Primer set for identifying authenticity and seed purity of cotton variety zhongmian 9001 and its application

Abstract

The application discloses a primer group for identifying the authenticity of cotton variety Zhongmian 9001 and seed purity and application thereof. The primer group is a composition of 21 primer pairs of QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20 and QS21. The composition of the 21 primer pairs can be used for identifying the purity of the cotton variety Zhongmian 9001 and the authenticity of the variety, constructing a fingerprint map of the cotton variety Zhongmian 9001, and is beneficial to the popularization and protection of Zhongmian 9001.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to primer sets and their applications for identifying the authenticity and seed purity of cotton variety Zhongmian 9001 in the field of crop molecular genetics and breeding. Background Technology

[0002] Zhongmian 9001 is a high-yield, high-quality transgenic cotton variety developed by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences, with the approval number Guoshenmian 20200009. Zhongmian 9001 is a mid-maturing conventional variety bred from Jimian 616 / ((Zhongmian Institute 25 × Zhongmian Institute 12) F2) using insect-resistant genes. In the Yellow River cotton-growing region, its spring-sown growth period is 115 days. It exhibits good emergence, a relatively loose plant type, long and spreading fruiting branches, robust stems, medium-sized and medium-colored leaves, and does not prematurely age. The bolls are oval, open smoothly, and it is resistant to Fusarium wilt (disease index 5.7), tolerant to Verticillium wilt (disease index 25.2), and resistant to bollworm. The plant height is 108.1 cm, the first fruiting branch node is at the 7.2 node, the number of bolls per plant is 19.2, the weight of a single boll is 5.8 g, the seed index is 11.5 g, the lint percentage is 39.0%, and the pre-frost flowering rate is 93.9%. The HVICC fiber has an average upper half length of 31.4 mm, a breaking strength of 32.2 centinewtons / tex, a micronaire value of 4.9, a breaking elongation of 5.4%, a reflectance of 79.6%, a yellowness depth of 7.4, a uniformity index of 85.1%, a spinning evenness index of 154, and is classified as type II fiber quality. In the 2017-2018 regional trials of medium-maturity conventional cotton varieties in the Yellow River Basin cotton-growing area, the average yields of seed cotton, lint, and pre-frost lint per mu (667 square meters) were 264.3 kg, 103.0 kg, and 96.7 kg, respectively, representing increases of 2.7%, 10.0%, and 10.4% compared to the control variety Shikang 126. In the 2019 production trials, the yields of seed cotton, lint, and pre-frost lint per mu were 268.0 kg, 111.1 kg, and 105.8 kg, respectively, representing increases of 5.0%, 5.4%, and 6.2% compared to the control variety Zhongmian Suo 100.

[0003] Due to its excellent overall traits, Zhongmian 9001 became the control variety for the national regional trials of medium-maturity conventional cotton in the Yellow River cotton-growing area in 2022.

[0004] To prevent the sale of substandard cotton variety Zhongmian 9001 in the market, a rapid and accurate method for identifying the authenticity of Zhongmian 9001 is needed to support the regulatory testing by seed enforcement agencies and the internal seed quality control of breeding units. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to identify the authenticity and / or purity of cotton variety Mian 9001.

[0006] To address the above technical problems, this invention provides a primer pair composition for identifying or assisting in the identification of cotton variety Mian 9001, which is a composition composed of 21 primer pairs: QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21.

[0007] The QS1 consists of two single-stranded DNA sequences shown in SEQ ID No. 1 and SEQ ID No. 2 of the sequence listing;

[0008] The QS2 consists of two single-stranded DNA sequences shown in SEQ ID No. 3 and SEQ ID No. 4 of the sequence listing;

[0009] The QS3 consists of two single-stranded DNA sequences shown in SEQ ID No. 5 and SEQ ID No. 6 of the sequence listing;

[0010] The QS4 consists of two single-stranded DNA sequences shown in SEQ ID No. 7 and SEQ ID No. 8 of the sequence listing;

[0011] The QS5 consists of two single-stranded DNA sequences shown in SEQ ID No. 9 and SEQ ID No. 10 of the sequence listing;

[0012] The QS6 consists of two single-stranded DNA sequences shown in SEQ ID No. 11 and SEQ ID No. 12 in the sequence listing;

[0013] The QS7 consists of two single-stranded DNA sequences shown in SEQ ID No. 13 and SEQ ID No. 14 in the sequence listing;

[0014] The QS8 consists of two single-stranded DNA sequences shown in SEQ ID No. 15 and SEQ ID No. 16 of the sequence listing.

