A set of ssr molecular marker primers for identifying 'ruixue' apple and application thereof

Abstract

The application belongs to the technical field of crop variety identification, and particularly relates to a primer set of SSR molecular markers for identifying 'Ruixue' apples and application. The primer set is composed of the following four pairs of primers: Hi01c11, CH02d10b, CH03a03 and Hi01e10. The nucleotide sequences of CH02d10b are shown in SEQ ID NO. 7 and SEQ ID NO. 8. The nucleotide sequences of CH03a03 are shown in SEQ ID NO. 9 and SEQ ID NO. 10. The nucleotide sequences of Hi01c11 are shown in SEQ ID NO. 49 and SEQ ID NO. 50. The nucleotide sequences of Hi01e10 are shown in SEQ ID NO. 51 and SEQ ID NO. 52. The primer set can accurately distinguish 'Ruixue' apples from other similar apple varieties.

Description

Technical Field

[0001] This invention belongs to the field of crop variety identification technology, specifically relating to an SSR molecular marker primer set for identifying 'Ruixue' apple and its application. Background Technology

[0002] Apples are one of the most widely planted and economically valuable deciduous fruit trees in my country. In recent years, domestically bred apple varieties such as 'Ruixue' have demonstrated excellent quality and strong adaptability, becoming core varieties promoted for optimizing and upgrading my country's apple industry structure, with their market promotion area expanding year by year. However, current domestic and international research on apple molecular markers mainly focuses on traditional introduced varieties such as Fuji, Gala, and Golden Delicious. Research on the molecular basis of domestically bred varieties such as Ruixue is relatively weak, lacking systematic SSR characteristic markers, fingerprint spectra, and standardized molecular identification.

[0003] Currently, the market circulation of new varieties is rife with problems such as mixed seedlings, misidentification, counterfeiting, and infringement. Due to the lack of specific molecular identification criteria, it is difficult to confirm the rights to new varieties and trace their origins, making it difficult to effectively protect the intellectual property rights of breeders. Traditional morphological identification methods are easily affected by climate, cultivation management, and tree development, resulting in strong subjectivity and low accuracy, failing to meet the industry requirements for batch identification of new varieties, germplasm preservation, variety rights registration, and commercial traceability.

[0004] SSR molecular markers possess advantages such as codominant inheritance, high stability, rich polymorphism, and good reproducibility, making them the mainstream technology for constructing germplasm fingerprints and developing molecular identity cards for fruit trees. Existing publicly available molecular identity cards are mostly constructed for traditional apple varieties or other fruit tree germplasm, without specific marker optimization and coding for the domestic new variety 'Ruixue'. This results in poor variety adaptability and insufficient specificity, making them unsuitable for direct and accurate identification of new varieties. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an SSR molecular marker primer set for identifying 'Ruixue' apples and its application.

[0006] A primer set for constructing 'Ruixue' apples using SSR molecular markers, the primer set consisting of the following 4 pairs of primers: Hi01c11, CH02d10b, CH03a03, and Hi01e10; The nucleotide sequence of CH02d10b is shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequence of CH03a03 is shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequence of Hi01c11 is shown in SEQ ID NO.49 and SEQ ID NO.50; The nucleotide sequence of Hi01e10 is shown in SEQ ID NO.51 and SEQ ID NO.52.

[0007] A kit for identifying 'Ruisue' apples, the kit comprising the primer set.

[0008] Preferably, the kit also contains reagents for extracting sample DNA and PCR amplification reagents.

[0009] Application of the primer set or the kit in the identification of 'Ruixue' apples.

[0010] Application of the primer set or the kit in constructing the molecular identity card of 'Ruixue' apple.

[0011] A method for constructing a molecular identity card for a 'snowy' apple includes the following steps: DNA was extracted from the sample to be tested, and PCR amplification was performed sequentially using the primer set described above. The PCR products were sequenced and the allele information at different loci was analyzed and statistically analyzed. The alleles amplified by each primer pair were arranged in ascending order of fragment size and numbered sequentially. Each number was concatenated to establish a unified coding rule. After being assigned a value, the numbers constituted the molecular identity card of the 'Ruixue' apple.

