SSR (Simple Sequence Repeat) molecular marker combination, primer and fingerprint spectrum for identifying potato germplasm resources and application of SSR molecular marker combination
By constructing SSR molecular marker combinations and primers, and using PCR amplification and sequencing to construct fingerprint maps, the problems of time consumption and accuracy in potato germplasm resource identification were solved, achieving efficient and accurate variety differentiation and identification.
Patent Information
- Application Number
- CN202511435487.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-09
- Publication Date
- 2025-12-12
AI Technical Summary
Existing technologies for identifying potato germplasm resources are time-consuming and susceptible to environmental factors, resulting in unclear synonyms and kinship, and low utilization rates.
Using SSR molecular marker technology, a molecular marker combination containing STSSRC1~STSSRC12 and its primers were constructed. A fingerprint pattern was constructed by PCR amplification and sequencing to achieve accurate identification of potato varieties.
It enables efficient and accurate differentiation and identification of potato varieties, shortens the identification process, and improves the identification accuracy rate to 100%, providing technical support for variety DNA molecular genotyping and genetic diversity analysis.
Smart Images
Figure CN121109639A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a combination of SSR molecular markers, primers, a fingerprint and application thereof for identifying potato germplasm resources. BACKGROUND
[0002] Potato (Solanum tuberosum L.) is rich in various nutrients, which can provide rich carbohydrates, vitamins and minerals, etc., and is of great significance to human health and nutritional balance. Compared with food crops such as corn and rice, potato can have high yield in a relatively short time and is not easily affected by adverse effects such as speculation. Solanum tuberosum With the challenges of population growth, extreme natural environment and changes in people's consumption concept, potato breeders are required to continuously breed new varieties. The lag in the preservation and utilization of potato germplasm resources, the problems of same name and unclear genetic relationship between materials, etc. make the utilization rate of germplasm resources low. The identification and evaluation of potato germplasm resources is the basis of breeding research work, and understanding the genetic background and genetic relationship of germplasm resources is an effective way to efficiently utilize potato germplasm resources. The identification method of potato mainly adopts traditional morphological identification (such as plant morphology, flower color, leaf color, disease resistance, and tuber type), however, morphological identification is time-consuming and easily affected by factors such as plant growth and development state, in addition, environmental factors can also lead to identification errors. Therefore, it is crucial to develop a rapid and reliable method to evaluate the genetic diversity between potato varieties.
[0003] With the development of modern biotechnology, molecular marker technology has been widely used in genetic analysis and breeding research of crops. The simple sequence repeat (SSR) molecular marker technology based on PCR is a tandem repeat sequence composed of 1-6 bases as a repeating unit, which has the characteristics of comprehensive plant gene coverage, co-dominance, stable frequency band, high polymorphism, and low price. This technology is widely used in potato hybrid identification, genetic difference analysis, marker development, etc.
[0004] At present, there are also studies on potato using molecular marker technology, but there are few studies on the development of SSR molecular markers for potato varieties and the construction of technical systems using SSR molecular markers.
[0005] SUMMARY
[0006] The purpose of this invention is to provide SSR molecular marker combinations, primers, fingerprint patterns, and their applications for the identification of potato germplasm resources, thereby addressing the problems existing in the prior art. The molecular marker combinations and primer combinations of this invention for identifying potato variety resources can accurately distinguish and identify multiple potato varieties, greatly shortening the identification process and providing technical support for DNA molecular genotyping of potato varieties and analysis of potato genetic diversity.
[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is an SSR molecular marker combination for constructing potato fingerprinting, wherein the molecular marker combination includes STSSRC1~STSSRC12, and its nucleotide sequence is shown in SEQ ID NO.25~36.
[0008] The second technical solution of the present invention is a primer for detecting the SSR molecular marker combination, wherein the primer includes sequences as shown in SEQ ID NO.1~24.
[0009] The third technical solution of the present invention is a kit for detecting the SSR molecular marker combination, wherein the kit contains the primers.
[0010] The fourth technical solution of the present invention is a chip for detecting the SSR molecular marker combination, the chip comprising the primers.
[0011] The fifth technical solution of the present invention is a method for constructing a potato fingerprint map, comprising the following steps: 1) Collect tissue samples; 2) Extract total DNA from the samples; 3) Perform PCR amplification using the primers described above, and sequence the amplification products; 4) Construct a potato fingerprint map based on the sequencing results.
