SNP (Single Nucleotide Polymorphism) molecular marker combination for identifying germplasm resources of red beans and application of SNP molecular marker combination
By developing a PCR detection system with 18 SNP sites and specific primer combinations, the problems of low efficiency and high cost in the identification of mung bean germplasm resources have been solved, and efficient and accurate germplasm resource identification and genetic analysis have been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-31
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies are insufficient for efficiently and accurately identifying soybean germplasm resources. Traditional morphological methods are greatly affected by the environment and have long cycles. Gene chips and resequencing are costly and inflexible. There is a lack of SNP marker sets suitable for different genetic backgrounds and lightweight detection systems.
Eighteen SNP sites with high polymorphism and significant resolution were developed, and primer combinations for specific detection were designed. Combined with optimized PCR detection conditions, a standardized molecular detection system was formed for the identification of soybean germplasm resources.
It enables clear and accurate identification of mung bean germplasm resources, is easy to operate and low in cost, and is suitable for rapid identification by breeding units and germplasm resource banks, providing abundant marker resources and technical support.
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Figure CN121852602A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and in particular to a combination of SNP molecular markers for the identification of soybean germplasm resources and its application. Background Technology
[0002] Red beans are an important coarse grain crop, highly nutritious and economically beneficial. Developing superior varieties is key to promoting the high-quality development of the red bean industry. Germplasm resources are a crucial material foundation for variety selection, genetic research, and agricultural production. As one of the origins of red beans, my country possesses a rich germplasm resource bank; however, the actual utilization rate of these resources in current breeding practices remains low. A major reason is that traditional identification methods based on morphological and agronomic traits are greatly affected by environmental factors and have long cycles, making it difficult to achieve accurate, efficient, and systematic evaluation of the genetic background of red bean germplasm.
[0003] As mung bean breeding efforts deepen, the number of new varieties and germplasm materials continues to increase, placing higher demands on high-throughput identification, precise identification, and innovative utilization of resources. Therefore, constructing an efficient, stable, and accurate germplasm resource identification technology system has become an urgent need to improve the efficiency of mung bean germplasm utilization and accelerate the process of variety innovation.
[0004] Molecular marker technology can directly reflect genetic variation at the DNA level and has become a reliable tool for germplasm resource identification and variety identification, especially suitable for distinguishing closely related materials. Among them, single nucleotide polymorphism (SNP) is a genetic polymorphism formed by a single nucleotide variation (such as substitution, insertion, or deletion) in the genome, and is a third-generation molecular marker. SNP markers have advantages such as large number, wide distribution, high genetic stability, and ease of automated detection, and have been widely used in crop genetic mapping, important gene localization, marker-assisted selection, and genome-wide association studies.
[0005] Currently, the large-scale development and application of SNP markers in plants mainly relies on two technology platforms: gene chips and high-throughput sequencing. While gene chip technology (such as Illumina and Affymetrix platforms) offers high throughput, its development and use are expensive, and it is highly dependent on specific equipment and reagents, lacking flexibility. Meanwhile, research on the soybean genome is relatively lagging, and commercially available chips are scarce, resulting in high customization costs and limiting its application in resource identification and breeding. Although whole-genome resequencing technology can comprehensively acquire genomic variation information, its detection efficiency is significantly affected by sequencing depth and coverage uniformity. The massive amounts of data generated by resequencing place high demands on storage, computing, and analysis capabilities, and the overall cost and time are still unsuitable for rapid, large-scale identification of germplasm in breeding practice. Neither of these two mainstream methods can simultaneously meet the multiple requirements of high accuracy, high flexibility, low cost, and ease of operation. The fundamental reason is that soybean lacks a systematically validated core SNP marker set applicable to different genetic backgrounds and breeding stages, and a corresponding lightweight, standardized detection system has not yet been established. Therefore, developing a set of SNP marker combinations with well-defined loci, high information content, and compatibility with conventional detection platforms, and establishing an efficient identification system suitable for soybean germplasm resources, has significant technical importance and application value. Summary of the Invention
[0006] The purpose of this invention is to provide a combination of SNP molecular markers for the identification of soybean germplasm resources and its application, thereby addressing the problems existing in the prior art. This invention has identified and screened 18 highly polymorphic and resolving SNP loci, and developed primer combinations for specific detection. Combined with optimized and standardized PCR detection conditions, a standardized molecular detection system has been formed. This system demonstrates excellent identification capabilities in soybean germplasm resource identification, clearly and accurately distinguishing soybean resources with different genetic backgrounds. It proves its high effectiveness and reliability in practical applications, providing abundant marker resources and reliable technical support for research such as soybean genetic map construction, important trait gene localization, fingerprint development, and marker-assisted selection.
