Polymorphic genome SSR molecular marker combination for identifying cotton varieties and application of polymorphic genome SSR molecular marker combination in development of molecular fingerprint spectrum and two-dimensional code recognition system

Abstract

The invention provides a polymorphic genome SSR molecular marker combination for identifying cotton varieties and application of the polymorphic genome SSR molecular marker combination in developing a molecular fingerprint spectrum and a two-dimensional code recognition system, and belongs to the technical field of molecular markers and bioinformatics. The invention provides a polymorphic genome SSR (Simple Sequence Repeat) molecular marker combination for identifying cotton varieties. The polymorphic genome SSR molecular marker combination for identifying the cotton varieties is NBRIHQ526730, NBRIHQ527820, HAU3071a, NAU3736b, NAU3913, NAU5172 and MONDPL0893. A core primer is screened from known cotton SSR primers positioned on a chromosome, and the polymorphic genome SSR molecular marker combination for identifying the cotton variety and the primers of the polymorphic genome SSR molecular marker combination are provided. And then constructing a DNA fingerprint spectrum and two-dimensional code recognition system of the Xinjiang cotton variety by using the SSR molecular marker combination.

Description

Technical Field

[0001] This invention relates to the fields of molecular markers and bioinformatics, and in particular to a polymorphic genome SSR molecular marker combinatorial system for identifying cotton varieties and its application in developing molecular fingerprinting and QR code recognition systems. Background Technology

[0002] cotton( Gossypium hirsutum ) belongs to the Malvaceae family ( Malvaceae ) Cotton ( Gossypium Cotton, an herbaceous plant, plays a vital role in the development of China's agricultural economy, with its planting area mainly distributed in Xinjiang and its northwestern regions. As the largest cotton-producing area in China, Xinjiang boasts diverse and complex varieties. Analyzing the phylogenetic relationships and genetic diversity of Xinjiang's cotton germplasm resources is essential, providing a reference for the protection, identification, and utilization of these resources.

[0003] Constructing DNA fingerprints of cotton varieties using molecular marker technology helps to assess the genetic diversity of cotton germplasm resources, locate functional genes related to important agronomic traits, lay a theoretical foundation for research fields such as germplasm innovation and genetic improvement, and provide technical support for molecular design breeding and efficient utilization of germplasm resources.

[0004] SSR molecular fingerprinting technology, characterized by its multi-site, high variability, and stable inheritance, is widely used in crop variety identification. Compared to traditional electrophoresis, capillary electrophoresis combined with multicolor fluorescence detection can achieve precise quantification and efficient analysis of DNA fragments, and has been successfully applied in crops such as rice. However, relying solely on molecular marker technology still has certain limitations. When faced with complex ecological environments and diverse germplasm resources, no single method can fully elucidate the complete characteristics of a variety.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of complex origins and difficulty in identification of cotton varieties in Xinjiang.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a polymorphic genome SSR molecular marker combination for identifying cotton varieties, wherein the SSR molecular marker combination is NBRI_HQ526730, NBRI_HQ527820, HAU3071a, NAU3736b, NAU3913, NAU5172 and MON_DPL0893.

[0008] Preferably, the primer set XP69 sequence of NBRI_HQ526730 is shown in SEQ ID NO: 137~138; The primer set XP71 sequence of NBRI_HQ527820 is shown in SEQ ID NO: 141~142; The primer set XP21 sequence of HAU3071a is shown in SEQ ID NO: 41~42; The primer set XP57 sequence of the NAU3736b is shown in SEQ ID NO: 113~114; The primer set XP60 sequence of NAU3913 is shown in SEQ ID NO: 119~120; The primer set XP63 sequence of NAU5172 is shown in SEQ ID NO: 125~126; The primer set XP42 sequence of MON_DPL0893 is shown in SEQ ID NO: 83~84.

[0009] Preferably, the cotton varieties identified include Xinjiang upland cotton, Xinjiang island cotton, and Xinjiang colored cotton.

[0010] This invention also provides a method for constructing a fingerprint map of Xinjiang cotton varieties using the aforementioned SSR molecular marker combination. Using the DNA of Xinjiang upland cotton, Xinjiang island cotton, and Xinjiang colored cotton as templates, PCR amplification is performed using primers from the aforementioned SSR molecular marker combination. The amplification products are then subjected to electrophoresis, and the electrophoresis data is analyzed to obtain the molecular fingerprint map.

[0011] The present invention also provides a molecular fingerprint spectrum of cotton varieties constructed by the method described above.

