp. Therefore, the mutation in the HvGS13 gene can be any mutation in the HvGS1-3 gene that results in encoding any of the mutant HvGS1-3 proteins described above.

[0408] This application also relates to the following implementation schemes:

[0409] 1. A barley plant having a mutation in a gene encoding HvGS1-3, wherein the mutant gene encodes a mutant HvGS1-3 protein with reduced enzyme activity.

[0410] 2. In barley plants according to implementation plan 1, regarding the synthesis of glutamyl hydroxamic acid from glutamate, hydroxylamine, and ATP, the mutated HvGS1-3 protein has a k cat The value of k is wild-type HvGS1-3 cat 1% to 20%.

[0411] 3. A barley plant according to any one of embodiments 1 to 2, wherein the HvGS13 form carries a mutation in the 287th amino acid residue of SEQ ID NO: 2.

[0412] 4. Malt prepared from barley plants according to any one of implementation schemes 1 to 3.

[0413] 5. A beverage prepared from barley plants according to any one of embodiments 1 to 3.

[0414] 6. A method for producing a beverage, the method comprising the following steps:

[0415] i. Provide barley grains from barley plants according to any one of implementation schemes 1 to 3;

[0416] ii. Optionally, at least a portion of the wheat grains are malted to obtain malt;

[0417] iii. Prepare an extract of the barley and / or the barley malt;

[0418] iv. Process the extract into a beverage.

[0419] 7. The method according to embodiment 6, wherein the beverage is beer.

[0420] Wheat plants carrying mutations in the GASR7 gene

[0421] In one embodiment, the present invention relates to a wheat plant carrying a mutation in a gene encoding GASR7 on the A genome. The gene may also be referred to as TaGASR7-A1, and its sequence is available in GenBank under NCBI number KJ000052, and its coding sequence is provided herein as SEQ ID NO: 25. The amino acid sequence of GASR7 is provided as SEQ ID NO: 8.

[0422] Preferably, the mutation is a mutation in the gene that imparts a sequence encoding a mutant GASR7 enzyme with reduced activity. More preferably, the mutation is a mutation that results in the complete loss of GASR7 function. Complete loss of GASR7 function can be, for example, the absence of the GASR7 polypeptide. Loss of GASR7 function can also lead to an increase in grain length.

[0423] Specifically, the preferred mutation is one that results in a mutation in the GASR7 gene encoding a truncated form of GASR7, wherein the truncated form of GASR7 contains up to 91 amino acids, such as the up to 91 consecutive amino acids of SEQ ID NO: 8. For example, wheat plants may contain mutations in the GASR7 gene that result in prematurely formed stop codons, such as mutations that result in a stop codon at the codon encoding Trp. In one embodiment, wheat plants may contain mutations in the GASR7 gene that result in prematurely formed stop codons at codon 91 (nucleotides 271-273) of SEQ ID NO: 25 or at any codon closer to the 5′ end.

[0424] In one implementation, the wheat plant may contain a mutation in the GASR7 gene that results in the premature formation of a stop codon at the position encoding the 91st amino acid residue of SEQ ID NO: 8.

[0425] This application also relates to the following implementation schemes:

[0426] 8. Wheat plants carrying mutations in the gene encoding GASR7.

[0427] 9. A wheat plant according to implementation scheme 8, wherein the wheat plant carries a mutation in the gene encoding GASR7 that results in the premature formation of a stop codon.

[0428] Plant products and their production methods

[0429] In one embodiment, the present invention relates to plant products from barley plants carrying mutations in the HvGS1-3 gene, such as any barley plant described in the "Barley Plants Carrying GS1-3 Gene Mutations" section above. The plant product can be the plant itself or a part thereof. For example, the plant product can be barley grains.

[0430] In one embodiment, the plant product is a malt composition. The malt composition may comprise, or consist of, a germinated barley plant or a portion thereof, such as a germinated barley grain, i.e., a barley grain from a barley plant carrying a mutation in the HvGS13 gene, such as any barley plant described in the "Barley Plants Carrying GS1-3 Gene Mutations" section above.

[0431] Germinated barley grains are barley grains that have undergone a germination process followed by a drying process. The malt composition may contain or consist of processed malt, for example, it may be "milled malt" or "flour". Therefore, the malt composition can be prepared by a method comprising the following steps:

[0432] - Soak grains, such as barley grains;

[0433] - To make grains germinate;

[0434] - Dry the germinated grains, for example, by drying them in a kiln at high temperatures.

[0435] This invention also relates to wort prepared from barley plants carrying mutations in the HvGS1-3 gene, such as any of the barley plants described in the "Barley Plants Carrying GS1-3 Gene Mutations" section above. Wort is a hydrous barley extract that can be prepared, for example, by crushing malt, ungerminated barley grains, and / or adjuvants. "Adjuvants" should be understood to include any source of carbohydrates other than malt, such as, but not limited to, cereals (e.g., barley, wheat, corn, or rice) – as whole grains or processed products such as coarse grains, syrup, or starch. All of the above adjuvants can be used primarily as an additional source of the extract (syrup is typically added after crushing). The malt is typically ground before crushing. Larger adjuvants, such as whole grains, are also typically milled.

[0436] Ungerminated barley grains lack or contain only limited amounts of enzymes beneficial for wort production, such as enzymes capable of degrading cell walls or depolymerizing starch into sugars. Therefore, in embodiments of the invention where ungerminated barley is used for pounding, it is preferable to add one or more suitable exogenous brewing enzymes to the wort. Even in embodiments of the invention where cereal malt is used for wort production, exogenous enzymes may be added during the pounding process. Suitable enzymes may be lipases, starch-degrading enzymes (e.g., amylase), glucanases [preferably (1-4)- and / or (1-3, 1-4)-β-glucanases], and / or xylanases (e.g., arabinoxylanase), and / or proteases, or enzyme mixtures containing one or more of the above enzymes, such as Cereflo, Ultraflo, or Ondea Pro (Novozymes).

[0437] Typically, the milled malt and / or barley grains and optional adjuvants are mashed by incubating with water at a predetermined elevated temperature. Mashing is usually carried out at a temperature of 40 to 80°C. The incubation temperature is typically either kept constant (isothermal mashing) or gradually increased. For example, it can be carried out sequentially. If the gelation temperature is higher than that typically observed during normal malt saccharification, the starch may gel and liquefy before being added to the malt mash. In either case, soluble substances in the malt / barley / adjuvants are released into the liquid portion. Subsequent filtration separates the wort from residual solid particles, also known as “waste wort.” The resulting wort can also be referred to as “primary wort.” During the “instruction washing of the waste wort” process, additional liquid, such as water, may be added to the waste wort. After washing the waste wort and filtration, “secondary wort” is obtained. Other worts can be prepared by repeating this process. Briggs et al. (ibid.) and Hough et al. (ibid.) describe non-limiting examples of suitable methods for preparing wort.

[0438] After crushing and / or washing the malt, the wort is brought to a boiling step—optionally in the presence of other compounds such as hops—to obtain boiled wort.

[0439] The wort according to the invention can be primary wort, secondary wort, other worts, or combinations thereof, as well as any of the boiling methods described above.

[0440] In a preferred embodiment of the invention, the plant product is a beverage made from a barley plant, such as barley-based beverages, including barley-based alcoholic and non-alcoholic beverages, wherein the barley plant has a mutation in the gene encoding HvGS1-3. For example, the grain-based alcoholic beverage could be beer or spirits.

[0441] The beer can be any kind of beer, such as lager or malt beer. Therefore, the beer can be selected from, for example, Altbier, Amber ale, Barley wine, Berliner weisse, Bière de Garde, Bitter, Blonde ale, Bock, Brown ale, California Common, Cream ale, Dortmunder Export, Doppelbock, Dunkel, Dunkelweizen, Eisbock, Fruitlambic, Golden ale, Gose, Gueuze, Hefeweizen, Helles, India pale ale, and Köln beer. Lambic beer, Light ale, Maibock, Maltliquor, Mild beer, March beer Old ale, Oud bruin, Pale ale, Pilsner, Porter, Redale, Roggenbier, Saison, Scotch ale, Steam beer, Stout, Schwarzbier, Lager, Witbier, Weissbier, and Weizenbock.

[0442] The distilled spirit can be any kind of distilled spirit. In particular, the distilled spirit can be based on barley plants carrying mutations in the HvGS1-3 gene, such as malted grains, such as barley malt. Non-limiting examples of such distilled spirits include whiskey and vodka.

[0443] The beverage can be a non-alcoholic beverage, such as a barley-based non-alcoholic beverage, like non-alcoholic beer or a non-alcoholic malt beverage, such as maltina.

[0444] Beverages can be prepared by any of the methods described below, for example.

[0445] Methods of producing beverages

[0446] In one embodiment, the present invention relates to a method for producing beverages. These methods may include the following steps:

[0447] - Provide barley plants carrying mutations in the HvGS1-3 gene, such as any mutations described in the "Barley Plants Carrying Mutations in the GS1-3 Gene" section above;

[0448] -Prepare an aqueous extract of the plant or a portion thereof;

[0449] - Optionally, the aqueous extract may be further processed into a beverage.

[0450] Therefore, in one embodiment of the present invention, a method for producing a beverage is provided, the method comprising the following steps:

[0451] a) Provide barley grains from barley plants carrying mutations in the gene encoding HvGS13, such as any mutations described in the “Barley Plants Carrying Mutations in the GS1-3 Gene” section above;

[0452] b) Optionally prepare a malt composition of at least a portion of the wheat grains to obtain a malt composition;

[0453] c) Prepare an aqueous extract of the barley and / or malt composition;

[0454] d) Process the extract into a beverage.

[0455] Step b) can be performed as described in the "Plant Products and Methods of Production Thereof" section above. And step c) can be, for example, a step of preparing wort, i.e., the aqueous extract can be wort. The wort can be any wort described in the "Plant Products and Methods of Production Thereof" section above; it can also be prepared according to the description in that section.

[0456] Step d) may include the step of fermenting the aqueous extract, for example, fermenting the wort with yeast. Step d) may include the following steps:

[0457] di) Heating extracts (e.g., in the presence of other ingredients such as hops);

[0458] d-ii) Extracts or heated wort fermented in the presence of yeast;

[0459] d-iii) Optionally add one or more other ingredients,

[0460] This is how beer is produced.

[0461] The other components may be, for example, CO2 or aromatic compounds.

[0462] Methods of producing beer are well known in the art, and therefore any conventional method of beer production involving the use of the barley plant of this invention as a starting material can be found, for example, in detailed descriptions of suitable methods for malting and brewing, including publications by Briggs et al. (1981) and Hough et al. (1982). Numerous regularly updated methods for analyzing barley, malt, and beer products exist, such as, but not limited to, those of the American Association of Cereal Chemists (1995), the American Society of Brewing Chemists (1992), the European Brewery Convention (1998), and the Institute of Brewing (1997). It is recognized that a given beer undergoes many specific procedures, with the most significant variations involving local consumer preferences. Any method of beer production can be used in this invention.

[0463] This application also relates to the following implementation schemes:

[0464] 10. A yeast carrying a mutation in the FDC1 gene, said mutation resulting in complete loss of fdc1 activity, wherein the mutation results in the formation of a stop codon at the codon encoding Trp.

[0465] 11. The yeast according to embodiment 10, wherein the yeast is Saccharomyces cerevisiae and the Saccharomyces cerevisiae carries a mutation that results in the formation of a stop codon at the codon encoding Trp159 of Saccharomyces cerevisiae ScFDC1.

[0466] 12. A method for producing a beverage, the method comprising the following steps:

[0467] i. Provide wort;

[0468] ii. Fermenting the wort with yeast according to any one of embodiments 10 to 11;

[0469] iii. Optionally, the fermented wort may be further processed into a beverage.

[0470] sequence list

[0471]

[0472]

[0473] Unless otherwise stated, all GenBank login numbers are for the GenBank database version as of July 1, 2016. Example

[0474] The invention is illustrated by way of description and examples in WS and should not be construed as limiting the invention. Unless otherwise stated, basic biochemical and molecular biological techniques are employed to process nucleic acids, proteins (including enzymes), and organisms.

[0475] All workflows described below include, for example, the specific quantity and concentration of grains used, and the specific PCR conditions. Those skilled in the art will be able to adjust the specific embodiments provided to use different quantities of grains, different concentrations, different PCR conditions, etc.

[0476] WS1: Preparation of randomly mutated cereal grains

[0477] Step 1.1: Mutagenesis Method

[0478] To induce mutations, according to details provided by Kleinhofs et al. (1978) and K. Breddam et al., U.S. 7,838,053, grains collected from barley plants are incubated in a solution of NaN3 mutagen. This method induces point mutations in the gDNA of barley grains, typically conferring randomly distributed codons for amino acid substitutions or translation termination of protein-coding DNA, resulting in protein changes and truncation in the protein encoded by the mutagenized DNA. However, the method of the present invention can also be used to produce cereal plants with point mutations in non-protein-coding regions of gDNA, such as promoters, terminators, and introns.

[0479] A total of 500g of mutated grains (all M0 generation) were sown in a 7.5m... 2 In some cases, M1 generation grains reproduce in the field, eventually producing M2 or M3 generation mutant plants (see [link to relevant documentation]). Figure 2A The mutation frequency in M3 generation grains is expected to be 0.9–2.3 per 10,000 grains (see Kleinhofs et al., ibid.).

[0480] WS2: Preparation of Ordered Grain Pools from Mutant Cereal Plants

[0481] Step 2.1: Harvest cereal crops and thresh the ears.

[0482] Each 7.5m in the field 2 The “plots” (FP#01, FP#02, etc.; see also) Figure 2B It is divided into several "small plots" (FSP#01, FSP#02, etc.); Figure 2B (The action marked as 1), each 0.45m2 It contains 300 individual plants. All ears of grain are harvested manually from a small plot using a sickle and then collected in individual bags. The contents of each bag are threshed separately to obtain bags labeled "Total Grains," each bag containing ~6000 grains (GT#01, GT#02, etc.); Figure 2B (The action marked as 2).

[0483] For the purpose of clarity and not as a limitation, the following embodiments detail the relevant topics of step 2:

[0484] Example 1: Growing barley in a “field”;

[0485] Example 2: Harvesting grains from a single “plot”.

[0486] WS3: Determine whether the library sample contains mutant grains.

[0487] Step 3.1: Divide the individual "Total Grain Count" grain bin into two parts.

[0488] The threshed grains in a single "Total Grain" bag were divided into two parts, with the "Total Sub-Grain" sample consisting of approximately 1500 grains (labeled as SGT#01, SGT#02, etc.; see [link]). Figure 2C Action 1), the processing of which is detailed in steps 3.2 to 3.4 below. Broadly speaking, the grain processing in the “Total Grain Count” section seeks to determine which portion of the grains contains the mutation of interest. For clear reasons, but unrelated to the actions in step 3, the remaining portion of the “Total Grain Count” consists of approximately 4500 grains, which are processed as detailed in the description relating to step 4.

[0489] For the purpose of clarity and not as a limitation, the relevant topics of step 3.1 are described in detail in the following embodiments:

[0490] Example 3: Description of grains in "Total Grain Quantity".

[0491] Step 3.2: Prepare flour samples from grains of "total grain size".

[0492] A sample containing 1500 grains per seed, designated as SGT#01, SGT#02, etc., was ground in a standard laboratory grinder (see [reference]). Figure 2B Action 2), thoroughly clean between applications to obtain the corresponding flour samples - denoted as SFT#01, SFT#02, etc. (see Action 2) Figure 2C ).

[0493] For the purpose of clarity and not as a limitation, the following steps 3.2 – related topics – are described in detail in the following embodiments:

[0494] Example 4: Preparation of grain flour from the "total amount of sub-flour" sample.