[0015] The QS9 consists of two single-stranded DNA sequences shown in SEQ ID No. 17 and SEQ ID No. 18 in the sequence listing;

[0016] The QS10 consists of two single-stranded DNA sequences shown in SEQ ID No. 19 and SEQ ID No. 20 of the sequence listing;

[0017] The QS11 consists of two single-stranded DNA sequences shown in SEQ ID No. 21 and SEQ ID No. 22 in the sequence listing;

[0018] The QS12 consists of two single-stranded DNA sequences shown in SEQ ID No. 23 and SEQ ID No. 24 in the sequence listing;

[0019] The QS13 consists of two single-stranded DNA sequences shown in SEQ ID No. 25 and SEQ ID No. 26 of the sequence listing;

[0020] The QS14 consists of two single-stranded DNA sequences shown in SEQ ID No. 27 and SEQ ID No. 28 of the sequence listing;

[0021] The QS15 consists of two single-stranded DNA sequences shown in SEQ ID No. 29 and SEQ ID No. 30 of the sequence listing;

[0022] The QS16 consists of two single-stranded DNA sequences shown in SEQ ID No. 31 and SEQ ID No. 32 in the sequence listing;

[0023] The QS17 consists of two single-stranded DNA sequences shown in SEQ ID No. 33 and SEQ ID No. 34 in the sequence listing;

[0024] The QS18 consists of two single-stranded DNA sequences shown in SEQ ID No. 35 and SEQ ID No. 36 in the sequence listing;

[0025] The QS19 consists of two single-stranded DNA sequences shown in SEQ ID No. 37 and SEQ ID No. 38 in the sequence listing;

[0026] The QS20 consists of two single-stranded DNA sequences shown in SEQ ID No. 39 and SEQ ID No. 40 of the sequence listing;

[0027] The QS21 consists of two single-stranded DNA sequences shown in SEQ ID No. 41 and SEQ ID No. 42 in the sequence listing.

[0028] In the primer pair composition for identifying or assisting in the identification of cotton variety Mian 9001, the 21 primer pairs can be of equal mass, and the concentration ratio of the two single-stranded DNAs after dilution of each primer pair can be 1:1.

[0029] To address the above technical problems, this invention provides a set of primers for obtaining the DNA fingerprint of the cotton variety Zhongmian 9001.

[0030] The primer set provided by this invention for obtaining the DNA fingerprint of cotton variety Zhongmian 9001 is the primer pair composition mentioned above for identifying or assisting in the identification of cotton variety Zhongmian 9001.

[0031] The present invention also provides a reagent or kit for identifying or assisting in the identification of the cotton variety Zhongmian 9001, wherein the reagent or kit contains the primer pair composition described above for identifying or assisting in the identification of the cotton variety Zhongmian 9001.

[0032] The present invention also provides a reagent or kit for identifying or assisting in the identification of the purity of cotton variety Zhongmian 9001 seeds, the reagent or kit containing the primer pair composition described above for identifying or assisting in the identification of cotton variety Zhongmian 9001.

[0033] The following applications also fall within the scope of protection of this invention:

[0034] X1) The application of the primer pair composition for identifying or assisting in the identification of cotton variety Mian 9001 or the set of primers for obtaining the DNA fingerprint of cotton variety Mian 9001 in the preparation of products for identifying or assisting in the identification of the authenticity of cotton variety Mian 9001.

[0035] X2) The application of the primer pair composition for identifying or assisting in the identification of cotton variety Zhongmian 9001 or the set of primers for obtaining the DNA fingerprint of cotton variety Zhongmian 9001 in the preparation of products for identifying or assisting in the identification of the purity of cotton variety Zhongmian 9001 seeds.

[0036] X3) The application of the primer pair composition for identifying or assisting in the identification of cotton variety Zhongmian 9001 or the set of primers for obtaining the DNA fingerprint of cotton variety Zhongmian 9001 in identifying or assisting in the identification of the authenticity of cotton variety Zhongmian 9001.

[0037] X4) The application of the primer pair composition for identifying or assisting in the identification of cotton variety Zhongmian 9001 or the set of primers for obtaining the DNA fingerprint of cotton variety Zhongmian 9001 in the identification or assistance in the identification of the purity of cotton variety Zhongmian 9001 seeds.

[0038] X5) The application of the primer pair composition for identifying or assisting in the identification of cotton variety Mian 9001 or the complete set of primers for obtaining the DNA fingerprint of cotton variety Mian 9001 in cotton breeding.

