CN102766697B - Molecular marking method for detecting imidazolone herbicide resisting gene of cabbage type rape - Google Patents

Abstract

The invention provides a three-primer molecular marker method for detecting imidazolinone herbicide-resistant genes of Brassica napus, belonging to the field of plant molecular breeding. The primer pairs AP15F/AP18R and AP15F/AP19R were added to two PCR reaction systems to amplify Brassica napus DNA, and to distinguish homozygotes without the resistance gene BnALS1R, homozygotes with the resistance gene BnALS1R, and those with the resistance gene BnALS1R hybrid. The invention can quickly and accurately detect whether the rapeseed germplasm resource or its breeding population contains the imidazolinone herbicide-resistant gene BnALS1R, more accurately distinguish whether the contained herbicide gene is homozygous or heterozygous, and improves the resistance gene BnALS1R The selection efficiency can accelerate the process of breeding rapeseed varieties resistant to imidazolinone herbicides.

Description

A molecular marker method for detecting genes resistant to imidazolinone herbicides in Brassica napus

technical field

The invention relates to the field of plant molecular breeding, in particular to a molecular marker method for detecting imidazolinone herbicide-resistant gene BnALS1R in Brassica napus .

Background technique

Weeds in farmland seriously affect the yield and quality of rapeseed, and have become one of the main biological disasters in rapeseed production. The popularization and application of herbicide-resistant rapeseed is an important way to overcome field weed damage. Compared with traditional rapeseed, herbicide-resistant rapeseed has three advantages: first, the farming operation is simple and efficient, and growers only need to spray herbicides once or twice at the seedling stage to effectively kill field weeds; Second, it can greatly reduce the cost of rapeseed production and increase profits; third, it can promote less-tillage and no-tillage, which is conducive to water and soil conservation and energy saving. However, herbicide-resistant rapeseed has not yet been approved for commercial planting in my country. There are two main reasons: (1) Herbicide-resistant rapeseed is generally a genetically modified crop, and commercial production must undergo strict safety evaluation (Cai Li et al. Science Bulletin, 2008, 53 (13): 1544-1551). (2) So far, domestic rapeseed herbicide resistance genes have all come from abroad, and resistance genes are protected by international intellectual property rights. Once they are commercially planted, expensive patent fees will be paid, which will greatly increase the production cost of rapeseed, which violates the principle of planting resistance. The original intention of herbicide crops (Pu Huiming et al. Ecological Journal, 2005, 25(3): 196-203).

Imidazolinone herbicides are currently one of the most widely used herbicides in rapeseed production in the world. They have a wide herbicidal spectrum (including grasses and broad-leaved weeds), low dosage, low toxicity to mammals and environmental friendliness. (Tan S, et al ., Pest Manag Sci , 2005, 61: 246-257). This kind of herbicide can be used as soil treatment agent or sprayed on stems and leaves. Its high weeding efficiency and convenient use are popular among users. Studies have shown that acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS), is the target enzyme of imidazolinone herbicides. Imidazolinone herbicides form a complex with ALS to block the pathway of substrates entering the active site of the enzyme, inhibiting the activity of ALS, causing the synthesis of branched-chain amino acids to be blocked, causing plant tissues to chlorosis, yellowing, and finally gradually die (Jennifer A M, et al ., PNAS , 2006, 103: 569-573). ALS is responsible for catalyzing two parallel reactions, catalyzing the condensation of two molecules of pyruvate to form 2-acetolactate to release CO ₂ , and finally generating valine and leucine, and catalyzing the condensation of one molecule of pyruvate and one molecule of 2-oxobutyric acid to form acetyl hydroxyl butyrate, eventually to isoleucine (Ronald G D, et al ., Plant Physiol Bioch , 2008, 46: 309-324). The cloning of the ALS gene of plants resistant to imidazolinone herbicides abroad shows that the emergence of resistant crops is due to the mutation of several base sites in the DNA sequence of the ALS gene of the mutant strain, resulting in mutations in the amino acid residues of the encoded protein, thereby It is caused by changes in the way herbicides bind to ALS (Lee H, et al. , PNAS , 2011, 108(21): 8909-8913).

