Method for screening of rice genotypes resistant to herbicides by mutagenesis and application thereof

Abstract

The present application relates to the technical field of nucleotide molecule screening, in particular to a mutagenesis screening method and application of a rice herbicide-resistant genotype. The present application designs a qPCR reaction system based on a specific SNP site region of a mutant ALS gene, realizes the effect of high-throughput and large-scale screening of a rice parent containing a mutant ALS gene, and can significantly improve the efficiency of rice screening and breeding. The present application designs a TaqMan-MGB probe based on a specific SNP site region of a mutant ALS gene, which can form a hybrid molecule with a specific SNP site with high specificity and high accuracy, ensuring the accuracy and reproducibility of qPCR.

Description

Technical Field

[0001] This invention relates to the technical field of nucleotide molecule screening, and in particular to a method and application for mutagenesis screening of herbicide-resistant rice genotypes. Background Technology

[0002] Weeds in paddy fields are one of the major threats to rice cultivation. Their presence leads to insufficient nutrient supply to rice plants, resulting in severe yield reductions. Furthermore, weeds act as intermediate hosts for pests and diseases, promoting their occurrence and spread. With the expansion of direct-seeded rice cultivation, the damage caused by paddy field weeds has become increasingly prominent, making control a very serious challenge.

[0003] Currently, weeds in paddy fields are mainly controlled by applying herbicides. However, improper use of herbicides can easily damage rice and affect its growth and development. To control weeds in paddy fields while protecting rice, breeding rice varieties with herbicide resistance is an effective method. Currently, the breeding of herbicide-resistant rice primarily relies on mutation breeding, which uses artificial physical or chemical methods to induce mutations in the plant genome. These mutants are then screened for herbicide resistance genes to obtain rice varieties with herbicide resistance genes.

[0004] Currently, the most common herbicides are ALS inhibitors, which target acetolactate synthase (ALS) and can control most common weeds in rice paddies. They primarily work by forming a complex with ALS in the plant, inhibiting ALS activity, thus blocking the synthesis of branched-chain amino acids, thereby inhibiting protein synthesis and ultimately causing plant death. Common ALS inhibitors include triazolopyrimidines, sulfonylureas, salicylate pyrimidines, and imidazolinones.

[0005] Although rice also possesses the ALS gene and is threatened by ALS inhibitor herbicides, the rice ALS gene coding region has only one exon, encoding 644 amino acids. Mutations in its conserved region reduce affinity for ALS inhibitor herbicides, thus leading to resistance. Current reports indicate that a mutation at position 1880 of the rice ALS gene can confer resistance to imidazolinone herbicides. Therefore, rapidly screening for rice mutants with this gene mutation is of great significance. Summary of the Invention

[0006] This invention relates to a method and application for screening rice herbicide-sensitive genotypes through mutagenesis. It utilizes primers and probes to perform high-throughput quantitative PCR on the whole genome DNA of mutagenized rice, achieving real-time detection and reporting of mutant ALS genes.

[0007] On the one hand, the present invention provides a method for mutagenesis screening of herbicide-resistant rice genotypes, comprising the following steps: S1. Radiation and / or chemical mutagenesis were performed on rice populations to obtain M1 generation seeds; S2. Plant M1 generation seeds to obtain M2 generation rice samples, and collect genomic DNA from the M2 generation rice samples; S3. Perform real-time PCR using primers specifically for detecting mutant ALS genes and the TaqMan-MGB probe; S4. Real-time detection of the fluorescence reporter signal of the TaqMan-MGB probe to determine the genotype of the ALS gene in the M2 generation rice sample, and screening out samples with the ALS gene as the mutant ALS gene. The last base at the 3' end of the nucleotide sequence of the TaqMan-MGB probe is thymine, and the mutant ALS gene is obtained by mutating the wild-type ALS gene at position 1880 to an adenine base.

[0008] In this invention, conventional rice refers to rice varieties with stable genetic traits and carrying the wild-type ALS gene, including but not limited to japonica rice Suxiu 852, japonica rice maintainer line Zhejing 7B, and wide-compatibility restorer line DR610. The wild-type ALS gene refers to the ALS gene of indica rice variety 9311.