[0495] Step 3.3: Preparation of gDNA from equal portions of flour sample

[0496] Distribute equal portions of the total sub-flour sample (each 25g, and designated as "sub-flour sample portions" (ASFT#01, ASFT#02, etc.)) into separate containers (see [reference]). Figure 2C Action 3).

[0497] Then gDNA was extracted from each flour aliquot sample (see...) Figure 2C (Step 4) and place each extracted gDNA sample into one well of a microtiter plate, labeled gDNA(GT#01), gDNA(GT#02), etc., to indicate the specific “total grain” source of the sample. Alternatively, extraction can be performed in multiple wells of the microtiter plate.

[0498] For the purpose of clarity and not as a limitation, the following embodiments detail the following topics related to step 3.3:

[0499] Example 5: Preparation of 25g flour samples (ASFT);

[0500] Example 6: Preparation of ASFT gDNA.

[0501] Step 3.4: Analyze samples containing gDNA from GT

[0502] Next, transfer the gDNA aliquots from each sample in the “Total Grain” range to a new 96-well microtiter plate (see [link]). Figure 2C Action 5) sometimes includes two negative control samples. Subsequent ddPCR analysis, such as detailed in Example 7, can help identify samples containing mutant plant gDNA.

[0503] For the purpose of clarity and not as a limitation, the following topics related to step 3.4 are described in detail in the Examples section:

[0504] Example 7: ddPCR-based experiment using gDNA from ASFT.

[0505] WS4: Discovering individual grains characterized by mutations of interest

[0506] Step 4.1: Details of the Grain Library Pool (GLP)

[0507] Given that the analysis described, for example, in step 3.4 above, generates a signal to indicate the presence of one or more mutated gDNAs in the 96 samples analyzed, i.e. Figure 2C In Chinese, it is represented as gDNA(GT#01), gDNA(GT#02), etc., and labeled as "Grain Library Pool" (GLP#01, GLP#02, etc.); Figure 2D The 4,500 grains in the "Total Grains" portion of Function 2) are considered highly likely to contain one or more grains with the same mutation of interest. In this application, the GLP processing will involve a final effort to discover specific mutant grains having the same mutation as the template molecule detected in processed aliquots from the "Total Grains" sample. Figure 2B , 2C And 2D), and it further induces mutant signals (e.g., in the corresponding ddPCR analysis) Figure 2C (As shown in action 5).

[0508] For clarity of description and not as a limitation, the following steps 4.1 - related topics are described in detail in the Embodiments section of this invention:

[0509] Example 8: Collecting grains as a “grain library pool”.

[0510] Step 4.2: Reduce "Grain Library Pool" to "A Part of Grain Library Pool"

[0511] When from a specific GT, a complete GLP sample containing ~4500 grains (and given gDNA analysis based on ddPCR, such as...) Figure 2C Action 5 shows that the gDNA contains a mutation. Under normal circumstances, for the same gene mutation as the specific gene mutation revealed in step 3.4, it is sufficient to screen fewer than 12 individual grains. Therefore, a sample of ~1200 grains of interest for the GLP was prepared. Figure 2D Action 2) generates a “part of the grain library pool”, abbreviated as FGLP. Finally, a total of 96 samples were separated, each consisting of 12 grains from a specific FGLP – each forming a secondary pool.

[0512] For the purpose of clarity and not as a limitation, the following steps 4.2 are described in detail in the following embodiments of the invention:

[0513] Example 9: Obtain a portion of the Grain Library from the Grain Library.

[0514] Step 4.3: Separate the grains and corresponding flour samples

[0515] Each of the 12 grains in the sample prepared as described in step 4.2 above was injured by drilling a small hole in the endosperm. The injured but surviving grains were then transferred to one well of a deep-well microtiter plate. Figure 2D Action 3) involves mixing the flour released through the same grains drilled and transferring it to a second microtiter plate. Figure 2D (Action 4)) Perform the procedure on the remaining 95 samples (keeping the same numbering system for both plates) to obtain a microtiter plate with a grain sample (named "Drilled Grain Pool from Part of the Library Pool", abbreviated as PDGLP; see [link]). Figure 2D ) and a microtiter plate with an endosperm powder sample (named "Flour Pool from Drilled Grains", abbreviated as PFGLP); Figure 2D ).

[0516] For the purpose of clarity and not as a limitation, the following embodiments detail the following topics related to step 4.3:

[0517] Example 10: Drilling holes in barley grains;

[0518] Example 11: Preparation of "Flour Pool from Drilled Grains".

[0519] Step 4.4: Preparation of gDNA from endosperm powder derived from mutant plants

[0520] The flour prepared as described in step 4.3 above, located in each well of the microtiter plate, was subjected to gDNA extraction. Figure 2D (Action 5). The extraction was performed using a semi-automated method based on the recommended method provided by the NucleoSpin 96Plant II kit (Machery-Nagel), producing a “gDNA pool” in which a single well of a microtiter plate contains gDNA from 12 barley grain flours.

[0521] For clarity of description and not as a limitation, the following topics related to step 4.4 are described in detail in embodiments of the invention:

[0522] Example 12: Purification of gDNA (“gDNA pool”) from flour of perforated grains.

[0523] Step 4.5: Analyze gDNA from mutant endosperm

[0524] Following Bio-Rad's recommendations regarding the use of the QX200 droplet reader and droplet generator, the gDNA samples prepared in step 4.4 above were subjected to standard ddPCR analysis, employing primer sets to detect specific mutations of interest. Only gDNA analysis from a single well should generate a signal indicating the presence of mutant DNA in the sample (see [link to documentation]). Figure 2D If action 6) is performed, then one or more grains from which the gDNA originates are highly likely to contain the mutation of interest.

[0525] For clarity of description and not as a limitation, the following steps related to topic 4.5 are described in detail in the Embodiments section of this invention:

[0526] Example 13: gDNA analysis of perforated grain flour based on ddPCR.

[0527] Step 4.6: Identify the mutant grains of interest

[0528] Although the sample in the microtiter plate designated PFGLP contains flour (see step 4.3), the matching wells in the plate designated PDGLP contain 12 corresponding live grains. Therefore, the next step is to separate the sample consisting of the 12 grains of interest. Figure 2D Action 7), then germinate these grains ( Figure 2D Action 8).

[0529] For clarity of description and not as a limitation, the following steps 4.6 - subject matter are described in detail in the Embodiments section of this invention:

[0530] Example 14: Germination of a potential mutant plant.

[0531] Step 4.7: Trait Verification

[0532] Those skilled in the art can use numerous methods to determine whether a potential mutant possesses the desired characteristic, i.e., the trait of interest. In this case, gDNA is extracted from germinating plantlets, identified and propagated as described in step 4.6 above, and combinatorial analysis using ddPCR and DNA sequencing is used to confirm which of the 12 potential mutants actually contains the mutation of interest (see [link to article]). Figure 2D Action 9).

[0533] For the purpose of clarity and not as a limitation, the following topics related to step 4.7 are described in detail in the Embodiments section of this invention:

[0534] Example 15: Analysis details, including ddPCR and DNA sequencing.

[0535] WS5: Generating and Using gDNA “Superpools”

[0536] Step 5.1: Ultra-high throughput identification of mutant barley grains

[0537] This invention also discloses the combined use of two different digital PCR analysis platforms, which improves the method for identifying low-incidence mutations. Here, RainDance Technologies' commercially available RainDrop platform is used to detect DNA mutations in complex gDNA samples from mutant plants, while Bio-Rad's product provides a method for identifying grain extracts from single barley mutants.

[0538] For clarity of description and not as a limitation, the following topics related to step 4.7 are described in detail in the Embodiments section of this invention:

[0539] Example 16: Preparation of gDNA “super pool”;

[0540] Example 17: ddPCR of gDNA from the “superpool”.

[0541] In summary, to provide a brief overview, the WS and corresponding embodiments are arranged as follows:

[0542] WS2:

[0543] Example 1: Growing barley in a “field”;

[0544] Example 2: Harvesting grains from a single “plot”.

[0545] WS3:

[0546] Example 3: Description of grains in "Total Grain Quantity";

[0547] Example 4: Preparation of grain flour from the "total amount of sub-flour" sample;

[0548] Example 5: Preparation of 25g flour samples (ASFT);

[0549] Example 6: Preparation of ASFT gDNA;

[0550] Example 7: ddPCR-based experiment using gDNA from ASFT.

[0551] WS4:

[0552] Example 8: Collecting grains as a "grain library";

[0553] Example 9: Obtain a portion of the Grain Library from the Grain Library;

[0554] Example 10: Drilling holes in barley grains;

[0555] Example 11: Preparation of "Flour Pool from Drilled Grains";

[0556] Example 12: Purification of gDNA (“gDNA pool”) from flour of perforated grains;

[0557] Example 13: gDNA analysis of perforated grain flour based on ddPCR;

[0558] Example 14: Germination of a potential mutant plant;

[0559] Example 15: Details of mutant plants identified by ddPCR-based screening.

[0560] WS5:

[0561] Example 16: Preparation of gDNA “super pool”;

[0562] Example 17: ddPCR of gDNA from the “superpool”.

[0563] Example

[0564] Example 1: Growing barley plants in a “field”.

[0565] As described in WS1 above, barley grains were mutagenized using NaN3. The mutagenized barley grains were 7.5 m in size. 2 Propagate in the fields. Sow 250g of grains in each field and let them grow until maturity.

[0566] Example 2: Harvesting grains from a single “plot”.

[0567] At harvest, the field is divided into 15-17 smaller plots, each containing approximately 300 plants. The plants are harvested with sickles, threshed, and bagged, with each plot handled individually. Therefore, the grains in a single "total grain" bag come from 300 plants.

[0568] Example 3: Description of grains in "Total Grain Quantity".

[0569] A grain sample comber is used to divide all the grains in a "total grains" sample into four equal-sized random portions. Each portion represents 25% of the total grains in a "total grains" sample. One of these portions constitutes a "sub-total grains".

[0570] Example 4: Preparation of grain flour from the "total amount of sub-flour" sample.

[0571] Place all grains from one “total sub-grain” sample into a grain mill (Retsch, GrindoMix GM200). Mill the grains at 10,000 RPM for 30 seconds. Place the resulting flour (“total sub-flour”; ~75 g) in a paper bag and store at 23°C.

[0572] Example 5: Preparation of 25g flour samples (ASFT).

[0573] Weigh out an equal sample of 25g of flour from a total flour sample. Transfer the 25g flour sample (ASTF) to a paper bag and store at 23°C.

[0574] Example 6: Preparation of purified ASF gDNA.

[0575] A 25g aliquot of flour (ASTF) was transferred to a 250mL glass bottle and resuspended in 30mL of H2O. Then, 70mL of 2% cetyltrimethylammonium bromide (CTAB) buffer (containing 1.4M NaCl, 20mM Na2EDTA, 100mM Tris-HCl, adjusted to pH 8.0 with 1M NaOH) heated at 65°C was added. After autoclaving, 250μl of 10mg / mL RNase A and 250μl of proteinase K were added, and the mixture was incubated under standard conditions to degrade RNA and protein. A 50mL aliquot was transferred to a 50mL Falcon tube, and the insoluble precipitate was removed by centrifugation at 4000rpm for 10 minutes. 24mL of the resulting supernatant was then transferred to a new 50mL tube.

[0576] After two consecutive extractions with chloroform (15 mL for the first extraction and 12.5 mL for the second), the 15 mL aqueous phase containing nucleic acids (including gDNA) was transferred to a 50 mL tube containing 30 mL of 0.5% CTAB buffer, prepared with 0.04 M NaCl and 50 mM Tris-HCl, and adjusted to pH 8.0 with 1 M NaOH. The tube was inverted at 6–8× and incubated at room temperature for 1 hour, followed by centrifugation at 4000 rpm for 10 minutes. The aqueous phase was discarded, and the precipitate containing gDNA was slowly dissolved in 10 mL of 1.2 M NaCl and incubated overnight at 8°C. Next, extraction was performed with 10 mL of chloroform, followed by centrifugation at 4000 rpm for 15 minutes. In the aqueous phase, 9.5 mL was combined with 5.5 mL of isopropanol, and the resulting sample was gently inverted at 6–8× and incubated at room temperature for 20 minutes, followed by centrifugation at 4000 rpm for 10 minutes to precipitate the gDNA.

[0577] The precipitate containing gDNA was washed with 5 mL of 70% ethanol, pre-centrifuged at 4000 rpm for 10 minutes, and the supernatant was discarded. Simultaneously, the gDNA precipitate was dried at room temperature for approximately 40 minutes. After resuspending the gDNA by adding 5 mL of H₂O and incubating at 65°C for 30 minutes, 500 μL of gDNA from the “total amount of sub-flour” was transferred to a specific 2 mL well of the corresponding microtiter plate. Plates containing different gDNA samples from “single SFT aliquots” were labeled “purified gDNA” and used for long-term storage or experimental analysis.

[0578] Example 7: ddPCR-based experiments using gDNA from ASFT.

[0579] ddPCR was performed using the Droplet Digital PCR QX200 system (Bio-Rad) according to the manufacturer's instructions. Sequence-specific primers and probes for wild-type and mutant alleles were purchased from Bio-Rad.

[0580] For analytical purposes, 5 μL of purified ASFT-only gDNA (see [link to analysis]) Figure 2C Add gDNA from yeast (such as that prepared in Yeast WS3) to 17 μL of a PCR mixture containing 11 μL of 2×ddPCR Supermix (No. dUTP; Bio-Rad) for probes, 900 nM of target-specific PCR primers, and 250 nM of mutant detection and reference probes labeled with 6-carboxyfluorescein-FAM and hexachlorofluorescein-HEX probes, respectively. The mutant detection probe specifically binds to the target sequence containing the mutation, while the reference detection probe specifically binds to the wild-type sequence. Load the reaction mixture onto an AutoDG Droplet Generator (Bio-Rad) and generate droplets according to the manufacturer's manual. Thermally cycle the droplet emulsion under standard PCR conditions: 40 cycles of denaturation at 95 °C for 10 min, followed by 30 sec at 94 °C and 1 min at 55 °C, and a final extension at 98 °C for 10 min, after which the microtiter plate is stored at 8 °C. Confirm PCR amplification in the droplets using a QX200 Droplet Reader (Bio-Rad). The analysis threshold was determined by comparing wild-type and non-template ddPCR results. All data were evaluated as exceeding the threshold.

[0581] The data was analyzed using QuantaSoft version v1.7 (Bio-Rad) software.

[0582] Example 8: Collecting grains as a "grain library".

[0583] A grain sample comber is used to divide all grains in a "total grains" sample into four equal-sized random portions. Each portion represents 25% of the total grains in a "total grains" sample. A "grain library pool" contains a collection of grains from three portions.

[0584] Example 9: Obtain a portion of the Grain Library from the Grain Library.

[0585] A portion of the grain library was created by sequentially removing 96 samples, each consisting of 12 grains from the "Grain Library Pool," with each sample of 12 grains forming a secondary pool.

[0586] Example 10: Drilling holes in barley grains

[0587] Grains from a portion of the "grain library pool" were divided into 96 equal aliquots, each consisting of 12 grains. Each 12-grain aliquot was placed on a weighing paper and held in place with a pair of pliers while a small, 2-3 mm deep hole was drilled in the endosperm using a carving machine (Marathon-3, Saeyang Microtech) equipped with a 1.6 mm drill bit. Rotational motion transferred flour from the endosperm to the weighing paper on top of and around the grain. The 12-grain drilled samples were then placed in individual 2 mL wells of a microtiter plate, creating a secondary pool of drilled barley grains, also known as a "drilled barley grain pool from a portion of the library pool." Figure 2D (Action 3). Store the drilled grains at 20°C until further analysis.

[0588] Example 11: Preparation of "Flour Pool from Drilled Grains".