Using chemical mutagenesis, foreign researchers obtained two rapeseed mutants P1 and P2 that are resistant to the imidazolinone herbicide imazethapyr. The currently commercialized imidazolinone-resistant rapeseed varieties are all bred from these two mutants (Swanson E B, et al ., Theor Appl Genet , 1989, 78: 525-530). In China, Gao Jianqin et al. reported a new rape material resistant to imidazolinone herbicides (Gao Jianqin et al. Journal of Plant Genetic Resources, 2010, 11 (3): 369-373), and further obtained resistance through molecular biology technology cloning Material M9 resistance gene BnALS1R . The gene can be introduced into other rapeseed varieties or lines that are not resistant to imidazolinone herbicides through conventional plant breeding methods such as hybridization and backcrossing, so as to improve the tolerance of the introduced target variety or line to imidazolinone herbicides (Hu Maolong et al., Brassica napus mutant gene resistant to imidazolinone herbicides and its application; Chinese patent ZL201010232607.4). If the resistance gene is introduced into the restorer line, the F ₁ true hybrids will have imidazolinone herbicide resistance characteristics, and all types of pseudo hybrids can be killed by spraying herbicides at one time, which improves the efficiency of seed production and Seed purity. However, the resistance trait is a dominant trait, and the F1 generation shows imidazolinone resistance, and it is separated from the F2 generation. In field breeding, it is necessary to start spraying herbicides for each generation for identification. If the herbicide dosage is used improperly or sprayed Inhomogeneity can easily lead to misselection or the death of required materials, and the conventional breeding in the field has a long selection period and a large workload. At the same time, continuous spraying of imidazolinone herbicides with excessive doses for many years may cause soil residues, which will have a certain impact on the next rotation crops. In addition, in the process of herbicide resistance gene research, it is sometimes necessary to save non-resistant materials for comparative research. For this reason, if molecular markers that are closely linked to resistance genes can be found and marker-assisted selection is performed, a complete set of materials can be preserved to guide field selection and speed up the breeding process. Researchers have also made bold attempts. Kadaru et al. designed PCR molecular markers to detect imidazolinone herbicide resistance genes in rice by using the single base variation of rice ALS gene (Kadaru S, et al., Euphytica, 2008, 160: 431-438). Lee et al. designed PCR molecular markers to detect imidazolinone herbicide resistance genes in barley using the single-base variation of the barley ALS gene (Lee H, et al. , PNAS , 2011, 108(21): 8909-8913).

Unlike the rice genome (2n=24) and barley (2n=14), Brassica napus is an allotetraploid species (2n = 38). The high degree of rearrangement in the genome during the evolution has led to the existence of Brassica napus-related genes in the form of gene families (Chen X, et al. , Plant Physiol , 2011, 155(2): 851-65). The ALS gene is no exception . There are 5 ALS genes in the Brassica napus genome. The homology between BnALS1 and BnALS3 at the nucleic acid and protein levels reaches 98% . The homology at the protein level is 85%, and the homology at the protein level is 75%. The coding regions of BnALS4 and BnALS5 are interrupted and are ALS genes with loss of function (Rutledge R G, et al ., Mol Gen Genet , 1991, 229: 31 -40). The high homology of DNA sequences in the ALS gene family makes it difficult to design specific molecular markers to detect mutations in Brassica napus resistant to imidazolinone herbicides. So far, there have been no public or published reports on the detection of imidazolinone herbicide resistance genes in Brassica napus at home and abroad. Although DNA sequencing technology can be used to detect the presence or absence of resistance genes, this technology is cumbersome to operate and relatively expensive. Therefore, establishing a PCR-based molecular marker to quickly and accurately detect the resistance gene BnALS1R is an urgent technical problem to be solved in molecular breeding of rapeseed herbicide resistance using this gene.

Contents of the invention

technical problem

The purpose of the present invention is to solve the shortcomings of using DNA sequencing technology to detect the resistance gene BnALS1R , such as cumbersome operation and high cost. The molecular marker linked with it can quickly and accurately detect the resistance gene BnALS1R through a simple method, and further perform marker-assisted selection.

Technical solutions

A molecular marker primer for detecting imidazolinone herbicide-resistant gene BnALS1R in Brassica napus, characterized in that:

Forward primer sequence AP15F: 5'-CTTTCGCTAGCAGGGCTAAA-3'

Reverse primer sequence AP18R: 5'-CATCTTTGAAAGTGCCACAAC-3'

Reverse primer sequence AP19R: 5'- CATCTTTGAAAGTGCCACAAT-3'

The PCR molecular marker method for rapid detection of imidazolinone herbicide resistance gene BnALS1R in Brassica napus using the above primers is as follows:

(1) Extraction of genomic DNA from Brassica napus plants (CTAB method), refer to (Murray M G, et al., Nucleic Acids Research , 1980, 8(19): 4321-4326).

(2) Add AP15F/AP18R and AP15F/AP19R of the three molecular marker primers to two PCR reaction systems respectively, and amplify the DNA of Brassica napus germplasm resources or breeding population plants;

PCR reaction system includes: 2.0 μL of DNA template (10 ng/μL), 2.0 μL of each primer (10 μmol/L), 2 μL of 10× enzyme reaction buffer, 1.2 μL of MgCl ₂ (25mmol/L), dNTP (2.5 mmol/L) 1.6 μL, Taq enzyme (5 U/L) 0.1 μL, add water to 20 μL;

The PCR reaction program was 94 ^o C pre-denaturation for 5 min, 94 ^o C denaturation for 30 s, 60 ^o C annealing for 30 s, 72 ^o C extension for 1 min, a total of 35 cycles; then 72 ^o C extension for 5 min, 12 ^o C cooling for 10 min Afterwards, the amplification product was added with sample buffer to terminate the reaction.