[0012] This invention also provides a method for constructing a QR code identification system for Xinjiang cotton varieties based on phenotypic traits and SSR molecular marker technology, comprising the following steps: (1) Extract DNA from cotton and perform fluorescent PCR amplification using the primer pair of the SSR molecular marker to obtain a specific expression map with the fluorescence expression level as the ordinate and the amplification product size as the abscissa. (2) Integrate basic information of cotton, important phenotypic traits and specific expression maps constructed from SSR molecular markers, and synthesize digital QR codes for the corresponding varieties by entering parameters of important phenotypic traits and specific expression maps of cotton into the advanced editor through the interconnected QR code generator platform.

[0013] Preferably, the SSR molecular marker is one of the SSR molecular marker combinations; the basic information includes: variety image, variety name, approval number, approval time, approval unit, pedigree, maturity characteristics, growth period, flowering rate, disease resistance and suitable planting area; the important phenotypic traits include: plant height, single boll weight, number of fruit branches, lint percentage and fiber strength.

[0014] The present invention also provides a QR code recognition system for Xinjiang cotton varieties constructed by the aforementioned construction method.

[0015] This invention also provides the application of the aforementioned molecular fingerprint spectrum or the aforementioned Xinjiang cotton variety QR code identification system in the identification and recognition of Xinjiang cotton varieties.

[0016] Preferably, using the DNA of the Xinjiang cotton variety to be tested as a template, PCR amplification is performed using primers of the SSR molecular marker combination, the amplification products are subjected to electrophoresis, the electrophoresis data are analyzed to obtain a molecular fingerprint map, and the fingerprint map is compared with the molecular fingerprint map of the known Xinjiang cotton varieties constructed in claim 5 to determine the variety of Xinjiang cotton to be tested; or using the DNA of the Xinjiang cotton variety to be tested as a template, PCR amplification is performed using primers of any marker in the SSR molecular marker combination to obtain the specific expression map, and then, combined with the phenotypic traits of the Xinjiang cotton variety to be tested, the variety of Xinjiang cotton to be tested is determined by comparing with the constructed Xinjiang cotton variety QR code identification system.

[0017] This invention screens core primers from known cotton SSR primers located on chromosomes, providing a combination of polymorphic genomic SSR molecular markers and their primers for identifying cotton varieties. Then, this SSR molecular marker combination is used to construct DNA fingerprints of Xinjiang cotton varieties. This method can effectively identify the authenticity of cotton varieties, with short detection time and high accuracy.

[0018] This invention also provides a QR code identification system for Xinjiang cotton varieties based on SSR molecular marker combinations. Based on traditional phenotypic data and SSR molecular marker technology, the system screens and analyzes the specificity of cotton varieties, achieving efficient and accurate identification. It boasts advantages such as short detection time, high accuracy, and ease of operation, making it suitable for large-scale sample processing and rapid on-site identification, providing technical support for variety management and market supervision in the Xinjiang cotton industry. The QR code identification system provided by this invention combines variety-specific phenotypic traits with SSR molecular marker technology as a core strategy for variety diversity assessment. By combining phenotypic and molecular marker methods, it not only overcomes the limitations of single technologies but also reveals the intrinsic relationship between phenotype and environment through significant loci related to traits. Furthermore, it combines DNA fingerprint data with variety phenotypic data, integrating the content into a recognizable digital QR code using modern digital technology, significantly improving the informatization and automation level of variety identification. Attached Figure Description

[0019] Figure 1 Fingerprint profiles of Xinjiang island cotton varieties; Figure 2Fingerprint maps of Xinjiang colored cotton varieties; Figure 3 This is the QR code for No. 55 Xinlu Middle School and the image information after scanning. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] Core primers were screened from known cotton SSR molecular markers located on chromosomes.

