A primer set for detecting the purity of herbicide-resistant three-line hybrid rice varieties and its application

Abstract

The present application relates to the field of biotechnology, and more particularly to a primer set for detecting the purity of herbicide-resistant three-line hybrid rice varieties and application, the present application develops the co-dominant KASP functional markers ALS-1642 and Rf4-503 aiming at the specific herbicide-resistant gene allele type and the wild failure type cytoplasmic male sterile restoration gene allele type of herbicide-resistant three-line hybrid rice varieties. The primer set composed of the above two markers can accurately and efficiently identify the purity of herbicide-resistant three-line hybrid rice varieties, compared with the universal primer, the detection method of the present application is strong in specificity, does not need primer screening and small sample experiment and other cumbersome steps, the number of markers is small, simple and fast, can high-throughput identify herbicide-resistant three-line hybrid rice varieties and its purity, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a primer set for detecting the purity of herbicide-resistant three-line hybrid rice varieties and its application. Background Technology

[0002] Weed infestation is a significant factor affecting rice yield. Breeding or improving rice varieties with specific herbicide resistance, and then combining them with chemical herbicides, has proven effective in controlling weeds. In recent years, various herbicides targeting acetolactate synthase (ALS), such as imidazolinones (IMs), sulfonylureas (SUs), triazolopyrimidines (TPs), and pyrimidinyl carboxyherbicides (PCs), have been widely used for weed control due to their broad spectrum of action, low toxicity, high efficiency, and strong selectivity. Research by Shenzhen Xingwang Biological Seed Industry Co., Ltd. has found that the ALS gene has a TG to AT mutation at bases 1642 and 1643 in the coding region, which causes the amino acid encoded at position 548 to change from tryptophan (Trp, W) to methionine (Met, M), thereby producing herbicide resistance (disclosed in CN102586215A), which has promoted the breeding 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 leads to a yield reduction of 4–5 kg / 667m². 2 A 10% decrease in purity will cause hybrid rice to lose its yield-increasing advantage. If a large number of non-herbicide varieties are mixed into herbicide-resistant varieties, phytotoxicity will occur, resulting in significant yield losses. Variety purity identification mainly includes traditional planting morphology methods, isoenzyme and seed storage protein methods, and molecular marker identification methods. Currently, the main types of molecular markers used are SSR markers, InDel or SNP markers, which are evenly distributed on chromosomes and exhibit polymorphism. Because the sources of mixed seeds affecting variety purity are complex, including parental lines (parental genetic segregation, maternal self-pollination, etc.), cross-pollination, mechanical mixing, and fallen grains, the selection of molecular markers directly affects the accuracy and reproducibility of purity identification results. Generally, the more markers, the more types of mixing can be identified, and the more accurate the results, but the workload also increases accordingly. Especially when the consistency of parental lines and seed isolation conditions are unknown, obtaining accurate purity identification results requires cumbersome steps such as primer screening and small-sample experiments, 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, so as 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] To address the aforementioned technical challenges, this invention first provides a primer set that can be used to detect the purity of herbicide-resistant three-line hybrid rice varieties, including sequences as shown in SEQ ID NO.1~6.

[0006] This invention targets the specific herbicide resistance gene alleles and the wild-type cytoplasmic male sterility restorer gene alleles in herbicide-resistant three-line hybrid rice varieties, and develops codominant KASP functional markers ALS-1642 and Rf4-503. The specific herbicide resistance gene allele refers to a TG-to-AT mutation at bases 1642 and 1643 of the ALS gene coding region. The cytoplasmic male sterility restorer gene allele refers to a T-to-A mutation at base 503 of the Rf4 gene coding region, representing a specific allelic variation found in the sterility allele.

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

[0008] Furthermore, the present invention provides reagents or kits containing the primer set described above.

[0009] Furthermore, the present invention provides the application of the primer set in product preparation.

[0010] In practice, the products include: chips, reagents, formulations, kits, or high-throughput screening platforms.

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

[0012] Meanwhile, this invention provides the application of the primer set, the reagent or kit, or the product prepared by the application in detecting the purity of herbicide-resistant three-line hybrid rice varieties.

[0013] Furthermore, this invention provides the application of the primer set, the reagent or kit, or the product prepared by the application in the breeding of herbicide-resistant three-line hybrid rice varieties.

[0014] Furthermore, the present invention provides a method for detecting the purity of herbicide-resistant three-line hybrid rice varieties, comprising: (1) extracting genomic DNA from the 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%, where normal individuals are those whose genotypes at both marker sites are heterozygous, and the total number of valid test samples = Total number of test samples - Number of genotype-deleted samples.

