Primer group for detecting purity of herbicide-resistant three-line hybrid rice variety and application

Abstract

The invention relates to the technical field of biology, in particular to a primer group for detecting the purity of a herbicide-resistant three-line hybrid rice variety and application of the primer group. According to the invention, co-dominant KASP functional markers ALS-1642 and Rf4-503 are developed for specific herbicide-resistant gene allelotype and wild abortion cytoplasmic male sterility restoring gene allelotype of a herbicide-resistant three-line hybrid rice variety. A primer group composed of the two markers can accurately and efficiently identify the purity of the herbicide-resistant three-line hybrid rice variety, compared with a universal primer, the detection method is high in variety pertinence, does not need complex steps such as primer screening and small sample experiments, is small in marker number, simple, convenient and rapid, and can be used for detecting the herbicide-resistant three-line hybrid rice variety. The herbicide-resistant three-line hybrid rice variety and the purity thereof can be identified in a high-throughput manner, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer set for detecting the purity of a herbicide-resistant three-line hybrid rice variety and an application thereof. Background Art

[0002] Weed damage is one of the important factors affecting rice yield. Breeding or improving rice varieties with specific herbicide resistance and combining them with chemical herbicides to control weed damage has a good effect and has become an effective method for weed control. In recent years, various herbicides targeting acetolactate synthase (ALS), such as imidazolinones (IMs), sulfonylureas (SUs), triazolopyrimidines (TPs), pyrimidinyl carboxy herbicides (PCs), etc., have been widely used in the elimination of crop weeds due to their advantages such as broad herbicidal spectrum, low toxicity, high efficiency, and strong selectivity. Shenzhen Xingwang Biological Seed Co., Ltd. found that the ALS gene underwent a TG to AT mutation at the 1642nd and 1643rd bases in the coding region, causing the 548th encoded amino acid to mutate from tryptophan (Trp, W) to methionine (Met, M), thereby producing herbicide resistance (disclosed in CN102586215A), which promoted the selection and promotion of a large number of herbicide-resistant three-line hybrid rice varieties.

[0003] Variety purity is one of the most important indicators for evaluating seed quality. Studies have shown that within a certain range, a 1% decrease in hybrid rice purity will result in a 4-5 kg / 667m2 reduction in yield. 2 , the purity drops by 10%, and hybrid rice will lose its yield-increasing advantage. If a large number of non-herbicide varieties are mixed into herbicide-resistant varieties, phytotoxicity will also occur, and the yield loss caused by it is even more obvious. Variety purity identification mainly includes traditional planting morphological identification methods, isozymes and seed storage proteins, and molecular marker identification methods. The main types of molecular markers currently used are SSR markers, InDel or SNP markers that are evenly distributed on chromosomes and have polymorphisms. Since the sources of mixed seeds that affect variety purity are complex, including seed production parents (parental genetic separation, maternal self-pollination, etc.), cross-pollination, mechanical mixing, and grounded valleys, the selection of molecular markers directly affects the accuracy and repeatability of purity identification results. Generally, the more markers there are, the more types of mixing that can be identified, and the more accurate the results, but the corresponding workload also increases. Especially when the consistency of seed production parents and seed production isolation conditions are not understood, in order to obtain accurate purity identification results, cumbersome steps such as primer screening and small sample experiments are required, which cannot meet the needs of rapid purity identification.

[0004] Therefore, how to develop an accurate and efficient method for detecting the purity of herbicide-resistant three-line hybrid rice varieties to provide more timely and effective support for seed production and sales has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0005] In order to solve the above technical problems, the present invention first provides a primer set that can be used to detect the purity of herbicide-resistant three-line hybrid rice varieties, including sequences shown in SEQ ID NOs. 1 to 6.

[0006] The present invention develops co-dominant KASP functional markers ALS-1642 and Rf4-503 for the specific herbicide resistance gene allele type and wild-type cytoplasmic male sterility restorer gene allele type of herbicide-resistant three-line hybrid rice varieties. The specific herbicide resistance gene allele type refers to the ALS gene undergoing a TG to AT mutation at the 1642nd and 1643rd bases in the coding region. The cytoplasmic male sterility restorer gene allele type refers to the Rf4 gene undergoing a T to A mutation at the 503rd base in the coding region, which is a specific allele variation contained in the sterile allele type.