[0039] X6) The application of the primer pair composition for identifying or assisting in the identification of cotton variety Zhongmian 9001 or the complete set of primers for obtaining the DNA fingerprint of cotton variety Zhongmian 9001 in the construction of the DNA fingerprint of cotton variety Zhongmian 9001.

[0040] This invention also provides a method for identifying or assisting in the identification of the cotton variety Zhongmian 9001.

[0041] The method for identifying or assisting in the identification of the cotton variety Zhongmian 9001 provided by this invention includes:

[0042] 1) Using the genomic DNA of the cotton variety to be tested and the cotton variety Zhongmian 9001 as templates, PCR amplification was performed using 21 primer pairs to obtain the PCR products of the cotton variety to be tested and the PCR products of the cotton variety Zhongmian 9001 using the 21 primer pairs.

[0043] 2) Perform electrophoresis on the PCR products of the cotton varieties to be tested and the PCR products of the cotton variety Zhongmian 9001 with the 21 primer pairs, and determine whether the cotton varieties to be tested and Zhongmian 9001 are the same variety based on the electrophoretic banding pattern.

[0044] The 21 primer pairs are QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21.

[0045] In the above-mentioned identification or auxiliary identification methods, the method for determining whether the cotton variety to be tested and the cotton variety Zhongmian 9001 are the same variety based on the electrophoretic banding pattern can be as follows: If the banding pattern of the PCR product of each primer pair of the 21 primer pairs of the cotton variety to be tested is consistent with the banding pattern of the corresponding primer pair of the PCR product of Zhongmian 9001 in the 21 primer pairs of the cotton variety to be tested, the cotton variety to be tested is determined to be cotton variety Zhongmian 9001 or a candidate cotton variety Zhongmian 9001; if the banding pattern of at least one primer pair of the PCR product of the cotton variety to be tested is inconsistent with the banding pattern of the corresponding primer pair of the PCR product of Zhongmian 9001 in the 21 primer pairs of the cotton variety to be tested, the cotton variety to be tested is not cotton variety Zhongmian 9001 or a candidate cotton variety Zhongmian 9001.

[0046] This invention also provides a method for identifying or assisting in the identification of the purity of cotton seeds of the variety Zhongmian 9001. This method can be used to test the purity of a specific batch of cotton seeds known to be Zhongmian 9001.

[0047] The method for identifying or assisting in the identification of seed purity of cotton variety Zhongmian 9001 provided by this invention may include:

[0048] 1) Using the genomic DNA of the cotton seeds to be tested as a template, PCR amplification was performed using primer pairs to obtain the PCR products of the cotton seeds to be tested using the primer pairs.

[0049] 2) Electrophoresis was performed on the PCR products of the cotton seeds to be tested using the primer pair to obtain the electrophoretic banding pattern of the PCR products of the cotton seeds to be tested using the primer pair.

[0050] 3) The purity of cotton variety Zhongmian 9001 seeds was determined by comparing the electrophoretic banding of the PCR product of the cotton seed to be tested with that of the Zhongmian 9001 seed.

[0051] The primer pairs are selected from at least eight of the following: QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21 as described in claim 1.

[0052] The electrophoretic banding of the PCR product of Zhongmian 9001 seed using the primer pair can be obtained by using Zhongmian 9001 seed genomic DNA as a template, performing PCR amplification with the above primer pair, and then electrophoresis the PCR product. Alternatively, it can be obtained directly using... Figure 2 The electrophoretic pattern shown.

[0053] In the above-mentioned identification or auxiliary identification methods, step 3) may include:

[0054] If the electrophoretic banding of the PCR product of the cotton seed to be tested for each primer pair is the same as that of the PCR product of Zhongmian 9001 seed with the same primer pair, or if the electrophoretic banding of the PCR product of only one primer pair is different, then the cotton seed to be tested is determined to be a Zhongmian 9001 seed; if the electrophoretic banding of the PCR product of the cotton seed to be tested for at least two primer pairs is different from that of the PCR product of Zhongmian 9001 seed with the same primer pair, then the cotton seed to be tested is determined to be a hybrid.

[0055] The purity of the cotton variety Zhongmian 9001 seeds is determined by calculating the percentage of Zhongmian 9001 seeds in the cotton seeds to be tested. This percentage is the purity of the cotton variety Zhongmian 9001 seeds.

