Sulfonylurea herbicide resistance gene of sesame and its application

Abstract

The present invention discloses a resistance gene of flax resistant to sulfonylurea herbicides and its application. The resistance gene is LusALS1R gene, the nucleotide sequence of which is a nucleotide sequence in which the 556th nucleotide in sequence 1 is T, and the SNP marker associated with it is the 556th nucleotide in sequence 1 in the sesame genome, which is C or T. The sulfonylurea herbicide resistance of TT genotype plants is higher than that of CT genotype plants, which is higher than that of CC genotype plants; the CC genotype is a homozygous type in which the SNP marker is C; the TC genotype is a heterozygous type in which the SNP marker is C and T; and the TT genotype is a homozygous type in which the SNP marker is T. The present invention provides SNP molecular markers for the cultivation of sulfonylurea herbicide-resistant sesame varieties for the first time. Plant resistance can be identified quickly and accurately without misjudgment due to genetic exchange.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to the creation of sulfonylurea herbicide-resistant sesame mutants, the separation of sulfonylurea herbicide-resistant gene nucleic acid sequences, and detection primers and applications of resistance genes. Background Art

[0002] Sesame is a densely planted crop, mainly cultivated in traditional ways. Sesame seeds are small, grow slowly in the seedling stage, and it is difficult to form a large area of ​​shade after mature plants. There are many types of weeds in the field, with high density and long symbiotic period, mainly dicotyledonous broad-leaved weeds. Effective control of field weeds is an important part of improving sesame yield and quality. However, manual weeding is time-consuming and labor-intensive, and commonly used herbicides have poor control effects on broad-leaved weeds and are prone to phytotoxicity. At present, there is a lack of safe and efficient herbicides in production.

[0003] Sulfonylurea herbicides are selective systemic herbicides with advantages such as low dosage, high efficiency, broad spectrum of weed control, low toxicity, and short soil residual period. They are one of the most widely used herbicides in production. Sulfonylurea herbicides (SU) and imidazolinones (IMI), triazole pyrimidines (TP), pyrimidine salicylates (PB), and sulfonamides (SCT) are collectively referred to as ALS herbicides, which target acetolactate synthase (ALS) in plants. ALS enzyme is a key enzyme in the synthesis of branched-chain amino acids leucine, valine, and isoleucine (McCourt et al .,. Amino Acids , 2006, 31:173-210). ALS herbicides form complexes with ALS enzymes, blocking the substrate from entering the enzyme active center, inhibiting the synthesis of branched-chain amino acids, leading to the accumulation of α-butanone, disrupting protein synthesis, and causing the plants to lose green color, turn yellow, and stunt their growth, leading to their gradual death (Jennifer et al ., PNAS , 2006, 103: 569-573). Sulfonylurea herbicides can effectively control dicotyledonous broadleaf weeds and are widely used in monocotyledonous crops such as rice and wheat in my country. However, sesame is a dicotyledonous crop and is sensitive to sulfonylurea herbicides and cannot be used directly. Therefore, creating new sesame germplasm with selective resistance to sulfonylurea herbicides is an economical and effective way to solve the problem of weed control in sesame fields.

[0004] Studies have shown that most weeds resistant to ALS herbicides have mutations in the target gene, which changes the target enzyme structure, weakening or hindering the binding of the herbicide to the target enzyme, thus producing resistance. At present, ALS genes have been found to mutate at 8 conserved sites in weeds, and substitutions of 29 different amino acids can make organisms resistant to ALS herbicides, namely Ala122, Pro197, Ala205, Asp376, Arg377, Trp574, Ser653 and Gly654 (calculated based on the amino acid positions of the model plant Arabidopsis, the same below), and there are multiple mutation types that can cause organisms to become resistant to ALS inhibitors (Powles et al., 2001). et al. , Annual Review of Plant Biology , 2010,61(1):317-347;Suzanne et al. , Weed Science , 2008, 56(6):p.797-806; Heap,2017), for example, when Pro197 is replaced by 9 amino acids such as histidine, serine, and threonine, the plant develops high resistance to sulfonylurea herbicides and cross-resistance to different types of ALS herbicides (Hai LC et al., Pesticide Biochemistry & Physiology , 2012, 102(3):229-232). Through mutagenesis and gene editing of target crops, a variety of corn and rice mutants resistant to ALS herbicides have been obtained, and mutant genes with resistance have been cloned one after another (Tan S et al. , Pest Manag Sci , 2005, 61: 246-257). For example, the rapeseed IMI herbicide resistance gene BnALS1R was first reported in China (Hu Maolong et al., Chinese Agricultural Science, 2012, 45 (20): 4326-4334); the rapeseed sulfonylurea herbicide resistance mutant M342 was obtained by EMS mutagenesis, and the resistance gene was cloned. BnALS3R (Hu Maolong et al., A gene for resistance to sulfonylurea herbicides in Brassica napus and its application; Chinese patent ZL201310111739.5). Summary of the invention