[0016] Preferably, the PCR amplification conditions include: pre-denaturation at 94℃ for 15 minutes; first amplification reaction: denaturation at 94℃ for 20 seconds, annealing and extension at 57℃~65℃ for 60 seconds, 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ in each cycle; second amplification reaction: denaturation at 94℃ for 20 seconds, annealing and extension at 57℃ 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 beneficial effects of the present invention are as follows:

[0019] The primer set of this invention can accurately and efficiently identify herbicide-resistant three-line hybrid rice varieties and their purity. Compared with general primers, the detection method of this invention is highly targeted, accurate and efficient, and does not require cumbersome steps such as primer screening and small sample experiments. It requires fewer markers, is simple and fast, and can identify herbicide-resistant three-line hybrid rice varieties and their purity in high throughput, and has broad application prospects. Attached Figure Description

[0020] Figure 1 This is the purity test result for "Leyou 966 (JT)".

[0021] Figure 2 This is the purity test result for "Leyou 456 (JT)".

[0022] Figure 3 These are photos of field testing. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0025] Example 1: Development of two specific KASP functional markers

[0026] Development of a marker for detecting herbicide-resistant ALS: Based on the wild-type and herbicide-resistant mutant ALS gene sequences of “Huang Huazhan” provided by Shenzhen Xingwang Biological Seed Industry Co., Ltd., flanking sequences were extracted targeting the TG-AT functional mutation at bases 1642 and 1643, and primer ALS-1642 was designed using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) (Table 1).

[0027] Development of a marker for detecting the nuclear restorer gene Rf4 in wild-type cytoplasmic male sterility: 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 male sterility gene and rf4-j japonica rice male sterility gene contained in “ZS97A” and “ZH11”, respectively, were downloaded. A T-to-A mutation was found at base 503 in the coding region of Rf4 with rf4-i and rf4-j. Flanking sequences of Rf4 and rf4-i at this site were extracted, and primers Rf4-503 were designed using the aforementioned online primer design website (Table 1). Each marker has three primers, with two specific primers having FAM and HEX fluorescent sequences linked to their 5' ends, respectively. Primers were synthesized by LGC.

[0028] Table 1

[0029]

[0030] Example 2: KASP functional markers used for purity detection in herbicide-resistant three-line hybrid rice varieties

[0031] 1. Genomic DNA extraction from experimental materials

[0032] The herbicide-resistant three-line hybrid rice varieties “Leyou 966 (JT)” and “Leyou 456 (JT)” and their parents were used as test materials. Genomic DNA of the parents was extracted by CTAB method, and genomic DNA of 90 seedlings of “Leyou 966 (JT)” and “Leyou 456 (JT)” was rapidly extracted by alkaline boiling method.

[0033] 2. PCR amplification reaction

[0034] The PCR amplification system was automatically assembled using the Meridian system of the LGC SNPline genotyping platform, as shown in Table 2 below. The 96-well modules in the 384-well plates contained DNA from two paternal parents, two maternal parents, two blank controls (water), and 90 individual plants of the test sample, respectively, as templates. The KASP Master mix was provided by LGC Ltd. (UK).

[0035] Table 2

[0036]

[0037] Hydrocycler using the LGC SNPline genotyping platform 2 The system was subjected to PCR amplification under the following conditions: pre-denaturation at 94℃ for 15 minutes; first amplification reaction: denaturation at 94℃ for 20 seconds, annealing and extension at 57℃~65℃ for 60 seconds, 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ per cycle; second amplification reaction: denaturation at 94℃ for 20 seconds, annealing and extension at 57℃ for 60 seconds, 33 cycles.

[0038] 3. Genotyping

[0039] After the PCR reaction, the fluorescence signal of the reaction system was scanned using the FLUOstar Ω microplate reader on the LGC SNPline genotyping platform, and genotyping and data analysis were performed using the Launch Kluster Caller software system. The genotyping results of ALS-1642 and Rf4-503 for the two herbicide-resistant three-line hybrid rice varieties “Leyou 966 (JT)” and “Leyou 456 (JT)” were clear. Figure 1 , Figure 2The alleles of the herbicide-resistant male parent and the wild-type female parent sterile line were ALS-1642:A:A; Rf4-503:T:T and ALS-1642:T:T; Rf4-503:A:A, respectively. The alleles of the hybrids (normal individuals) were ALS-1642:A:T; Rf4-503:T:A, respectively. The allele data of each individual plant in the tested samples are shown in Table 3.

[0040] 4. Calculation of rice variety purity

[0041] The purity of the sample to be tested is calculated based on the marker genotype. The formula for calculating the purity is: Purity (%) = Number of normal individuals / Total number of valid test samples × 100%, where normal individuals are samples with heterozygous genotypes at both marker loci, and the total number of valid test samples = Total number of test samples - Number of genotype-deficient samples.