[0023] The known cotton molecular markers located on chromosomes in this embodiment include primer markers constructed by Huazhong Agricultural University, which are located on a genetic linkage map containing 5152 markers on chromosomes (https: / / www.cottongen.org / data / download). Preferably, the above primers were screened, including screening for polymorphic markers and screening for marker genetic diversity, resulting in 71 primer pairs with high polymorphism, good stability, and uniform distribution on the 26 cotton chromosomes: BNL2449, BNL2535b, BNL2865, BNL3031, BNL3043, CCRI303, CCRI596a, CK988221-PR-ss, DPL0238, Gh107, Gh330, HAU0875, HAU0938, HAU1496, H... AU1952b, HAU1968a, HAU2481, HAU2588, HAU2770, HAU2846, HAU3071a, HAU3193, HAU3371, HAU4022, HAU4483, HAU4 748, HAU-SNP085, HAU-SNP113, MGHES31, MON_CGR5007, MON_CGR5447, MON_CGR5732, MON_CGR6012, MON_CGR6812, M ON_DPL0010, MON_DPL0024, MON_DPL0375c, MON_DPL0502, MON_DPL0504a, MON_DPL0521, MON_DPL0811b, MON_DPL0 893, MON_SHIN-1481, MON_SHIN-1494b, MON_SHIN-1584, MON_SHIN-1585, MUSS422a, NAU2126, NAU2240, NAU2631, N The primer sequences for the following primers are listed in Table 1: AU2713a, NAU2985, NAU3298, NAU3346b, NAU3419c, NAU3433, NAU3736b, NAU3774, NAU3827, NAU3913, NAU4022, NAU4089, NAU5172, NAU5323, NAU6734, NAU6997, NAU7049, NAU7182, NBRI_HQ526730, NBRI_HQ527566, and NBRI_HQ527820.

[0024] Table 1 Primer information for 71 SSR molecular markers

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] PCR amplification was performed on Xinjiang cotton varieties shown in Table 2 using 71 primer pairs. The total PCR system was 10 μL, including 5 μL of 2×Fine Taq Mix premix, 0.25 μL each of 2 μmol / L forward and reverse primers, and 0.5 μL of DNA template. The DNA template concentration was diluted to 50–100 ng / μL. Finally, ddH2O was added to bring the total volume to 10 μL. The PCR amplification program included: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s (depending on the primers), 72℃ extension for 20 s, for 28 cycles. The final extension was at 72℃ for 5 min. After amplification, capillary electrophoresis was performed, and the data were analyzed to obtain cotton variety banding diagrams. The genetic diversity parameters associated with each primer pair were calculated using GenAIex software, as shown in Table 3. The parameters included the number of amplified polymorphic sites, Shannon genetic information index (I), number of alleles (Na), effective number of alleles (Ne), and polymorphic information content (PIC). The polymorphic information content (PIC) was calculated using the formula of Nie et al. (1997). As shown in Table 3, a total of 180 polymorphic sites were detected by 71 pairs of SSR primers distributed on 26 chromosomes, with an average of 2.53 polymorphic sites per primer. The average effective number of alleles (Ne) for each SSR marker was 1.414, ranging from 1.023 to 1.8983. The average Shannon diversity index (I) was 0.3986, ranging from 0.0622 to 0.6653. The average polymorphic information content (PIC) for each SSR marker was 0.5227, ranging from 0.0949 to 0.7661. When the PIC is greater than 0.5, it indicates that the locus has high polymorphism; a value between 0.25 and 0.5 indicates moderate polymorphism; and a value less than 0.25 indicates low polymorphism. Among the 71 primer pairs described in this invention, 47 primer pairs have high polymorphism, and 18 primer pairs have moderate polymorphism. Among them, primer XP69 has the highest PIC value of 0.7661. The number of alleles (Na) is 2.0000, the effective number of alleles (Ne) is 1.8983, the average Nei's gene diversity index H′ is 0.4726, and Shannon's polymorphism information index I is 0.6653. Among them, six primers, namely XP71, XP21, XP57, XP60, XP63 and XP42, have high PIC (>0.4), H′ (>0.4) and Shannon's index I (>0.6) values, indicating that these primers have high polymorphism detection efficiency.

[0031] Table 2 Information on the types of test materials

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] Table 3 Genetic diversity parameters of 71 SSR primer pairs

[0047]

[0048]