[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a combination of SNP molecular markers for the identification of soybean germplasm resources, comprising 18 SNP molecular markers, the SNP site information corresponding to the SNP molecular markers being as follows: The genome version number of the SNP site is GCA_016808095.1.
[0008] The present invention also provides a primer combination for detecting SNP molecular marker combinations as described above, the primer combination comprising the following primers:
[0009] The present invention also provides a kit for detecting the above-mentioned SNP molecular marker combination, the kit containing the above-mentioned primer combination.
[0010] The present invention also provides a chip for detecting the above-mentioned SNP molecular marker combinations, the chip comprising the above-mentioned primer combinations.
[0011] This invention also provides a method for constructing a molecular marker fingerprint of soybean germplasm resources, comprising the following steps: (1) Extracting the genome of soybean germplasm resources; (2) The genome of the soybean germplasm resource was detected by PCR using the above primer combination to obtain the genotype of the site where the above SNP molecular marker combination is located; (3) Use the genotype to construct a molecular marker fingerprint map of soybean germplasm resources.
[0012] Furthermore, the PCR detection system consists of: 1 μL of genomic DNA, 1.4 μL of primer mixture, 5 μL of HiGeno 2xProbe Mix B, and 2.6 μL of sterile water; in the primer mixture, the final concentrations of primer 1-FAM, primer 2-HEX, and universal primer C are 12 μM, 12 μM, and 30 μM, respectively. The PCR detection procedure was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s; annealing and extension at 61~55℃ for 40 s, 10 cycles, with the temperature decreasing by 0.6℃ per cycle; 95℃ denaturation for 20 s, amplification at 55℃ for 40 s, 30~34 cycles, and storage at 4℃.
[0013] The present invention also provides the application of the above-mentioned SNP molecular marker combination, the above-mentioned primer combination, the above-mentioned kit, the above-mentioned chip, or the molecular marker fingerprint spectrum of soybean germplasm resources constructed as described above in the identification of soybean germplasm resources.
[0014] Furthermore, the soybean germplasm resources include TL01, Jilin 373, TL02, TL03, Jian 265, Extremely Drought Red Bean, Bei 6 JN003, Longken Early Red, TL05, and Bei 13. F015, QH1, QH4, QH7, LJ01, LJ02, LJ03, GN01, Longxiaodou No. 2, GN04, Jilin 362, XD11, LZX022, Baoqinghong 1, Dacha 1, Mianjin, Nong'anhong, Zaluan 2, Jihong 352, Zhenzhuhong, Baihong No. 3, Jihong 0001, Liaohong 7, Liaohong No. 1, Liaohong No. 3, Pinhong 2011-18, Baihong No. 8, Liaohong 08712, Baohong 200831-2, Tanghong 2010-23, Tanghong 2010-12, JHPX01, Yuhong No. 2, Zhuangyuanhong Jia Hong No. 1, Tianjin Red, Bai Hong No. 2, 012-8, 012-25, Red Bean, IIE100058, IIE100103, IIE100074, IIE100003, IIE100006, IIE100007, IIE100018, IIE100023, IIE100024, IIE100029, IIE100033, IIE100041, IIE100042, IIE100049, IIE100051, IIE100055.
[0015] This invention also provides a method for identifying mung bean germplasm resources, characterized by comprising the following steps: (1) Extract the genome of the soybean to be tested; (2) The genome was subjected to PCR detection using the above primer combination to obtain the genotype of the site where the above SNP molecular marker combination was located; (3) Compare the genotype with the molecular marker fingerprint map of soybean germplasm resources constructed by the above method to determine the variety of soybean to be tested.