[0056] The cotton seeds to be tested can be selected from cotton seeds known to be Zhongmian 9001, which can be sampled according to national standards. For example, 24 seeds can be randomly selected from cotton seeds known to be Zhongmian 9001, DNA can be extracted, and PCR amplification can be performed using primer pairs to obtain the PCR products of the cotton seeds to be tested. Electrophoresis is performed, and the data is read. If a seed has two or more primer pairs (including two pairs) with band patterns different from Zhongmian 9001, it is defined as a hybrid. If the band patterns are completely the same or one primer pair has a different band pattern, it is considered as Zhongmian 9001. The percentage of Zhongmian 9001 seeds in the total number of tests is the purity of the test batch, that is, the purity of the cotton seeds known to be Zhongmian 9001 in the test batch.

[0057] The application of any eight or more primer pairs from the 21 primer pairs QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20 and QS21 in identifying or assisting in the identification of the purity of cotton 9001 seeds is also within the scope of protection of this invention.

[0058] This invention screened and obtained a composition composed of 21 primer pairs: QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21. This composition of 21 primer pairs can be used to identify the purity of the cotton variety Zhongmian 9001, verify the authenticity of the Zhongmian 9001 cotton variety, and construct the fingerprint spectrum of the Zhongmian 9001 cotton variety, which is beneficial for the promotion and protection of Zhongmian 9001. Attached Figure Description

[0059] Figure 1 This is an electrophoresis pattern of PCR products from 21 primer pairs in different cotton varieties. Each primer pair amplifies three band patterns: types 1 and 2 appear in conventional cotton varieties, while type 3 is a co-occurring band pattern, generally found in hybrid cotton varieties. The varieties identified in this patent are conventional varieties and will not exhibit type 3 patterns. In the figure, M represents the marker, and the eight bands from top to bottom are 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, and 100bp in size. Each primer pair has six lanes: the first two lanes from left to right represent band pattern 1, the middle two lanes represent band pattern 2, and the last two lanes represent band pattern 3.

[0060] Figure 2The image shows the electrophoretic patterns of PCR products from 21 primer pairs of Zhongmian 9001. In the image, M is the marker, and the sizes of the eight bands from top to bottom are 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, and 100bp, respectively. Each primer pair has four lanes, representing four individual plants.

[0061] Figure 3 The image shows the electrophoretic patterns of PCR products from 21 primer pairs for Zhongmian 9001 and Zhongmian Institute 100. In the image, M is the marker, and the eight bands from top to bottom are 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, and 100bp, respectively. Each primer pair has four lanes, and from left to right, they represent two individual plants, Zhongmian 9001 and Zhongmian Institute 100.

[0062] Figure 4 This image shows the electrophoretic patterns of PCR products from Zhongmian 9001 amplified using primers QS1, QS5, QS6, and QS12. Purity was determined using these primers. In the image, M represents the marker, and the eight bands from top to bottom are 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, and 100bp in size. Each primer pair has 24 lanes, representing 24 individual plants of Zhongmian 9001. Figure 4 There were no differential bands in the amplification products of the primers.

[0063] Figure 5 This is an electrophoresis pattern of the PCR products of Zhongmian 9001 amplified with primers QS13, QS16, QS18, and QS20. Purity was determined. In the figure, M represents the marker, and the eight bands from top to bottom are 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, and 100bp, respectively. Each primer pair has 24 lanes, representing 24 individual plants of Zhongmian 9001 (and...). Figure 4 (The same 24 individual plants), the boxes indicate seeds with different band patterns. Detailed Implementation

[0064] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0066] Example 1: Identification of cotton variety Zhongmian 9001

[0067] To screen a set of core primers for identifying the authenticity and purity of the cotton variety Zhongmian 9001 (hereinafter referred to as Zhongmian 9001 or Zhongmian 9001), this embodiment used the control varieties Zhongmian 100, Zhongmian 9001, and 28 other nationally approved varieties as materials. Molecular marker technology was employed, and 102 pairs of cotton SSR primers were amplified by PCR. After initial screening and reselection, 21 pairs of primers with clear amplification bands, good repeatability, and strong resolution were finally obtained. These 21 pairs of primers can be used to detect the authenticity of the cotton variety Zhongmian 9001 and to construct the fingerprint spectrum of the cotton variety Zhongmian 9001. The specific experimental methods and results are as follows:

[0068] 1. Preparation of primer pair composition for identifying cotton variety Zhongmian 9001