[0012] Preferably, the PCR amplification system contains 100 ng / μL of the reaction mixture per 20 μL. -1 2 μL of genomic DNA, 0.5 μL each of 10 μM forward and reverse primers, and 5 U / μL of... -1 0.1 μL Taq DNA polymerase, 2 μL 10-fold buffer, 1.6 μL 25 mM MgCl2, and 1.2 μL 2.5 mM dNTPs.

[0013] A method for identifying 'Ruixue' apples involves obtaining a molecular ID card for the 'Ruixue' apple based on the method for constructing a molecular ID card for the 'Ruixue' apple, comparing the molecular ID card of the 'Ruixue' apple with the molecular ID card of the apple to be identified, and if the two are completely identical, then the apple to be identified is 'Ruixue'; if they are inconsistent, then the apple to be identified is not 'Ruixue'.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) It provides a unique molecular identity system for 'Ruixue', clarifies the characteristic genetic loci of the new variety, and provides authoritative molecular basis for the germplasm identification of the new variety.

[0015] (2) The new varieties are highly identifiable and specific: By screening the main characteristic bands, the unique genetic bands of the new varieties are accurately preserved and the non-specific interference bands are weakened. The constructed molecular identity card is highly unique and distinguishable, and can accurately distinguish Ruixue and other similar apple varieties.

[0016] (3) Standardized coding and easy germplasm archiving: The natural number assignment method is adopted, the format is uniform, concise and clear, and can be entered into the germplasm resource database for a long time, so as to establish a standardized genetic archive for apple varieties independently bred in my country.

[0017] (4) Adapting to the supervision of new variety industrialization: The generated barcode can be directly used for the identification of new variety seedlings, traceability of fruits, and publicity of property rights, effectively curbing the circulation of counterfeit seedlings and protecting the intellectual property rights of breeders.

[0018] (5) Strong representativeness of genetic information: The main effect bands obtained by screening are highly stable and can truly reflect the genetic characteristics of the new Ruixue variety, avoiding identification errors caused by interference from mixed bands. The identification results are reliable and highly repeatable. Attached Figure Description

[0019] Figure 1 The results are for screening 25 pairs of primers from 30 pairs of SSR primers that stably amplify clear bands.

[0020] Figure 2 This is the barcode for the "Ruixue" molecule. Detailed Implementation

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0022] The defects of existing technology and their causes are as follows: (1) The existing marker system does not cover domestic new varieties, and the genetic information of new varieties is missing. Causes: Existing apple molecular marker systems are mostly constructed from imported, established varieties, bred in earlier periods, and the experimental materials do not include new nationally approved varieties such as Ruiyang, Ruixue, and Ruixianghong. Defects: New varieties lack specific molecular markers and standardized fingerprint profiles, making market identification difficult and counterfeit seedlings hard to distinguish quickly.

[0023] (2) Without graded screening of the bands, the characteristic bands of new varieties are easily obscured by impurities. Cause: Current technology does not distinguish between major stable bands and minor non-specific bands, and all bands are entered and coded uniformly. Defects: The specific and stable genetic bands of new varieties are interfered with by weak heterogeneous bands and conserved common bands, failing to highlight the unique genetic characteristics of the new variety, resulting in poor specificity and weak stability of the identification bands.

[0024] (3) The coding format is not uniform, which makes it difficult to establish a new variety database. Causes: Traditional coding generally uses two-digit codes with leading zeros, leading to inconsistent assignment rules across different studies. Drawbacks: The non-standard coding format makes it impossible to establish a unified, long-term archiveable database of new variety germplasm, hindering the systematic preservation of domestic new variety resources.

[0025] (4) Primer combinations were not optimized for new varieties, resulting in poor compatibility. Causes: Existing universal primer combinations for screening do not use new varieties as the core experimental material. Drawbacks: Primer pairs for Ruiyang, Ruixue, and Daphne exhibit unstable amplification effects, low polymorphism, and difficulty in generating highly identifiable molecular identifiers.