[0016] Furthermore, the PCR detection system consists of: 1 μL of genomic DNA, 1.4 μL of primer mixture, 5 μL of HiGeno 2xProbe Mix B, and 2.6 μL of sterile water; in the primer mixture, the final concentrations of primer 1-FAM, primer 2-HEX, and universal primer C are 12 μM, 12 μM, and 30 μM, respectively. The PCR detection procedure was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s; annealing and extension at 61~55℃ for 40 s, 10 cycles, with the temperature decreasing by 0.6℃ per cycle; 95℃ denaturation for 20 s, amplification at 55℃ for 40 s, 30~34 cycles, and storage at 4℃.
[0017] The present invention discloses the following technical effects: This invention, through simplified genome sequencing of 180 soybean germplasm resources and comparative analysis with a reference genome, successfully identified and screened 18 highly polymorphic and significantly resolvable SNP loci, forming a core molecular marker combination for identification. Based on this molecular marker combination, this invention developed a primer combination for specific detection, coupled with optimized unified PCR detection conditions, forming a standardized molecular detection system. This system demonstrates excellent identification capabilities in soybean germplasm resource identification, clearly and accurately distinguishing soybean resources with different genetic backgrounds, proving its high effectiveness and reliability in practical applications. Compared to traditional morphological identification methods and molecular techniques relying on microarrays or resequencing, the molecular marker combination, primer combination, and identification method of this invention have significant advantages such as ease of operation, rapid detection, and low cost. It is suitable for testing applications in breeding units, germplasm resource banks, and seed quality testing institutions, and also provides abundant marker resources and reliable technical support for subsequent research such as soybean genetic map construction, important trait gene localization, fingerprint development, and molecular marker-assisted selection. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 This is a cluster analysis diagram of 65 soybean germplasm resources using 18 SNP sites from this invention. Detailed Implementation
[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0025] Example 1: Screening of molecular markers for mung bean germplasm resources 1. Extraction of genomic DNA from soybeans (1) Place the mung bean leaves in a mortar, add liquid nitrogen in three batches, grind thoroughly, and then add to a centrifuge tube.
[0026] (2) Add 600 μL of 2% CTAB preheated at 65℃, 15 μL of proteinase K and 10 μL of RNase, vortex to mix, and place in a 60℃ water bath for 30 min. Invert the container every 10 min to mix.
[0027] (3) After the water bath is finished, place the centrifuge tube on ice for 3 minutes, then add 500 μL of chloroform and vortex.
[0028] (4) 4℃, 12000rpm for 10min.
[0029] (5) Take the supernatant into a 1.5 mL centrifuge tube, add 500 μL of -20 °C pre-cooled isopropanol, mix by inverting (gently), and store at -20 °C for more than 2 hours.
[0030] (6) Centrifuge at 4℃ and 12000rpm for 15min.
[0031] (7) Pour off the supernatant, add 500 μL of 75% ethanol aqueous solution pre-cooled at -20℃, and blow and agitate several times.
[0032] (8) Centrifuge at 4℃ and 12000rpm for 5min.
[0033] (9) Pour off the supernatant, add 500 μL of 75% ethanol aqueous solution pre-cooled at -20℃, and blow and agitate several times.
[0034] (10) Centrifuge at 4℃ and 12000rpm for 5min.
[0035] (11) Pour off the supernatant and invert the centrifuge tube onto absorbent paper and air dry in a clean bench (40 min).
[0036] (12) Add an appropriate amount of 200 μL ddH2O to the centrifuge tube and store it at -20℃ for later use.
[0037] (13) The quality of the extracted soybean genomic DNA was detected by electrophoresis.
[0038] 2. Detection of variant sites in soybeans Following the steps outlined in "1. Extraction of Mung Bean Genomic DNA," genomic DNA was extracted from 180 broadly representative mung bean germplasm resources (provided by the National Engineering Research Center for Coarse Grains) and sequenced. The sequencing data were then compared with the mung bean reference genome (genome of mung bean variety Longxiaodou 4, genome version number GCA_016808095.1). Based on the alignment results of the sample sequencing data and the reference genome, SNP detection was performed using the Haplotypecaller module of GATK4 software. To reduce the error rate of SNP detection, a QD>=2.0 criterion was used for filtering, retaining only mutation sites that simultaneously met this condition. QD is the ratio of mutation quality to coverage depth, i.e., the mutation quality per unit depth; most false positive mutations have a QD value less than 2. Further filtering was performed using vcftools, as follows: (1) Reads support sites with a depth of not less than 4; (2) Delete sites with a minimum allele frequency (MAF) of less than 0.01; (3) Retain 80% of the individuals’ genotypes.