(3) The amplified product was electrophoresed on an agarose gel with a mass ratio concentration of 1.2%, stained with ethidium bromide, observed and recorded under a gel imaging system.

If the amplified product of primer pair AP15F/AP18R has a characteristic band of 828bp and AP15F/AP19 has no characteristic band, it is a homozygote that does not contain the herbicide resistance gene BnALS1R ; if the amplified product of primer pair AP15F/AP18R has no characteristic band And AP15F/AP19 has a characteristic band of 828bp, which means that it is a homozygote containing the herbicide resistance gene BnALS1R ; if the two primer pairs AP15F/AP18R and AP15F/AP19 have a characteristic band of 828bp, it is a herbicide-resistant gene. The agent gene BnALS1R heterozygote.

Beneficial effect

The invention provides a PCR molecular marker method for detecting imidazolinone herbicide-resistant gene BnALS1R in Brassica napus. Compared with the prior art, the invention has the following advantages and effects:

(1) For the first time, the present invention provides a PCR molecular marker for the breeding of imidazolinone herbicide-resistant rapeseed in China. The molecular marker is not a molecular marker linked to the imidazolinone-resistant gene BnALS1R , but is designed according to the mutation site of the gene Functional markers can directly reflect the resistance of plants, and there is no misidentification caused by genetic exchange.

(2) The molecular marker method provided by the present invention can be effectively used for assisted breeding of rapeseed that is resistant to imidazolinone herbicides caused by the imidazolinone-resistant gene BnALS1R . The conventional breeding method is to use the M9 containing the BnALS1R gene to carry out a series of crosses with the main cultivars promoted in production, and then spray herbicides in each segregated progeny for identification, and finally select the resistant single containing the imidazolinone-resistant gene BnALS1R . strain. In the process of herbicide identification for each segregated offspring, if the herbicide dose is used improperly or sprayed unevenly, it may easily lead to misselection or death of the desired material. In addition, continuous spraying of excessive imidazolinone herbicides for many years may cause soil residues, which will have a certain impact on the next crop rotation. Sometimes in the process of herbicide resistance gene research, it is necessary to save non-resistant materials for comparative research. Therefore, by using PCR molecular markers directly related to the BnALS1R gene to detect plant DNA, single plants homozygous for the resistance mutation gene and non-resistant single plants with excellent traits can be identified at any time in rapeseed, and other single plants can be eliminated. It not only saves breeding costs and reduces soil residues, but also greatly improves the selection process of imidazolinone-resistant rapeseed varieties.

(3) Compared with detecting the presence or absence of resistance genes by DNA sequencing technology, the molecular marker method provided by the present invention only needs 2 PCR reactions to identify different resistance genotypes, and can not only effectively distinguish pure BnALS1R Synthesis and heterozygosity, and because DNA sequencing is not involved, there are no disadvantages such as cumbersome steps and high costs, and the PCR molecular marker detection method is more efficient and faster.

Description of drawings

Fig. 1—Clone electrophoresis of acetolactate synthase family gene BnALS3 in Brassica napus

M, DNA molecular weight standard, fragments from small to large are 500bp, 800bp, 1200bp, 2000bp, 3000bp, 4500bp; 1, Ningyou 16 (NY16) genome as template; 2, Ningyou 18 (NY18) genome as template; 3. The genome of Brassica napus mutant M9 resistant to imidazolinone herbicides was used as a template.

Figure 2 - Nucleic acid sequence comparison of the coding regions of rapeseed acetolactate synthase genes BnALS1 and BnALS3 from different sources and the positions of the three primers. The triangles indicate the single nucleotide difference between the wild-type gene BnALS1 and the resistance mutant gene BnALS1R (G/A) , the arrow indicates the position of the primer and the direction of amplification.