[0026] This invention presents a method for constructing molecular identity cards for apple varieties based on SSR molecular markers. Using the nationally approved new apple variety Ruixue as the core research object and other major cultivated apple varieties as auxiliary controls, a series of standardized experimental procedures are employed to construct a unique molecular identity card for Ruixue, filling a technological gap related to the precise identification and intellectual property protection of this variety. Different apple varieties exhibit variations in the length of their genomic microsatellite sequences. Using specific primers, DNA fragments of varying lengths are amplified via conventional PCR. Gel electrophoresis is then used to screen for stable and specific characteristic alleles for the Ruixue variety, while conserved sites with no identifying function are removed. The fragments are uniformly assigned natural numbers according to their length, spliced ​​together to form the unique molecular identity card for Ruixue, and then converted into a standard barcode. This method leverages genetic differences to achieve precise variety identification, seedling traceability, and protection of variety rights.

[0027] Overall technical process: Material preparation → Genomic DNA extraction and quality control → SSR primer screening for new varieties → PCR amplification → Electrophoresis pattern acquisition → Band correction and determination → Allele summarization and sorting → Unified natural number assignment → Screening of major characteristic bands → Unit coding combination → Complete molecular ID card assembly → Barcode generation → New variety identification and germplasm archiving.

[0028] Example 1 1. Genomic DNA extraction and quality testing Healthy young leaves of Ruixue and various control apple varieties were collected. Apple sample information is shown in Table 1. Samples were stored at low temperatures to prevent DNA degradation. All samples were collected from the Northwest A&F University Baishui Apple Experimental Station (35°2'N, 9°6'E) in Baishui County, Weinan City, Shaanxi Province. Genomic DNA was extracted using a modified CTAB method. DNA integrity was detected by agarose gel electrophoresis to ensure intact bands without degradation. Sample purity was determined using a spectrophotometer. Qualified samples with acceptable absorbance ratios were screened, uniformly prepared to appropriate experimental concentrations, and stored at low temperatures for later use.

[0029] Table 1: Information on the apple samples used 2. Conventional PCR amplification system and procedure Reagents: 2×PCR Mix, 10μmol / L SSR primers, Taq DNA polymerase, 10×PCR buffer, 25 mmol / L MgCl2, 2.5 mmol / L dNTPs, sterile deionized water.

[0030] This invention employs touchdown PCR for gene amplification throughout the entire process, using a standardized amplification reaction system containing DNA template, upstream and downstream primers, polymerase, buffer, substrate, and other reagents, with sterile water added to make up the system volume. A consistent 20 μL reaction system is used, containing 100 ng / μL of each primer. -1 2 μL of genomic DNA, 0.5 μL each of 10 μM forward and reverse primers, and 5 U / μL of... -10.1 μL Taq DNA polymerase, 2 μL 10-fold buffer, 1.6 μL 25 mM MgCl2, and 1.2 μL 2.5 mM dNTPs were added, with the remaining volume made up with sterile deionized water. The Touchdown PCR program was as follows: pre-denaturation at 95℃ for 5 min, followed by a falling-down cycle of denaturation at 94℃ for 20 s. The first stage was annealing starting at 68℃ for 6 cycles, with each cycle decreasing the annealing temperature by 2℃ for 20 seconds, followed by an extension at 72℃ for 30 seconds. Then, the second stage began with denaturation at 94℃ for 20 seconds, followed by annealing starting at 58℃, with each cycle decreasing the annealing temperature by 1℃ for 20 seconds, followed by an extension at 72℃ for 30 seconds, for a total of 8 cycles. After the two gradient cooling cycles are completed, the amplification phase at a fixed temperature begins. The parameters are: denaturation at 94℃ for 20 seconds, annealing at 50℃ for 20 seconds, and extension at 72℃ for 30 seconds, for a total of 20 cycles. Finally, a final extension at 72℃ for 5 minutes is performed, followed by long-term incubation at 16℃ for later use. Touchdown PCR utilizes a gradual decrease in annealing temperature. A higher annealing temperature is used initially to ensure amplification specificity and reduce non-specific bands, followed by a suitable fixed annealing temperature to continue amplification and ensure product efficiency and yield. This approach effectively balances specificity and amplification efficiency.