[0012] The sixth technical solution of this invention is a method for identifying potato varieties, comprising the following steps: 1) Collect tissue samples of the sample to be identified; 2) Extract total DNA from the sample to be identified; 3) Identify the genotype of the SSR molecular marker combination described in the genome of the sample to be identified; 4) Compare the individual genotype of the sample to be identified with the constructed fingerprint map; 5) Determine the variety of the sample to be identified based on the comparison results of step 4).
[0013] The seventh technical solution of the present invention is the application of the SSR molecular marker combination, the primers, the reagent kit, and / or the chip in the identification of potato varieties.
[0014] Based on the above technical solution, the present invention has the following technical effects: This invention discloses molecular marker combinations and primer combinations for identifying potato varietal resources. Using only 11 pairs of specific primers, different molecular markers can be amplified, enabling effective differentiation and identification of potato varieties with an accuracy rate of up to 100%. The molecular marker combinations of this invention have great potential for identifying a large number of potato germplasm resources. Compared with existing conventional morphological identification methods, they offer advantages such as better results and savings in time and cost. This invention can provide technical support for DNA molecular genotyping of potato varieties and analysis of potato genetic diversity. Attached Figure Description
[0015] Figure 1 Electrophoresis images of SSR primers for different varieties. Note: M is Marker (DL2000), 1 is Xinyu No. 3, 2 is Desiree, 3 is Favorita, 4 is Wanshu No. 9, and 5 is Eugene.
[0016] Figure 2 To utilize the method of detecting and recording DNA fingerprint patterns, M stands for Marker, and 01-26 are electrophoretic band patterns of 26 potato materials. Detailed Implementation
[0017] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0018] This invention provides an SSR molecular marker combinatorial array for constructing potato fingerprinting, the molecular marker combinatorial array comprising STSSRC1~STSSRC12, the nucleotide sequences of which are shown in SEQ ID NO.25~36.
[0019] Embodiments of the present invention also provide primers for detecting the SSR molecular marker combination, the primers comprising sequences as shown in SEQ ID NO. 1~24.
[0020] This invention also provides a kit for detecting the SSR molecular marker combination, the kit comprising the primers described above.
[0021] This invention also provides a chip for detecting the SSR molecular marker combination, the chip comprising the primers described above.
[0022] This invention also provides a method for constructing a potato fingerprint map, comprising the following steps: 1) Collect tissue samples; 2) Extract total DNA from the samples; 3) Perform PCR amplification using the primers described above, and sequence the amplification products; 4) Construct a potato fingerprint map based on the sequencing results.
[0023] In some specific implementations, the PCR amplification in step 3 is a multiplex PCR amplification.
[0024] In some specific implementations, the PCR amplification reaction system comprises: 1 μL DNA, 0.5 μL of 10 pmol / L upstream primer, 0.5 μL of 10 pmol / L downstream primer, 2 μL of 10×PCR buffer, and 25 mmol / L MgSO4. 2+ 1 μL of 10 mmol / L dNTP, 0.5 μL of 5 U / μL Taq enzyme, and 13.5 μL of ddH2O; the PCR buffer is Mg-free. 2+ ; The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 33 cycles; 72℃ extension for 8 min, and storage at 4℃.
[0025] In some specific implementation schemes, the method for sequencing the amplified products is as follows: The amplification products were detected by 3% agarose gel electrophoresis. The electrophoresis results were recorded according to the standard DNA molecular weight and the migration rate of the primers. The band with the highest migration rate for each primer pair was recorded as 1, the band with the second highest migration rate was recorded as 2, and no band was recorded as 0. The fingerprint code obtained by encoding according to the primer sequence is the characteristic fingerprint map of the variety. The feature fingerprint spectrum includes the following SSR fingerprint spectrum:
[0027] This invention also provides a method for identifying potato varieties, comprising the following steps: 1) Collect tissue samples of the sample to be identified; 2) Extract total DNA from the sample to be identified; 3) Identify the genotype of the SSR molecular marker combination described in the genome of the sample to be identified; 4) Compare the individual genotype of the sample to be identified with the constructed fingerprint map; 5) Determine the variety of the sample to be identified based on the comparison results of step 4).
[0028] This invention also provides the application of the SSR molecular marker combination, the primers, the kit, and / or the chip in the identification of potato varieties.