[0007] The primer set composed of the above two markers can accurately and efficiently identify the purity of herbicide-resistant three-line hybrid rice varieties, and the primer set includes Primer Allele X, Primer Allele Y and a universal primer. Among them, the sequences of Primer Allele X, Primer Allele Y and the universal primer in the primer set of the ALS-1642 molecular marker are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in the sequence table, respectively. The sequences of Primer Allele X, Primer Allele Y and the universal primer in the primer set of the Rf4-503 molecular marker are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 in the sequence table, respectively.

[0008] Furthermore, the present invention provides a reagent or a kit containing the primer set.

[0009] Furthermore, the present invention provides the use of the primer set in preparing products.

[0010] In the specific implementation process, the product includes: a chip, a reagent, a preparation, a kit or a high-throughput screening platform.

[0011] Furthermore, the present invention provides the use of the primer set, the reagent or the kit, or the product prepared by the use in identifying herbicide-resistant three-line hybrid rice varieties.

[0012] At the same time, the present invention provides the use of the primer set, the reagent or the kit, or the product prepared by the use in detecting the purity of herbicide-resistant three-line hybrid rice varieties.

[0013] In addition, the present invention provides the use of the primer set, the reagent or the kit, or the product prepared by the use in the breeding of herbicide-resistant three-line hybrid rice varieties.

[0014] Furthermore, the present invention provides a method for detecting the purity of a herbicide-resistant three-line hybrid rice variety, comprising: (1) extracting genomic DNA of a sample to be tested; (2) performing PCR amplification using the primer set to detect the genotype of the sample to be tested at the ALS-1642 and Rf4-503 marker sites; and (3) calculating the purity of the sample to be tested based on the genotype of the marker sites.

[0015] Preferably, the purity is calculated as follows: Purity (%) = number of normal individuals / total number of valid test samples × 100%, wherein a normal individual is a test sample whose genotypes at two marker sites are heterozygous, and the total number of valid test samples = total number of test samples - number of genotype-missing samples.

[0016] Preferably, the conditions for PCR amplification include: pre-denaturation at 94°C for 15 minutes; a first amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C to 65°C for 60 seconds, 10 cycles, and the temperature of annealing and extension is reduced by 0.8°C in each cycle; a second amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, 33 cycles.

[0017] Preferably, the herbicide-resistant three-line hybrid rice varieties include but are not limited to "Leyou 966 (JT)" and "Leyou 456 (JT)".

[0018] Compared with the prior art, the present invention has the following beneficial effects: The primer set of the present invention can be used to accurately and efficiently identify herbicide-resistant three-line hybrid rice varieties and their purity. Compared with universal primers, the detection method of the present invention is highly targeted, accurate and efficient, does not require tedious steps such as primer screening and small sample experiments, has a small number of markers, is simple and fast, and can be used for high-throughput identification of herbicide-resistant three-line hybrid rice varieties and their purity, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the purity test result of "Leyou 966 (JT)".

[0020] Figure 2 This is the purity test result of "Leyou 456 (JT)".

[0021] Figure 3These are test photos of field identification. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0024] Example 1 Development of two specific KASP functional markers Development of ALS detection marker for herbicide-resistant gene: Based on the wild-type and herbicide-resistant mutant gene sequences of ALS of "Huanghuazhan" provided by Shenzhen Xingwang Biological Seed Co., Ltd., the functional mutation from TG to AT at bases 1642 and 1643 was detected, the flanking sequences were extracted, and the primer ALS-1642 was designed using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) (Table 1).