[0031] All reactions were performed on Eppendorf-authorized PCR instruments, and all amplification experiments were replicated to ensure stable and reliable results. Amplification products were stored at low temperatures. The overall procedure is simple, universal, and compatible with routine experimental operations.

[0032] 3. Stepwise screening of SSR primers Reagents: 0.5M EDTA (pH=8) formula: EDTA-Na2 18.61g, ddH2O 100ml.

[0033] 30% PAGE formulation: Bis-Acrylamide 10g, Acrylamide 290g, H2O to 1L, heat to dissolve and then filter.

[0034] 10×TBE formula: Tris 108g, Boric Acid 55g, 0.5M EDTA (pH=8) 40ml, H2O to 1L.

[0035] The 1xTBE formulation is: 10×TBE 100mL plus ddH2O to 1L 8% PAGE formula: 30% PAGE 300ml, 10xTBE ​​100ml, H2O 600ml.

[0036] 6×Loading Dye Formula: Bromophenol Blue 25mg, Xylene Cyanol FF 25mg, Glycerin 3ml, H2O to 1L.

[0037] 10% Ammonium Persulfate Formula: 10g ammonium persulfate, H2O to 100ml.

[0038] 0.13% AgNO3 formulation: AgNO3 1.3g, H2O to 1L.

[0039] Using Qin Fu No. 1 and Pink Lady apple DNA as templates, PCR was performed to amplify the 30 pairs of SSR primers shown in Table 2. The amplification products were screened by polyacrylamide gel electrophoresis and silver staining. The steps are as follows: (1) Experimental preparation and gel preparation: Prepare electrophoresis glass plates, clamps, combs, pipettes and other experimental equipment in advance, and take out the refrigerated 10% ammonium persulfate and TEMED reagent. Prepare 8% polyacrylamide gel mixture (8% PAGE), mix thoroughly and quickly inject into the gap between the aligned and clamped glass plates, remove air bubbles and insert the comb, and let stand at room temperature for 15-20 minutes until the gel is completely solidified.

[0040] (2) Sample preparation: Mix the PCR amplification products with 6× loading buffer (6× Loading Dye) in the specified ratio, and simultaneously prepare the marker. Centrifuge briefly before use. Assemble and fix the solidified gel in the electrophoresis tank, add 1× TBE buffer, gently remove the comb, and load the samples and markers in sequence. Set the electrophoresis to a constant voltage of 180V for 45 minutes. Stop the electrophoresis when the indicator migrates to the bottom of the gel, remove the gel, and peel and wash it.

[0041] (3) Silver staining and results: First, the gel was stained with silver nitrate solution by shaking. After staining, the staining solution was recovered, and the gel was quickly rinsed with deionized water. Then, a mixture of sodium hydroxide and formaldehyde was added and the gel was shaken to develop the color until the bands were clearly visible. The reaction was terminated by washing away the residual staining solution. The gel was then flattened, wrapped in plastic wrap to remove air bubbles, and placed in an imaging device to take pictures and record the results. Based on the clarity of the bands and the amplification stability, the SSR primer screening was completed.

[0042] A screening process was conducted from thirty pairs of candidate apple SSR primers. After preliminary amplification screening, twenty-five pairs of primers capable of stably amplifying clear bands were selected. For example... Figure 1As shown, based on the gel images obtained by electrophoresis after amplification with 30 pairs of primers, the leftmost band is the marker; 1 and 2 are the bands of 'Qinfu No. 1' and 'Pink Lady' after amplification with the first pair of primers; 3 and 4 are the bands of 'Fuji' and 'Pink Lady' after amplification with the second pair of primers; 5 and 6 are the bands of 'Fuji' and 'Pink Lady' after amplification with the third pair of primers, and so on, for a total of 60 bands, of which 25 pairs of primers can amplify clear and stable bands.