[0039] A total of 4360 high-quality SNP variants were identified across the entire genome. Based on these 4360 SNPs, equidistant sampling was used to screen 78 loci evenly distributed on the chromosome for specific primer design. Primers for genotyping were successfully designed for 55 of these loci. Genotyping of these 55 loci was then performed, and 18 clearly genotyped SNPs were selected as the core marker combination for identifying soybean germplasm resources. Information on the SNP molecular markers is shown in Table 1.
[0040] Table 1. 18 SNP molecular markers of soybean 3. Primer design for detecting variant sites in soybeans (1) Primer design was performed using DNAMAN software.
[0041] (2) Download 300bp sequences upstream and downstream of the variant site from the soybean reference genome.
[0042] (3) Copy the downstream sequence of the SNP site (excluding the SNP site bases), open DNAMAN software, input the sequence, and save the file.
[0043] (4) Generate a reverse complementary sequence.
[0044] (5) Add complementary bases to the SNP mutation sites at the end.
[0045] (6) Select the 25bp upstream sequence of the mutation site, input it into the software, input the SNP site base at the 3' end, and check whether the primer sequence meets the requirements. It needs to be without complementary sequence. Adjust the primer Tm value to about 60℃ by adding or removing the 5' end base.
[0046] (7) The reverse primer is located downstream of the complementary strand of the forward primer. Randomly select and copy a sequence approximately 25 bp upstream of the SNP site (this sequence can be adjacent to the SNP site, but cannot contain the SNP site bases), input it into the software, and design primers. No complementary sequence is required; adjust the primer Tm value to approximately 60℃ by adding or removing bases.
[0047] The primer sequences for the 18 SNP molecular markers are shown below: Vig8511446698 (product length 86bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTGGGATGCTTTTTCACGGCGTAG (SEQ ID NO. 1); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTGGGATGCTTTTTCACGGCGTAA (SEQ ID NO. 2); Universal primer C (5'-3'): ATCCAGGATCATCGGCACCT (SEQ ID NO.3); Vig8516159407 (product length 49bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTGAGGAATGGAGCTGGTTGTGG (SEQ ID NO. 4); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTGAGGAATGGAGCTGGTTGTGA (SEQ ID NO. 5); Universal primer C (5'-3'): TGTGTGATAACTACCTGATAACACGTG (SEQ ID NO.6); Vig8533821711 (product length 132bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTGACTGCAGCAGAAGAACGTGCT (SEQ ID NO.7); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTGACTGCAGCAGAAGAACGTGCC (SEQ ID NO. 8); Universal primer C (5'-3'): GGGTAGCCAAGCATGTGGAT (SEQ ID NO.9); Vig8559772262 (Product length 90bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTCAAGATTGCAGGAGGTTCTTCTG (SEQ ID NO. 10); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTCAAGATTGCAGGAGGTTCTTCTA (SEQ ID NO. 11); Universal primer C (5'-3'): CTCCCCCATCAACTCCAAGG (SEQ ID NO.12); Vig8611666615 (product length 104bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTACACCGGGTTCTCTGTCTTCG (SEQ IDNO.13); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTACACCGGGTTCTCTGTCTTCA (SEQ ID NO. 14); Universal primer C (5'-3'): GTGGTGGAGGCCTAATTCGT (SEQ ID NO.15); Vig8620555894 (product length 71bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTAGCGTAAACAAAGAAATAACCATATCAAT (SEQ ID NO. 16); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTAGCGTAAACAAAGAAATAACCATATCAAA (SEQ ID NO. 17); Universal primer C (5'-3'): CTGAGTAACTTTGGAACTACTAATCATTTTG (SEQ ID NO.18); Vig8631878983 (product length 138bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTAGGGGCAATGTATGTTGGCACGC (SEQ ID NO. 19); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTAGGGGCAATGTATGTTGGCACGT (SEQ ID NO. 20); Universal primer C (5'-3'): GCAGGGTTTGCCAGAGTGAT (SEQ ID NO.21); Vig8635619590 (product length 97bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTACCTGTTTCTAAAACCGGAAAG (SEQ ID NO. 22); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTACCTGTTTCTAAAACCGGAAAA (SEQ ID NO. 23); Universal primer C (5'-3'): AGCACAAGACAGGTCAGATGT (SEQ ID NO. 24); Vig8640126984 (product length 56bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTACGTGGGTATGCTCAGGCAAT (SEQ IDNO.25); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTACGTGGGTATGCTCAGGCAAA (SEQ ID NO. 26); Universal primer C (5'-3'): AGCATCTCCGGTCTTCAGATCAA (SEQ ID NO.27); Vig8729267157 (product length 97bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTACATGCCTAAAACCTTGACCAC (SEQ ID NO. 28); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTACATGCCTAAAACCTTGACCAT (SEQ ID NO. 29); Universal primer C (5'-3'): TGGTCCAACATGGTGAGCATT (SEQ ID NO.30); Vig8734464702 (product length 137bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTACTGGTGCAGAAACCAACACCC (SEQ ID NO. 31); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTACTGGTGCAGAAACCAACACCT (SEQ ID NO. 32); Universal primer C (5'-3'): AGGGCCCAGAAGGCATAGTA (SEQ ID NO.33); Vig8834161606 (product length 120bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTTGAATCAGCAAACAGCTGCAAA (SEQ ID NO. 34); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTTGAATCAGCAAACAGCTGCAAC (SEQ ID NO. 35); Universal primer C (5'-3'): AGCTCTTCATTCCATGACTGCT (SEQ ID NO.36); Vig8923862544 (product length 140bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTGTTGGGTCTGGAGTATCAGTCT (SEQ ID NO. 37); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTGTTGGGTCTGGAGTATCAGTCC (SEQ ID NO. 38); Universal primer C (5'-3'): TGGTCTCCTCAACTGCCATTC (SEQ ID NO.39); Vig8933746474 (product length 102bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTAAGATTCTTGAAGCCGTGTCAA (SEQ ID NO. 40); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTAAGATTCTTGAAGCCGTGTCAC (SEQ ID NO. 41); Universal primer C (5'-3'): GAGACCCACTCTGGAACGTC (SEQ ID NO.42); Vig9032696138 (product length 71bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTGAAGCCTAGGTGCCTGCAAGC (SEQ ID NO. 43); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTGAAGCCTAGGTGCCTGCAAGT (SEQ ID NO. 44); Universal primer C (5'-3'): ACAAGCACATTTCAATGGTTACA (SEQ ID NO.45); Vig914574631 (product length 82bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTCACAGCAGCTCAGGGCACATA (SEQ ID NO. 46); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTCACAGCAGCTCAGGGCACATG (SEQ ID NO. 47); Universal primer C (5'-3'): ATGTCCCGCATCTGGAATGG (SEQ ID NO.48); Vig9216967638 (product length 99bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTTTCTTGTGGTTTCCTTCGACG (SEQ IDNO.49); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTTTCTTGTGGTTTCCTTCGACA (SEQ IDNO.50); Universal primer C (5'-3'): CCACTGGTGCAATAGCTGGA (SEQ ID NO.51); Vig9327976532 (product length 101bp): Primer 1-FAM (5'-3'): GAAGGTGACCAAGTTCATGCTGGCCCACGAGTGAGTATCTAGAT (SEQ ID NO. 52); Primer 2-HEX (5'-3'): GAAGGTCGGAGTCAACGGATTGGCCCACGAGTGAGTATCTAGAC (SEQ ID NO. 53); Universal primer C (5'-3'): AACACCGCACAAGATCCGAA (SEQ ID NO.54).
[0048] 4. Genotyping detection of variant sites in soybeans (1) Primer mixture preparation and PCR system The primer powder dilution and mixing method is as follows: First, dilute the two upstream primers and the universal primer powder to 100 μM. Then, mix 120 μL of primer 1-FAM, 120 μL of primer 2-HEX, 300 μL of universal primer C and 460 μL of sterile water to prepare a 1000 μL primer mixture.
[0049] The PCR system volume was 10 μL, including 1 μL template DNA, 1.4 μL primer mixture, 5 μL HiGeno 2x Probe MixB, and 2.6 μL sterile water.