[0009] Mutagenesis in rice populations is a well-known and conventional method in the field of genetics, aiming to induce mutations in the rice genome to obtain mutants with target traits. Conventional mutagenesis methods include, but are not limited to, those described above. Mutagenesis can be induced by radiation, ion beams, ethyl methanesulfonate (EMS), or sodium azide. Of course, those skilled in the art can also perform multiple mutagenesis simultaneously on the same rice plant to increase the probability of genomic variation. This invention does not limit the mutagenesis methods used on rice populations.

[0010] The methods for extracting genomic DNA from rice samples are well known to those skilled in the art, such as the CTAB method, SDS method, PVP method, and the use of commercially available plant DNA extraction kits.

[0011] In this invention, the primers for specifically detecting the mutant ALS gene are a pair of oligonucleotide sequences capable of specifically amplifying a short DNA fragment containing the characteristic single nucleotide polymorphism (SNP) site of the mutant ALS gene via polymerase chain reaction (PCR). This primer pair includes an upstream primer and a downstream primer, which are complementary to the sequences at both ends of the target DNA fragment, respectively. The characteristic SNP site of the mutant ALS gene refers to a single base difference present in the coding or regulatory region of the ALS gene, capable of clearly distinguishing the ALS allele from other alleles within the same gene cluster. As a non-limiting embodiment, the primer pair can be designed to amplify a fragment between 60 and 150 base pairs in length to ensure high efficiency of the quantitative real-time PCR reaction.

[0012] The TaqMan-MGB probe for specifically detecting the mutant ALS gene is an oligonucleotide sequence with a fluorescent reporter group (such as FAM, VIC, etc.) labeled at its 5' end and a quencher group and minor groove binder (MGB) labeled at its 3' end. The nucleotide sequence of this probe needs to specifically hybridize with the DNA target sequence containing the characteristic SNP site amplified by the primer pair, and the last base at its 3' end must be completely complementary to the specific base of the mutant ALS gene at that SNP site. In one specific embodiment, the SNP site is located at the 1880th base of the mutant ALS gene, and this base is not guanine.

[0013] In quantitative real-time PCR, if the template DNA originates from a sample carrying the Pigm gene, the TaqMan-MGB probe of this invention will bind perfectly to the target sequence. When the DNA polymerase encounters the bound probe during extension, it uses its 5'→3' exonuclease activity to hydrolyze the probe, separating the fluorescent reporter group from the quencher group, thereby generating a detectable fluorescent signal. Conversely, if the template DNA originates from a sample carrying other alleles, base mismatches will occur at key SNP sites, resulting in no detectable fluorescent signal. The genotype of the ALS gene in the rice sample can be determined by detecting changes in the fluorescent signal.

[0014] In some embodiments, the 3' end base of the nucleotide sequence of the TaqMan-MGB probe is completely complementary to the mutant base at position 1880 of the mutant ALS gene.

[0015] In some embodiments, the nucleotide sequence of the TaqMan-MGB probe is as shown in SEQ ID NO: 1, or is a sequence that has at least 90% homology with SEQ ID NO: 1 and is capable of specifically hybridizing with the mutant ALS gene.

[0016] In some embodiments, in step S4, the genotype of the ALS gene is automatically determined by a real-time PCR instrument based on the fluorescence signal growth curve and Ct value.

[0017] In another aspect, the present invention provides a TaqMan-MGB probe for specifically detecting the herbicide-resistant ALS genotype in rice, the nucleotide sequence of which is shown in SEQ ID NO: 1, or is a sequence having at least 90% homology with SEQ ID NO: 1 and capable of specifically hybridizing with the mutant ALS gene.

[0018] In another aspect, the present invention provides a primer-probe combination comprising an upstream primer, a downstream primer, and the aforementioned TaqMan-MGB probe for amplifying a region containing a specific SNP site of the mutant ALS gene.

[0019] In some embodiments, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2.

[0020] In some embodiments, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3.

[0021] In another aspect, the present invention provides the application of the above-described screening method for rice blast-resistant rice, or the above-described TaqMan-MGB probe, or the above-described primer-probe combination in screening rice with mutant ALS genes.

[0022] In another aspect, the present invention provides the application of the above-mentioned screening method for rice blast-resistant rice, or the above-mentioned TaqMan-MGB probe, or the above-mentioned primer-probe combination in herbicide-resistant rice breeding.

[0023] The present invention has the following advantages and effects: 1. This invention designs a qPCR reaction system based on the specific SNP site region of the mutant ALS gene, achieving high-throughput, large-scale screening of rice parents containing the mutant ALS gene, and significantly improving the efficiency of rice screening and breeding.