[0043] Based on the microsatellite selection criteria, a number of effective alleles greater than four loci is required for effective assessment and analysis of population genetic diversity (Takezak IN 1996). From 25 primer pairs, 12 pairs meeting the criteria were selected, and further screening yielded twelve pairs of highly polymorphic primers. The 12 primer pairs are named CH02d10b, CH03a03, CH03g06, CH04f10, CH05d08, CH05g07, CH05g08, CHO5d02, Hi01c11, Hi01e10, Hi03d06, and GD162.

[0044] Table 2: Complete primer sequences Table 2: Complete primer sequences (continued) 4. Fingerprint mapping and feature site selection Using the 12 primer pairs obtained from the screening, PCR amplification was performed on all the experimental varieties shown in Table 1. Amplified bands were obtained by polyacrylamide gel electrophoresis, and an SSR molecular fingerprint was constructed with *Ruixue* as the core. As shown in Table 3, the experiments yielded abundant allele loci, indicating good population genetic diversity, which can meet the needs of variety identification.

[0045] In the experiment, information was recorded based on the presence or absence of bands. Common loci for all varieties were eliminated, and specific characteristic loci that were stably expressed only in the Ruixue variety were selected. Only characteristic loci were used for subsequent coding, and ordinary observation bands were only retained as experimental data.

[0046] Table 3: Amplification results of 12 selected SSR primer pairs for 12 apple varieties This table presents 12 effective primer pairs selected from 30 total primer pairs, providing core amplification primers for the subsequent construction of apple variety fingerprint profiles.

[0047] 5. Standardized Assignment and Molecular Identity Card Construction Based on the characteristic bands corresponding to each selected SSR primer, fingerprint profiles of 12 apple varieties were constructed, as shown in Table 4-6. The selection process utilized the principle that differences in the characteristic bands amplified by the same primer could identify varieties. The selection strategy involved first finding the primer that could distinguish the most varieties, then grouping and progressively selecting primers for different combinations of varieties, ultimately obtaining a primer combination that could distinguish all apple varieties. Through screening 12 primer pairs, four primer combinations were obtained: CH02d10b, CH03a03, Hi01c11, and Hi01e10.

[0048] Analysis of the table shows that Hi01c11 is the first key primer. After amplification with this primer, the characteristic bands of Qin Fu No. 1, Qin Feng, Qin Yue, Gala, Hua Hong, Jazz, and Wang Lin are 170bp and 180bp, respectively. The characteristic bands of Rui Xue and Pink Lady are 180bp, and Qin Guan is 350bp. Therefore, Qin Guan can be directly distinguished during screening. The remaining 11 varieties are divided into two groups: Rui Xue and Pink Lady are classified into the first group, and Qin Fu No. 1, Qin Feng, Qin Yue, Gala, Hua Hong, Jazz, and Wang Lin are classified into the second group.

[0049] CH02d10b is the second key primer. Its characteristic band is 170bp in Ruixue, Qinguan, and Huahong, and 140bp and 170bp in Pink Lady, Qinfeng, Gala, and Wanglin. The characteristic bands of Qinfu No. 1, Qinyue, and Jazz are all unique, thus directly distinguishing the first group containing Ruixue and Pink Lady. In the second group, Qinfu No. 1, Jazz, and Qinyue can be directly distinguished from other varieties due to their specific bands. The characteristic bands of the remaining varieties and Huahong are 170bp, while Qinfeng, Gala, and Wanglin are 140bp and 170bp. Based on the band characteristics, they are further divided into smaller combinations to narrow the identification range.

[0050] Then, primer CH03a03 was used for identification. Ruixue, Gala, and Jazz all amplified 220bp and 250bp characteristic bands under the action of this primer, while most other varieties only amplified a single 250bp characteristic band. Therefore, Gala containing the 220bp characteristic band can be effectively distinguished from Jazz and Wanglin, and previously difficult-to-distinguish similar varieties can be reclassified and verified.