[0025] Development of detection markers for wild-type cytoplasmic male sterility nuclear restorer gene Rf4: Using the NCBI website, the CDS sequence KJ680248.1 of the Rf4 restorer gene contained in "Minghui63" and the CDS sequences KJ680247.1 and KJ680250.1 of the rf4-i indica rice sterility gene and rf4-j japonica rice sterility gene contained in "ZS97A" and "ZH11" respectively were downloaded. It was found that Rf4, rf4-i and rf4-j had a T-to-A mutation at the 503rd base of the coding region. The flanking sequences of Rf4 and rf4-i at this site were extracted, and the primer Rf4-503 was designed using the above-mentioned online primer design website (Table 1). Each marker has three primers, and the 5' ends of two of the specific primers are connected to FAM and HEX fluorescent sequences respectively. The primers were commissioned to be synthesized by LGC.

[0026] Table 1

[0027] Example 2 Application of KASP functional markers in purity detection of herbicide-resistant three-line hybrid rice varieties 1. Extraction of genomic DNA from test materials The herbicide-resistant three-line hybrid rice varieties "Leyou 966 (JT)" and "Leyou 456 (JT)" and their parents were used as test materials. The genomic DNA of the parents was extracted by CTAB method, and the genomic DNA of 90 seedlings of "Leyou 966 (JT)" and "Leyou 456 (JT)" was quickly extracted by alkaline boiling method.

[0028] 2. PCR amplification reaction The Meridian system of the LGC SNPline genotyping platform was used to automatically assemble the PCR amplification system, as shown in Table 2. The 96-well module in the 384-well plate was used to place DNA from 2 male parents, 2 female parents, 2 blank controls (water), and 90 individual strains of the sample to be tested as templates. Among them, KASP Master mix was provided by LGC, UK.

[0029] Table 2

[0030] Hydrocycler using the LGC SNPline genotyping platform 2 The system performed PCR amplification under the following conditions: pre-denaturation at 94°C for 15 min; first step amplification reaction, denaturation at 94°C for 20 s, annealing and extension at 57°C-65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.8°C in each cycle; second step amplification reaction, denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 33 cycles.

[0031] 3. Genotyping After the PCR reaction was completed, the reaction system fluorescence signal was scanned using the FLUOstar Ω microplate reader of the LGC SNPline genotyping platform, and the Launch Kluster Caller software system was used for genotyping and data analysis. The genotyping results of ALS-1642 and Rf4-503 for two herbicide-resistant three-line hybrid rice "Leyou 966 (JT)" and "Leyou 456 (JT)" were clear ( Figure 1 , Figure 2 ), the alleles of the herbicide-resistant male parent and the wild-type female sterile line were ALS-1642: A: A; Rf4-503: T: T and ALS-1642: T: T; Rf4-503: A: A, and the alleles of the hybrids (normal individuals) were ALS-1642: A: T; Rf4-503: T: A. The allele data of each plant of the sample to be tested are shown in Table 3.

[0032] 4. Calculation of rice variety purity The purity of the sample to be tested is calculated according to the marker genotype. The purity calculation formula is: Purity (%) = number of normal individuals / total number of valid test samples × 100%, wherein a normal individual is a sample with heterozygous genotypes at both marker sites, and the total number of valid test samples = total number of test samples - number of samples with missing genotypes.

[0033] As shown in Table 3, among the 90 samples to be tested of "Leyou 966 (JT)", the number of invalid single plants is 4 (genotype deletion at the Rf4-503 marker site), the total number of valid test samples is 86, the number of normal individuals (heterozygous genotype) is 78, and its purity is 78 / 86×100%=90.70%. Among the 90 samples to be tested of "Leyou 456 (JT)", the number of invalid single plants is 2 (genotype deletion at the ALS-1642 and Rf4-503 marker sites, 1 each), the total number of valid test samples is 88, the number of normal individuals (heterozygous genotype) is 84, and its purity is 84 / 88×100%=95.45%.

[0034] Table 3 5. Use traditional testing methods to detect the purity of the above-mentioned "Leyou 966 (JT)" and "Leyou 456 (JT)" samples, the steps are as follows: According to the requirements of "6.4 Field plot planting identification" in the "Authenticity and variety purity identification of agricultural crop seeds inspection procedures" (GB / T 3543.5-1995), in the summer of 2024, at the Hefei rice production base, select fields with uniform soil, consistent fertility, and no similar crops and weeds in the previous crop, and plant 200 individual plants of the same batch of "Leyou 966 (JT)" and "Leyou 456 (JT)" respectively, carry out appropriate cultivation management, and mark the mutant strains through observation of characteristics throughout the growth period ( Figure 3 ), and the sample purity was statistically analyzed (Table 4).