BnALS1R.seq, partial nucleic acid sequence of the BnALS1 acetolactate synthase gene BnALS1 of Brassica napus mutant M9;

BnALS1-NY16.seq, the partial nucleic acid sequence of the acetolactate synthase gene BnALS1 of Ningyou 16;

BnALS1-NY18.seq, the partial nucleic acid sequence of the acetolactate synthase gene BnALS1 of Ningyou 18;

The above sequences BnALS1R.seq, BnALS1-NY16.seq (ie NY16.seq), and BnALS1-NY18.seq (ie NY18.seq) are obtained from previously applied patents ((Hu Maolong et al., Imidazolinone herbicide-resistant cabbage Rapeseed mutant gene and its application; Chinese patent ZL201010232607.4);

Z11524.seq, Genbank download sequence (accession number: Z11524);

BnALS3-M9.seq, partial nucleic acid sequence of acetolactate synthase gene BnALS3 of Brassica napus mutant M9;

BnALS3-NY16.seq, the partial nucleic acid sequence of the acetolactate synthase gene BnALS3 of Ningyou 16;

BnALS3-NY18.seq, the partial nucleic acid sequence of the acetolactate synthase gene BnALS3 of Ningyou 18;

Z11526.seq, Genbank download sequence (accession number: Z11526);

Figure 3— Detection of resistance gene BnALS1R in different Brassica napus varieties or lines by PCR molecular markers

M, DNA molecular weight standard, fragments from small to large are 500bp, 800bp, 1200bp, 2000bp, 3000bp, 4500bp; 1~5 and the corresponding 1'~5', the conventional rape variety Ningyou 12 that does not contain the resistance gene BnALS1R No., Ningyou No. 14, Ningyou No. 16, Ningyou No. 18, and Ningyou No. 20 as templates, PCR products amplified with primers AP15F/18R and AP15F/19R, respectively; 6 and 6', compared with H ₂ O As a template, the PCR product amplified with primers AP15F/18R and AP15F/19R; 7 to 11 and the corresponding 7' to 11' were used to imidazolinone-resistant Brassica napus lines M9 and M9-2 containing the resistance gene BnALS1R , M9-3, M9-11, and M9-14 as templates, PCR products amplified with primers AP15F/18R and AP15F/19R, respectively.

Figure 4—Genotype detection of resistance gene BnALS1R in F2 (3075R/M9-2) population by PCR molecular markers

M, DNA molecular weight standard, fragments from small to large are 500bp, 800bp, 1200bp, 2000bp, 3000bp, 4500bp; 1, 3075R; 2, F1 (3075R/M9-2); 3, M9-2; 4～24, part F2 individual plants, 6, 11, 14, 17, and 20 are homozygous individual plants that do not contain the resistance gene BnALS1R , and 4, 5, 7-9, 13, 15, 18, 19, 21-23 are homozygous individual plants that contain The heterozygous individual plants for the resistance gene BnALS1R , 10, 12, 16, and 24 were the homozygous individual plants for the resistance gene BnALS1R .

Figure 5—Genotype detection of resistance gene BnALS1R in BC1[(3075R/M9-2)/3075R] population by PCR molecular markers

M, DNA molecular weight standard, fragments from small to large are 200bp, 500bp, 800bp, 1200bp, 2000bp, 3000bp, 4500bp; 1, 3075R; 2, M9-2; 3, F1 (3075R/M9-2); 4～24 , part of the BC1 individuals, of which 4, 6-8, 11, 13, 16-18, 21-22, and 24 were heterozygous individuals containing the resistance gene BnALS1R , 5, 9-10, 12, 14- 15, 19-20, and 23 were homozygous individual plants that did not contain the resistance gene BnALS1R .

Figure 6—Genotype detection of resistance gene BnALS1R in BC2[(3075R/M9-2)/M9-2] population by PCR molecular markers

M, DNA molecular weight standard, fragments from small to large are 200bp, 500bp, 800bp, 1200bp, 2000bp; 1, 3075R; 2, M9-2; 3, F1 (3075R/M9-2); 4～24, part of BC2 Individual plants, of which 5, 8, 10-11, 14-15, 18-20, and 23 are heterozygous individual plants containing the resistance gene BnALS1R , 4, 6-7, 9, 12-13, 16-17, 21-22 and 24 were homozygous individual plants containing the resistance gene BnALS1R .

Figure 7—PCR molecular marker PCR detection electrophoresis of the imidazolinone-resistant DH strain resistance gene BnALS1R

M, DNA molecular weight standard, fragments from small to large are 500bp, 800bp, 1200bp, 2000bp, 3000bp, 4500bp; 1, M9; 2, Ningyou No. 18; 5, negative control water as template; 3~4, 6~24 , an imidazolinone-resistant DH line containing a homozygous form of the resistance gene BnALS1R .