[0024] 2. This invention designs the TaqMan-MGB probe based on the specific SNP site region of the mutant ALS gene, which can form hybrid molecules with specific SNP sites with high specificity and accuracy, ensuring the accuracy and reproducibility of qPCR. Attached Figure Description

[0025] Figure 1 Comparison chart of rice growth. Detailed Implementation

[0026] To make the objectives, features, and advantages of this invention more apparent and understandable, the invention will be further described in detail below with reference to embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of the appended claims.

[0027] Unless otherwise specified, all experimental reagents and materials used in this invention are commercially available.

[0028] Example 1: Method for mutagenesis screening of rice with mutant ALS genes 1. Mutagenesis pass Conventional japonica rice variety Suxiu 852 was subjected to dual mutagenesis using X-rays and EMS chemical mutagen to obtain M1 generation seeds. M1 generation seeds were then planted to obtain M2 generation rice samples.

[0029] Young leaves of M2 generation rice seedlings that have grown for about 10 days are taken. A leaf tissue disc with a diameter of about 2 mm is punched into the bottom of each well of a 96-well plate using a puncher. Up to 96 different rice samples can be tested at the same time.

[0030] 2. DNA extraction Add to each well of the 96-well plate Plant genomic DNA rapid extraction solution (MolPure) ® The Plant DNA Kit (purchased from Yisheng Biotechnology) was sealed, and the plate was placed on a shaker and shaken vigorously for 2 minutes. Then, the 96-well plate was placed in a PCR instrument and heated at 95°C for 10 minutes. After removal, the plate was centrifuged, and the supernatant was collected and diluted with Tris-HCl buffer at a volume ratio of 1:5 (pH 8.0) to obtain rice genomic DNA.

[0031] 3. qPCR detection 3.1 Primers and Probes Upstream primer sequence: ALS-F: 5'-TCGCCCAAACCCAGAAAC-3' (SEQ ID NO: 2); Downstream primer sequence: ALS-R: 5'-ATGCCAAGCACATCAAACAA-3' (SEQ ID NO: 3); TaqMan-MGB probe sequence: 5'-FAM-ACCGATGCCCTGCAT-MGB-NFQ-3' (SEQ ID NO: 1).

[0032] 3.2 qPCR reaction Prepare the reaction mixture; information about the mixture is shown in Table 1. Table 1 Information on the Reaction Mixture ; Set the PCR cycling conditions, as detailed in Table 2: Table 2 PCR Cycling Conditions Information ; Place the 96-well plate with the sample added in QuantStudio TM The reaction was performed in a 1 Plus real-time PCR instrument, and the FAM channel fluorescence signal was acquired at the end of the annealing / extension step of each cycle.

[0033] 4. Interpretation of Genotype Results After the qPCR reaction, automated analysis was performed using Applied Biosystems QuantStudio Design & Analysis Software. The Ct value was automatically calculated based on the fluorescence growth curve of each sample. A Ct value ≤ 30, with a typical S-shaped amplification curve and strong fluorescence signal, indicated a mutant ALS gene; a Ct value > 35 or no Ct value, with no significant increase in fluorescence signal, indicated a wild-type ALS gene or other mutants.

[0034] Example 2: Specificity test of the qPCR detection method Leaves from standard rice plants of known genotypes were selected, and qPCR reactions were performed according to the method in Example 1. Sample information is as follows: Positive group: Zhexinxiang No. 2 carrying the mutant ALS gene; Negative group A: Japonica rice Suxiu 852 carrying the wild-type ALS gene; Negative group B: Indica rice variety 9311 carrying the wild-type ALS gene; Blank control group: sterile water.

[0035] The genotype results are shown in Table 3: Table 3. qPCR genotype interpretation results ; Table 3 shows that the TaqMan-MGB probe and primers provided by this invention only produce specific fluorescent amplification signals for rice samples carrying the mutant ALS gene with adenine mutation at position 1880, and the Ct values ​​are early, stable, and highly efficient. However, for rice samples carrying other alleles, the TaqMan-MGB probe and primers provided by this invention do not produce specific fluorescent amplification signals, and the amplification curves are no different from the blank control group. The above demonstrates that the TaqMan-MGB probe and primers of this invention are specific and can be used for high-throughput molecular screening of rice samples with mutant ALS genes, significantly improving the breeding efficiency of herbicide-resistant rice samples.