[0042] As shown in Table 3, among the 90 samples of "Leyou 966 (JT)", there were 4 invalid plants (genotype deletion at the Rf4-503 marker site), a total of 86 valid samples, and 78 normal individuals (heterozygous genotype). Therefore, the purity is 78 / 86 × 100% = 90.70%. Among the 90 samples of "Leyou 456 (JT)", there were 2 invalid plants (1 each at the ALS-1642 and Rf4-503 marker sites), a total of 88 valid samples, and 84 normal individuals (heterozygous genotype). Therefore, the purity is 84 / 88 × 100% = 95.45%.

[0043] Table 3

[0044]

[0045]

[0046]

[0047] 5. The purity of the above-mentioned "Leyou 966 (JT)" and "Leyou 456 (JT)" samples was tested using traditional testing methods. The steps are as follows: According to the requirements of "6.4 Field Plot Planting Identification" in the "Regulations for the Inspection of Crop Seeds: Authenticity and Variety Purity Identification" (GB / T 3543.5-1995), in the summer of 2024, in the Hefei rice production base, fields with uniform soil, consistent fertility, and no similar crops or weeds in the previous crop were selected. 200 individual plants of each of the above-mentioned batches of "Leyou 966 (JT)" and "Leyou 456 (JT)" were planted, and appropriate cultivation management was carried out. Through observation of the characteristics throughout the entire growth period, variant plants were marked (…). Figure 3 ), and the purity of the samples was statistically analyzed (Table 4).

[0048] Table 4

[0049]

[0050] Test results show that the varietal purity of "Leyou 966 (JT)" is 91.41%, and the varietal purity of "Leyou 456 (JT)" is 96.43%. Comparison with the purity test results of this invention shows that the field identification results are slightly higher than the test results of this invention's method, but the difference is within the allowable error range (GB / T 3543.5-1995), indicating that the detection method of this invention has extremely high accuracy.

[0051] Comparative Example

[0052] This comparative example provides the detection of the purity of the above-mentioned "Leyou 966 (JT)" and "Leyou 456 (JT)" samples using universal SSR primers. The steps are as follows: According to the "Detection of Authenticity and Purity of Major Crop Varieties by SSR Molecular Markers in Rice" (GB / T39917-2021), fingerprint profiles of "Leyou 966 (JT)" and "Leyou 456 (JT)" and their parents were constructed using 48 SSR markers. Two SSR markers that differed between the parents were randomly selected (Table 5). The purity of 96 seedlings of each of the same batch of "Leyou 966 (JT)" and "Leyou 456 (JT)" samples as in Example 2 was detected.

[0053] Table 5

[0054]

[0055] The test results (Table 6) show that the purity of "Leyou 966 (JT)" at the RM471 and RM311 marker sites was 94.68% and 96.81%, respectively, while the purity of "Leyou 456 (JT)" at the RM471 and RM311 marker sites was 98.92% and 100%, respectively. Compared with the above-mentioned field plot planting identification method, the purity results are higher, and due to different hybrid types, the purity results at different SSR marker sites are inconsistent, making it difficult to determine which site's result is more accurate. Therefore, when using a universal SSR marker combination for the purity detection of herbicide-resistant three-line hybrid rice varieties, not only is primer screening necessary, but when the number of markers used is small, it is difficult to obtain uniform detection results, resulting in low accuracy. However, increasing the number of markers further increases the workload and significantly affects work efficiency.

[0056] Table 6

[0057]

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of primer set in identifying herbicide-resistant three-line hybrid rice variety; the sequence of the primer set is shown in SEQ ID NO. 1-6; the herbicide-resistant three-line hybrid rice variety is Leyou 456 or Leyou 966.

2. Application of primer set in detecting purity of herbicide-resistant three-line hybrid rice variety; the sequence of the primer set is shown in SEQ ID NO. 1-6; the herbicide-resistant three-line hybrid rice variety is Leyou 456 or Leyou 966.

3. Application of primer set in breeding herbicide-resistant three-line hybrid rice variety; the sequence of the primer set is shown in SEQ ID NO. 1-6; the herbicide-resistant three-line hybrid rice variety is Leyou 456 or Leyou 966.

4. Application of reagent or kit containing primer set in identifying herbicide-resistant three-line hybrid rice variety; the sequence of the primer set is shown in SEQ ID NO. 1-6; the herbicide-resistant three-line hybrid rice variety is Leyou 456 or Leyou 966.

5. Application of reagent or kit containing primer set in detecting purity of herbicide-resistant three-line hybrid rice variety; the sequence of the primer set is shown in SEQ ID NO. 1-6; the herbicide-resistant three-line hybrid rice variety is Leyou 456 or Leyou 966.

6. Application of reagent or kit containing primer set in breeding herbicide-resistant three-line hybrid rice variety; the sequence of the primer set is shown in SEQ ID NO. 1-6; the herbicide-resistant three-line hybrid rice variety is Leyou 456 or Leyou 966.

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