[0050] (2) PCR procedure The PCR program was set as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s; annealing and extension at 61~55℃ for 40 s, 10 cycles (decreasing by 0.6℃ per cycle); 95℃ denaturation for 20 s, 55℃ amplification for 40 s, 30~34 cycles, 4℃ Forever.
[0051] (3) Detection of PCR products The amplification results were obtained by using the fluorescence typing results of the Allelic Discrimination option in Bio-Rad CFX Manager 3.0 software to genotype the SNP sites of soybean germplasm resources. The genotype of each SNP site was calculated based on the typing results and the terminal bases of the primer sequences.
[0052] Example 2: Detection of soybean germplasm resources 1. Genotyping of 18 SNP loci in mung bean germplasm resources Using the primers and genotyping method with 18 molecular markers designed in Example 1, genotyping was performed on 65 soybean germplasm resources (provided by the National Coarse Grains Engineering Technology Research Center). The genotyping results are shown in Table 2. As can be seen from the table, the genotyping of 65 soybean germplasm resources covering different genetic backgrounds using the 18 molecular marker detection primers successfully obtained SNP locus genotyping data for all resources. The genotyping data constituted the molecular marker fingerprint map of the soybean resources, verifying the accuracy, stability, and efficiency of this marker combination in practical applications. For example, the sequence of the 18 SNP sites corresponding to the Xiaodou resource "TL01" is as follows: Vig8933746474-Vig9032696138-Vig8923862544-Vig8834161606-Vig8734464702-Vig8729267157-Vig8559772262-Vig8511446698-Vig8635619590-Vig8533821711-Vig9327976532-Vig9216967638- The variants Vig914574631-Vig8631878983-Vig8611666615-Vig8640126984-Vig8620555894-Vig8516159407 have the following base types at their mutation sites: AA-CC-CC-AA-CC-TT-GG-AA-GG-CT-TT-GG-AA-TT-AA-AA-AA-CC. The fingerprint of this variety is AACCCCAACCTTGGAAGGCTTTGGAATTAAAAAACC. And so on.
[0053] Table 2. Genotyping information of 18 SNP loci from 65 soybean resources. 2. Evolutionary Tree Analysis of Red Bean Resources Genotypes for each SNP locus were determined based on the typing results and primer sequence terminal bases. The genotype data was then processed using Excel to generate an input file format compatible with MEGA X64 software. Based on the genotype data of 18 markers, cluster analysis was performed using the NJ method in MEGA X64, and cluster analysis diagrams were generated using iTOL. The results are as follows: Figure 1 As shown.
[0054] Figure 1The first group of soybean resources includes: Jilin373; the second group includes: TL02, Jiahong 1hao, Nonganhong, Jian265, Zhuangyuanhong, Zhenzhuhong; the third group includes: IIE10007, LJ02, 012-25, QH1, Bei6JN003, Jihong0001, GN01, Jilin 362, TL01, Longkenzaohong, Baihong. 8hao (Baihong No. 8), LZX002, Mianjin (Mianjin), Yuhong2hao (Yuhong No. 2), Pinhong2011-18 (Pinhong 2011-18), QH7, Liaohong7 (Liaohong 7), Dacha1 (Dacha 1), LJ03; The fourth group of small bean resources includes four subgroups. The first subgroup includes IIE100042, GN04, Baihong2hao (Baihong No. 2), JHPX01, Liaohong 3hao (Liaohong No. 3), Liaohong 1hao (Liaohong No. 1); The second subgroup includes IIE100074, 012-8, IIE100051, Baoqinghong1 (Baoqinghong 1), IIE100006, LJ01, Longxiaodou2hao (Longxiaodou No. 2), Zaluan 2 (Miscellaneous 2), Chixiaodou (Red Bean), Tianjinhong (Tianjin Red); Subgroup 3 includes IIE100049, QH4, Tanghong 2010-23 (Tanghong 2010-23), Jihong 352 (Jihong 352), Tanghong 2010-12 (Tanghong 2010-12), Jihanhongxiaodou (Extremely Drought Red Bean), Baohong 200831-2 (Baohong 200831-2), IIE100029, IIE100024, IIE100033, IIE100023; Subgroup 4 includes XD11, IIE100055, IIE100103, IIE100058, IIE100003, Bei13 F015 (Bei 13 F015), Baihong 3hao (Baihong 3), IIE100018, Liaohong 08712 (Liaohong 08712), IIE100041, TL05, TL03. Therefore, it can be seen that the SNP molecular marker combinations of this invention can distinguish soybean germplasm resources with different genetic backgrounds, and genetic analysis can be performed accordingly.