[0036] Example 3: Identification of Rice Traits Twenty-five rice plants carrying the mutant ALS gene and twenty-five rice plants carrying the wild-type ALS gene were sown. The entire seedbed was tilled, ridged, and irrigated. Pesticides and herbicides (2-(4-isopropyl-4-methyl-5-oxo-2-imidazoline-2-yl)nicotinic acid) were applied. The field was sun-dried for 1-2 days before sowing. Seeds were soaked in warm water for three days, changing the water twice daily. After the seeds showed signs of germination, germination was initiated for one and a half to two days. Sowing was carried out when the seed buds reached 5mm in length. Germinated seeds were sown in the center of the grid until they grew into seedlings. Two weeks later, the seedlings were pulled out according to their numbers, and markers were tied to the seedlings with straw or nylon rope, gently inserting the roots into the soil. The 50 samples were planted in two areas according to genotype, with five rows per area, 25cm apart; five plants were planted per row, 20cm apart. The rice growth was observed and recorded after five days. Results are as follows: Figure 1 As shown.

[0037] Figure 1 The results showed that rice carrying the mutant ALS gene grew normally and was not affected by herbicides; rice carrying the wild-type ALS gene showed yellowing and even wilting, indicating its sensitivity to herbicides. This confirms that the rice plants obtained through the molecular marker-assisted mutagenesis breeding technology of this application possess herbicide resistance.

[0038] In this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0039] Although specific embodiments of the invention have been described for illustrative purposes, various modifications or alterations can be made by those skilled in the art without departing from the spirit and scope of the invention. All such modifications or alterations should fall within the scope of the appended claims.

Claims

1. A method for mutagenesis screening of herbicide-resistant rice genotypes, characterized in that, Includes the following steps: S1. Radiation and / or chemical mutagenesis were performed on rice populations to obtain M1 generation seeds; S2. Plant M1 generation seeds to obtain M2 generation rice samples, and collect genomic DNA from the M2 generation rice samples; S3. Perform real-time PCR using primers specifically for detecting mutant ALS genes and the TaqMan-MGB probe; S4. Real-time detection of the fluorescence reporter signal of the TaqMan-MGB probe to determine the genotype of the ALS gene in the M2 generation rice sample, and screening out samples with the ALS gene as the mutant ALS gene. The last base at the 3' end of the nucleotide sequence of the TaqMan-MGB probe is thymine, and the mutant ALS gene is obtained by mutating the wild-type ALS gene at position 1880 to an adenine base.

2. The screening method according to claim 1, characterized in that, The 3' end base of the nucleotide sequence of the TaqMan-MGB probe is completely complementary to the 1880th mutant base of the mutant ALS gene.

3. The screening method according to claim 1 or 2, characterized in that, The nucleotide sequence of the TaqMan-MGB probe is shown in SEQ ID NO: 1, or is a sequence that has at least 90% homology with SEQ ID NO: 1 and can specifically hybridize with the mutant ALS gene.

4. The screening method according to claim 1, characterized in that, In step S4, the genotype of the ALS gene is automatically determined using a real-time PCR instrument based on the fluorescence signal growth curve and Ct value.

5. A TaqMan-MGB probe for specifically detecting the herbicide-resistant ALS genotype in rice, characterized in that, Its nucleotide sequence is shown in SEQ ID NO: 1, or is a sequence that has at least 90% homology with SEQ ID NO: 1 and can specifically hybridize with the mutant ALS gene.

6. A primer-probe combination, characterized in that, It includes upstream primers, downstream primers, and the TaqMan-MGB probe as described in claim 5, for amplifying regions containing SNP sites specific to mutant ALS genes.

7. The primer-probe combination according to claim 6, characterized in that, The nucleotide sequence of the upstream primer is shown in SEQ ID NO:

2.

8. The primer-probe combination according to claim 6, characterized in that, The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

3.

9. The application of the screening method according to any one of claims 1-4, or the TaqMan-MGB probe according to claim 5, or the primer-probe combination according to any one of claims 6-8 in screening rice with mutant ALS genes.

10. The application of the screening method according to any one of claims 1-4, or the TaqMan-MGB probe according to claim 5, or the primer-probe combination according to any one of claims 6-8 in the breeding of rice resistant to herbicides.

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