[0051] Finally, primer Hi01e10 was used for identification. Ruixue and Qinguan showed characteristic bands of 150 and 60 bp under this primer amplification, distinct from the 140, 150, and 160 bp bands of Qinfu No. 1, Pink Lady, Qinfeng, and Qinyue, and the 140 and 150 bp characteristic bands of Gala, Huahong, and Jazz. Thus, the 10 apple varieties could be completely distinguished.

[0052] Table 4: Fingerprint patterns of apple varieties The amplified alleles from each primer pair were arranged in ascending order of fragment size and numbered sequentially. Each number was concatenated to establish a unified coding rule, with all varieties following the same assignment standard, thus obtaining a molecular identity card composed of numbers (Table 6).

[0053] Table 5: Assignment Table Note: 1-9 in the table are assigned values, and the corresponding data are the site bp values.

[0054] Based on the primer sequence of CH02d10b, CH03a03, Hi01c11, and Hi01e104, representative characteristic sites of Ruixue were selected to complete the unit coding combination, and continuous splicing was used to form a unique digital molecular ID card for Ruixue. The codes of other control varieties were only used for comparison.

[0055] Table 6: Variety ID Cards for Apple Varieties 6. Barcode Visualization Applications The molecular identification digital code of the Ruixue variety is converted into a universal one-dimensional barcode. The barcode information corresponds one-to-one with the genetic characteristics of the variety and can be printed on seedling labels, germplasm archives, and fruit packaging to realize full-process traceability and rapid authenticity identification of the Ruixue variety.

[0056] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0057] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0058] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An SSR molecular marker primer set for identifying 'Ruixue' apple, characterized in that, The primer set consists of the following four pairs of primers: Hi01c11, CH02d10b, CH03a03, and Hi01e10. The nucleotide sequence of CH02d10b is shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequence of CH03a03 is shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequence of Hi01c11 is shown in SEQ ID NO.49 and SEQ ID NO.50; The nucleotide sequence of Hi01e10 is shown in SEQ ID NO.51 and SEQ ID NO.

52.

2. A reagent kit for identifying 'Ruixue' apples, characterized in that, The kit comprises the primer set of claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also contains reagents for extracting DNA from samples and PCR amplification reagents.

4. The use of the primer set of claim 1 or the kit of any one of claims 2 to 3 in the identification of 'Ruixue' apples.

5. The use of the primer set of claim 1 or the kit of any one of claims 2 to 3 in constructing the molecular identity card of 'Ruisue' apple.

6. A method for constructing a molecular identity card for a 'snowy' apple, characterized in that, Includes the following steps: DNA was extracted from the sample to be tested and PCR amplification was performed sequentially using the primer set described in claim 1. The PCR products were sequenced and the allele information at different loci was analyzed and statistically analyzed. The alleles amplified by each primer pair were arranged in ascending order of fragment size and numbered sequentially. Each number was concatenated and assigned a value to obtain a number that constitutes the molecular identity card of the 'Ruixue' apple.

7. The method according to claim 6, characterized in that, The PCR amplification system consisted of 100 ng / μL of the reaction mixture per 20 μL. -1 2 μL of genomic DNA, 0.5 μL each of 10 μM forward and reverse primers, and 5 U / μL of... -1 0.1 μL Taq DNA polymerase, 2 μL 10-fold buffer, 1.6 μL 25 mM MgCl2 and 1.2 μL 2.5 mM dNTPs.

8. A method for identifying 'Ruixue' apples, characterized in that, According to the method described in claim 7, the molecular identification of the 'Ruixue' apple is obtained. The molecular identification of the 'Ruixue' apple is compared with the molecular identification of the apple to be identified. If the two are completely consistent, the apple to be identified is 'Ruixue'. If they are inconsistent, the apple to be identified is not 'Ruixue'.

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