[0069] The primer pair composition prepared in this step for identifying the cotton variety Zhongmian 9001 consists of 21 primer pairs: QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21. QS1 consists of two single-stranded DNA pairs as shown in SEQ ID No. 1 and SEQ ID No. 2 of the sequence listing; QS2 consists of two single-stranded DNA pairs as shown in SEQ ID No. 3 and SEQ ID No. 4 of the sequence listing; QS3 consists of two single-stranded DNA pairs as shown in SEQ ID No. 5 and SEQ ID No. 6 of the sequence listing; QS4 consists of two single-stranded DNA pairs as shown in SEQ ID No. 7 and SEQ ID No. 8 of the sequence listing; and QS5 consists of two single-stranded DNA pairs as shown in SEQ ID No. 9 and SEQ ID No. 2 of the sequence listing. QS6 consists of two single-stranded DNA molecules as shown in SEQ ID No. 10; QS7 consists of two single-stranded DNA molecules as shown in SEQ ID No. 13 and SEQ ID No. 14; QS8 consists of two single-stranded DNA molecules as shown in SEQ ID No. 15 and SEQ ID No. 16; QS9 consists of two single-stranded DNA molecules as shown in SEQ ID No. 17 and SEQ ID No. 18; QS10 consists of two single-stranded DNA molecules as shown in SEQ ID No. 19 and SEQ ID No. 20; QS11 consists of two single-stranded DNA molecules as shown in SEQ ID No. 21 and SEQ ID No. 22; QS12 consists of two single-stranded DNA molecules as shown in SEQ ID No. 23 and SEQ ID No. 24; QS13 consists of two single-stranded DNA molecules as shown in SEQ ID No. 25 and SEQ ID No. 26; QS14 consists of two single-stranded DNA molecules as shown in SEQ ID No. 27 and SEQ ID No. 28. QS15 consists of two single-stranded DNA sequences as shown in SEQ ID No. 28; QS16 consists of two single-stranded DNA sequences as shown in SEQ ID No. 31 and SEQ ID No. 32; QS17 consists of two single-stranded DNA sequences as shown in SEQ ID No. 33 and SEQ ID No. 34; QS18 consists of two single-stranded DNA sequences as shown in SEQ ID No. 35 and SEQ ID No. 36; QS19 consists of two single-stranded DNA sequences as shown in SEQ ID No. 37 and SEQ ID No. 38.The primer pair compositions for identifying the cotton variety Zhongmian 9001 are as follows: QS20 consists of two single-stranded DNA molecules shown in SEQ ID No. 38 and SEQ ID No. 40 of the sequence listing; QS20 consists of two single-stranded DNA molecules shown in SEQ ID No. 41 and SEQ ID No. 42 of the sequence listing (Table 1). In these primer pair compositions, each primer pair is individually packaged, and the 21 primer pairs are of equal mass. The concentration ratio of the two single-stranded DNA molecules after dilution of each primer pair is 1:1.

[0070] Table 1. Sequences of primer pair combinations for identifying cotton variety Zhongmian 9001

[0071]

[0072] 2. Identify the authenticity of the cotton variety Zhongmian 9001 and construct its fingerprint spectrum.

[0073] The cotton varieties Zhongmian 9001 and the control variety Zhongmian Suo 100 were both from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. The varieties used in the examples were all cotton varieties approved by the state in recent years, and the samples came from various breeding units (Table 2). DNA was extracted from field samples.

[0074] Table 2. Sources of 28 Nationally Approved Cotton Varieties

[0075]

[0076] Genomic DNA was extracted from individual plants of cotton variety Zhongmian 9001, control variety Zhongmian 100, and the 28 nationally approved varieties listed in Table 2. Amplification was performed using any one of the 21 primer pairs (QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21) from step 1. The PCR products for each of these 21 primer pairs were obtained for cotton variety Zhongmian 9001, control variety Zhongmian 100, and the other 28 nationally approved varieties listed in Table 2. The PCR products were then subjected to 8% polyacrylamide gel electrophoresis and silver staining.

[0077] The PCR system for the 21 primer pairs (QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21) was 10.0 μL, with a 5 μL mix containing Taq enzyme and dNTPs, a concentration of 0.5 μM for both the forward and reverse primers, 1 μL of DNA template, and the remainder being double-distilled water. The amplification program for the 21 primer pairs QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21 was as follows: pre-denaturation at 94℃ for 3 min, followed by 30 cycles of denaturation at 94℃ for 45 s, annealing at 55℃ for 45 s, extension at 72℃ for 1 min, and finally extension at 72℃ for 10 min, followed by storage at 4℃.