[0005] The purpose of the present invention is to use EMS to induce mutagenesis of the main sesame variety Longya No. 10, screen mutants resistant to SU herbicides, obtain SU herbicide-resistant sesame mutant materials, clone the nucleic acid sequence of SU herbicide-resistant genes, and design molecular markers.

[0006] The present invention claims to protect the detection of whether the sesame genome contains genes LusALS1R The use of a substance in the preparation of a product for identifying or assisting in identifying resistance to sulfonylurea herbicides in sesame, wherein the gene LusALS1RThe nucleotide sequence in which the 556th nucleotide in sequence 1 is T.

[0007] Wherein, the detection of whether the sesame genome contains genes LusALS1R The substance is used to detect the LusALS1R A substance related to a gene-related SNP marker C556T, wherein the SNP marker C556T is the 556th nucleotide of SEQ ID No. 1 in the sesame genome, which is C or T.

[0008] Wherein, the substance for detecting the polymorphism or genotype of CAPS molecular marker C556T in sesame genome includes: restriction endonuclease Bsa I or restriction endonuclease Eco O109I and the sequences shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing constitute a primer pair.

[0009] The present invention claims a method for detecting whether a plant is resistant to sulfonylurea herbicides, comprising the step of detecting the genotype of a SNP marker C556T in the genome of the plant to be tested, wherein the sulfonylurea herbicide resistance of a TT genotype plant is higher than that of a CT genotype plant, which is higher than that of a CC genotype plant; the CC genotype is a homozygous type in which the SNP marker C556T is C; the TC genotype is a heterozygous type in which the SNP marker C556T is C and T; and the TT genotype is a homozygous type in which the SNP marker C556T is T.

[0010] The method for detecting the genotype of the SNP marker C556T in the genome of the plant to be tested is as follows: using the DNA of the plant to be tested as a template, performing PCR amplification with the primer pair, and PCR amplification products with restriction endonucleases. Bsa I digestion, if the enzyme digestion product shown by electrophoresis contains two bands of 405 bp and 177 bp, the genotype of SNP marker C556T is TT genotype, showing homozygous resistance to sulfonylurea herbicides;

[0011] If the enzyme digestion product shown by electrophoresis contains three bands of 582 bp, 405 bp and 177 bp, the genotype of the SNP marker C556T is the CT genotype, indicating a heterozygous resistant plant to sulfonylurea herbicides;

[0012] If the enzyme cleavage product shown by electrophoresis contains only one band of 582 bp, the genotype of the SNP marker C556T is the CC genotype, which indicates a homozygous sensitive plant that is not resistant to sulfonylurea herbicides.

[0013] The method for detecting the genotype of the SNP marker C556T in the genome of the plant to be tested is as follows: using the DNA of the plant to be tested as a template, performing PCR amplification with the primer pair, and PCR amplification products with restriction endonucleases. Eco O109I digestion, if the enzyme digestion product shown by electrophoresis contains only one band of 582bp, then the genotype of SNP marker C556T is TT genotype, showing homozygous resistance to sulfonylurea herbicides;

[0014] If the enzyme digestion product shown by electrophoresis contains three bands of 582 bp, 399 bp and 183 bp, the genotype of the SNP marker C556T is the CT genotype, which indicates heterozygous resistance to sulfonylurea herbicides.