[0049] Example 2

[0050] Seven primers with high detection efficiency were selected using molecular marker technology: NBRI_HQ526730, NBRI_HQ527820, HAU3071a, NAU3736b, NAU3913, NAU5172, and MON_DPL0893. Based on these primers, the fingerprint information of Xinjiang colored cotton and Sea Island cotton materials was further analyzed. Among the sea island cotton varieties, the following were selected: Xin Hai No. 1, Xin Hai No. 2, Xin Hai No. 3, Xin Hai No. 4, Xin Hai No. 5, Xin Hai No. 6, Xin Hai No. 7, Xin Hai No. 8, Xin Hai No. 9, Xin Hai No. 10, Xin Hai No. 11, Xin Hai No. 12, Xin Hai No. 13, Xin Hai No. 14, Xin Hai No. 15, Xin Hai No. 16, Xin Hai No. 17, Xin Hai No. 18, Xin Hai No. 19, Xin Hai No. 20, Xin Hai No. 21, Xin Hai No. 22, Xin Hai No. 23, Xin Hai No. 24, Xin Hai No. 25, Xin Hai No. 26, and Xin Hai No. 27. Xinhai No. 28, Xinhai No. 29, Xinhai No. 30, Xinhai No. 31, Xinhai No. 32, Xinhai No. 33, Xinhai No. 34, Xinhai No. 35, Xinhai No. 36, Xinhai No. 37, Xinhai No. 38, Xinhai No. 39, Xinhai No. 40, Xinhai No. 41 No., Xinhai No. 42, Xinhai No. 43, Xinhai No. 44, Xinhai No. 45, Xinhai No. 46, Xinhai No. 47, Xinhai No. 48, Xinhai No. 49, Xinhai No. 50, Xinhai No. 51, Xinhai No. 52, Xinhai No. 53, Xinhai No. 54, XinHai No. 55, XinHai No. 56, XinHai No. 57, XinHai No. 58, XinHai No. 59, XinHai No. 60, XinHai No. 61, XinHai No. 62, and XinHai No. 63 were used for identification. Seven pairs of primers with high polymorphism were used, and any single primer could be used to distinguish the materials. Among colored cotton, XinCaiMian No. 1, XinCaiMian No. 2, XinCaiMian No. 3, XinCaiMian No. 4, XinCaiMian No. 5, XinCaiMian No. 6, XinCaiMian No. 7, XinCaiMian No. 8, XinCaiMian No. 10, and XinCaiMian No. 11 were selected. Identification was performed using primers No. 12, No. 13, No. 14, No. 15, No. 16, No. 17, No. 18, No. 19, No. 20, No. 21, No. 22, No. 23, No. 24, No. 25, No. 26, No. 27, and No. 28. Using seven pairs of primers with high polymorphism, it was also possible to distinguish some materials using any single primer.

[0051] Fingerprint profiles of Xinjiang island cotton varieties, as shown below Figure 1 As shown, the fingerprint spectrum of Xinjiang colored cotton varieties is as follows: Figure 2 As shown. By Figure 1 , 2 It can be seen that, based on the molecular weight in the amplification spectrum, the absence of other molecular weight bands under the primer-specific fragment is defined as 0; the presence of a special molecular weight band is defined as 1. By sequentially encoding with 7 pairs of highly efficient polymorphic primers, the specific coding sequence type of each variety was obtained, which can be used to distinguish variety differences and was used to construct the fingerprint spectrum of Xinjiang cotton.

[0052] Example 3

[0053] Important data on cotton varieties were collected and identified, including basic information about the varieties, key phenotypic traits, and specific expression maps. Basic information includes: variety illustration, variety name, approval number, approval date, approval authority, pedigree, maturity characteristics, growth period, flowering rate, disease resistance, and suitable planting area.

[0054] Important phenotypic traits include: plant height, single boll weight, number of fruit branches, lint percentage, and fiber strength.

[0055] The specific expression map was obtained by fluorescent PCR amplification using primer pairs with SSR molecular markers, and plotted with the fluorescence expression level as the ordinate and the size of the amplified product as the abscissa.

[0056] Collect basic information and important phenotypic traits of Xinluzhong 55: Basic information includes: illustration; variety name: Xinluzhong 55; approval number: Xin Shenmian 2012 No. 53; approval date: 2012; approval unit: Xinjiang Dafengshou Seed Industry; pedigree: hybrid of Zhongmian 35 × 0582. This variety is an early-maturing conventional upland cotton with a growth period of 121 days. It is resistant to wilt and Verticillium wilt; suitable for planting in the early-to-mid-maturing cotton areas of southern Xinjiang.

[0057] Key phenotypic traits include: pyramidal plant shape, vigorous and compact growth, type II fruiting branches, sturdy and hairy stems, and a plant height of approximately 66.5 cm. The bolls are oval, medium-sized, and exhibit strong boll-setting ability; the leaves are dark green and medium to small in size. The average boll weight is 5.1 grams, with a lint percentage of 42.1%, indicating excellent fiber quality and concentrated lint opening. In trials conducted from 2008 to 2011, the yield of seed cotton was 350-400 kg per mu (approximately 0.067 hectares), with a maximum yield of 430 kg per mu.

[0058] Fluorescent PCR amplification was performed using the specific primer XP71 (No. 55) from Xinluzhong, and a specific expression map was constructed. The PCR amplification program included: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 20 s, for 28 cycles. A final extension at 72℃ for 5 min was performed.