[0589] Ninety-six flour samples (each containing flour from 12 borehole barley grains (as detailed in Example 10)) were transferred to individual wells of a 1.5 mL microtiter plate (“Flour Pool from Borehole Grains”; see also…) Figure 2D Action 4) Maintain the matching of the sample numbering system with the numbering of the drilled grains.

[0590] Example 12: Purification of gDNA (“gDNA pool”) from flour of drilled grains.

[0591] gDNA was extracted from the flour pools of the perforated barley grains prepared as detailed in Example 11 using a semi-automatic DNA extraction method, as described in the instructions for the NucleoSpin 96Plant II kit (Macherey-Nagel). Therefore, each well of the microtiter plate contained gDNA from the flour of 12 grains. Figure 2D Action 5).

[0592] Example 13: gDNA analysis of perforated grain flour based on ddPCR.

[0593] Although the WS3 test (see Figure 2C Examples 3-7) were designed to identify which individual sample among 96 gDNA aliquots (generally representing gDNA from ~6000 mutant grains) contained the nucleotide mutation of interest, followed by WS4-specific work (see Examples 3-7). Figure 2D It was designed to precisely locate specific mutant grains.

[0594] like Figure 2D As shown in Action 6, the main task is to determine which gDNA sample from the 12 different grains will produce a positive test result in the ddPCR analysis. Therefore, by using the same reaction conditions and analytical parameters as provided in Example 7, it is possible to distinguish which well in the microtiter plate contains the gDNA of interest. A sample containing one heterozygous mutant grain is defined as having a fractional abundance of 5% (±2.5%), and a sample with 10% (±2.5%) is defined as containing a homozygous mutant grain.

[0595] Based on the above analysis, potential flour pools can be identified and selected, and thus corresponding grain pools with mutations of interest in nucleotides can be identified. Homozygous and heterozygous mutants were transferred to soil in large pots and then to a greenhouse for propagation. All negative-negative plantlets were discarded.

[0596] Example 14: Germination of a potential mutant plant.

[0597] As long as analysis of gDNA from 12 barley grain flours yields a mutation signal (see...) Figure 2D Action 6) causes the corresponding grains in the "drilled grain pool from a portion of the library pool" to germinate. Figure 2D Actions 7 and 8).

[0598] Example 15: Details of mutant plants identified by ddPCR-based screening.

[0599] The contents of a "drilled grain pool from a portion of the library pool," consisting of 12 drilled grains, were placed in a 9 mm Petri dish (made with two sheets of filter paper (8.5 mm; Whatman)) with 2 mL of H2O added. After a 96-hour germination period in the dark at 16°C, the grains were placed in soil and grown for another 7 days under controlled conditions. Then, 2 × 5 mm tissue sections were extracted from the first batch of leaves and placed in 0.2 mL tubes. 50 μL of extraction solution (Sigma) was pipetted onto each leaf and incubated at 95°C for 10 minutes. The samples were cooled to room temperature and mixed with 50 μL of dilution solution (Sigma). 10 μL of the extraction-dilution mixture was combined with 30 μL of H2O to finalize the gDNA sample.

[0600] For analytical purposes, 5 μL of purified gDNA was added to 17 μL of PCR mixture (containing 11 μL of 2×ddPCR Supermix (No. dUTP; Bio-Rad) for probes, 900 nM target-specific PCR primers, and 250 nM mutant-specific (6-carboxyfluorescein-FAM) and wild-type-specific (hexachlorofluorescein-HEX) probes). The reaction mixture was loaded onto an AutoDGDroplet Generator (Bio-Rad), and droplets were generated according to the manufacturer's manual. The droplet emulsion was thermally cycled under standard PCR conditions: 40 cycles of denaturation at 95 °C for 10 min, followed by 30 sec at 94 °C and 1 min at 55 °C, and a final extension at 98 °C for 10 min. The microtiter plate was stored at 8 °C. PCR amplification in the droplets was confirmed using a QX200 Droplet Reader (Bio-Rad). The test threshold was determined by comparing wild-type and template-free ddPCR results. All data obtained were evaluated as being above the threshold. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). A single plantlet was considered a heterozygous mutant when 50% of the total positive ddPCR events originated from the mutant probe (FAM). A single plantlet was considered a homozygous mutant when 100% of the total positive ddPCR events originated from the mutant probe (FAM). Each mutant plantlet was then grown to maturity (see [link to documentation]). Figure 1D Action 9).

[0601] Example 16: Preparation of gDNA “super pool”.

[0602] Simply put, aliquots (e.g., 50 μL) of each gDNA extract from all 94 “sub-pools” of a library plate are combined into a single “super-pool”.

[0603] In some implementations, a gDNA superpool is further enriched prior to ddPCR analysis.

[0604] Therefore, for example, 2 μl of gDNA from a superpool can be amplified in a standard PCR reaction, including, for example, 10 μl of 5X Q5 reaction buffer (New England Biolabs), 200 μM dNTPs, 0.02 U / μl Q5 high-fidelity DNA polymerase, 100 nM target-specific forward PCR primers, 100 nM target-specific reverse PCR primers, 100 nM blocking probe, and water. The PCR mixture is then thermally cycled for approximately 20 PCR cycles under standard PCR conditions. This produces an enriched gDNA superpool.

[0605] Example 17: ddPCR analysis of gDNA from the “super pool”.

[0606] To improve yields and reduce chemical usage in screening mutant barley grains, a combination of two technologies was developed: one from RainDance Technologies (excellent in droplet fabrication) and the other from Bio-Rad (outstanding in processing a large number of samples simultaneously). Generally, RainDance technology is used to identify specific but complex samples containing gDNA templates from numerous mutant grains (detailed in Section A below), while subsequent Bio-Rad-based analysis helps identify individual grains characterized by the mutation of interest (described in Section B below).

[0607] Program A: RainDance-based ddPCR with a 16-gDNA "superpool".

[0608] First, droplets for each “superpool” (e.g., 4 to 8 superpools) are generated using a RainDrop Source instrument. The gDNA used for RainDance-based ddPCR can be a combined gDNA extract or an enriched gDNA superpool prepared as described in Example 16. For example, 20 μL aliquots of gDNA from one “superpool” or 10 μL aliquots of enriched gDNA superpools are mixed with 25 μL of Supermix (Bio-Rad) for probes; droplet stabilizer (RainDance); 900 nM target-specific forward primer; 900 nM target-specific reverse primer; 120–250 nM wild-type detection probe (VIC); 250–440 nM mutation detection probe (FAM); and H2O to adjust the total reaction volume to 50 μL. A total of 4-8 separate reaction mixtures (representing 4-8 separate gDNA “superpools”) are added to an 8-channel source chip (RainDance) and processed on a Source Instrument (RainDance). The entire process described above can be repeated for additional “superpools”, resulting in a total of 8 to 16 samples for subsequent analysis.

[0609] Two 8-well strips containing droplets generated from the reaction mixture (as described above) were sealed, and the contents were amplified as described in Example 9 of this document. The amplified mixture was then transferred to a Sense Instrument (RainDance) and analyzed using RainDrop Analyst data analysis software.

[0610] Those "superpools" that showed higher signals associated with mutations of interest, compared to the average signal of mutation events across all 16 "superpools," were defined as containing gDNA derived from potential mutants.

[0611] Program B: ddPCR analysis based on Bio-Rad's "super pool".

[0612] As detailed in Procedure A above, ddPCR analysis was performed on aliquots of each of the 96 gDNA samples constituting the “superpool” of interest, indicating the presence of a mutant template. The analysis was performed as described above for WS3 and WS4.

[0613] Specifically, the data analysis aims to select grain pools containing predetermined mutations of interest. Data sets with individually defined thresholds are plotted against all data points across the entire plate. Analysis categories—including but not limited to graphs of target concentration, fractional abundance, and identified mutation events—are evaluated separately regarding candidate selection.

[0614] Fractional abundance can be determined as: [(signal of the mutation detection probe)] divided by [(signal of the reference detection probe + the mutation detection probe)].

[0615] If the following properties are observed, the sample is assumed to contain mutant DNA:

[0616] -Increased fractional abundance;

[0617] - The mutant droplet level increases, or;

[0618] - The number of mutant events increases to more than 50% of the average level or higher.

[0619] Example 18: Screening of barley mutants with specific mutations in the glutamine synthase GS1-3 gene based on ddPCR.

[0620] The methods described in the workflow and examples above are used to identify barley plants carrying specific mutations. As mentioned above, this method can be used to identify any mutant. The specific mutation G→A is identified, which results in the substitution of the Gly residue at position 287 of the barley glutamine synthetase 1 isotype 3 (HvGS1-3) sequence with an Asp residue.

[0621] Glutamine synthase

[0622] Glutamine synthase 1 (GS1) is a key enzyme involved in nitrogen assimilation in higher plants by catalyzing the condensation of ammonium or ammonia with glutamate to glutamine. In nitrogen-rich soils, excess nitrogen can lead to increased nitrogen accumulation during barley grain filling, negatively impacting malt quality. Knockout mutations of GS1 can result in severe growth defects, highlighting the enzyme's importance for overall plant health (Tomoyuki Yamaya and Miyako Kusano, 2014). Three GS1 isotypes (HvGS1-1, HvGS1-2, and HvGS1-3) are known in barley. Of these three barley isotypes, HvGS1-3 is primarily active in developing grains and is upregulated under high ammonium conditions. A mutational strategy was designed to maintain optimal plant development while reducing grain nitrogen accumulation capacity. This mutational strategy focused on amino acid substitutions to reduce GS1-3 enzyme activity. A suitable amino acid substitution was identified at position 287 of the protein sequence (Protein Seq ID, NCBI: AFX60877.1, provided herein as SEQ ID NO: 2). Changing codon GGC (nucleotides 859, 860, 861; GenBank ID NCBI CDS: JX878491.1 – provided herein as SEQ ID NO: 1) to GAC resulted in a change from glycine to aspartic acid at position 287 of the protein sequence (Protein Seq ID, NCBI: AFX60877.1). This amino acid change introduces a negatively charged amino acid into the protein sequence. GS1-3 is a decameric compound consisting of two rings containing five subunits, with amino acid residue 287 located at the interface between the two rings and far from the active site. Therefore, the introduction of a negatively charged amino acid is not considered to affect the overall ability to catalyze its reaction, but structural changes in the decameric assembly can reduce enzyme activity.

[0623] ddPCR assay

[0624] A unique ddPCR assay was designed to distinguish between the mutant and wild-type alleles of HvGS1-3 at nucleotide 860 of the wild-type coding sequence (GenBank ID NCBI: JX878491.1). The mutation detection probe is complementary to the coding sequence and contains an A base at nucleotide 860. The reference detection probe is also complementary to the coding sequence and contains a G base at nucleotide 860. Two flanking primers were designed to amplify the genomic sequence surrounding nucleotide 860 of the coding sequence.

[0625] The following primers and probes are specifically designed for the HvGS1-3 locus:

[0626] - Target-specific forward primer (SEQ ID NO: 3):

[0627] 5′-GTGATCAAGAGGGCGATCAA-3′;

[0628] - Target-specific reverse primer (SEQ ID NO: 4):

[0629] 5′-CAAGTCTCAACTCGCCGTAT-3′;

[0630] - Mutant-specific detection probe (SEQ ID NO: 5):

[0631] 5′-AAGACAACGAGCGC-3′–labeled with 6-carboxyfluorescein (FAM);

[0632] - Reference specific detection probe (SEQ ID NO: 6):

[0633] 5′-AAGGCAACGAGCGC-3′- is labeled with hexachlorofluorescein (HEX).

[0634] As described in WS1 above, a pool of randomly mutated barley grains was prepared, and then an ordered library was prepared.

[0635] Determine whether the library sample contains mutant grains (WS3).

[0636] Typically, the next step is to determine whether the library contains mutant grains, which is basically as described in WS3 and Example 7 above, with the following details:

[0637] - Screening was performed using a total of 376 GLPs (i.e., 376 sub-pools), representing approximately 120,000 mutant barley plants. ddPCR was performed essentially as described in Example 7;

[0638] Add 5 μL of gDNA sample (GT#377-GT#470) to each well containing 17 μL of PCR reaction mixture and mix thoroughly by aspiration.

[0639] A microtiter plate for PCR was loaded onto a QX200 droplet reader (Bio-Rad) for droplet analysis. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). Thresholds were determined using 2-D curves, with amplitudes for channels 1 and 2 set to 2700 and 1500, respectively, for amplification. Comparison of individual fractional abundance values ​​showed that gDNA (GT#380) provided a higher signal than any other sample relative to mutant detection. The fractional abundance of gDNA (GT#380) was 0.089% compared to 0.0077%, where 0.0077% represents the average fractional abundance of gDNA samples across all 94 tests.

[0640] A single grain (WS4) characterized by the mutation of interest was discovered.

[0641] The identification of individual barley grains carrying gene mutations is basically as described above in WS4, including details of the following sequential order:

[0642] 1. Based on HvGS1-3 specific ddPCR analysis of gDNA from GT#377-GT#470, 4500 grains of GLP#380 [corresponding to positive sample gDNA (GT#380)] were considered to be highly likely to contain one or more grains with mutations in the gene of interest.

[0643] 2. An FGLP#380 was established by sequentially removing 96 × 12 grain samples from a GLP#380. Each 12-grain aliquot was placed on a weighing paper and held in place continuously with a pair of pliers while a small, 2–3 mm deep hole was drilled in the endosperm using a burr cutter (Marathon-3, Saeyang Microtech) with a 1.6 mm drill bit. Rotational motion transferred flour from the endosperm to the weighing paper on top of and around the grain. The 12 drilled samples were placed in individual 2 mL wells of a microtiter plate to obtain a secondary cell (PDGLP#380) for the drilled barley grains. 96 flour samples (each containing flour from 12 drilled barley grains) were transferred to individual wells of a 1.5 mL microtiter plate (PFGLP#380), maintaining the sample numbering system consistent with the numbering of the drilled grains.

[0644] 3. Next, gDNA was extracted from the PFGLP#380 using a semi-automated DNA extraction method detailed in the instructions for the NucleoSpin 96Plant II kit (Macherey-Nagel). Therefore, each well of the microtiter plate contained gDNA from 12 grain flour samples.

[0645] 4. The gDNA from PFGLP#380 was analyzed as described above. Data was analyzed using QuantaSoft software (version v1.7, Bio-Rad). Thresholds were determined using 2-D curves, with an amplitude of 2700 for channel 1 and 1500 for channel 2. Comparison of fractional abundance values ​​showed that none of the PFGLP#380 samples contained mutant grains. Since GLP#380 was considered likely to contain mutant grains, it was decided to prepare and analyze other samples by establishing a second FGLP (FGLP#380-2). Thus, PDGLP#380-2 and PFGLP#380-2 were prepared. The gDNA from PFGLP#380-2 was analyzed as described above. Data was analyzed using QuantaSoft software (version v1.7, Bio-Rad). Thresholds were determined using 2-D curves, with an amplitude of 2700 for channel 1 and 1500 for channel 2. Three separate wells (C02, F04, F05) were identified in the microtiter plate, showing fractional abundances of 4.02%, 4.11%, and 5.55%, respectively, all indicating the presence of three separate heterozygous mutants in the three independent wells of PDGLP#380-2.

[0646] 5. Germinated all 12 grains from wells C02 and F04 of PDGLP#380-2. Leaf material from all 24 plantlets was collected and DNA was extracted using REDExtract (Sigma Aldrich). gDNA from the leaf samples was analyzed as described above. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). Thresholds were determined using 2-D curves, with amplitudes set to 2700 for channel 1 and 1500 for channel 2. One plantlet from well C02 of PDGLP#380-2 showed a fractional abundance of 41%, confirming the presence of a heterozygous mutant. One plantlet from well F04 of PDGLP#380-2 showed a fractional abundance of 39.6%, confirming the presence of a heterozygous mutant.