[0015] If the electrophoresis test shows that the enzyme digestion product contains two bands of 399 bp and 183 bp, the genotype of the SNP marker C556T is the CC genotype, which indicates homozygous sensitive sesame that is not resistant to sulfonylurea herbicides.

[0016] The present invention claims a product for detecting whether a plant is resistant to sulfonylurea herbicides, the product comprising a primer pair shown as SEQ ID No. 3 and SEQ ID No. 4 in the sequence list.

[0017] The application of the above method in plant breeding should also be within the protection scope of the present invention.

[0018] The plant breeding is to cultivate plants resistant to sulfonylurea herbicides.

[0019] The plant is sesame.

[0020] Application of this molecular marker in breeding herbicide-resistant sesame varieties:

[0021] The application is characterized in that the above primers are used to isolate Longya No. 10 from the female parent and a gene containing resistance to sulfonylurea herbicides. LusALS1R The plants in the F2 population obtained by self-pollination of the F1 single plant obtained by hybridization of the mutant material with the male parent are subjected to PCR amplification detection of resistance genes, and the resistant individuals in the F2 population are screened by the size of the enzyme cutting product band.

[0022] The application can also be used as other flax varieties as female parents, containing genes for resistance to sulfonylurea herbicides. LusALS1R The mutant material is used as the male parent for hybridization and breeding, and molecular markers are used for genotype detection. The heterozygous offspring are backcrossed multiple times, and combined with molecular marker identification, single plants with homozygous resistance genes are screened out.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention is the first to discover SU herbicide resistance genes and their related molecular markers (SNP sites) in sesame; the SU herbicide resistance genes of the sesame of the present invention can be applied to other plants sensitive to SU herbicides to improve their tolerance; the present invention provides two methods for detecting sulfonylurea herbicide resistance genes LusALS1R

[0025] The CAPS labeling method has reliable results and simple operation; the molecular marker of the present invention is located at +552 to +557 downstream of the first nucleotide of the start codon of the positive chain of the acetolactate synthase gene of the resistant sesame, and the corresponding sequence is "GGTCTC", which can be restricted by restriction endonucleases. Bsa I recognizes and cuts, and cannot be recognized by restriction endonucleases Eco O109I is recognized. The corresponding sequence of the sensitive genotype from +552 to +557 is "GGTCCC", which cannot be recognized by restriction endonucleases. Bsa I recognition, can be restricted by restriction enzymes Eco O109I recognizes and splices, thus two genes that can detect flax resistance to sulfonylurea herbicides can be developed LusALS1R CAPS molecular markers Bsa I-ALS1R and Eco O109I-ALS1R.

[0026] The molecular marker provided by the present invention is a co-dominant marker, and the detection method can be effectively applied to the sulfonylurea herbicide resistance gene. LusALS1R The marker-assisted selection breeding of flax resistant to sulfonylurea herbicides is produced. The present invention provides PCR molecular markers for the breeding of flax varieties resistant to sulfonylurea herbicides for the first time. The markers can quickly and accurately identify plant resistance without misjudgment due to genetic exchange. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The resistance of the herbicide-resistant mutant R10 (left) and the wild type Longya 10 (right) to different doses of the SU herbicide bensulfuron-methyl.

[0028] Figure 2 The F2 population was obtained by self-pollination of F1 individual plants with Longya No. 10 as the female parent and the herbicide-resistant mutant R10 as the male parent. Spraying the field-recommended dose of bensulfuron-methyl showed resistance segregation: R was the resistant type, M was the moderately resistant type, and S was the sensitive type (non-resistant).

[0029] Figure 3 Sesame acetolactate synthase gene LusALS1Comparison of amino acid sequences: LusALS1R-R10 represents the amino acid sequence of wild-type Longya 10 (LY10); LusALS1-LY10 represents the amino acid sequence of SU-resistant sesame mutant R10. “△” represents a mutant amino acid.

[0030] Figure 4 Molecular marker identification of resistance genes in mutant R10, wild-type Longya 10 and F1 plants resistant to SU herbicides (using restriction endonuclease Bsa I digestion).