[0078] All 21 primer pairs were verified to be polymorphic in cotton varieties, exhibiting three banding patterns (see...). Figure 1 To make it more intuitive, the amplification patterns of the same primer pair are generally classified according to the molecular weight of the smallest amplified product. When the molecular weights of the smallest products are the same, they are classified according to the second largest molecular weight, and so on. The type of low molecular weight band is defined as 1, the type of high molecular weight band is defined as 2, and the type of heterozygous co-occurrence is defined as 3. The corresponding pattern type is converted into a unique code like an ID number. The difference between varieties is distinguished by the code, which is different from the traditional method of reading 1 for bands and 0 for no bands. The reading method is simple and fast [Fu Xiaoqiong, SSR fingerprint pattern of cotton regional trial control varieties in the Yellow River Basin in 2011, China Cotton 2011.12:29-32]. The electrophoretic pattern of the PCR amplification products of 21 primer pairs of cotton variety Zhongmian 9001 is as follows. Figure 2 As shown, the comparative electrophoresis patterns of PCR amplification products from 21 primer pairs for cotton varieties Zhongmian 9001 and Zhongmian Institute 100 are as follows. Figure 3 As shown in Table 3, the number of differential sites between the two varieties is 9.

[0079] Table 3. Coding table of the spectra of Zhongmian 9001 and the control variety Zhongmian 100

[0080]

[0081]

[0082] From 2019 to 2021, 28 conventional cotton varieties were approved by the state. Among the 21 pairs of core primers used, the fingerprint patterns of the corresponding 28 varieties were different from those of Zhongmian 9001. At least three pairs of primers produced amplification products that were different from those of Zhongmian 9001 (Table 4).

[0083] Table 4. Coding of fingerprint profiles of 28 nationally approved cotton varieties

[0084]

[0085]

[0086]

[0087] The above experimental results show that, among the PCR products of the 21 primer pairs for all cotton varieties tested (numbered 1-28 in Table 2), at least four primer pairs show inconsistent banding patterns with the corresponding primer pairs in the PCR product of cotton variety Zhongmian 9001. (Table 4)

[0088] Primer pair compositions consisting of 21 primer pairs (QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21) can distinguish cotton variety Zhongmian 9001 from cotton varieties listed in Table 2. These compositions can be used to identify the authenticity of cotton variety Zhongmian 9001 and to determine the purity of its seeds.

[0089] The above experimental results show that primer compositions consisting of 21 primer pairs (QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21) can be used to construct fingerprint profiles of the cotton variety Zhongmian 9001, identify the authenticity of the cotton variety Zhongmian 9001, and determine the seed purity of the cotton variety Zhongmian 9001.

[0090] Methods for verifying the authenticity of the cotton variety Zhongmian 9001 may include:

[0091] 1) Using genomic DNA from the cotton varieties to be tested (e.g., any one of the cotton varieties listed in Table 2 (numbered 1-28) and Table 3) and the cotton variety Zhongmian 9001 (hereinafter referred to as Zhongmian 9001) as templates, PCR amplification was performed using 21 primer pairs: QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21. The PCR products of the cotton varieties to be tested and the PCR products of Zhongmian 9001 using the 21 primer pairs were obtained. The PCR products of the cotton varieties tested were measured, including the PCR products of Zhongmian 9001 (QS1), Zhongmian 9001 (QS2), Zhongmian 9001 (QS3), Zhongmian 9001 (QS3), Zhongmian 9001 (QS4), Zhongmian 9001 (QS4), Zhongmian 9001 (QS5), Zhongmian 9001 (QS5), Zhongmian 9001 (QS6), Zhongmian 9001 (QS6), Zhongmian 9001 (QS7), and Zhongmian 9001 (QS8). CR products and QS8 Zhongmian 9001 PCR products, QS9 test cotton variety PCR products and QS9 Zhongmian 9001 PCR products, QS10 test cotton variety PCR products and QS10 Zhongmian 9001 PCR products, QS11 test cotton variety PCR products and QS11 Zhongmian 9001 PCR products, QS12 test cotton variety PCR products and QS12 Zhongmian 9001 PCR products, QS13 test cotton variety PCR products and QS13 Zhongmian 9001 PCR products, QS14 test cotton variety PCR products and QS14 Zhongmian 9001 PCR products, QS15 test cotton variety PCR products and QS14 Zhongmian 9001 PCR products, QS15 test cotton variety PCR products and QS12 Zhongmian 9001 PCR products, QS13 test cotton variety PCR products and QS13 Zhongmian 9001 PCR products, QS14 test cotton variety PCR products and QS15 Zhongmian 9001 PCR products, QS15 test cotton variety PCR products and QS12 ... PCR products of cotton varieties and PCR products of Zhongmian 9001 in QS15, PCR products of cotton varieties to be tested in QS16 and PCR products of Zhongmian 9001 in QS16, PCR products of cotton varieties to be tested in QS17 and PCR products of Zhongmian 9001 in QS17, PCR products of cotton varieties to be tested in QS18 and PCR products of Zhongmian 9001 in QS18, PCR products of cotton varieties to be tested in QS19 and PCR products of Zhongmian 9001 in QS19, PCR products of cotton varieties to be tested in QS20 and PCR products of Zhongmian 9001 in QS20, PCR products of cotton varieties to be tested in QS21 and PCR products of Zhongmian 9001 in QS21;