[0647] 6. Further phenotypic verification was performed using direct sequencing of the identified mutants and reference samples. DNA was extracted from leaf material using the REDExtract DNA extraction method (Sigma Aldrich). For sequencing analysis, 50 μl PCR reactions were prepared containing 1 μl purified gDNA, 20 μl REDExtract (Sigma Aldrich), 500 nM target-specific forward primer, 500 nM target-specific reverse primer, and water. Samples were thermally cycled using the following PCR conditions: 94 °C for 2 min denaturation, 38 cycles of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min, after which the PCR plates were stored at 8 °C. Individual PCR products were purified using NucleoSpin Gel and a PCR washing kit. All samples were sequenced using target-specific forward and reverse primers.

[0648] 7. Two plantlets identified as mutation-positive in ddPCR analysis relative to their genotype at a predetermined genomic location were heterozygous. The reference sample showed a homozygous wild-type genotype at the same location.

[0649] Example 19: Analysis of recombinant barley glutamine synthase.

[0650] Three recombinant HvGS1-3 variants were synthesized using *E. coli* host cells, and the enzymes were then enriched to high purity via affinity purification. The following variants were expressed in *E. coli*:

[0651] -Q: Wild-type reference -SEQ ID NO: 1;

[0652] -3864: SEQ ID NO: 1 with G287D amino acid exchange, corresponding to the mutant protein expressed by mutant barley plants (as identified in Example 18).

[0653] -LR: SEQ ID NO: 1 with amino acid exchange D300N, i.e., an enzyme variant for which the expected change has little effect on enzyme activity.

[0654] The activities of three HvGS1-3 variants were determined by synthesizing glutamyl hydroxamic acid ester from glutamate, hydroxylamine, and ATP in the presence of the enzyme. Enzyme kinetics of all three HvGS1-3 variants were measured at different concentrations of glutamate and hydroxylamine, and the results are shown in Tables 1 and 2.

[0655] Although HvGS1-3 Q and LR's k cat and k cat / K MSimilar to the other two variants, HvGS1-3 3864 consistently showed values ​​one or two orders of magnitude lower than the other two variants. Kinetic data indicate that the mutation identified in HvGS1-3 3864, namely the change from Gly 287 to Asp, has a significant impact on the binding and utilization of any substrate in this variant. Due to the high variability of absorbance at high substrate concentrations of nucleotides, kinetic values ​​for ATP binding and utilization could not be determined.

[0656] Table 1. Enzyme kinetics of HvGS1-3 variants in the presence of different concentrations of glutamate. For Q, LR, and 3864, the numbers in parentheses refer to the total amount of protein reacted, in μg.

[0657]

[0658] Table 2. Enzyme kinetics of HvGS1-3 variants in the presence of different concentrations of the substrate hydroxylamine. For Q, LR, and 3864, the numbers in parentheses refer to the total amount of protein reacted, in μg.

[0659]

[0660] To determine whether the oligomerization of the GS1-3 complex was affected in either of the two variants compared to Q, purified HvGS1-3 was first passed through an exclusion column. All three variants exhibited a main peak at the same elution volume.

[0661] The following describes a detailed explanation of the six steps involved in obtaining the results described in this embodiment.

[0662] Step 1: Design the HvGS1-3 gene sequence for heterologous gene expression

[0663] The coding sequence of barley glutamine synthase isotype GS1-3 (HvGS1-3), deposited in GenBank with accession number JX878491.1, was used as the basis for synthesizing three corresponding gene sequences (GenScript, China), representing wild-type HvGS1-3(Q) and two mutant forms (3864 or LR). The wild-type GS1-3 gene and protein sequences are provided in this paper as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The protein-coding DNA sequences of all three variants were inserted into the E. coli expression plasmid pET-28a(+) purchased from Merck Millipore (Germany), and then transformed into E. coli strain BL21(DE3) using standard methods.

[0664] Step 2: Heterologous expression of HvGS1-3

[0665] Escherichia coli BL21(DE3) cells were transformed with HvGS1-3Q or its variants from pET-28a(+). The bacteria were cultured in LB medium containing 30 μg / mL kanamycin under standard conditions. Gene expression was induced by adding 250 μM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma-Aldrich). Expression was performed at 37°C, 120 rpm for 4 hours. Cells were harvested by centrifugation at 8°C, 4,000 × g for 15 minutes. The supernatant was discarded, and the cell pellet was resuspended in 10 mL of buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM MgCl2). The resuspended cells were frozen at -20°C until further use.

[0666] Step 3: Cell lysis of E. coli BL21(DE3) cells after recombinant gene expression

[0667] First, the transformed frozen *E. coli* BL21(DE3) cells were thawed and then, on moist ice, lysed using a Vibra cell sonicator (Sonics & Materials Inc., USA) at 30% amplitude for 90 seconds, with 5-second pulses and 5-second rests. After sonication, the DNA was digested with 5 μg / mL DNase I (Sigma-Aldrich) for 10 minutes. The lysed cells were removed by centrifugation at 13,000 × g and 4 °C for 10 minutes. The supernatant was injected into a new tube through a 0.45 μm filter in a syringe.

[0668] Step 4: Enrichment of HvGS1-3 by immobilized metal affinity chromatography

[0669] Wash 1 mL of immobilized metal affinity chromatography (IMAC) containing nickel solution with 5 column volumes of double-distilled water and equilibrate with the same volume of buffer A (composition as described above). Manually inject the filtered soluble fraction into the equilibrated column using a syringe, collect the unbound material, and maintain at 4°C. Elute the loosely bound column material with 10 column volumes (CV) of buffer A containing 50 mM imidazole (Sigma-Aldrich). Under similar conditions, elute with 10 CVs of buffer A containing 400 mM imidazole and collect HvGS1-3. Permeate the eluted protein solution 4× through a centrifuge filter unit made of regenerated cellulose with a nominal molecular cutoff of 10,000 (Millipore, Ireland).

[0670] Step 5: In vitro activity assay of recombinant HvGS1-3

[0671] The activity assay described by Wellner and Meister (1966) was appropriately modified to determine the changes in activity between the two HvGS1-3 variants (compared to the activity of HvGS1-3 Q). 50 μL of the reaction mixture contained 100 mM Tris-HCl, pH 8.0, 50 mM MgCl2, 20 mM ATP, and varying concentrations of γ-glutamate hydroxylamine (when L-glutamate was varied; otherwise 50 mM), and varying concentrations of L-glutamate (when glutamate hydroxylamine was varied; otherwise 50 mM). The reaction was placed in half-area, flat-bottomed 96-well microplates (Corning, USA) and equilibrated to 37°C. The reaction was initiated by adding purified HvGS1-3 to a final concentration of 1 or 5 μg / mL. After 30 minutes, the reaction was terminated by adding 50 μL of 370 mM Fe(III)Cl3, 200 mM TCA, and 670 mM HCl. Then, A was measured from the bottom using a SpectraMax 340PC384 microplate reader (Molecular Devices, USA). 540 nm, using path correction to account for small differences in volume. Data was plotted using GraphPad Prism (version 4, GraphPad Software, USA).

[0672] Step 6: Size exclusion chromatography

[0673] Equal amounts of the three HvGS1-3 variants were loaded onto 10 / 300 size resistor arrays containing Superdex 200 (GE Life Sciences, USA), and then... Equilibrate in buffer A on an FPLC (GE Life Sciences) instrument. Elution of bound proteins was performed at 0.5 mL / min, and protein A was measured after elution. 280 .

[0674] Example 20: ddPCR analysis of gDNA from mutagenized yeast cells.

[0675] For the purpose of clarity and not as a limitation, the following topics are described in detail in this embodiment:

[0676] Part 1: Preparation of yeast cultures for random mutagenesis

[0677] Part 2: Preparation of Ordered Libraries

[0678] Part 3: Identification of wells containing gDNA with a predetermined mutation

[0679] Part 4: Enrichment of yeast cells characterized by predetermined mutations

[0680] WS1: Preparation of yeast cultures for random mutagenesis

[0681] Step 1.1: Mutagenesis Procedure

[0682] To induce mutations, the yeast strain was treated with MNNG (methylnitrosoguanidine) according to the protocol described in Methods in Molecular Biology, Yeast Protocols, V.313, 2006.

[0683] MNNG is a mutagen known to alkylate guanidine or thymine into gDNA, subsequently converting the G·C pair to A·T during gDNA replication. The method described in this section aims to identify point mutations in genes of interest that alter defined amino acid codons to premature stop codons. Those skilled in the art will be able to adapt this method to the identification of other types of mutations. Thus, point mutations result in truncated and correspondingly nonfunctional or defective proteins. The desired point mutation is defined based on the sequence of the target gene / protein to produce the stop codon. Other point mutations affecting regulatory regions of genes of interest can also be identified using this method.

[0684] Mutagenesis was induced in yeast cultures, for example, when the total cell count was 2 × 10⁻⁶. 7 The mutant state of the yeast cells used for mutagenesis is determined and adjusted to ensure the desired mutation rate. Mutagenesis conditions are typically adjusted to provide a survival rate of 60-70%. Mutagenic agent concentration and exposure time vary depending on the yeast strain.

[0685] WS2: Preparation of Ordered Libraries

[0686] Step 2.1: Cell Viability

[0687] Live cell titers were determined, and all mutagenic yeast cells were aliquoted into 96-well plates equipped with YPD using a Biomek FXp robot. 3000-5000 live cells were seeded into each well.

[0688] WS3: Identification of wells containing gDNA with a predetermined mutation

[0689] Step 3.1: Preparation of a mutagenic yeast cell library

[0690] The mutagenic yeast cells inoculated into 96-well plates in step 1.1 were considered to contain a total library of the targeted mutant yeast. The yeast cells were incubated for 3 days to allow for growth saturation. The library was then re-inoculated into minimal growth medium using a Biomek FXp robot (replication step) to amplify the library, and after 3 days of growth, it was used for gDNA isolation. The remaining yeast cell suspension in the 96-well plates was preserved at -80°C with glycerol (15% final concentration) for downstream applications to isolate the desired mutant yeast.

[0691] Step 3.2: Isolate gDNA from yeast

[0692] To prepare gDNA, yeast cells were transferred to DNA isolation 96-well plates, and the DNA isolation step was performed using robotic technology (Biomek FXp, Agencourt DNAdvance kit, and a protocol from Beckman Coulter). The procedure followed the manufacturer's instructions and included an additional step of digesting the yeast cell wall and disrupting the cells using a lysin. The protocol employed magnetic beads for gDNA precipitation. The gDNA in the 96-well plates was used for downstream applications to identify positive pools for predetermined mutations. DNA concentration was measured using a 96-well plate reader, adjusted to 25 ng / 5 μL, and used directly for ddPCR. The raw, concentrated DNA in the 96-well plates was stored for later use.

[0693] Step 3.3: Analyze the sample by ddPCR

[0694] The gDNA prepared in step 2.2 in 96-well plates was quantified, and the concentration was adjusted to 25 ng / 5 μL. This included positive and negative control DNA samples. Each gDNA sample was subsequently analyzed by ddPCR, essentially as described in Example 7. At this stage, probe arrays that identify different point mutations at several or all possible locations in the gene sequence can be used, generating premature stop codons to increase the chance of mutant identification. This method allows for the identification of wells that are positive for the desired mutation, i.e., wells containing yeast carrying the desired mutation.

[0695] WS4: Enrichment of yeast cells characterized by predetermined mutations

[0696] Step 4.1: Preparation of yeast library pool

[0697] Thaw the 96-well plates containing the mutant-positive wells and resuscitate the yeast cells by inoculating the contents of the positive wells into fresh YPD broth (1:10) and incubating by rotation at room temperature for 4–6 hours. To ensure that the yeast cells do not proliferate further, store the resuscitated yeast culture in a freezer until downstream plating and successful isolation of pure mutant cultures. Before plating, count the live yeast cells and dilute with PBS containing 1 mM EDTA, then plate them on Qpix square dishes with YPD agar to obtain 2000–3000 colonies per plate. The number of plates prepared in this manner depends on the titer of live yeast cells resuscitated from the frozen primary culture and the progress of mutant identification. For example, 10–12 Qpix plates can be inoculated with up to 50,000 single cells that produce single colonies. The positive pool identification described in Part 3 (where the wild-type:mutant ratio is up to 1:5000) indicates that at least 10 cells out of 50,000 single cells are target mutants. Colony growth was monitored to ensure appropriate size and spacing between colonies so that individual colonies could be picked using the Qpix robot. Once colonies were ready for collection, libraries of YPD-grown yeast cells were generated in 96-well plates by randomly collecting colonies from all Qpix plates into 10 wells, thus creating pools of 50 colonies per well with a minimum potential ratio of wild-type to mutant of 1:50. These plates were further processed as described in step 3.1 (WS3) to isolate gDNA and prepare a cryogenic stock solution, which was stored at -80°C for further downstream applications.

[0698] Step 4.2: Isolate yeast gDNA from the library pool

[0699] To prepare gDNA from 50 isolated colony pools, the yeast culture from the 96-well plates obtained in step 3.1 was re-inoculated into fresh minimal growth medium. Once growth was sufficient, DNA was isolated from the yeast culture using robotic techniques as described in step 3.2 (WS3). The DNA concentration was measured using a 96-well plate reader and adjusted to 25 ng / 5 μl, and aliquots were used directly for ddPCR. The original concentrated gDNA was retained in 96-well plate form. The gDNA in the 96-well plates was used for downstream applications to identify positive pools / wells with predetermined mutations. A total of 10 96-well plates containing gDNA were prepared.

[0700] Step 4.3: Analyze the sample using ddPCR

[0701] The gDNA prepared in step 3.2 in 96-well plate form was quantified, and the concentration was adjusted to 25 ng / 5 μL. This included positive and negative control DNA samples. Each gDNA sample was subsequently analyzed by ddPCR as described in Example 7. If multiple probes were used in step 3.3, only the probes used for positive identification of the positive pools in step 3.3 were employed at this stage. This method allows for the identification of positive sub-pools with predetermined mutations.

[0702] Step 4.4: Preparation of single-colony yeast libraries

[0703] The frozen 96-well plates with positive wells identified in step 4.3 were used for plate-stacking for single colony growth. Yeast cultures from the positive wells were inoculated into fresh YPD broth and incubated overnight. 1000 cells were seeded onto YPD agar (Qpix square plates) to generate single colonies. The grown colonies were selected into 10 96-well plates using a Qpix robot, with YPD broth as the growth medium. These plates were further processed as described in step 3.1 (WS3) to isolate gDNA and prepare frozen raw materials for further downstream applications.

[0704] Step 4.5: Isolate gDNA from single colony library

[0705] Prepare gDNA as described in step 4.2.

[0706] Step 4.6: Analyze the sample by ddPCR

[0707] The gDNA prepared in step 4.5 in 96-well plate form was quantified, and the concentration was adjusted to 25 ng / 5 μL. Positive and negative control DNA samples were included. Each gDNA sample was subsequently analyzed by ddPCR as described in Example 7. At this stage, only the probes given for positive identification in step 3.1 were applied. This method allows for the identification of pure yeast cultures with predetermined mutations. This method allows for the determination of the homozygous or heterozygous status of isolates.

[0708] Step 4.7: Isolate pure cultures of mutant yeast

[0709] To prepare the primary strain of mutant yeast, a yeast suspension from positive wells was plated onto YPD agar to allow isolated colonies to grow. Three colonies were manually picked and further processed as bioreplicas.

[0710] Step 4.8: Sequencing of the target gene or the gDNA segment of interest

[0711] The yeast mutant isolate with the identified mutation from step 4.7 (WS4) was used to isolate gDNA, followed by specific PCR, cloning of the obtained DNA fragment, and DNA sequencing to confirm the identity of the mutation.