[0031] Figure 5 Molecular marker identification of resistance genes in mutant R10, wild-type Longya 10 and F1 plants resistant to SU herbicides (using restriction endonuclease Eco O109I digest). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0033] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0034] Humalongya No. 10 in the following examples was cultivated by the Crop Research Institute of Gansu Academy of Agricultural Sciences, and the public can obtain it from Gansu Academy of Agricultural Sciences or purchase it directly.

[0035] Example 1 Obtaining the SU herbicide-resistant mutant R10

[0036] The promoted variety Longya No. 10 was used as the original material. The seeds were treated with 0.9% EMS for 18 hours, and the treated seeds were sown in the field and mixed. The harvested M1 generation seeds were sown in the field and sprayed with 0.5 times the recommended field dose of sulfonylurea herbicide bensulfuron-methyl to harvest potential resistant seeds. The harvested M2 generation seeds were sown in the laboratory and sprayed with 0.5 times bensulfuron-methyl for re-screening. Materials with obvious resistance were selected for southern breeding. The M3 generation seeds were harvested from individual plants and partly sown in the field. They were sprayed with 2 times the recommended field dose of sulfonylurea herbicide bensulfuron-methyl to select strains with stable resistance and similar agronomic traits to Longya No. 10, named R10, planted in the greenhouse and sprayed with different doses of bensulfuron-methyl to measure their resistance levels (such as Figure 1 and Table 1).

[0037] Table 1. Spraying dosage of sulfonylurea herbicide bensulfuron-methyl

[0038]

[0039] Example 2 SU herbicide resistance gene LusALS1R Acquisition

[0040] The F2 population was obtained by self-pollination of F1 plants from a hybrid of Longya 10 as the female parent and the herbicide-resistant mutant R10 as the male parent. After spraying the recommended field dose of bensulfuron-methyl, the F2 population (203 plants) showed three phenotypes: resistant type R (40 plants), moderately resistant type M (103 plants), and sensitive type S (60 plants) ( Figure 2 ), the trait separation ratio is 1:2:1. Among them, the plants with no obvious difference in growth point and normal growth are indicated as resistant type R, the plants with curled leaves and slightly slow growth are indicated as moderately resistant type M, and the plants with wilting and yellowing and stagnant growth are indicated as sensitive type S (non-resistant type). Take 10 plants with different resistance phenotypes, and use the Kangwei Century DNA extraction kit to extract leaf genomic DNA. According to the Arabidopsis thaliana acetolactate synthase protein sequence (Gene ID: 824015), it was compared in the sesame database (NCBI: txid4005), and amplification primers were designed according to the sequence differences. According to the instructions of the high-fidelity DNA polymerase of Nanjing Novozyme Biotechnology Co., Ltd., a 50μL PCR reaction system was prepared and amplified in a PCR instrument. LusALS1 For example, the amplification primers are sequence 5: 5'-ATGGCCGCCGCTAATTC-3' and sequence 4: 5'-CTTAGGAACGTTAATCAACACAGGG-3'. The amplification program is 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension, a total of 35 cycles. After the product is separated by 1% agarose gel electrophoresis, the band of the correct size is taken for product recovery and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0041] The sequencing results showed that there were consistent differential sites among F2 plants with different resistance phenotypes, namely LusALS1 The gene mutation C556T causes the proline at position 186 (position 197 in Arabidopsis) of the protein sequence to be changed to serine ( Figure 3 The mutation site is named SNP site C556T, which corresponds to position 556 of the nucleotide sequence shown in sequence 1 in the sequence table, which is C or T, and is represented by Y in the sequence table. LusALS1R , i.e., the SU herbicide resistance gene, the sequence is a sequence in which the 556th nucleotide in sequence 1 in the sequence list is T, and the other sequences remain unchanged, consisting of 744 bases, and the encoded amino acid residue sequence is sequence 2.