Detailed ways

All methods in the following implementation are conventional methods unless otherwise specified, and the specific implementation steps are as follows:

(1) Test materials

Conventional rape varieties Ningyou No. 12, Ningyou No. 14, Ningyou No. 16, Ningyou No. 18, and Ningyou No. 20 are approved varieties in Jiangsu Province; MICMS double-low restorer line 3075R (Pu Huiming et al., 2002, Jiangsu Agricultural Science , 4:33-34); imidazolinone herbicide-resistant mutant M9 (Hu Maolong et al., Imidazolinone herbicide-resistant Brassica napus mutant gene and its application; Chinese patent ZL201010232607.4) and offspring selection line M9 -2, M9-3, M9-11, M9-14 (Pu Huiming et al., Inheritance and application of imidazolinone resistance traits in rapeseed. Chinese Journal of Oil Crops, 33 (1): 15-19)

BnALS1R genotype segregation population: F2 population (3075R/M9-2), 3075R is the female parent, M9-2 is the male parent crossing and selfing; BC1 population [(3075R/M9-2)/3075R] and BC2 population [ (3075R/M9-2)/M9-2] was obtained by backcrossing F1 (3075R/M9-2) with 3075R and M9-2 respectively. Imidazolinone-resistant DH strains are obtained from F1 (3075R/M9) plant pollen through microspore culture (Hu Maolong et al., Brassica napus mutant genes resistant to imidazolinone herbicides and their application; Chinese patent ZL201010232607.4).

(2) Molecular marker development

Brassica napus is an allotetraploid species formed by the fusion of non-heading Chinese cabbage and cauliflower. During the evolution process, the genes of Brassica napus mostly exist in the form of gene families due to the high degree of rearrangement in the genome (Chen X, et al. , Plant Physiol , 2011, 155(2): 851-65). The ALS gene is no exception. There are 5 ALS genes in the Brassica napus genome, of which BnALS1 and BnALS3 have a homology of 98% at the nucleic acid and protein levels. Homology at the level of 85%, BnALS4 and BnALS5 are loss-of-function ALS genes (Rutledge R G, et al ., Mol Gen Genet , 1991, 229: 31-40). In our previous patent application, we found that the imidazolinone herbicide-resistant mutant M9 had a single base variation in the nucleotide sequence encoding BnALS1 in the ALS family of conventional rapeseed Ningyou No. 16 and Ningyou No. 18, namely M9 The resistance gene BnALS1R gene sequence was mutated from G to A in 1933, so that the 638-position serine (AGT) residue of the coding protein sequence was replaced by asparagine (AAT) (Hu Maolong et al., resistance to imidazolinone herbicides Brassica napus mutant gene and its application; Chinese patent ZL201010232607.4). Therefore, molecular markers can be developed based on this single base variation to detect the resistance gene BnALS1R . However, due to the high homology of the nucleic acid sequences of the ALS gene family, especially the 98% homology of BnALS1 and BnALS3 at the nucleic acid level, it is difficult to develop primers to specifically amplify the gene fragments of BnALS1 and BnALS3 in the coding region of the gene. . To this end, it is first necessary to find the sequence differences between these two genes between M9, Ningyou 16, and Ningyou 18, and design primers based on the sequence differences between BnALS1 and BnALS3 to specifically amplify the BnALS1 gene sequence fragment and exclude the BnALS3 gene sequence pair. Detection of interference with resistance genes. In the patent (Hu Maolong et al., Imidazolinone herbicide-resistant brassica napus mutant gene and its application; Chinese patent ZL201010232607.4), we have obtained the complete BnALS1 gene of M9, Ningyou 16, and Ningyou 18. Long coding sequences (BnALS1R.seq, NY16.seq, NY18.seq). In order to obtain the full-length coding sequences of BnALS3 genes of M9, Ningyou 16, and Ningyou 18, according to the known BnALS3 gene sequence (accession number Z11526) in the NCBI database, on both sides of the gene coding segment and non-identical to BnALS1 Specific primers were designed for the source region to amplify the full-length BnALS 3 gene. Its forward primer F: 5'CGCGGTACCCTCTCTCTCTCTCATCTAACCAT 3'' and reverse primer R: 5'CGCACTAGTCTCTCAGTACTTAGTGCGACC 3'.

Genomic DNA was extracted from M9, Ningyou 16 and Ningyou 18 rapeseed leaves at seedling stage by CTAB method (Murray M G, et al., Nucleic Acids Research , 1980, 8(19): 4321-4326), and the steps were as follows:

(1) Take about 0.2g of leaves and put them in a mortar, add liquid nitrogen to grind them, and transfer them to a 1.5ml centrifuge tube;

(2) Add 700 μl extraction buffer (2% CTAB, 100mM Tris pH8.0, 20mM EDTA pH8.0, 1.4M NaCl, add 1% β-Me before use), mix well, and bathe in 65℃ water for 1.5h;

(3) Cool on ice, centrifuge at 12000rpm for 5min;

(4) Aspirate the supernatant, add RNase (final concentration 20 μg/ml), and incubate at 37°C for 0.5h;

(5) Add 600 μl of phenol: chloroform: isoamyl alcohol (25:24:1), mix up and down;

(6) Centrifuge, 12000rpm, 15min;

(7) Take the supernatant, add 600 μl of chloroform:isoamyl alcohol (24:1), and mix up and down;

(8) Centrifuge, 12000rpm, 15min;

(9) Take the supernatant, add 300 μl -20°C pre-cooled isopropanol, mix up and down, -20°C for 0.5h;

(10) centrifugation, 10000rpm, 10min;

(11) The precipitate was suspended and washed twice with 600 μl 70% ethanol;

(12) Discard ethanol, vacuum dry, dissolve DNA with 200μl 1×TE buffer,

(13) Use 1% agarose gel electrophoresis to test the quality of the extracted DNA, and store the samples at -20°C for later use.