[0712] Step 4.9: Functionality of the mutation

[0713] Yeast mutants with homozygous mutations of interest are used directly to confirm that the mutation has the desired effect. If heterozygosity is determined and found to be insufficient to provide the desired effect, mutagenesis is repeated, or the yeast isolate undergoes sporulation, and the mutant is then isolated to confirm homozygosity and the mutant phenotype. Yeast isolates with mutations of interest are used directly for applied purposes or for yeast breeding to control the desired phenotype.

[0714] Example 21

[0715] In brewer's yeast, the ferulic acid decarboxylase Fdc1 is essential for the decarboxylation of aromatic carboxylic acids, including cinnamic acid or coumaric acid. The decarboxylation reaction converts the substrate into its corresponding vinyl derivative, some of which are known to be flavor-active compounds. For example, during beer fermentation, Fdc1 converts ferulic acid from wort into 4-vinylguaiacol, which imparts a distinctive clove-like aroma to the final beer. While these aromas are typical of some beers, particularly German wheat beers, they are considered phenolic off-flavors (POFs) in other beers, including lager. This article describes the identification of a brewer's yeast carrying a nonsense mutation in the ScFDC1 gene using non-GMO methods. The identified yeast strain was expected to be POF-negative.

[0716] In the food industry, it is undesirable to use molecular genetic techniques to modify selected organisms, and the identification of strains of interest relies on classical random mutagenesis techniques and time-consuming screening. The method described below describes a less time-consuming technique for identifying strains carrying selected mutations of interest within a population of randomly mutagenized organisms. More specifically, in this embodiment, a yeast strain carrying a specific nonsense mutation (W159*) is described, which changes position +476 of the ScFDC1 gene in the Saccharomyces cerevisiae var. diastaticus strain FS0105 from G to A. The sequence of the ScFDC1 gene is available in GenBank under NCBI: NM_001180847, and the cDNA sequence is provided herein as SEQ ID NO: 26, and the amino acid sequence of fdc1 is provided as SEQ ID NO: 7.

[0717] WS1: Preparation of randomly mutated FS0105 yeast cell populations.

[0718] Step 1.1: EMS mutation of strain FS0105.

[0719] To induce mutations, yeast strain FS0105 was treated with ethyl mesylate (EMS) according to the protocol described in "Methods in Yeast Genetics" (CSHL Press, 2000). Briefly, strain FS0105 entered the stationary phase overnight in YPD medium. Cells were collected by centrifugation, washed once with sterile distilled water, and once with 0.1 M sodium phosphate buffer (pH 7). Finally, the cells were resuspended in 0.1 M phosphate buffer to approximately 2 × 10⁻⁶ cells / day. 8 Cells / ml. 30 μl of EMS was added to 1 ml of cells in a 2 ml safety lock reaction tube, and the cells were incubated in an Eppendorf Thermomixer (1.5 ml) at 30 °C and 1000 rpm for approximately 75 minutes, which typically resulted in a kill rate of approximately 60-80% for strain FS0105. To stop mutagenesis, the cells were briefly centrifuged to precipitate the cells, washed three times with freshly prepared sterile 5% sodium thiosulfate solution, and once with sterile distilled water. Finally, the cells were resuspended in 1 ml of YPD and incubated in an Eppendorf Thermomixer (1.5 ml) at 30 °C and 1000 rpm for 1 hour. Cells from this step can be stored at 4 °C or used immediately for downstream processes, for example, by determining the kill rate through plating on complex media.

[0720] Literature indicates that immediately subjecting mutagenized yeast cells to selection pressure after mutagenesis leads to a significant increase in the number of mutations per cell (Lada et al., 2013; Den Abt et al., 2016), which may significantly reduce the number of yeast cells selected for identification of mutations of interest. Therefore, approximately 2 × 10⁶ cells per plate are used. 7 Live, but mutagenic cells were inoculated onto SD agar plates containing 2 μg / ml of the herbicide metsulfuron-methyl, and the plates were incubated at 30°C for several days until herbicide-resistant colonies appeared on selected plates. Our goal was approximately 50,000 herbicide-resistant colonies in total. To further increase the number of mutations per cell, we eluted the herbicide-resistant cells from the plates with sterile 0.1M sodium phosphate buffer (pH 7) and diluted them to ~2 × 10⁻⁶. 8 Cells / ml, EMS mutagenesis was repeated as described above. This mutagenesis and selection cycle was repeated a total of four times.

[0721] WS2: Preparation of randomized libraries of randomly mutated FS0105 yeast cells.

[0722] Step 2.1: Generate a total randomized library of mutagenic FS0105 yeast cells.

[0723] Following the fourth and final rounds of EMS mutagenesis (see step 1.1), viable cell titers were determined by plate-diluenting individual cells onto composite culture plates. Approximately 1,200–1,500 viable cells / plate were then plated onto YPD plates and incubated at 30°C for 3 days. Cells were dissociated from 96 individual plates, each plate washed with 3 ml of 0.1 M sodium phosphate buffer, forming 96 “library pools.” Approximately 5 × 10⁶ cells from these library pools were then… 7 One cell was used for DNA isolation (see below), while the remaining cells were centrifuged, resuspended in 40% glycerol / 10% YP, and frozen at -80°C to establish a “total random pool” of 96 libraries containing a total of approximately 120,000-150,000 EMS-mutated FS0105 clones.

[0724] WS3: Identify the library pool containing clones with selected Scfdc1 nonsense mutations in the FS0105 total random pool.

[0725] Step 3.1: Isolate yeast genomic DNA from the FS0105 total random library.

[0726] According to the manufacturer's instructions, use Pro 96 Genomic DNA Kit (Invitrogen) and EveryPrep TM Genomic DNA was isolated from each pool of the total randomized library using a Universal Vacuum Manifold (Invitrogen). The DNA concentration of each sample was determined using a NanoDrop 1000 3.8.1, and the DNA solution was adjusted to a final concentration of 5 ng DNA / μl with sterile, DNase-free water. DNA samples were stored in 96-well PCR plates at -20°C until further use.

[0727] Step 3.2: Analyze the gDNA samples from the library pool using ddPCR.

[0728] A unique ddPCR assay was designed specifically to distinguish the ScFDC1 mutant allele at nucleotide +476 of the wild-type coding sequence in strain FS0105 from the wild-type allele. The corresponding TGG codon is identical to the ScFDC1 sequence of the common laboratory reference strain S288C at this position (GenBank ID NCBI: NM_001180847). The mutation detection probe is complementary to the coding sequence and contains an A base at nucleotide +476. The reference detection probe is complementary to the coding sequence and contains a G base at nucleotide +476. Two flanking primers were designed to amplify the genomic sequence surrounding nucleotide +476 of the coding sequence. The assay was designed using the BioRads Droplet Digital PCR Assays design tool to detect the mutation. The following primers and probes were developed for the specific ScFDC1 locus:

[0729] Target-specific forward primer (5′-CATGTTTCAGACGGTGG-3′) (SEQ ID NO: 13)

[0730] Target-specific reverse primer (5′-CATACCTCTAGCAATTGACC-3′) (SEQ ID NO: 14)

[0731] Mutation detection probe (5′-ACGTACGGAATGTAGATTCT-3′) (SEQ ID NO: 15) - labeled with 6-carboxyfluorescein-FAM

[0732] The reference detection probe (5'-ACGTACGGAATGTGGATT-3') (SEQ ID NO: 16) was labeled with hexachlorofluorocarbon-HEX.

[0733] Typically, the next step is to determine whether the library contains mutant yeast strains. Screening is performed using a total of 96 yeast library pools, each containing 1200-1500 mutagenized yeast colonies, representing approximately 120,000-150,000 mutant yeast strains in total. ddPCR is performed essentially as described in Example 7. 5 μL of gDNA sample from each of the 96 mutagenized yeast strain pools is added to each well of a microtiter plate containing 17 μL of PCR reaction mixture, and thoroughly mixed by pipetting.

[0734] A microtiter plate for PCR was loaded onto a QX200 droplet reader (Bio-Rad) for droplet analysis. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). Thresholds were determined using 2-D curves, with amplitudes for channels 1 and 2 set to 2700 and 1500, respectively, for amplification. Comparison of individual fractional abundance values ​​indicated that, for mutant detection, gDNA at plate coordinate G01 had a higher signal than any other sample. The fractional abundance of gDNA pool G01 was 0.190% compared to 0.030%, where 0.030% represents the average fractional abundance of all 96 tested gDNA samples.

[0735] WS4: Identify individual yeast clones carrying selected Scfdc1 mutations by creating subsequent sub-pools.

[0736] Step 4.1: Create a 100er sub-pool from the FS0105 main random pool.

[0737] Based on the analysis of 96 gDNA library pool samples determined by ScFDC1-specific ddPCR, 1,200–1,500 yeast strains in pool G01 were considered likely to contain one or more clones carrying mutations of the gene of interest.

[0738] Cell titers of the positive library pool G01 identified in step 3.2 were determined by seeding individual dilutions onto YPD agar plates. Subsequently, 48 15ml culture tubes containing 3ml of liquid YPD medium were seeded, each with approximately 100 cells from library pool G01. The tubes were incubated at 30°C for 3 days in a rotary incubator, yielding 48 100er sub-pools from library pool G01, representing approximately 4800 individual clones from library pool G01.

[0739] Step 4.2: Isolate yeast genomic DNA from the FS0105 100er sub-pool.

[0740] Separate genomic DNA from each of the 48 100er sub-pools according to step 3.1 (see previous steps), and adjust the final concentration again to approximately 5 ng gDNA / μl.

[0741] Step 4.3: Analyze the 100er sub-pool gDNA sample by ddPCR.

[0742] gDNA from 48 100er sub-pools (each containing approximately 100 colonies from pool G01) was analyzed as described above (see step 3.2). Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 2000 and channel 2 amplitude set to 2500. Four individual wells (G07, C08, C09, G10) in the microtiter plate were identified as showing fractional abundances greater than 1%, indicating the presence of at least one mutant colony in a specific pool of 100 colonies.

[0743] Step 4.4: Create a 10er sub-pool from the FS0105 total random pool.

[0744] In the ddPCR analysis, the 100er subpool C09 showed the highest fractional abundance (2.99%) and was selected for further analysis.

[0745] Similar to step 4.1, the cell titers of the positive 100er sub-pool C09 identified in step 3.2 were determined by seeding individual dilutions onto YPD agar plates. Subsequently, 24 15ml culture tubes containing 3ml of liquid YPD medium were seeded, each with approximately 10 cells from the 100er sub-pool C09. The tubes were incubated at 30°C for 3 days in a rotary incubator to obtain 24 100er sub-pools from library C09, representing approximately 240 individual clones from the 100er sub-pool G01.

[0746] Step 4.5: Isolate yeast genomic DNA from the FS0105 100er sub-pool.

[0747] Genomic DNA was isolated from each of the 24 10er sub-pools according to step 3.1 (see step 4.4), and the final concentration was adjusted again to approximately 5 ng gDNA / μl.

[0748] Step 4.6: Analyze the 10er sub-pool gDNA sample by ddPCR.

[0749] gDNA from 24 separate 10er sub-pools was analyzed as described above. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad) as described above (see step 3.2). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 1500 and channel 2 amplitude set to 2000. Three separate pools (A02, CO3, and D03) showed fractional abundances greater than 10%, confirming the presence of at least one mutant colony in each of the three pools with 10 colonies.

[0750] Step 4.7: Identify individual clones from the FS0105 10er sub-pool carrying the introduced mutation of interest.

[0751] The 10er sub-pool D03, which showed the highest fractional abundance in the ddPCR analysis (step 4.6), was selected for further analysis. gDNA was isolated from 16 individual yeast colonies from the 10er sub-pool D03 and subjected to the same analyses as described above (see steps 4.3 and 4.6). Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 2000 and channel 2 amplitude set to 2500. Both individual pools (CO2 and D02) showed fractional abundances above 99.9%, confirming the presence of two mutant yeast colonies among the 16 tested individual colonies.

[0752] Step 4.8: Isolate pure cultures of mutant yeast.

[0753] Strain FS0105 showed a tendency to form larger cell aggregates. To ensure that future primary strains of the mutant yeast of interest originated from single cells rather than cell clumps, single cells were isolated from two separate pools, CO2 and DO2, using a Singer MSM 400 Dissecting microscope. For this purpose, cell suspensions from each pool were diluted 1000-fold in TE buffer, which supports the release of some cell clumps, and 10 μl of the dilution was spotted onto a composite medium agar plate. Single cells were then isolated according to the manufacturer's recommendations / manual.

[0754] Step 4.9: Identify the selected mutation of interest through DNA sequence analysis.

[0755] Genomic DNA was isolated from the single-cell isolates obtained in step 4.8 using a standard yeast genomic DNA preparation protocol, and the region covering the target gene ScFDC1 was amplified using standard PCR technology employing ScFDC1-specific oligonucleotides. The recovered DNA fragments were cleaned using an SV gel and a PCR clean-up system (Promega), and the region surrounding the nucleotide of interest (+476 of ScFDC1) was sequenced (LGC Genomics). Analysis of the recovered DNA sequences revealed that single-cell clones derived from individual pools CO2 and D03 (see 4.7 and 4.8) contained the desired nonsense mutation W159*, caused by a G-to-A transition at nucleotide +476 of the ScFDC1 gene.

[0756] Example 22

[0757] Wheat plants carrying specific mutations were identified using the workflow and methods described in the examples above. Various pools, etc., were named using the nomenclature shown in Figure 2. Although barley is a self-pollinating diploid species with 14 chromosomes, polyploidy is common in wheat. This example demonstrates that the method of the present invention can even be used for polyploid organisms, such as wheat. As described above, this method can be used to identify any mutant. This example describes the identification of a specific mutation (guanine to adenine) that causes the codon region of the wheat gene GASR7 (TaGASR7-A1) at position 91 of the A genome to change from the amino acid tryptophan to translation termination. The sequence of the GASR7-A1 gene is available in GenBank under number NCBI: KJ000052, the cDNA sequence is provided herein as SEQ ID NO: 25, and the amino acid sequence of GASR7 is provided as SEQ ID NO: 8.

[0758] ddPCR assay

[0759] A specific ddPCR assay was designed to specifically distinguish between the mutant and wild-type alleles of TaGASR7-A1 at nucleotide position 273 in the wild-type coding sequence (GenBank ID NCBI: KJ000052). The mutation detection probe is complementary to the coding sequence containing adenine at nucleotide position 273. The reference detection probe is complementary to the coding sequence containing guanine at nucleotide position 273. Two flanking primers were designed to amplify the genomic sequence surrounding nucleotide 273 in the coding sequence. The following primers and probes were developed for the specific TaGASR7-A1 locus:

[0760] Target-specific forward primer (5′-CGCCTGCCCCTGCTA-3′) (SEQ ID NO: 9)

[0761] Target-specific reverse primer (5′-AGAAGAAGAAGAAGAAGAAGAA AACCAAGAA-3′) (SEQ ID NO: 10)

[0762] Mutation detection probe (5′-CAACAACTGAAAGACCA-3′) (SEQ ID NO: 11) - labeled with 6-carboxyfluorescein-FAM

[0763] Reference detection probe (5'-CAACAACTGGAAGACCA-3') (SEQ ID NO: 12) - labeled with the fluorophore VIC

[0764] Wheat grain pools for random mutagenesis were prepared by soaking grains in 0.6% EMS for 17 hours. The grains were then rinsed with water and dried on filter paper for 45 minutes. The mutagenesis grains were planted immediately after drying.

[0765] WS3: Determine whether the library sample contains mutant grains.

[0766] Then, it was determined whether the library contained mutated grains, basically as described in WS3 and Example 7 above, with the following details:

[0767] A total of 94 GLPs (94 sub-pools) were screened, representing approximately 30,000 mutant wheat plants. ddPCR was performed as per Example 7.

[0768] Add a 5 μl gDNA sample from gDNA (GT#10001-GT#10094) to each well containing 17 μl of PCR reaction mixture and mix thoroughly by up-and-down pipetting.