[0042] Example 3 Detection of flax genes resistant to sulfonylurea herbicides LusALS1R Development of CAPS molecular markers

[0043] By comparing the sequences and restriction sites of mutant R10 and wild-type LY10 (Longya No. 10), it was found that the mutant R10 (i.e., the genotype containing the SNP site T) LusALS1R The corresponding sequence from +552 to +557 is "GGTCTC", which can be restricted by restriction endonucleases. Bsa I recognizes and cuts, and cannot be recognized by restriction endonucleases Eco O109I is recognized. The wild genotype (i.e. when the SNP site is C) has a corresponding sequence of "GGTCCC" from +552 to +557, which cannot be recognized by restriction endonucleases. Bsa I recognition, can be restricted by restriction enzymes Eco O109I recognizes and cuts. LusALS1 A conservative primer pair (sequence 3: 5'-TTCCTTCCCAATGCTACCAC-3' and sequence 4: 5'-CTTAGGAACGTTAATCAACACAGGG-3') was designed on the gene, and the PCR product length was expected to be 582bp. The genomic DNA of the mutant, wild type and hybrid F1 strains was extracted respectively, and a 50μL PCR reaction system was configured. The amplification program was 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56.5℃ annealing for 30s, and 72℃ extension for 45s, for a total of 35 cycles. Part of the amplified product was sent for sequencing, and part was purified and recovered using the product recovery kit of Nanjing Novozyme. The recovered products were respectively Bsa I or Eco O109I was used for enzyme digestion. The enzyme digestion reaction system included: 1 μg of PCR product, 10u of endonuclease, 5 μL of 10× enzyme reaction buffer, and water to 50 μL. Reaction procedure: 37℃ 60min, 65℃ 20min. Part of the enzyme digestion product was detected by 1.5% agarose gel electrophoresis.

[0044] Use restriction enzyme Bsa I digestion, if the electrophoresis detection shows that the enzyme digestion product contains two bands of 405bp and 177bp, then the sulfonylurea herbicide resistance gene LusALS1R -C556T is a homozygous mutation (the genotype corresponding to the SNP site is the homozygous TT type); if the electrophoresis test shows that the enzyme digestion product contains three bands of 582bp, 405bp and 177bp, then the sulfonylurea herbicide resistance gene LusALS1R-C556T is a heterozygous mutation (the genotype corresponding to the SNP site is a heterozygous CT type); if the electrophoresis test shows that the enzyme digestion product contains only one band of 582bp, then the sulfonylurea herbicide resistance gene LusALS1R -C556T has not mutated (the genotype corresponding to the SNP site is homozygous for CC) (e.g. Figure 4 As shown, among which: lane 1 is 2kMarker, lanes 2 and 3 are sensitive LY10, lanes 4 and 5 are mutant R10, and lanes 6 and 7 are F1 (LY10×R10) hybrid strains).

[0045] The above results show that if the SNP site C556T is TT genotype, it can be restricted by restriction endonuclease Bsa I digestion resulted in two bands of 405 bp and 177 bp; SNP site C556T was CT genotype and could be digested by restriction endonuclease Bsa I digestion resulted in three bands of 582 bp, 405 bp and 177 bp; SNP site C556T was CC genotype and could not be digested by restriction endonuclease Bsa I digestion, the enzyme digestion product contains only one band of 582bp. Combined with the phenotype of sesame, it can be found that the phenotype of TT genotype sesame is resistant type R, the phenotype of CT genotype sesame is moderately resistant type M, and the phenotype of CC genotype sesame is sensitive type S; that is, the sulfonylurea herbicide resistance of TT genotype plants is higher than that of CT genotype plants, and the sulfonylurea herbicide resistance of CC genotype plants is higher than that of CC genotype plants.

[0046] Use restriction enzyme Eco O109I digestion, if the electrophoresis detection shows that the enzyme digestion product contains only a 582bp band, then the sulfonylurea herbicide resistance gene LusALS1R -C556T is a homozygous mutation; if the electrophoresis test shows that the enzyme digestion product contains three bands of 582bp, 399bp and 183bp, then the sulfonylurea herbicide resistance gene LusALS1R -C556T is a heterozygous mutation; if the electrophoresis test shows that the enzyme digestion product contains two bands of 399bp and 183bp, then the sulfonylurea herbicide resistance gene LusALS1R - C556T has not mutated ( Figure 5 Lane 1 is 2k Marker, lanes 2 and 3 are sensitive LY10 (corresponding phenotype is sensitive type S), lanes 4 and 5 are mutant R10 (corresponding phenotype is resistant type R), and lanes 6 and 7 are F1 (LY10×R10) hybrid strains (corresponding phenotype is moderately resistant type M)).