Using the extracted rapeseed genomic DNA as a template, using the designed forward primer F and reverse primer R, the M9, Ningyou 16 and Ningyou 18 BnALS3 genes were amplified by PCR on a MJ Research PTC-200 PCR instrument. long coding sequence. PCR system contains 2 μL of DNA template, 5 μL of 10× enzyme reaction buffer, 3 μL of MgSO ₄ (25 mmol L), 5 μL of dNTP (2 mmol L ), 5 μL of each primer (10 mmol L ), KOD-Plus Taq enzyme (1 U/L) 2μL, add water to 50uL. The reaction program was pre-denaturation at 94 ^° C for 5 min, denaturation at 94 ^° C for 30 s, annealing at 55 ^° C for 30 s, and extension at 72 ^° C for 2.5 min, a total of 35 cycles. The blunt end of the PCR product was added with A kit (Dalian, China) by TaKaRa Bao Bioengineering Co., Ltd., and then separated by 1.2% (V/W) agarose gel electrophoresis. The electrophoresis results are shown in Figure 1. The PCR product was purified and recovered according to the steps of the ordinary agarose gel DNA recovery kit (catalogue number: DP209) produced by Beijing Tiangen Company, connected to the cloning vector pEASY-T1, and transformed into DH5α by heat shock. After blue-white screening and colony PCR identification, positive clones were sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing. The differences of BnALS1 and BnALS3 genes in M9, Ningyou 16 and Ningyou 18 were analyzed by DNAMAN6.0, Sequencher, DNASTAR and other bioinformatics software. The sequence alignment results are shown in Figure 2.

It can be seen from Figure 2 that the DNA sequences of the BnALS1 and BnALS3 gene coding regions of the three rapeseed species are highly homologous, with only one insertion/deletion of 9 bases and some single nucleotide variations. The 9-base insertion/deletion is mainly a repetitive sequence, which is not suitable as a primer sequence. Therefore, we found a single base variation (G/A ), the primer sequences were set on the single nucleotide variant sequences of BnALS1 and BnALS3 (to eliminate the interference of the BnALS3 gene sequence on PCR amplification), and a total of 30 molecular marker primers were designed (Table 1). Among them, 12 forward primers AP3F-AP14F were paired with 3 reverse primers AP1R, AP2R, A5R respectively, and 3 forward primers AP15F-AP17F were paired with 12 reverse primers AP18R-AP29R respectively. The template was amplified by PCR on the MJ Research PTC-200 PCR instrument, and the polymorphism of the amplification products of 72 pairs of molecular marker primers was screened. The three molecular marker primers obtained through screening can detect the resistant gene BnALS1R and the sensitive gene BnALS1 . The positions of these three PCR molecular marker primers on the gene sequence are shown in Figure 2. The primer sequences are:

Forward primer sequence AP15F: 5'-CTTTCGCTAGCAGGGCTAAA-3'

Reverse primer sequence AP18R: 5'-CATCTTTGAAAGTGCCACAAC-3'

Reverse primer sequence AP19R: 5'- CATCTTTGAAAGTGCCACAAT-3'

Table 1 Molecular marker primer sequences designed for the detection of resistance genes (bold underlines indicate mismatched bases, bold italics indicate mutant bases)