[0769] PCR plates were loaded onto a QX200 Droplet Reader (Bio-Rad Laboratories) for droplet analysis. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad Laboratories). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 3000 and channel 2 amplitude set to 2000. Comparison of individual fractional abundance values ​​showed that the signal from the mutant-detected gDNA (GT#10072) was higher than any other sample. The fractional abundance of gDNA (GT#10072) was 0.130% compared to 0.024%, where 0.024% represents the average fractional abundance of all 94 tested gDNA samples.

[0770] WS4: Discovering individual grains characterized by mutations of interest

[0771] The identification of individual wheat grains carrying the mutation is basically as described in WS4 above, with the following details:

[0772] Based on gDNA analysis of subpools (GT#10001-GT#10094) determined by TaGASR7-A1-specific ddPCR, the 4500 grains of GLP#10072 (corresponding to positive sample gDNA (GT#10072)) are highly likely to contain one or more grains with the same mutation of interest.

[0773] FGLP#10072 was established by sequentially removing 10 grains from GLP#10072. Ten aliquots were placed on a weighing paper and held in place with a pair of pliers while a small, 2-3 mm deep hole was drilled in the endosperm using a burr cutter (Marathon-3, Saeyang Microtech) with a 1.6 mm drill bit. Rotational motion transferred flour from the endosperm to the weighing paper on top of and around the grain. Ten drilled samples were placed in a single 2 ml well of a microtiter plate, creating a secondary cell for the drilled wheat grain PDGLP#10072. Ninety-six flour samples (each containing flour from 10 drilled wheat grains) were transferred to individual wells (PFGLP#10072) of a 1.5 ml microtiter plate, maintaining the sample numbering system consistent with the drilled grain numbering.

[0774] gDNA was extracted from the PFGLP#10072 using the semi-automated DNA extraction method detailed in the instructions for the NucleoSpin 96Plant II kit (Macherey-Nagel). Therefore, each well of the microtiter plate contained gDNA from 10 grains of cereal flour.

[0775] DNA derived from PFGLP#10072 was analyzed as described above. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad Laboratories). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 2500 and channel 2 amplitude set to 1700. Two separate wells (B10 and G05) in the microtiter plate were identified, showing fractional abundances of 7.6% and 5.2%, respectively, both indicating the presence of two separate mutants in the two separate wells of PDGLP#10072.

[0776] All 10 grains from well B10 of PDGLP#10072 were germinated. Leaf material was harvested from all 10 plantlets, and DNA was extracted using REDExtract (Sigma Aldrich). gDNA from the leaf samples was analyzed as described above. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad Laboratories). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 2000 and channel 2 amplitude set to 1600. One plantlet from well C03 of PDGLP#10072 showed a fractional abundance of 99%, confirming the presence of a homozygous mutant.

[0777] Further phenotypic validation was performed by direct sequencing of identified mutants and reference samples. DNA was extracted from leaf material using the REDExtract DNA extraction method (Sigma Aldrich). For sequencing analysis, 50 μl PCR reactions were prepared containing 1 μl purified gDNA, 20 μl REDExtract (Sigma Aldrich), 500 nM target-specific forward primer, 500 nM target-specific reverse primer, and water. Samples were thermally cycled using the following PCR conditions: denaturation at 94 °C for 2 min, 38 cycles at 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min, and then the PCR plates were stored at 8 °C. Individual PCR products were purified using NucleoSpin Gel and a PCR washing kit. All samples were sequenced using target-specific forward and reverse primers.

[0778] In ddPCR analysis, the mutant-positive plantlets also showed a heterozygous mutant genotype at the predetermined genomic location. The reference sample showed a homozygous wild-type genotype at the same location.

[0779] Example 23

[0780] Barley plants carrying specific mutations were identified by combining two technologies, one provided by RainDance Technologies and the other by Bio-Rad Laboratories. Generally, RainDance technology was used to identify specific but complex samples containing gDNA templates from numerous mutant grains, while subsequent BioRad-based analysis helped identify individual grains characterized by the mutation of interest. Nomenclature for various pools, etc., was completed using the nomenclature shown in Figure 2.

[0781] As described above, this method can be used to identify any mutant. This example describes the identification of a specific mutation (guanine to adenine) that causes a translational termination at position 31 of the codon region encoding the putative BAHD acyltransferase (HvBADH1) in the barley gene.

[0782] ddPCR assay

[0783] A specific ddPCR assay was designed to specifically distinguish the mutant and wild-type alleles at nucleotide position 93 of HvBADH1. The wild-type coding sequence (cDNA sequence) is provided herein as SEQ ID NO: 17, and the mutant coding sequence is provided herein as SEQ ID NO: 18. The amino acid sequence of HvBADH1 is provided herein as SEQ ID NO: 19. The mutation detection probe is complementary to the coding sequence portion containing adenine at nucleotide position 93 of SEQ ID NO: 17. The reference detection probe is complementary to the coding sequence portion containing guanine at nucleotide position 93. Two flanking primers were designed to amplify the genomic sequence surrounding nucleotide position 93 in the coding sequence. Additionally, a blocking probe was developed using the nucleotide sequence of the reference detection probe, supplemented with a 3' spacer, which prevents the extension of the reference sequence by DNA polymerase during PCR. The following primers and probes were developed for the specific HvBADH1 locus:

[0784] Target-specific forward primer (5'-CCCGACCACACGC-3') (SEQ ID NO: 20)

[0785] Target-specific reverse primer (5'-ACTCCACCAGGCCG-3') (SEQ ID NO: 21)

[0786] Mutation detection probe (5′-CTGGCGTGAGTGGAC-3′) (SEQ ID NO: 22) - labeled with 6-carboxyfluorescein-FAM

[0787] Reference detection probe (5′-CTGGCGTGGGTGGA-3′)(SEQ ID NO: 23) - labeled with tetrachlorofluorescein - TET

[0788] The blocking probe (5′-CTGGCGTGGGTGGA-3′) (SEQ ID NO: 24) is labeled with a 2′,3′-dideoxy C spacer.

[0789] Preparation of gDNA "super pool".

[0790] Simply put, 50 μl of each gDNA extract from all 94 “Aliquots of Individual Sub-Flour Total (ASFT)” represented by a 96-well plate is combined into a “superpool”.

[0791] Determine whether the "super pool" contains mutated grains.

[0792] Then it was determined whether the “super pool” containing DNA from 94 ASFTs contained mutated grains, essentially as described in Example 17 above, with the following details:

[0793] First, 2 μl of gDNA from the superpool was added to 50 μl of enrichment and blocking PCR mixture, which contained 10 μl of 5×Q5 reaction buffer (New England Biolabs), 200 μM dNTPs, 0.02 U / μl Q5 high-fidelity DNA polymerase, 100 nM target-specific forward PCR primers, 100 nM target-specific reverse PCR primers, 100 nM blocking probe (2',3'-dideoxyC spacer), and water. The PCR mixture was thermally cycled using standard PCR conditions: 20 cycles of denaturation at 98 °C for 2 min, followed by 20 cycles of denaturation at 98 °C for 10 s, 57 °C for 20 s, and 72 °C for 10 s, and a final incubation at 72 °C for 5 min, followed by storage at 8 °C.

[0794] Next, droplets were generated for four individually enriched and blocked “superpools” using the RainDrop Source instrument. Ten-tenths of the enriched and blocked PCR product from each “superpool” were bound to the following: 25 μl Supermix (Bio-Rad) for probes; 1× droplet stabilizer (RainDance); 900 nM target-specific forward primer; 900 nM target-specific reverse primer; 120 nM reference detection probe (TET); 440 nM mutation detection probe (FAM); and H₂O to adjust the total reaction volume to 50 μl. Four separate reaction mixtures (representing four separate “superpool” gDNAs) were added to the source chip (RainDance) and processed on the Source Instrument (RainDance).

[0795] Eight-well PCR strips containing droplets generated from the reaction mixture by Source Instrument (RainDance) were sealed and thermally cycled under standard PCR conditions: 40 cycles of denaturation at 95°C for 10 min, followed by 15 seconds at 95°C and 1 minute at 55°C, and a final extension at 98°C for 10 min, then stored at 8°C. The amplified mixture was then transferred to Sense Instrument (RainDance) and analyzed using RainDrop Analyst software.

[0796] The average fractional abundance of the four superpools was 0.0115%. The fractional abundance of superpool SP-gDNA#05 was 0.0228%, indicating that this superpool is very likely to contain DNA from the mutant grain.

[0797] Determine whether the library sample contains mutated grains.

[0798] Then, it was determined whether the 94 ASFT plates corresponding to SP-gDNA#05 contained mutated grains, basically as described in Example 7 above, with the following details:

[0799] A total of 94 SGTs (total number of seed grains) were screened, representing approximately 30,000 mutant barley plants. The gDNA used for screening was purified gDNA from ASFT—i.e., unenriched DNA. For analytical purposes, 5 μl of purified gDNA from gDNA (GT#377-GT#470) was added to a 17 μl PCR mixture containing 11 μl of 11 μl of lddPCR Supermix for probes (No. dUTP; Bio-Rad Laboratories), 900 nM target-specific PCR primers, a 250 nM mutation detection probe (6-carboxyfluorescein-FAM), and a wild-type detection probe (tetrachlorofluorescein-TET). The reaction mixture was loaded onto an AutoDG Droplet Generator (Bio-Rad), and droplets were generated according to the manufacturer's manual. The droplet emulsion was thermally cycled using standard PCR conditions: 40 PCR cycles were performed, including denaturation at 95°C for 10 minutes, followed by 30 seconds at 94°C and 1 minute at 55°C, and a final extension at 98°C for 10 minutes. The microtiter plate was then stored at 8°C. PCR amplification in the droplets was confirmed using a QX200 DropletReader (Bio-Rad Laboratories).

[0800] PCR plates were loaded onto a QX200 Droplet Reader (Bio-Rad Laboratories) for droplet analysis. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad Laboratories). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 2200 and channel 2 amplitude set to 2700. Comparison of individual fractional abundance values ​​showed that gDNA (GT#416) contained more signal from mutant detection compared to any other sample. The fractional abundance of gDNA (GT#416) was 0.4% compared to 0.073%, where 0.073% represents the average fractional abundance of all 94 tested gDNA samples.

[0801] Discover individual grains characterized by mutations of interest

[0802] The identification of individual barley grains carrying mutations, as basically described in Examples 9-15 above, has the following details:

[0803] Based on the analysis of gDNA (GT#416) with HvBADH1-specific ddPCR assay, 4500 GLP#416 grains (corresponding to positive sample gDNA (GT#416)) were considered to likely contain one or more grains with the same mutation of interest.

[0804] FGLP#416 was established by sequentially removing 96 samples of 12 grains each from GLP#416. Each 12-grain aliquot was placed on a weighing paper and held in place with a pair of pliers while a small, 2-3 mm deep hole was drilled in the endosperm using a sculptor (Marathon-3, Saeyang Microtech) fitted with a 1.6 mm drill bit. Rotational motion moved the flour from the endosperm onto the weighing paper on top of and around the grain. Ten drilled samples were placed in individual 2 ml wells of a microtiter plate to obtain a secondary cell for the drilled barley grain PDGLP#416. The 96 flour samples (each from 12 drilled barley grains) were transferred to individual wells of a 1.5 ml microtiter plate (PFGLP#416), maintaining the sample numbering system to match the numbering of the drilled grains.

[0805] gDNA was extracted from PFGLP#416 using the semi-automated DNA extraction method detailed in the instructions for the NucleoSpin 96Plant II kit (Macherey-Nagel). Therefore, each well of the microtiter plate contained gDNA from flour extracted from 12 grains.

[0806] DNA from PFGLP#416 was analyzed using the same PCR mixture and PCR reaction conditions (to determine whether the library sample contained mutated grains). Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad Laboratories). Thresholds were determined using 2-D curves, with channel 1 amplitude set to 3000 and channel 2 amplitude set to 2500. Upon identification, two separate wells (D10 and F02) in the microtiter plate showed fractional abundances of 3.4% and 13.7%, respectively, indicating the presence of two separate mutants in the two independent wells of PDGLP#416.

[0807] All 12 grains from well F02 of PDGLP#416 were germinated. Leaf material was collected from all 12 plantlets and DNA was extracted using REDExtract (Sigma Aldrich). gDNA from the leaf samples was analyzed using the same PCR mixture and PCR reaction conditions as described above (to determine if the library samples contained mutant grains). Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad Laboratories). Thresholds were determined using 2-D curves with channel 1 amplitude set to 5000 and channel 2 amplitude set to 3200. One plantlet from well F02 of PDGLP#416 showed a fractional abundance of 39%, confirming the presence of a heterozygous mutant.

[0808] References

[0809] Andrew J.Goodall,Pankaj Kumar and Alyson K.Tobin(2013).Identification and expression analyzes of cytosolic glutamine synthetase genes in barley(Hordeum vulgare L.).Plant Cell Physiol.54:492-505.

[0810] Botticella,E.,Sestili,F.,Hernandez-Lopez,A.,Phillips,A.,&Lafiandra,D.(2011).High resolution melting analysis for the detection of EMS inducedmutations in wheat SbeIIa genes.BMC Plant Biology 11:156.

[0811] Inoue,H.,Nojima,H.and Okayama,H.,(1990).High efficiency transformation of Escherichia coli with plasmids.Gene 96,23-28.

[0812] Jiang,F.et al.(2016).Structues of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.Science 351:867-871.

[0813] Pleasance et al. (2010). Acomprehensive catalog of somatic mutations from a human cancer genome. Nature 463:191-196.

[0814] B.and Lehner,B.(2012).Chromatin organization is a major influence on regional mutation rates in uman cancer cells.Nature 488:504-507.

[0815] Yamaya,T.and Kusano,M.(2014).Evidence supporting distinct functions of three cytosolic glutamine synthetases and two NADH-glutamate synthases inrice.J.Exp.Bot.65:5519-5525.