[0092] 2) Perform electrophoresis on the PCR products of the cotton variety to be tested using the 21 primer pairs and the PCR products of the cotton variety Zhongmian 9001 using the 21 primer pairs. Determine whether the cotton variety to be tested and the cotton variety Zhongmian 9001 are the same variety based on the electrophoretic banding: If the banding pattern of the PCR product of the cotton variety to be tested using each primer pair in the 21 primer pairs is consistent with the banding pattern of the corresponding primer pair in the PCR product of the cotton variety Zhongmian 9001 using the 21 primer pairs, then the cotton variety to be tested is determined to be cotton variety Zhongmian 9001; if the banding pattern of the PCR product of the cotton variety to be tested using at least one primer pair in the 21 primer pairs is inconsistent with the banding pattern of the corresponding primer pair in the PCR product of the cotton variety Zhongmian 9001 using the 21 primer pairs, then the cotton variety to be tested is not cotton variety Zhongmian 9001.

[0093] Example 2: Identification of seed purity of cotton variety Zhongmian 9001

[0094] Cotton seeds of the known variety Zhongmian 9001 were tested to determine the purity of a batch. Electrophoretic patterns of PCR amplification products from 21 primer pairs (QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21) for the cotton variety Zhongmian 9001 were obtained. Figure 2 Any eight or more primer pairs from these 21 primer pairs can be used to identify the seed purity of the cotton variety Zhongmian 9001. The following example, using eight primer pairs, illustrates the specific method for identifying the seed purity of the cotton variety Zhongmian 9001.

[0095] Purity was determined using eight primer pairs (QS1, QS5, QS6, QS12, QS13, QS16, QS18, and QS20) from Example 1. Seeds were sampled according to national standards, and 24 seeds were randomly selected from the sample. DNA was extracted, and PCR amplification, electrophoresis, and data reading were performed according to the method in Example 1. Banding patterns were defined as in Example 1. Results are as follows: Figure 4 and Figure 5 As shown, one of the 24 seeds has a different banding pattern. Figure 5 The seed numbered 4 was amplified by QS13 and QS16, and the band pattern was 1. The band pattern of the rest was 2. The SSR label purity of Zhongmian 9001 was (24-1) / 24*100%=95.8%.