[0047] That is to say, if the SNP site C556T is TT genotype, it cannot be restricted by restriction endonuclease EcoO109I digestion, the digestion product contains only a 582bp band; if the SNP site C556T is CT genotype, it can be digested by restriction endonuclease Eco O109I digestion is 582bp, 399bp and 183bp three bands; if the SNP site C556T is CC genotype, it can be restricted by restriction endonuclease Eco O109I digestion was two bands of 399 bp and 183 bp. Combining the phenotype of sesame, it can be found that the phenotype of TT genotype sesame is resistant type R, the phenotype of CT genotype sesame is moderately resistant type M, and the phenotype of CC genotype sesame is sensitive type S; that is, the sulfonylurea herbicide resistance of TT genotype plants is higher than that of CT genotype plants, and the sulfonylurea herbicide resistance of CC genotype plants is higher than that of CC genotype plants.

[0048] Example 4: Molecular markers BsaI - ALS1R and EcoO109I - ALS1R Resistance genes in segregating populations LusALS1R Genotype detection and identification

[0049] With Longya No. 10 as the female parent and herbicide-resistant mutant R10 as the male parent, the F1 hybrid single plant was self-pollinated to obtain the F2 population. The leaves of the seedling plants were taken to extract genomic DNA for PCR amplification. The amplified products were amplified using restriction endonucleases. Bsa I enzyme digestion, electrophoresis detection, the operation method is the same as above.

[0050] There were 203 strains in the F2 population, and 10 strains of different resistance phenotypes (resistant type R, moderately resistant type M and sensitive type S) were selected. The sequencing was performed and the above two molecular markers were used for detection. The results showed that:

[0051] The drug-resistant R plant C556T is a homozygous mutation, and the corresponding SNP gene is TT. The product amplified using the above primer pair can be cleaved by restriction endonuclease Bsa I digestion resulted in two bands: 405 bp and 177 bp;

[0052] The moderately resistant M plant C556T is a heterozygous mutation, and the corresponding SNP gene is CT. The product amplified using the above primer pair can be cleaved by restriction endonuclease Bsa I digestion resulted in three bands: 582 bp, 405 bp, and 177 bp;

[0053] The sensitive S plant had no mutation at position 556, and the corresponding SNP gene was CC. The product amplified using the above primer pair could not be cleaved by restriction endonuclease. Bsa I digestion, the digestion product contained only one band of 582 bp.

[0054] With Longya No. 10 as the female parent and herbicide-resistant mutant R10 as the male parent, the F1 hybrid single plant was self-pollinated to obtain the F2 population. The leaves of the seedling plants were taken to extract genomic DNA for PCR amplification. The amplified products were amplified using restriction endonucleases. Eco O109I digestion and electrophoresis detection were performed in the same manner as above.

[0055] There were 203 strains in the F2 population, and 10 strains of different resistance phenotypes (resistant type R, moderately resistant type M and sensitive type S) were selected. The sequencing was performed and the above two molecular markers were used for detection. The results showed that:

[0056] The drug-resistant R plant C556T is a homozygous mutation, and the corresponding SNP gene is TT. The product amplified using the above primer pair cannot be cleaved by restriction endonuclease Eco O109I digestion, so the digestion product contains only one band of 582 bp;

[0057] The moderately resistant M plant C556T is a heterozygous mutation, and the corresponding SNP gene is CT. The product amplified using the above primer pair can be cleaved by restriction endonuclease Eco O109I digested three bands: 582 bp, 399 bp, and 183 bp;

[0058] The sensitive S plant had no mutation at position 556, and the corresponding SNP gene was CC. The product amplified using the above primer pair could be restricted by restriction endonuclease Eco O109I digestion resulted in two bands: 399 bp and 183 bp.