serial number sequence (5' to 3') serial number sequence (5' to 3') AP1R CGTCTGGGAACAACCAAAGT AP15F CTTTCGCTAGCAGGGCTAAA AP2R GCATTGAGTCCCAAACATATGA AP16F ATCCTCGACGAGCTAACCG A5R CGAGTACGTCTGGGAACAA AP17F CTGTCGTCATCAGGCCTCG AP3F TGTGTTACCGATGATCCC C A G AP18R CATCTTTGAAAGTGCCAC A A C AP4F TGTGTTACCGATGATCCC C A A   AP19R CATCTTTGAAAGTGCCAC A A T AP5F TGTGTTACCGATGATCCC T A G AP20R CATCTTTGAAAGTGCCAC T A C ' AP6F TGTGTTACCGATGATCCC T A A   AP21R CATCTTTGAAAGTGCCAC T A T AP7F TGTGTTACCGATGATCCC G A G AP22R CATCTTTGAAAGTGCCAC G A C AP8F TGTGTTACCGATGATCCC G A A AP23R CATCTTTGAAAGTGCCAC G A T AP9F TGTGTTACCGATGATCC A A A G   AP24R CATCTTTGAAAGTGCCA A CA C ' AP10F TGTGTTACCGATGATCC A A A A ' AP25R CATCTTTGAAAGTGCCA A CA T AP11F TGTGTTACCGATGATCC T AA G   AP26R CATCTTTGAAAGTGCCA T CA C AP12F TGTGTTACCGATGATCC T A A A AP27R CATCTTTGAAAGTGCCA T CA T AP13F TGTGTTACCGATGATCC G AA G   AP28R CATCTTTGAAAGTGCCA G CA C   AP14F TGTGTTACCGATGATCC G A A A   AP29R CATCTTTGAAAGTGCCA G CA T

(3) Molecular marker verification

Fresh leaves were taken at the seedling stage and stored at -70°C for later use. The total DNA of the rapeseed genome was extracted using the same method as above. Using the extracted rapeseed genomic DNA as a template, the screened three molecular marker primers AP15F/AP18R and AP15F/AP19R were respectively added to two PCR reaction systems, and amplified on a MJ Research PTC-200 PCR instrument. The PCR reaction system includes: 2.0 μL of DNA template (10 ng/μL), 2.0 μL of each primer (10 μmol/L), 2 μL of 10× enzyme reaction buffer, 1.2 μL of MgCl ₂ (25 mmol/L), dNTP ( 2.5 mmol/L ) 1.6 μL, Taq enzyme (5 U/L) 0.1 μL, add water to 20uL; the PCR reaction program is 94 ^o C pre-denaturation for 5 min, 94 ^o C denaturation for 30 s, 60 ^o C annealing for 30 s, 72 ^o C extension for 1 min, a total of 35 cycles; then 72 ^o C extension for 5 min, 12 ^o C cooling for 10 min, the amplification product was added to the sample buffer to terminate the reaction.

The amplified products were electrophoresed on an agarose gel with a mass ratio concentration of 1.2%, stained with ethidium bromide, observed and recorded under a gel imaging system, and the electrophoresis results are shown in Figure 3. The primer pair AP15F/AP18R specifically amplifies the allelic site at the 1933rd nucleotide on the ALS1 site with G, that is, the unmutated rapeseed wild-type BnALS1 gene, which is sensitive to herbicides; the primer pair AP15F/AP19R specifically amplifies What is added is the allelic site with A at the 1933rd nucleotide on the ALS1 site, that is, the mutated resistance gene BnALS1R gene, which is resistant to herbicides. Therefore, the primers using the conventional rapeseed Ningyou No. 12, Ningyou No. 14, Ningyou No. 16, Ningyou No. 18, and Ningyou No. 20 containing the sensitive gene BnALS1 as templates have 828bp characteristic bands for the amplification products of AP15F/AP18R However, the primer pair AP15F/AP19 had no characteristic bands, while the AP15F/AP18R amplification of the imidazolinone-resistant Brassica napus lines M9, M9-2, M9-3, M9-11, and M9-14 containing the resistance gene BnALS1R were used as templates. The amplification product has no characteristic band, but AP15F/AP19 has a characteristic band of 828bp. In order to verify the reliability of PCR molecular marker detection of single base variation, the vegetable seedlings were treated with imidazolinone herbicide 5.0% "Dou Xerox" water agent produced by Shandong Xianda Chemical Co., Ltd. at the 3-5 leaf stage, and the treatment concentration was 90ga .i./hm2, investigate the resistance phenotype after 15 days. The results showed that the 5 conventional rapeseed without resistance gene all died at 15 days, and the 5 lines with resistance gene grew normally. The PCR results were consistent with the field resistance identification phenotype, indicating that the PCR molecular markers of the two pairs of primers could accurately detect the imidazolinone resistance gene BnALS1R in rapeseed germplasm resources.