[0096] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The primer pair composition for identifying or assisting in the identification of cotton variety Mian 9001 is a composition consisting of 21 primer pairs: QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20, and QS21. The QS1 consists of two single-stranded DNA sequences shown in SEQ ID No. 1 and SEQ ID No. 2 of the sequence listing; The QS2 consists of two single-stranded DNA sequences shown in SEQ ID No. 3 and SEQ ID No. 4 of the sequence listing; The QS3 consists of two single-stranded DNA sequences shown in SEQ ID No. 5 and SEQ ID No. 6 of the sequence listing; The QS4 consists of two single-stranded DNA sequences shown in SEQ ID No. 7 and SEQ ID No. 8 of the sequence listing; The QS5 consists of two single-stranded DNA sequences shown in SEQ ID No. 9 and SEQ ID No. 10 of the sequence listing; The QS6 consists of two single-stranded DNA sequences shown in SEQ ID No. 11 and SEQ ID No. 12 in the sequence listing; The QS7 consists of two single-stranded DNA sequences shown in SEQ ID No. 13 and SEQ ID No. 14 in the sequence listing; The QS8 consists of two single-stranded DNA sequences shown in SEQ ID No. 15 and SEQ ID No. 16 of the sequence listing; The QS9 consists of two single-stranded DNA sequences shown in SEQ ID No. 17 and SEQ ID No. 18 in the sequence listing; The QS10 consists of two single-stranded DNA sequences shown in SEQ ID No. 19 and SEQ ID No. 20 of the sequence listing; The QS11 consists of two single-stranded DNA sequences shown in SEQ ID No. 21 and SEQ ID No. 22 in the sequence listing; The QS12 consists of two single-stranded DNA sequences shown in SEQ ID No. 23 and SEQ ID No. 24 in the sequence listing; The QS13 consists of two single-stranded DNA sequences shown in SEQ ID No. 25 and SEQ ID No. 26 of the sequence listing; The QS14 consists of two single-stranded DNA sequences shown in SEQ ID No. 27 and SEQ ID No. 28 of the sequence listing; The QS15 consists of two single-stranded DNA sequences shown in SEQ ID No. 29 and SEQ ID No. 30 of the sequence listing; The QS16 consists of two single-stranded DNA sequences shown in SEQ ID No. 31 and SEQ ID No. 32 in the sequence listing; The QS17 consists of two single-stranded DNA sequences shown in SEQ ID No. 33 and SEQ ID No. 34 in the sequence listing; The QS18 consists of two single-stranded DNA sequences shown in SEQ ID No. 35 and SEQ ID No. 36 in the sequence listing; The QS19 consists of two single-stranded DNA sequences shown in SEQ ID No. 37 and SEQ ID No. 38 in the sequence listing; The QS20 consists of two single-stranded DNA sequences shown in SEQ ID No. 39 and SEQ ID No. 40 of the sequence listing; The QS21 consists of two single-stranded DNA sequences shown in SEQ ID No. 41 and SEQ ID No. 42 in the sequence listing.

2. A reagent or kit for identifying or assisting in the identification of the authenticity or seed purity of the cotton variety Zhongmian 9001, characterized in that: The reagent or kit contains the primer pair composition of claim 1.

3. Any of the following applications: X1) The use of the primer pair composition according to claim 1 in the preparation of products for identifying or assisting in the identification of the authenticity of cotton variety 9001; X2) The use of the primer pair composition according to claim 1 in the preparation of products for identifying or assisting in the identification of the purity of cotton 9001 seeds in cotton varieties; X3) The application of the primer pair composition according to claim 1 in identifying or assisting in the identification of the authenticity of cotton variety cotton 9001; X4) The application of the primer pair composition according to claim 1 in identifying or assisting in the identification of the purity of cotton 9001 seeds in cotton varieties; X5) The application of the primer pair composition according to claim 1 in constructing the DNA fingerprint of cotton variety Zhongmian 9001.

4. Methods for identifying or assisting in the identification of the cotton variety Zhongmian 9001, including: 1) Using the genomic DNA of the cotton variety to be tested and cotton variety Zhongmian 9001 as templates, PCR amplification was performed using 21 primer pairs to obtain the PCR products of the cotton variety to be tested and the PCR products of cotton variety Zhongmian 9001 using the 21 primer pairs. 2) Perform electrophoresis on the PCR products of the cotton varieties to be tested and the PCR products of the cotton variety Zhongmian 9001 with the 21 primer pairs, and determine whether the cotton varieties to be tested and Zhongmian 9001 are the same variety based on the electrophoretic banding pattern. The 21 primer pairs are the 21 primer pairs QS1, QS2, QS3, QS4, QS5, QS6, QS7, QS8, QS9, QS10, QS11, QS12, QS13, QS14, QS15, QS16, QS17, QS18, QS19, QS20 and QS21 described in claim 1.

5. The identification or auxiliary identification method according to claim 4, characterized in that: The method for determining whether the cotton variety to be tested and cotton variety Zhongmian 9001 are the same variety based on electrophoretic banding is as follows: If the banding pattern of the PCR product of each primer pair of the 21 primer pairs of the cotton variety to be tested is consistent with the banding pattern of the corresponding primer pair of the PCR product of cotton variety Zhongmian 9001 in the 21 primer pairs of the cotton variety Zhongmian 9001, the cotton variety to be tested is determined to be cotton variety Zhongmian 9001 or a candidate cotton variety Zhongmian 9001; if the banding pattern of at least one primer pair of the PCR product of the cotton variety to be tested is inconsistent with the banding pattern of the corresponding primer pair of the PCR product of cotton variety Zhongmian 9001 in the 21 primer pairs of the cotton variety Zhongmian 9001, the cotton variety to be tested is not cotton variety Zhongmian 9001 or a candidate cotton variety Zhongmian 9001.

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