[0059] The above F2 population molecular marker detection results are consistent with the phenotype and sequencing results of the sulfonylurea herbicide bensulfuron-methyl sprayed at the seedling stage. Bsa I-ALS1R (restriction endonuclease Bsa 1 and a primer pair consisting of sequence 3 and sequence 4) and Eco O109I-ALS1R (restriction endonuclease Eco O109I and a primer pair consisting of sequence 3 and sequence 4) and the detection method can accurately and effectively distinguish sesame sulfonylurea herbicide resistance genes LusALS1R Or three different genotypes at the SNP site to improve the screening efficiency of resistant genes.

[0060] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Detect whether the sesame genome contains LusALS1R The invention discloses a method for preparing a reagent for identifying or assisting in identifying safflower's resistance to sulfonylurea herbicides, wherein: Said LusALS1R The nucleotide sequence of the gene is shown in SEQ ID NO. 1, wherein the 556th nucleotide is T; The substances include restriction endonucleases and primer pairs; The restriction endonuclease is a restriction endonuclease Bsa I or restriction endonuclease Eco O109I; The sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. Detect whether the sesame genome contains LusALS1R The use of a gene reagent in cultivating flax resistant to sulfonylurea herbicides is characterized in that: Said LusALS1R The nucleotide sequence of the gene is shown in SEQ ID NO. 1, wherein the 556th nucleotide is T; The reagents include restriction endonucleases and primer pairs; The restriction endonuclease is a restriction endonuclease Bsa I or restriction endonuclease Eco O109I; The sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.

4.

3. A method for detecting whether flax is resistant to sulfonylurea herbicides, characterized in that: The method comprises the steps of detecting the genotype of the SNP marker C556T in the genome of the sesame to be tested, wherein the SNP marker C556T is a polymorphic site located at the 556th position in the sequence shown in SEQ ID NO.1; wherein the sulfonylurea herbicide resistance of the TT genotype sesame is higher than that of the CT genotype sesame, and the CC genotype sesame is not resistant to the sulfonylurea herbicide; The step of detecting the genotype of the SNP marker C556T in the genome of the sesame to be tested is any one of the following 1) or 2): 1) Using the DNA of the sesame to be tested as a template, PCR amplification was performed using the primer pair for detecting the SNP marker C556T, and the PCR amplification product was cleaved with a restriction endonuclease Bsa I digestion and electrophoresis detection, When the electrophoresis detection result contains two bands of 405 bp and 177 bp, the genotype of the SNP marker C556T is the TT genotype, and the sesame to be tested is identified as homozygous resistant sesame to sulfonylurea herbicides; When the electrophoresis detection result contains three bands of 582 bp, 405 bp and 177 bp, the genotype of the SNP marker C556T is the CT genotype, and the sesame to be tested is identified as heterozygous resistant sesame to sulfonylurea herbicides; When the electrophoresis detection result contains only one band of 582 bp, the genotype of the SNP marker C556T is the CC genotype, and the sesame to be tested is identified as homozygous sensitive sesame that is not resistant to sulfonylurea herbicides; 2) Using the DNA of the sesame to be tested as a template, PCR amplification was performed using the primer pair for detecting the SNP marker C556T, and the PCR amplification product was cleaved with a restriction endonuclease Eco O109I digestion and electrophoresis detection, When the electrophoresis detection result contains only one band of 582 bp, the genotype of the SNP marker C556T is the TT genotype, and the sesame to be tested is identified as homozygous resistant sesame to sulfonylurea herbicides; When the electrophoresis detection result contains three bands of 582 bp, 399 bp and 183 bp, the genotype of the SNP marker C556T is the CT genotype, and the sesame to be tested is identified as heterozygous resistant sesame to sulfonylurea herbicides; When the electrophoresis detection result contains two bands of 399 bp and 183 bp, the genotype of the SNP marker C556T is the CC genotype, and the sesame to be tested is identified as homozygous sensitive sesame that is not resistant to sulfonylurea herbicides; In 1) and 2), the sequences of the primer pair are shown as SEQ ID NO.3 and SEQ ID NO.

4.

4. Use of the method according to claim 3 in cultivating flax resistant to sulfonylurea herbicides.

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