[0816] Wellner, VP and Meister, A. (1966). Binding of Adenosine Triphosphate and Adenosine Diphosphate by Glutamine Synthetase. Biochemistry 5:872-879. sequence list <110> Carlsberg A / S <120> Methods for screening mutants within a biological population by applying a hybrid fission method <130> 2205435DK01 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 1089 <212> DNA <213> Hordeum (vulgar) <400> 1 atgtctcggc tcgccgacct tctcagcctc gacctgtccg gctgcaccgg caatcatc 60 gccgagtaca tatgggtcgg cggcaccggg atggacgtca ggagcaagc caggacgctt 120 cccggacccg tggacgaccc cagcaagctt ccaagtgga atttcgacgg ctccagcacc 180 ggccaagcca cgggcgacga cagcgaagtc atcctccgac cccaagccat cttcagggac 240 ccgttcagga aagggaacaa catcctggtc atctgtgact gctatgcgcc taccggag 300 ccgattccga gcacaagcg gtacaacgcg gcgaggatat tcggccatcc tgatgtcaag 360 tctgaagaac catggtatgg gattgagcag gagtacaccc ttctccagaa ggacaccac 420 tggcccattg gctggccact agggggttac cctggccctc aggggcctta ctactgcgcc 480 gcgggtgcgg agaatctta cgggcgcgac atcgtcgacg cccactacaa ggcctgccctc 540 tacgccggca tcacatcgg cggcatcaat gcagaagtca tgccagggca gtgggaggttc 600 caagtcggcc cttccgtcgg yatctccgcc ggcgacgagc tctggcggc tcgctacatt 660 ctcgagagga tcactgagat cgccggcgtc gtcgtctcct tcgaccccaa accgatcccg 720 ggagagtgga acggtgccgg tgcccacaca aactacagca ccaagtcgat gaggagcgag 780 ggcgggtacg aggtgatcaa gagggcgatc aagaagctcg aggcgcggca cacggagcac 840 atagccgcct acggggaagg caacgagcgc cggctcaccg gccgccacga gaccgccgac 900 atcaacacct tcgtatgggg cgtggcaaac cgcggcgcgt cggtgcgggt ggggcgcgac 960 accgagaagg aaggcagggg ctacttcgag gaccggaggc cggcgtccaa catggatccc 1020 tacgtcgtca cctccatgat cgccgagacc accatcctct ggaaggccgg tctctccaat 1080 ggcaagtag 1089 <210> 2 <211> 362 <212> PRT <213> Hordeum vulgare <400> 2 Put Ser Arg Leu Ala Asp Leu Leu Ser Leu Asp Leu Ser Gly Cys Thr 1 5 10 15 Gly Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Thr Gly Met Asp 20 25 30 Val Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Asp Asp Pro Ser 35 40 45 Lys Leu Pro Lys Trp Asn Phe Asp Gly Ser Ser Thr Gly Gln Ala Thr 50 55 60 Gly Asp Asp Ser Glu Val Ile Leu Arg Pro Gln Ala Ile Phe Arg Asp 65 70 75 80 Pro Phe Arg Lys Gly Asn Asn Ile Leu Val Ile Cys Asp Cys Tyr Ala 85 90 95 Pro Thr Gly Glu Pro Ile Pro Ser Asn Lys Arg Tyr Asn Ala Ala Arg 100 105 110 Ile Phe Gly His Pro Asp Val Lys Ser Glu Glu Pro Trp Tyr Gly Ile 115 120 125 Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Thr Asn Trp Pro Ile Gly 130 135 140 Trp Pro Leu Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala 145 150 155 160 Ala Gly Ala Glu Lys Ser Tyr Gly Arg Asp Ile Val Asp Ala His Tyr 165 170 175 Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Gly Gly Ile Asn Ala Glu 180 185 190 Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205 Ser Ala Gly Asp Glu Leu Trp Ala Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220 Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro 225 230 235 240 Gly Glu Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255 Met Arg Ser Glu Gly Gly Tyr Glu Val Ile Lys Arg Ala Ile Lys Lys 260 265 270 Leu Glu Ala Arg His Thr Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285 Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe 290 295 300 Val Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly Arg Asp 305 310 315 320 Thr Glu Lys Glu Gly Arg Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335 Asn Met Asp Pro Tyr Val Val Thr Ser Met Ile Ala Glu Thr Thr Ile 340 345 350 Leu Trp Lys Ala Gly Leu Ser Asn Gly Lys 355 360 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Primers <400> 3 gtgatcaaga gggcgatcaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Primers <400> 4 caagtctcaa ctcgccgtat 20 <210> 5 <211> 14 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> 5 aagacaacga gcgc 14 <210> 6 <211> 14 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> 6 aaggcaacga gcgc 14 <210> 7 <211> 503 <212> PRT <213> Saccharomyces cerevisiae <400> 7 Met Arg Lys Leu Asn Pro Ala Leu Glu Phe Arg Asp Phe Ile Gln Val 1 5 10 15 Leu Lys Asp Glu Asp Asp Leu Ile Glu Ile Thr Glu Glu Ile Asp Pro 20 25 30 Asn Leu Glu Val Gly Ala Ile Met Arg Lys Ala Tyr Glu Ser His Leu 35 40 45 Pro Ala Pro Leu Phe Lys Asn Leu Lys Gly Ala Ser Lys Asp Leu Phe 50 55 60 Ser Ile Leu Gly Cys Pro Ala Gly Leu Arg Ser Lys Glu Lys Gly Asp 65 70 75 80 His Gly Arg Ile Ala His His Leu Gly Leu Asp Pro Lys Thr Thr Ile 85 90 95 Lys Glu Ile Ile Asp Tyr Leu Leu Glu Cys Lys Glu Lys Glu Pro Leu 100 105 110 Pro Pro Ile Thr Val Pro Val Ser Ser Ala Pro Cys Lys Thr His Ile 115 120 125 Leu Ser Glu Glu Lys Ile His Leu Gln Ser Leu Pro Thr Pro Tyr Leu 130 135 140 His Val Ser Asp Gly Gly Lys Tyr Leu Gln Thr Tyr Gly Met Trp Ile 145 150 155 160 Leu Gln Thr Pro Asp Lys Lys Trp Thr Asn Trp Ser Ile Ala Arg Gly 165 170 175 Met Val Val Asp Asp Lys His Ile Thr Gly Leu Val Ile Lys Pro Gln 180 185 190 His Ile Arg Gln Ile Ala Asp Ser Trp Ala Ala Ile Gly Lys Ala Asn 195 200 205 Glu Ile Pro Phe Ala Leu Cys Phe Gly Val Pro Pro Ala Ala Ile Leu 210 215 220 Val Ser Ser Met Pro Ile Pro Glu Gly Val Ser Glu Ser Asp Tyr Val 225 230 235 240 Gly Ala Ile Leu Gly Glu Ser Val Pro Val Val Lys Cys Glu Thr Asn 245 250 255 Asp Leu Met Val Pro Ala Thr Ser Glu Met Val Phe Glu Gly Thr Leu 260 265 270 Ser Leu Thr Asp Thr His Leu Glu Gly Pro Phe Gly Glu Met His Gly 275 280 285 Tyr Val Phe Lys Ser Gln Gly His Pro Cys Pro Leu Tyr Thr Val Lys 290 295 300 Ala Met Ser Tyr Arg Asp Asn Ala Ile Leu Pro Val Ser Asn Pro Gly 305 310 315 320 Leu Cys Thr Asp Glu Thr His Thr Leu Ile Gly Ser Leu Val Ala Thr 325 330 335 Glu Ala Lys Glu Leu Ala Ile Glu Ser Gly Leu Pro Ile Leu Asp Ala 340 345 350 Phe Met Pro Tyr Glu Ala Gln Ala Leu Trp Leu Ile Leu Lys Val Asp 355 360 365 Leu Lys Gly Leu Gln Ala Leu Lys Thr Thr Pro Glu Glu Phe Cys Lys 370 375 380 Lys Val Gly Asp Ile Tyr Phe Arg Thr Lys Val Gly Phe Ile Val His 385 390 395 400 Glu Ile Ile Leu Val Ala Asp Asp Ile Asp Ile Phe Asn Phe Lys Glu 405 410 415 Val Ile Trp Ala Tyr Val Thr Arg His Thr Pro Val Ala Asp Gln Met 420 425 430 Ala Phe Asp Asp Val Thr Ser Phe Pro Leu Ala Pro Phe Val Ser Gln 435 440 445 Ser Ser Arg Ser Lys Thr Met Lys Gly Gly Lys Cys Val Thr Asn Cys 450 455 460 Ile Phe Arg Gln Gln Tyr Glu Arg Ser Phe Asp Tyr Ile Thr Cys Asn 465 470 475 480 Phe Glu Lys Gly Tyr Pro Lys Gly Leu Val Asp Lys Val Asn Glu Asn 485 490 495 Trp Lys Arg Tyr Gly Tyr Lys 500 <210> 8 <211> 101 <212> PRT <213> Wheat (Triticum aestivum) <400> 8 Met Ala Lys Ile Ser Phe Leu Leu Val Ala Leu Leu Val Leu Ala Val 1 5 10 15 Gly Phe Pro Val Glu Val Met Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Gly Gly Asn Leu Lys Pro Trp Glu Cys Ser Ser Lys Cys Ser 35 40 45 Ser Arg Cys Ser Gly Thr Gln Tyr Lys Lys Ala Cys Leu Thr Tyr Cys 50 55 60 Asn Lys Cys Cys Ala Thr Cys Leu Cys Val Pro Pro Gly Thr Tyr Gly 65 70 75 80 Asn Lys Gly Ala Cys Pro Cys Tyr Asn Asn Trp Lys Thr Lys Glu Gly 85 90 95 Gly Pro Lys Cys Pro 100 <210> 9 <211> 15 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Primers <400> 9 cgcctgcccc tgcta 15 <210> 10 <211> 31 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Primers <400> 10 agaagaagaa gaagaagaag aaaaccaaga a 31 <210> 11 <211> 17 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> 11 caacaactga aagacca 17 <210> 12 <211> 17 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> 12 caacaactgg aagacca 17 <210> 13 <211> 17 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Primers <400> 13 catgtttcag acggtgg 17 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Primers <400> 14 catacctcta gcaattgacc 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> 15 acgtacggaa tgtagattct 20 <210> 16 <211> 18 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> 16 acgtacggaa tgtggatt 18 <210> 17 <211> 1260 <212> DNA <213> Barley (Hordeum vulgare) <400> 17 atggcgtcgt cgagcttcaa ggtgacgcgg atctcggagg gcgcggtgaa gccggcgtcg 60 gagacgcccg accacacgct gccgctggcg tgggtggacc ggtacccgac ccaccgcggc 120 ctggtggagt cgatgcacat cttccggtcc ggcgccgacg cggcccccgg cgtgatccgc 180 gaggcgctgg gcaaggcgct ggccttcttc tacccgctgg cggggcgcat cgtggagcag 240 ccggagaagg ggtgccccgc catccgctgc accgccgacg gcgtctactt cgcggaggcc 300 gtcgccgagt gcagcctgga ggacgtccgg ttcctggagc gccccctgct gctccccaag 360 gaggacctcg tcccctaccc cgccgccgat ctctgggccg tcgagcccca caacaccatc 420 atgatgatgc agatcacgaa attcacatgc ggcgggttcg tgatgggcct ccggttcaac 480 cacgcgtcgg cggacggcat gggcgcggcg cagttcatca aggcggtcgg cgacatggcc 540 cgggggctcc cggagccgac ggtgaagccg gtgtgggaca gggagaagtt ccccaacccg 600 agcatcaagc cgggccctct cccggagctc ccggtgctgg cgctggacta catcgtgctc 660 gacttcccca cgggctacat cgacgggctc aagacgcagt acaaggcgca cagcggcaag 720 ttctgctccg gcttcgacgt gctgacggcc aagctgtggc agtgccgtac ccgggcgctg 780 aacctggagc cggacgccac ggtgaagctg tgctttcttg ccagcgtgcg ccacctgctg 840 aagctggacg ccgggtacta cggcaactcc atcttccccg tgaagatgtc cgggacgagc 900 aagaaggtgc tggagtcgtc ggtgatggag gtgatcgaca tgatccgggga ggccaagcag 960 cggatggcgg tggagttctt ccagttcgcc aaggaggaga cgcggcagga ccccttccag 1020 atgaccttcg actacgagtc catctacgtc tccgactgga gcaagctggg gttctccgac 1080 gtggactacg gcttcggccc gcccatgttc gccggaccgc tcgtcaacaa cgacttcatc 1140 gcctccgtcg tcatcctcaa ggcgccgctg ccgctggacg gcaccaggat gctcgccagc 1200 tgcgtcacca aggagcactc gcaggagttc gcccgcggca tgaaggagga cctgccatga 1260 <210> 18 <211> 1260 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Mutant cDNA <400> 18 atggcgtcgt cgagcttcaa ggtgacgcgg atctcggagg gcgcggtgaa gccggcgtcg 60 gagacgcccg accacacgct gccgctggcg tgagtggacc ggtacccgac ccaccgcggc 120 ctggtggagt cgatgcacat cttccggtcc ggcgccgacg cggcccccgg cgtgatccgc 180 gaggcgctgg gcaaggcgct ggccttcttc tacccgctgg cggggcgcat cgtggagcag 240 ccggagaagg ggtgccccgc catccgctgc accgccgacg gcgtctactt cgcggaggcc 300 gtcgccgagt gcagcctgga ggacgtccgg ttcctggagc gccccctgct gctccccaag 360 gaggacctcg tcccctaccc cgccgccgat ctctgggccg tcgagcccca caacaccatc 420[[ID=!7]] atgatgatgc agatcacgaa attcacatgc ggcgggttcg tgatgggcct ccggttcaac 480 cacgcgtcgg cggacggcat gggcgcggcg cagttcatca aggcggtcgg cgacatggcc 540 cgggggctcc cggagccgac ggtgaagccg gtgtgggaca gggagaagtt ccccaacccg 600 agcatcaagc cgggccctct cccggagctc ccggtgctgg cgctggacta catcgtgctc 660 gacttcccca cgggctacat cgacgggctc aagacgcagt acaaggcgca cagcggcaag 720 ttctgctccg gcttcgacgt gctgacggcc aagctgtggc agtgccgtac ccgggcgctg 780 aacctggagc cggacgccac ggtgaagctg tgctttcttg ccagcgtgcg ccacctgctg 840 aagctggacg ccgggtacta cggcaactcc atcttccccg tgaagatgtc cgggacgagc 900 aagaaggtgc tggagtcgtc ggtgatggag gtgatcgaca tgatccgggga ggccaagcag 960 cggatggcgg tggagttctt ccagttcgcc aaggaggaga cgcggcagga ccccttccag 1020 atgaccttcg actacgagtc catctacgtc tccgactgga gcaagctggg gttctccgac 1080 gtggactacg gcttcggccc gcccatgttc gccggaccgc tcgtcaacaa cgacttcatc 1140 gcctccgtcg tcatcctcaa ggcgccgctg ccgctggacg gcaccaggat gctcgccagc 1200 tgcgtcacca aggagcactc gcaggagttc gcccgcggca tgaaggagga cctgccatga 1260 <210> 19 <211> 419 <212> PRT <213> Hordeum vulgare <400> 19 Met Ala Ser Ser Ser Phe Lys Val Thr Arg Ile Ser Glu Gly Ala Val 1 5 10 15 Lys Pro Ala Ser Glu Thr Pro Asp His Thr Leu Pro Leu Ala Trp Val 20 25 30 Asp Arg Tyr Pro Thr His Arg Gly Leu Val Glu Ser Met His Ile Phe 35 40 45 Arg Ser Gly Ala Asp Ala Ala Pro Gly Val Ile Arg Glu Ala Leu Gly 50 55 60 Lys Ala Leu Ala Phe Phe Tyr Pro Leu Ala Gly Arg Ile Val Glu Gln 65 70 75 80 Pro Glu Lys Gly Cys Pro Ala Ile Arg Cys Thr Ala Asp Gly Val Tyr 85 90 95 Phe Ala Glu Ala Val Ala Glu Cys Ser Leu Glu Asp Val Arg Phe Leu 100 105 110 Glu Arg Pro Leu Leu Leu Pro Lys Glu Asp Leu Val Pro Tyr Pro Ala 115 120 125 Ala Asp Leu Trp Ala Val Glu Pro His Asn Thr Ile Met Met Met Gln 130 135 140 Ile Thr Lys Phe Thr Cys Gly Gly Phe Val Met Gly Leu Arg Phe Asn 145 150 155 160 His Ala Ser Ala Asp Gly Met Gly Ala Ala Gln Phe Ile Lys Ala Val 165 170 175 Gly Asp Met Ala Arg Gly Leu Pro Glu Pro Thr Val Lys Pro Val Trp 180 185 190 Asp Arg Glu Lys Phe Pro Asn Pro Ser Ile Lys Pro Gly Pro Leu Pro 195 200 205 Glu Leu Pro Val Leu Ala Leu Asp Tyr Ile Val Leu Asp Phe Pro Thr 210 215 220 Gly Tyr Ile Asp Gly Leu Lys Thr Gln Tyr Lys Ala His Ser Gly Lys 225 230 235 240 Phe Cys Ser Gly Phe Asp Val Leu Thr Ala Lys Leu Trp Gln Cys Arg 245 250 255 Thr Arg Ala Leu Asn Leu Glu Pro Asp Ala Thr Val Lys Leu Cys Phe 260 265 270 Phe Ala Ser Val Arg His Leu Leu Lys Leu Asp Ala Gly Tyr Tyr Gly 275 280 285 Asn Ser Ile Phe Pro Val Lys Met Ser Gly Thr Ser Lys Lys Val Leu 290 295 300 Glu Ser Ser Val Met Glu Val Ile Asp Met Ile Arg Glu Ala Lys Gln 305 310 315 320 Arg Met Ala Val Glu Phe Phe Gln Phe Ala Lys Glu Glu Thr Arg Gln 325 330 335 Asp Pro Phe Gln Met Thr Phe Asp Tyr Glu Ser Ile Tyr Val Ser Asp 340 345 350 Trp Ser Lys Leu Gly Phe Ser Asp Val Asp Tyr Gly Phe Gly Pro Pro 355 360 365 Met Phe Ala Gly Pro Leu Val Asn Asn Asp Phe Ile Ala Ser Val Val / / 370 375 380 Ile Leu Lys Ala Pro Leu Pro Leu Asp Gly Thr Arg Met Leu Ala Ser 385 390 395 400 Cys Val Thr Lys Glu His Ser Gln Glu Phe Ala Arg Gly Met Lys Glu 405 410 415 Asp Leu Pro <210> 20 <211> 13 <212> DNA <213> Artificial sequence / / <220> <221> <222> <223> Primer <400> 20 cccgaccaca cgc 13 <210> 21 <211> 14 <212> DNA <213> Artificial sequence / / <220> <221> <222> <223> Primers <400> twenty one actccaccag gccg 14 <210> twenty two <211> 15 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> twenty two ctggcgtgag tggac 15 <210> twenty three <211> 14 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> twenty three ctggcgtggg tgga 14 <210> twenty four <211> 14 <212> DNA <213> Artificial sequence <220> <221> <222> <223> probe <400> twenty four ctggcgtggg tgga 14 <210> 25 <211> 306 <212> DNA <213> Wheat (Triticum aestivum) <400> 25 atggccaaga tctccttcct cctcgtggcg ctcctcgtcc tcgccgtcgg gttccccgtg gaggtgatgg gaggtggggg cggcggcggc ggtggcggtg gcggcggcaa cctcaagcca 120 180. tgggagtgct cgtccaagtg ctcgtcgcgg tgctcgggga cgcagtacaa gaaggcgtgc ctgacctact gcaacaagtg ctgcgccacc tgcctctgcg tgccgccggg cacctacggc 240 aacaagggcg cctgcccctg ctacaacaac tggaagacca aggagggagg ccccaagtgc ccctag 306 <210> 26 <211> 1512 <212> DNA <213> Spices(Saccharomyces cerevisiae) <400> 26 atgaggaagc taaatccagc tttagaattt agagacttta tccaggtctt aaaagatgaa gatgacttaa tcgaaattac cgaagagatt gatccaaatc tcgaagtagg tgcaattatg aggaaggcct atgaatccca cttaccagcc ccgttattta aaaatctcaa aggtgcttcg aaggatcttt tcagcatttt aggttgccca gccggtttga gaagtaagga gaaaggagat catggtaga ttgcccatca tctggggctc gacccaaaaa caactatca ggaaatcata gattattgc tggagtgtaa ggagaaggaa cctctccccc caatcactgt tcctgtgtca 360 tctgcacctt gtaaaacaca tatactttct gaagaaaaaa tacatctaca aagcctgcca 420 acaccatatc tacatgtttc agacggtggc aagtacttac aaacgtacgg aatgtggatt 480 cttcaaactc cagataaaaa atggactaat tggtcaattg ctagaggtat ggttgtagat 540 gacaagcata tcactggtct ggtaattaaa ccacaacata ttagacaaat tgctgactct 600 tgggcagcaa ttggaaaagc aaatgaaatt cctttcgcgt tatgttttgg cgttccccca 660 gcagctattt tagttagttc catgccaatt cctgaaggtg tttctgaatc ggattatgtt 720 ggcgcaatct tgggtgagtc ggttccagta gtaaaatgtg agaccaacga tttaatggtt 780 cctgcaacga gtgagatggt atttgagggt actttgtcct taacagatac acatctggaa 840 ggcccatttg gtgagatgca tggatatgtt ttcaaaagcc aaggtcatcc ttgtccattg 900 tacactgtca aggctatgag ttacagagac aatgctattc tacctgtttc gaaccccggt 960 cttgtacgg atgagacaca taccttgatt ggttcactag tggctactga ggccaaggag 1020 ctggctattg aatctggctt gccaattctg gatgccttta tgccttatga ggctcaggct 1080 ctttggctta tcttaaaggt ggatttgaaa gggctgcaag cattgaagac aacgcctgaa 1140 gaattttgta agaaggtagg tgatatttac tttaggacaa aagttggttt tatagtccat 1200 gaaataattt tggtggcaga tgatatcgac atatttaact tcaaagaagt catctgggcc 1260 tacgttacaa gacatacacc tgttgcagat cagatggctt ttgatgatgt cacttctttt 1320 cctttggctc cctttgtttc gcagtcatcc agaagtaaga ctatgaaagg tggaaagtgc 1380 gttactaatt gcatatttag acagcaatat gagcgcagtt ttgactacat aacttgtaat 1440 tttgaaaagg gatatccaaa aggattagtt gacaaagtaa atgaaaattg gaaaaggtac 1500 ggatataaat aa 1512