[0029] In some specific implementation schemes, the potato variety resources include Kexin No. 1, Xinyu No. 3, Atlantic, Desiree, Rustet Burbank, Favorita, Gabriela, Shaza No. 15, Sangre, Wanshu No. 9, Yukon Gold, Ningshu No. 1, Gaoyuan No. 5, Magnum, Shepody, Lionheart, Markie, Eugene, Dongnong 310, Innovator, Dokota, Alvery, Daisy, H18-24012, Jinkengbai, and Jinshu No. 5.
[0030] The molecular marker combination and primer combination of the present invention for identifying potato varietal resources can accurately distinguish and identify multiple potato varieties, greatly shorten the identification process of potato varietal resources, and provide technical support for the DNA molecular genotyping of potato varieties and the analysis of potato genetic diversity.
[0031] Example 1 1. Experimental materials Using 26 potato varieties as sample materials, fingerprint data of potato varieties were constructed based on their developed SSR molecular markers to achieve variety identification. Variety information for the 26 potato materials is shown in Table 1. In the following examples, the variety names and resource bank numbers of the potato materials were obtained from the Potato Germplasm Resource Bank of Keshan Branch of Heilongjiang Academy of Agricultural Sciences and the resource numbers preserved by the Potato Research Institute of Heilongjiang Bayi Agricultural Reclamation University.
[0032] Table 1 Information on 26 potato varieties
[0033] 2. Construction of standard DNA fingerprint libraries for different potato varieties 2.1 Total DNA extraction from various potato varieties Virus-free potato plantlets, after reaching 8-10 cm in height, had their leaves collected, frozen in liquid nitrogen, and then stored at -80°C for later use. 0.2 g of young leaves from each potato variety were placed in a 2.0 mL centrifuge tube, added to liquid nitrogen, and ground into powder. DNA was extracted using the Coolaber Plant Genomic DNA Rapid Extraction Kit, following the kit's instructions. The total DNA quality was assessed using agarose gel electrophoresis and spectrophotometry. The total DNA was then diluted to 100 ng / μL and stored at -20°C for later use.
[0034] 2.2 Primer screening a. SSR marker synthesis: The potato genome sequence (SolTub_3.0) was downloaded from the ensembl website. The amplification primers for the SSR molecular markers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The purity requirement for primer synthesis was PAGE purification, and the primers were aliquoted into 10D / tube. The concentrations of both upstream and downstream primers were diluted to 10 pmol / L.
[0035] b. Screening methods for SSR molecular marker primer combinations SSR screening primers that exhibit polymorphism, clear banding patterns, and stable reproducibility among different potato varieties were selected. The primers obtained through screening are shown in Table 2, and their corresponding molecular markers, primer sequences, and positional information are also presented.
[0036] Table 2. Molecular markers, sequences, and positional information corresponding to the primers.
[0037] The sequences of each molecular marker are shown in SEQ ID NO.25~36.
[0038] STSSRC1 (SEQ ID NO. 25): TGAAGAGGGACTGTCCAGCTTAGAAAAGGACACAAGATAACCTCTCAAACTATTCTAAACAGAGGAACAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAACAAGAAGGGACCTGGTCAACAGGGGAATAAGCAGAAGAATGGACCTGGTCATGCTCCTGGTCCTA; STSSRC2 (SEQ ID NO. 26): TCAGGGAGTGTTTTTCCGCTTCTTACCTTATTATTATTATTATTATTATTATTATTAAGTATTGGGGACAATGCTAATTTTCAGTTGGGGTTGCTTATGTGGTCA; STSSRC3 (SEQ ID NO. 27): TGAAGAGGATGAAGACGAGGAATACGAAGAAATTGAGGTTGAAGAGGAGATTGAAGTTGAAGAAGAAATAGAGGTAGAAGAAGAAGAGGAAGAAGAAATGGAGGTAGAAGAAGAGGTAAAGGAAGAGGGGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAAGAAGTAGGAGGTGAAGGATCA; STSSRC4(SEQ ID