[0059] The basic information, important phenotypic traits, and specific expression maps of Xinluzhong 55 were input into the advanced editor via the Internet QR code generator platform (https: / / www.hlcode.cn / ), along with the corresponding parameters of the material, and integrated into a digital QR code. The QR code for Xinluzhong 55 and its scanned image information are shown below. Figure 3 As shown, the text information is the same as above.

[0060] By using modern technology to scan corresponding QR codes and obtain relevant information, a new approach to targeted identification of Xinjiang cotton varieties is provided. Under the Internet technology, cotton varieties can be identified on a large scale, which not only saves costs and time, but also significantly improves the informatization and automation level of variety identification.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polymorphic genome SSR molecular marker combination for identifying cotton varieties, characterized in that, The SSR molecular marker combination is NBRI_HQ526730, NBRI_HQ527820, HAU3071a, NAU3736b, NAU3913, NAU5172 and MON_DPL0893.

2. The polymorphic SSR molecular marker combination for identifying cotton varieties as described in claim 1, characterized in that, The primer set XP69 sequence of NBRI_HQ526730 is shown in SEQ ID NO: 137~138; The primer set XP71 sequence of NBRI_HQ527820 is shown in SEQ ID NO: 141~142; The primer set XP21 sequence of HAU3071a is shown in SEQ ID NO: 41~42; The primer set XP57 sequence of the NAU3736b is shown in SEQ ID NO: 113~114; The primer set XP60 sequence of NAU3913 is shown in SEQ ID NO: 119~120; The primer set XP63 sequence of NAU5172 is shown in SEQ ID NO: 125~126; The primer set XP42 sequence of MON_DPL0893 is shown in SEQ ID NO: 83~84.

3. A polymorphic genomic SSR molecular marker combination for identifying cotton varieties as described in claim 1 or 2, characterized in that, The cotton varieties identified include Xinjiang upland cotton, Xinjiang island cotton, and Xinjiang colored cotton.

4. A method for constructing fingerprint profiles of Xinjiang cotton varieties using the SSR molecular marker combinations described in any one of claims 1 to 3, characterized in that, Using DNA from Xinjiang upland cotton, Xinjiang island cotton, and Xinjiang colored cotton as templates, PCR amplification was performed using primers of the aforementioned SSR molecular marker combination. The amplification products were then subjected to electrophoresis, and the electrophoresis data were analyzed to obtain molecular fingerprints.

5. A molecular fingerprint of a cotton variety constructed by the method of claim 4.

6. A method for constructing a QR code identification system for Xinjiang cotton varieties based on phenotypic traits and SSR molecular marker technology, characterized in that, Includes the following steps: (1) Extract DNA from cotton and amplify it by PCR using primers with the SSR molecular marker to obtain a specific expression graph with fluorescence expression level as the ordinate and amplification product size as the abscissa. (2) Integrate basic information of cotton, important phenotypic traits and specific expression maps constructed from SSR molecular markers, and synthesize digital QR codes for the corresponding varieties by entering parameters of important phenotypic traits and specific expression maps of cotton into the advanced editor through the interconnected QR code generator platform.

7. The construction method as described in claim 6, characterized in that, The SSR molecular marker is one of the SSR molecular marker combinations described in any one of claims 1 to 3; the basic information includes: variety image, variety name, approval number, approval time, approval unit, pedigree, maturity characteristics, growth period, flowering rate, disease resistance and suitable planting area; the important phenotypic traits include: plant height, single boll weight, number of fruit branches, lint percentage and fiber strength.

8. A QR code recognition system for Xinjiang cotton varieties constructed using the construction method described in claim 6 or 7.

9. The application of the molecular fingerprint spectrum of claim 5 or the Xinjiang cotton variety QR code identification system of claim 8 in the identification and recognition of Xinjiang cotton varieties.

10. The application as described in claim 9, characterized in that, Using DNA extracted from the Xinjiang cotton variety to be tested as a template, PCR amplification is performed using primers of the SSR molecular marker combination. The amplification products are then subjected to capillary electrophoresis, and the electrophoresis data is analyzed to obtain a molecular fingerprint. This fingerprint is then compared with the molecular fingerprint of a known Xinjiang cotton variety constructed according to claim 5 to identify the variety of the Xinjiang cotton to be tested. Alternatively, using DNA from the Xinjiang cotton variety to be tested as a template, PCR amplification is performed using primers of any marker in the SSR molecular marker combination to obtain the specific expression map. This map is then combined with the phenotypic traits of the Xinjiang cotton variety to be tested and compared with the constructed Xinjiang cotton variety QR code identification system to identify the variety of the Xinjiang cotton to be tested.

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