Claims

1. A method for identifying a subpool of a predetermined species organism or its reproductive portion, wherein the subpool comprises a predetermined species organism carrying one or more predetermined substitution and / or deletion mutations of a nucleotide of interest (NOI) in a target sequence, wherein the species is a plant, fungus, algae, or unicellular organism, the method comprising the following steps: a) Provide a pool of organisms or reproductive portions of the species representing multiple genotypes, wherein the pool of organisms is prepared by subjecting organisms or reproductive portions of the species to random mutagenesis; b) Divide the pool into one or more sub-pools of organisms or their reproductive parts, wherein each sub-pool includes more than one single organism or its reproductive part of each genotype, wherein all offspring of an organism or its reproductive part from the pool are included in one sub-pool; c) Each subpool is randomly divided into multiple parts in such a way that each part includes an organism or its reproductive portion representing each genotype of the subpool; d) Prepare genomic DNA (gDNA) samples from one of the plurality of portions of each subpool while preserving the remaining portion of the organism or its reproductive portion while maintaining its reproductive potential; e) Perform multiple compartmentalized PCR amplifications, each compartmentalized PCR amplification containing gDNA samples from a sub-pool, one or more sets of primers and PCR reagents to amplify the target sequence, each of the one or more sets of primers being located on the flanking side of the target sequence. f) Detect PCR amplification products containing one or more target sequences, said target sequences containing mutations in NOI, thereby identifying subpools containing said mutations.

2. The method according to claim 1, wherein the species is a single-celled organism.

3. The method according to claim 1, wherein the species is yeast.

4. The method of claim 1, wherein the species is a plant, and steps a) and b) of the method comprise the following steps: I. Provides multiple reproductive parts for the plant; II. Randomly mutate the reproductive portion to obtain the M0 generation reproductive portion; III. Allow the reproductive portion of the M0 generation to grow into a mature plant, and obtain a reproductive portion from the mature plant, wherein the reproductive portion is the reproductive portion of the M1 generation; IV. Obtain the M1 generation reproductive portion from the mature plant to obtain a reproductive portion pool; V. Divide the reproductive portion pool obtained in step IV. into sub-pools, wherein all reproductive portions from a given mature plant are placed in the same sub-pool.

5. The method of claim 4, further comprising repeating step III. X times to obtain a plant containing M(1+X) generation propagation portions, and obtaining M(1+X) generation propagation portions from the mature plant in step IV. to obtain a propagation portion pool.

6. A method for identifying a subpool of a plant of a predetermined plant species or a propagating portion thereof, wherein the subpool comprises a plant carrying one or more predetermined substitution and / or deletion mutations of a nucleotide of interest (NOI) in a target sequence, the method comprising the steps of: - Provides multiple reproductive parts for the plant; - Randomly mutate the reproductive portion to obtain the M0 generation reproductive portion; - The reproductive portion of the M0 generation is grown into a mature plant, and a reproductive portion is obtained from the mature plant, wherein the reproductive portion is the M1 generation reproductive portion; - Obtain the M1 generation reproductive portion from the mature plant to obtain a reproductive portion pool; - Divide the propagation pool into sub-pools, wherein all propagation portions from a given mature plant are placed in the same sub-pool. - Prepare genomic DNA (gDNA) samples, each sample containing gDNA from each genotype in a subpool, while maintaining the proliferative potential of plants of each genotype in the subpool; - Perform multiple PCR amplifications, each PCR amplification containing gDNA samples from a sub-pool, one or more sets of primers and PCR reagents, thereby amplifying the target sequence, each of the one or more sets of primers being located on the flanking side of the target sequence. - Detect PCR amplification products containing one or more target sequences, said target sequences containing mutations in NOI, thereby identifying subpools containing said mutations.

7. The method of claim 6, further comprising repeating the third step X times to obtain a plant containing M(1+X) generation propagation portions, and in the fourth step obtaining M(1+X) generation propagation portions from the mature plant to obtain a propagation portion pool.

8. The method according to any one of claims 1-4 and 6, wherein the species is a plant, and steps a) and b) or the first and second steps of the method comprise the following steps: I. Provides multiple reproductive parts for the plant; II. Randomly mutate the reproductive portion to obtain the M0 generation reproductive portion; III. Allow the reproductive portion of the M0 generation to grow into a mature plant, and obtain a reproductive portion from the mature plant, wherein the reproductive portion is the reproductive portion of the M1 generation; V. Obtain the reproductive portion of the M1 generation from the mature plant, thereby obtaining a reproductive portion pool; VI. Divide the reproductive portion pool obtained in step V. into sub-pools, wherein all reproductive portions from a given mature plant are placed in the same sub-pool.

9. The method of claim 1, wherein the species is a plant.

10. The method according to any one of claims 1, 4 and 6, wherein the species is barley.

11. The method according to any one of claims 1, 4 and 6, wherein the reproductive portion is a seed.

12. The method according to any one of claims 1, 4, and 6, wherein the step of dividing the breeding pool of generation M0 into sub-pools comprises the following steps: - M0 generation propagation parts are cultivated in different fields and grown into mature plants, wherein the propagation parts of the mature plants constitute a propagation part pool; - Divide the field into smaller fields; - Harvest all the reproductive parts of all plants in a small plot to obtain a reproductive part sub-pool.

13. The method of claim 12, wherein the reproductive portion is cultured in separate containers.

14. A method for identifying a subpool of organisms of a predetermined single-celled species, wherein the subpool comprises organisms of a predetermined single-celled species carrying one or more predetermined substitution and / or deletion mutations of a nucleotide of interest (NOI) in a target sequence, the method comprising the steps of: a) Provide a pool of organisms representing multiple genotypes of the single-celled organism species, wherein the pool of organisms is prepared by subjecting the organisms of the single-celled organism species to random mutagenesis. b) Divide the pool into one or more sub-pools for different organisms; c) Allow each sub-pool to undergo a breeding step; d) Divide each subpool into multiple parts in such a way that each part includes an organism representing each genotype of the subpool; e) Prepare genomic DNA (gDNA) samples from one of the plurality of portions of each subpool while preserving the remaining portion of the organism while maintaining its reproductive potential; f) Perform multiple PCR amplifications, each of which includes a gDNA sample from a sub-pool, one or more sets of primers and PCR reagents to amplify the target sequence, wherein each of the one or more sets of primers is located on the flanking side of the target sequence. g) Detect PCR amplification products containing one or more target sequences, said target sequences containing mutations in NOI, thereby identifying subpools containing said mutations.

15. The method according to any one of claims 1-4, 6 and 14, wherein the predetermined mutation is a substitution of a single nucleotide.

16. The method according to any one of claims 1-4, 6 and 14, wherein 10 to 50% of the organisms or their reproductive portions in each sub-pool are used to prepare gDNA samples.

17. The method according to any one of claims 1-4, 6 and 14, wherein the organisms or their reproductive portions in the sub-pool are randomly divided into 2, 3 or 4 portions.

18. The method according to any one of claims 1-4, 6 and 14, wherein the PCR amplification in step e), the seventh step or the sixth step is performed by a method comprising the following steps: - Prepare one or more PCR amplifications, which include a gDNA sample, one or more sets of primers and PCR reagents, wherein each of the one or more sets of primers is located flanking the target sequence; - The PCR amplification is divided into multiple spatially separated compartments; - Perform PCR amplification; - Detect PCR amplification products.

19. The method of claim 18, wherein the spatially separated compartment is a droplet.

20. The method of claim 19, wherein the average volume of each droplet ranges from 0.1 to 10 nL.

21. The method according to any one of claims 1-4, 6 and 14, wherein each PCR compartment is divided into spatially separated compartments in the range of 1,000 to 100,000.

22. The method according to any one of claims 1-4, 6 and 14, wherein the pool of organisms comprises at least 50,000 organisms or their reproductive portions having different genotypes.

23. The method according to any one of claims 1-4, 6 and 14, wherein the pool of organisms comprises at least 100,000 organisms or their reproductive portions having different genotypes.

24. The method according to any one of claims 1-4, 6 and 14, wherein the pool of organisms comprises at least 500,000 organisms or their reproductive portions having different genotypes.

25. The method according to any one of claims 1-4, 6 and 14, wherein the pool of organisms comprises at least 1,000,000 organisms or their reproductive portions having different genotypes.

26. The method according to any one of claims 1-4, 6 and 14, wherein the pool of organisms comprises 1,000,000 to 100,000,000 organisms or their reproductive portions having different genotypes.

27. The method according to any one of claims 1-4, 6 and 14, wherein each subpool contains at least 100 organisms or their reproductive portions having different genotypes.

28. The method according to any one of claims 1-4, 6 and 14, wherein each subpool contains at least 500 organisms or their reproductive portions having different genotypes.

29. The method according to any one of claims 1-4, 6 and 14, wherein the method further comprises the following steps performed prior to the step of performing multiple PCR amplifications on gDNA samples from a sub-pool: - Obtain a portion of each gDNA sample from each sub-pool; - Combine portions of multiple gDNA samples into a superpool to obtain a gDNA superpool containing gDNA samples from multiple subpools; - Perform multiple PCR amplifications, each PCR amplification containing a gDNA sample superpool, wherein each PCR amplification contains multiple compartmentalized PCR amplifications, each PCR compartmentalized amplification containing a portion of the gDNA sample, one or more sets of primers and PCR reagents, thereby amplifying the target sequence, each of the one or more sets of primers being located flanking the target sequence. - Detect PCR amplification products containing one or more target sequences, said target sequences containing mutations in NOI, thereby identifying superpools containing said mutations.

30. The method of claim 29, wherein the PCR amplification of the gDNA sample superpool is performed by a method comprising the following steps: - Preparation of PCR amplification, comprising a gDNA sample, one or more sets of primers and PCR reagents, wherein each of the one or more sets of primers is located flanking the target sequence; - The PCR amplification is divided into multiple spatially separated compartments, each with an average volume of 0.1 to 10 pL; - Perform PCR amplification; - Detect PCR amplification products.

31. The method according to any one of claims 1-4, 6 and 14, wherein the method further comprises the step of: - The organisms or their reproductive parts in the identified sub-pools are classified into secondary sub-pools; - Prepare gDNA samples, each sample containing gDNA from each genotype in the secondary pool, while maintaining the proliferation potential of each genotype organism in the secondary pool; - Perform multiple PCR amplifications, each PCR amplification containing gDNA samples from a sub-pool, one or more sets of primers and PCR reagents, thereby amplifying the target sequence, each of the one or more sets of primers being located flanking the target sequence. - Detect PCR amplification products containing one or more target sequences, said target sequences containing a predetermined mutation of NOI, thereby identifying the secondary pool containing said mutation in step i); - Identify the organisms or their reproductive portions within the secondary pool carrying the mutation.

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