NO.28):TACAACTCCGTCAGCACCTGCACTCCCAGCACCAGCCCCAGCCGCAGAACCATCTGACCCCTCCGAAGAGGAAGAGGAAGAGGAAGAGGAAGAGGAGGAGGAAGAAGAAGAAGAAGAAGAAGAAGAGGAAGAGGAGCAACAATTGAAATCTCCTCCTCGTTCACAACC; STSSRC5(SEQ ID NO.29):GGCACTTTCAAAGTACAAACCGTAACAACTTTAAATATGATCTAAAATATCTTATTATTATTATTATTATTATTATTATTATTTTAAATCCTTATCAAGAGAAGGATAAAATAGTCATTTGAAAATGTTACAATTCATGATATTAAGTGTCAACCAAACTCTCTTAAATTCGTTAGGGGGA; STSSRC6(SEQ ID NO.30):ATTGTCACCAACCCCCTCACCCACTTCTTTGCCAAATGCGGTTTGTGCAGCATCCTCCTTGGTAGCATTAACTCCACCTCCACCTCCACCTCCACCTCCACCTCCACATGCACCCACTGCACCTAAGCTAGAAAGTCTTAGAATATCCACTCCACCA; STSSRC7(SEQ ID NO.31):GGAAGCTTCCTCTTGAACCCTAGCTTGAATCTCCTTCTTCTTCTCCTTCTCCTTCTCCTTCTCCTTCTCCTTCTCCTTCTCCTTCTTCTTCTTCTTCTTCTAAAGAGAGAATATTTGGAGAGGAGTTGGTTTTCATTTGAGAATTTGGAAAAATGAATGACATGCGGTGAGTTTGGGGGTTAA; STSSRC8 (SEQ ID NO.32): TGGGATTGGACTCCATGAACTAATGCACTTTGTTTGGATGGTTGTTACATATTGTTTCATAATGTATCATATTATATTGTGTTGTGTTGTGTTGTGTTGTGTTGTGTTGTATTGTATCAATACTGTGTTGTTTTAATGAATACAATGTTTGAATAGATTGTATCATTCTCCGTCGTTACAGAATGTCACA; STSSRC9 (SEQ ID NO.33): GAACCTCGTGTCAATTTGGCACAGCCAGGTATTACCAATAATAACAATGTTAATGTTAATGTTAATGTTAATGTTAATGTTACTGCACCTTCTACGAATAGTACTTGGATTATAGATAGTGGAGCATCTGATCACATGACC; STSSRC\(10\) (SEQ ID NO.34): TGGCGCTGTCATGTAGCATTTTTTCTTCTCCTATTATATATATAGAGAGAGAGAGAGAGAGAGATTTTTCTTATTATTATTATTATTATTATTATTATTATTATTTTTCTCTACATTTGTTTTCTTTAAATGAGGAGGTTGTGGAAAGATGG; STSSRC\(11\) (SEQ ID NO.35): TGGATGGTCGAAAAGCACCGGACCTCCACCTCCACCTCCACCTCCACCTCCAATCGTATCGTTGGTTGGATCATAAGCGGTTGTTGAGTATTCCGGCTCTCCGGTGGTGGTGTAGCCATGTGCTCCACCATGTCT; [[ID=~7]] ~ STSSRC\(12\) (SEQ ID NO.36): CCCTCACAATGGTTGCAACAATTGGAGGAATTTGACCTAGCTTGACCCTCTCATCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTAGAGAGAATTTTTTAGAGGAAGAACTTTCGGGATCTTTTGGGATTTTCAAATTAATGACTTAAATGGGGCGGTTAAAGGCT.
[0039] c. Screening of SSR marker amplification primer combinations Using 26 potato varieties as materials, PCR amplification of DNA from each potato variety was performed using synthesized SSR molecular marker amplification primers (as shown in Table 2). The PCR reaction system used in the experiment was a 20 μL system: 1 μL DNA template, 0.5 μL F-primer (10 pmol / L), 0.5 μL R-primer (10 pmol / L), and 10× PCR buffer (without Mg). 2+ 2μL, Mg 2+ 1 μL of (25 mmol / L), 1 μL of dNTP (10 mmol / L), 0.5 μL of Taq enzyme (5 U / μL), 13.5 μL of ddH2O, for a total of 20.0 μL.
[0040] The PCR reaction program was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 33 cycles; 72℃ extension for 8 min, and storage at 4℃.
[0041] d. Agarose gel electrophoresis and detection of electrophoresis results The amplification products were detected by 3% agarose gel electrophoresis. Electrophoresis was performed on a Sequi-Gen GT nucleic acid electrophoresis system (Bio-Rad, USA) for 30 minutes. After electrophoresis, the gel was photographed and recorded using a gel imaging (Bio-red) system. The electrophoresis results were recorded according to the standard DNA molecular weight and primer migration rate. For each primer pair among the 26 amplification products, the band with the highest migration rate was recorded as 1, the band with the second highest migration rate as 2, and so on, with no band recorded as 0. SSR genotype information data was established, and the fingerprint code was encoded according to the primer sequence. The obtained fingerprint code is the characteristic fingerprint map of this variety. The recording method is described in [link to documentation]. Figure 2 As can be seen, the electrophoretic band migration rates of the amplification products of the SSR molecular markers in the 26 potato materials differed. Based on the migration rates, the bands were numbered to obtain the codes for different potato materials under that molecular marker. This was then further analyzed according to... Figure 2 The numbering method shown allows for the sequential generation of fingerprints of potato materials under 12 molecular markers.