(4) Detection of molecular markers in isolated populations

In order to further verify the detection effect of two pairs of primer PCR molecular markers on the heterozygous genotype of the imidazolinone resistance gene BnALS1R in Brassica napus, three isolated populations of BnALS1R genotype were extracted at the seedling stage of Brassica napus: F2 population (3075R/M9-2) , BC1 population [(3075R/M9-2)/3075R], BC2 population [(3075R/M9-2)/M9-2] plant leaf DNA for PCR amplification. DNA extraction, PCR reaction, and product detection methods are the same as above. The results of electrophoresis showed three kinds of characteristic bands, that is, the characteristic bands of MICMS double low restorer line 3075R without the resistance gene (primer pair AP15F/AP18R amplification product has a characteristic band of 828bp, while AP15F/AP19 has no characteristic band), and contains the anti- The M9-2 characteristic band of the sex gene (primer pair AP15F/AP18R amplification product has no characteristic band but AP15F/AP19 has a characteristic band of 828bp), and has the heterozygous band type of the parental band type (primer pair AP15F/AP18R amplification product The amplification product has a characteristic band of 828bp and AP15F/AP19 also has a characteristic band of 828bp) (Figure 4). A total of 154 individual plants were detected in the F2 population, 35 individual plants with 3075R band, 39 individual plants with M9-2 band type, and 80 individual plants with heterozygous band type, and the three genotypes met 1: 2:1 (χ ² =0.32, 0.5<P<0.75). Simultaneously, 227 individual plants of the BC1 population and 201 individual plants of the BC2 population were amplified by PCR, and the results showed that there were 110 individual plants with the 3075R band in the BC1 population, and 117 individual plants with the M9-2 band type (Fig. 5), the two genotypes corresponded 1:1 (χ ² =0.45, 0.25<P<O.50). In the BC2 population, there were 98 individual plants with 3075R band, and 113 individual plants with M9-2 band type (Figure 6), and the two genotypes matched 1:1 (χ ² =0.37, 0.50<P<O.75 ). The PCR detection results of the above three populations were completely consistent with the phenotypic identification results of the imidazolinone herbicide "Dou Xerox" sprayed at the seedling stage. Therefore, PCR molecular markers with 2 pairs of primers can effectively distinguish three different genotypes of imidazolinone resistance gene BnALS1R in rapeseed, improve the selection efficiency of resistance genes, and accelerate the breeding process.

(5) Imidazolinone-resistant DH strains obtained by molecular marker identification

In order to verify the accuracy of PCR molecular marker detection again, we extracted imidazolinone-resistant DH strains at the seedling stage (Hu Maolong et al., Brassica napus mutant genes resistant to imidazolinone herbicides and their application; Chinese patent ZL201010232607.4 ) DNA was amplified by PCR. The results showed that all imidazolinone-resistant DH strains contained the characteristic band of the resistance gene BnALS1R (primer pair AP15F/AP18R amplification product had no characteristic band but AP15F/AP19 had a 828bp characteristic band) (Figure 7), which further proved The accuracy of 2 pairs of primers PCR molecular markers to detect resistance genes.

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Claims (2)

1. a molecular marker primer for detecting imidazolinone herbicide-resistant gene BnALS1R of Brassica napus, characterized in that: Forward primer sequence AP15F: 5'-CTTTCGCTAGCAGGGCTAAA-3' Reverse primer sequence AP18R: 5'-CATCTTTGAAAGTGCCACAAC-3' Reverse primer sequence AP19R: 5'- CATCTTTGAAAGTGCCACAAT-3'. 2. the described primer of claim 1 is used for detecting the method for Brassica napus anti-imidazolinone herbicide gene BnALS1R , it is characterized in that: (1) Extraction of Brassica napus plant genomic DNA, (2) 3 molecular marker primers AP15F/AP18R, AP15F/AP19R described in claim 1 are added to 2 PCR reaction systems respectively, and the DNA of Brassica napus germplasm resources or breeding population plants is amplified; PCR reaction system includes: 10 ng/μL DNA template 2.0 μL, 10 μmol/L primers 2.0 μL each, 10× enzyme reaction buffer 2 μL, 25 mmol/L MgCl ₂ 1.2 μL, 2.5 mmol/L dNTP1.6 μL , 5 U/L Taq enzyme 0.1 μL, add water to 20 μL; The PCR reaction program was 94 ^o C pre-denaturation for 5 min, 94 ^o C denaturation for 30 s, 60 ^o C annealing for 30 s, 72 ^o C extension for 1 min, a total of 35 cycles; then 72 ^o C extension for 5 min, 12 ^o C cooling After 10 min, the amplification product was added with sample buffer to terminate the reaction; (3) The amplified product was electrophoresed on an agarose gel with a mass ratio concentration of 1.2%, stained with ethidium bromide, observed and recorded under a gel imaging system: If the primer pair AP15F/AP18R amplification product has 828bp characteristic band and AP15F/AP19 has no characteristic band, it is the homozygote that does not contain the herbicide resistance gene BnALS1R ; if the primer pair AP15F/AP18R amplification product has no characteristic band but AP15F/AP19 has a 828bp characteristic band, which means it is a homozygote containing the herbicide resistance gene BnALS1R ; if both primer pairs AP15F/AP18R and AP15F/AP19 have a 828bp characteristic band, it means that it contains a herbicide resistance gene Hybrid of BnALS1R .

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