[0035] Table 4

[0036] The test results show that the purity of "Leyou 966 (JT)" is 91.41%, and the purity of "Leyou 456 (JT)" is 96.43%. Compared with the purity test results of the present invention, the field identification results are slightly higher than the test results of the present invention, and the difference is within the allowable error range (GB / T 3543.5-1995), indicating that the detection method of the present invention has extremely high accuracy.

[0037] Comparative Example This comparative example provides the use of universal SSR primers to detect the purity of the above-mentioned "Leyou 966 (JT)" and "Leyou 456 (JT)" samples, and the steps are as follows: According to "SSR Molecular Marker Detection of Authenticity and Purity of Major Crop Varieties" (GB / T39917-2021), 48 SSR markers are used to construct the fingerprints of "Leyou 966 (JT)" and "Leyou 456 (JT)" and their parents, and 2 SSR markers that are different between the parents are randomly screened (Table 5), and the purity of 96 seedlings of each of the "Leyou 966 (JT)" and "Leyou 456 (JT)" samples from the same batch as in Example 2 is detected.

[0038] Table 5

[0039] The test results show (Table 6) that the purity of "Leyou 966 (JT)" at the RM471 and RM311 marker sites is 94.68% and 96.81%, respectively, and the purity of "Leyou 456 (JT)" at the RM471 and RM311 marker sites is 98.92% and 100%, respectively. Compared with the above-mentioned field plot planting identification method, its purity result is higher, and due to the different types of hybrid plants, the purity results at different SSR marker sites are inconsistent, and it is difficult to determine which site has a more accurate result. Therefore, when using a universal SSR marker combination for the purity detection of herbicide-resistant three-line hybrid rice varieties, it is not only necessary to screen primers, but also when the number of markers used is small, it is difficult to obtain a unified test result, and the accuracy is low. However, increasing the number of markers will further increase the workload and significantly affect the work efficiency.

[0040] Table 6

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set, characterized in that: Including sequences shown as SEQ ID NO.1~6.

2. A reagent or kit containing the primer set according to claim 1.

3. Use of the primer set according to claim 1 in preparing a product.

4. The use according to claim 3, characterized in that: The products include: chips, reagents, preparations, kits or high-throughput screening platforms.

5. Use of the primer set according to claim 1, the reagent or kit according to claim 2, or the product prepared by the use of claim 3 or 4 in identifying herbicide-resistant three-line hybrid rice varieties.

6. Use of the primer set according to claim 1, the reagent or kit according to claim 2, or the product prepared by the application according to claim 3 or 4 in detecting the purity of herbicide-resistant three-line hybrid rice varieties.

7. Use of the primer set according to claim 1, the reagent or kit according to claim 2, or the product prepared by the application according to claim 3 or 4 in the breeding of herbicide-resistant three-line hybrid rice varieties.

8. A method for detecting the purity of herbicide-resistant three-line hybrid rice varieties, characterized in that: include: (1) Extracting genomic DNA from the sample to be tested; (2) Performing PCR amplification using the primer set described in claim 1 to detect the genotype of the sample to be tested at the ALS-1642 and Rf4-503 marker sites; (3) Calculating the purity of the sample to be tested based on the genotype of the marker site.

9. The detection method according to claim 8, characterized in that: The calculation formula of the purity is: Purity (%) = number of normal individuals / total number of valid test samples × 100%, wherein a normal individual is a test sample whose genotypes at two marker sites are heterozygous, and the total number of valid test samples = total number of test samples - number of samples with missing genotypes.

10. The detection method according to claim 8, characterized in that: The conditions of the PCR amplification include: pre-denaturation at 94°C for 15 minutes; a first amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C-65°C for 60 seconds, 10 cycles, and the temperature of annealing and extension is reduced by 0.8°C in each cycle; a second amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, 33 cycles.

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