[0042] Recording method as follows Figure 2 The numbers are shown in the table. The DNA fingerprint profiles of each potato variety are shown in Table 3.
[0043] Table 3. Molecular marker fingerprint data of potato varieties
[0044] Table 3 shows that the DNA molecular marker fingerprints of any two varieties are different, which indicates that the 20 tested potato samples belong to different varieties, and the corresponding DNA molecular fingerprint codes are fingerprints unique to each variety.
[0045] The results showed that the 12 primer pairs can be used for plant genotype identification and can be applied to identify the genetic characteristics of potato varieties, thus protecting these variety rights from infringement.
[0046] This invention utilizes the aforementioned technical system and 12 pairs of potato SSR polymorphic primers to construct the DNA molecular genotypes of 26 potato germplasm resources, which can distinguish the 26 potato germplasm resources and will provide technical support for the identification of potato germplasm resources and the protection of variety rights.
[0047] Example 2 To verify the specificity of the developed SSR molecular marker, DNA from five varieties—Xinyu No. 3, Desiree, Favorita, Wanshu No. 9, and Youjin—was amplified by PCR using STSSRC12 primers. The pPCR reaction system was 20 μL: 1 μL DNA template, 0.5 μL F-primer (10 pmol / L), 0.5 μL R-primer (10 pmol / L), 2 μL 10×PCR buffer (without Mg2+), 1 μL Mg2+ (25 mmol / L), 1 μL dNTP (10 mmol / L), 0.5 μL Taq enzyme (5 U / μL), and 13.5 μL ddH2O, for a total of 20.0 μL.
[0048] PCR results showed that the bands amplified from the five varieties were specific, such as Figure 1 As shown.
[0049] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. An SSR molecular marker combinatorial system for constructing potato fingerprinting, characterized in that, The molecular marker combination comprises STSSRC1~STSSRC12, whose nucleotide sequences are shown in SEQ ID NO.25~36.
2. Primers for detecting the SSR molecular marker combination of claim 1, characterized in that, The primers include sequences as shown in SEQ ID NO.1~24.
3. A kit for detecting the SSR molecular marker combination of claim 1, characterized in that, The kit contains the primers as described in claim 2.
4. A chip for detecting the SSR molecular marker combination of claim 1, characterized in that, The chip contains the primers as described in claim 2.
5. A method for constructing a potato fingerprint spectrum, characterized in that, It includes the following steps: 1) Collect tissue samples; 2) Extract total DNA from the samples; 3) Perform PCR amplification using the primers described in claim 2, and sequence the amplification products; 4) Construct a potato fingerprint map based on the sequencing results.
6. The method as described in claim 5, characterized in that, The PCR amplification described in step 3 is a multiplex PCR amplification.
7. The method according to claim 5, characterized in that, The method for sequencing the amplified products is as follows: The amplification products were detected by 3% agarose gel electrophoresis. The electrophoresis results were recorded according to the standard DNA molecular weight and the migration rate of the primers. The band with the highest migration rate for each primer pair was recorded as 1, the band with the second highest migration rate was recorded as 2, and no band was recorded as 0. The fingerprint code obtained by encoding according to the primer sequence is the characteristic fingerprint map of the variety. The feature fingerprint spectrum includes the following SSR fingerprint spectrum: 。 8. A method for identifying potato varieties, characterized in that, It includes the following steps: 1) Collect tissue samples of the sample to be identified; 2) Extract total DNA from the sample to be identified; 3) Identify the genotype of the SSR molecular marker combination as described in claim 1 on the genome of the sample to be identified; 4) Compare the individual genotype of the sample to be identified with the fingerprint pattern constructed according to any one of claims 5-7; 5) Determine the variety of the sample to be identified based on the comparison results of step 4).
9. The application of the SSR molecular marker combination of claim 1, the primers of claim 2, the kit of claim 3, and / or the chip of claim 4 in the identification of potato varieties.