[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A combination of SNP molecular markers for the identification of soybean germplasm resources, characterized in that, It includes 18 SNP molecular markers, and the SNP site information corresponding to the SNP molecular markers is as follows: The genome version number of the SNP site is GCA_016808095.
1.
2. A primer combination for detecting the SNP molecular marker combination as described in claim 1, characterized in that, The primer combination includes the following primers: 。 3. A kit for detecting the SNP molecular marker combination as described in claim 1, characterized in that, The kit contains the primer combination as described in claim 2.
4. A chip for detecting SNP molecular marker combinations as described in claim 1, characterized in that, The chip comprises the primer combination as described in claim 2.
5. A method for constructing a molecular marker fingerprint map of soybean germplasm resources, characterized in that, Includes the following steps: (1) Extracting the genome of soybean germplasm resources; (2) The genome of the soybean germplasm resource was detected by PCR using the primer combination described in claim 2 to obtain the genotype of the site where the SNP molecular marker combination described in claim 1 was located; (3) Use the genotype to construct a molecular marker fingerprint map of soybean germplasm resources.
6. The method according to claim 5, characterized in that, The PCR detection system consisted of: 1 μL of genomic DNA, 1.4 μL of primer mixture, 5 μL of HiGeno 2x Probe Mix B, and 2.6 μL of sterile water; the final concentrations of primer 1-FAM, primer 2-HEX, and universal primer C in the primer mixture were 12 μM, 12 μM, and 30 μM, respectively. The PCR detection procedure was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s; annealing and extension at 61~55℃ for 40 s, 10 cycles, with the temperature decreasing by 0.6℃ per cycle; 95℃ denaturation for 20 s, amplification at 55℃ for 40 s, 30~34 cycles, and storage at 4℃.
7. The application of the SNP molecular marker combination of claim 1, the primer combination of claim 2, the kit of claim 3, the chip of claim 4, or the molecular marker fingerprint spectrum of soybean germplasm resources constructed by the method of claim 5 or 6 in the identification of soybean germplasm resources.
8. The application according to claim 7, characterized in that, The mung bean germplasm resources include TL01, Jilin 373, TL02, TL03, Jian 265, Extremely Drought Red Bean, Bei 6 JN003, Longken Early Red, TL05, Bei 13 F015, QH1, QH4, QH7, LJ01, LJ02, LJ03, GN01, Longxiaodou No. 2, GN04, Jilin 362, XD11, LZX022, Baoqing Red 1, Dacha 1, Mianjin, Nongan Red, Zaluan 2, Jihong 352, Zhenzhu Red, Baihong No. 3, Jihong 0001, Liaohong 7, Liaohong No. 1, Liaohong No. 3, Pinhong 2011-18, Baihong No. 8, Liaohong 08712, Baohong 200831-2, Tanghong 2010-23, Tanghong 2010-12, JHPX01, Yuhong No. 2, Zhuangyuan Red. Jia Hong No. 1, Tianjin Red, Bai Hong No. 2, 012-8, 012-25, Red Bean, IIE100058, IIE100103, IIE100074, IIE100003, IIE100006, IIE100007, IIE100018, IIE100023, IIE100024, IIE100029, IIE100033, IIE100041, IIE100042, IIE100049, IIE100051, IIE100055.
9. A method for identifying mung bean germplasm resources, characterized in that, Includes the following steps: (1) Extract the genome of the soybean to be tested; (2) The genome is subjected to PCR detection using the primer combination described in claim 2 to obtain the genotype of the site where the SNP molecular marker combination described in claim 1 is located; (3) Compare the genotype with the molecular marker fingerprint map of soybean germplasm resources constructed by the method described in claim 5 or 6 to determine the variety of soybean to be tested.
10. The method according to claim 9, characterized in that, The PCR detection system consisted of: 1 μL of genomic DNA, 1.4 μL of primer mixture, 5 μL of HiGeno 2x Probe Mix B, and 2.6 μL of sterile water; the final concentrations of primer 1-FAM, primer 2-HEX, and universal primer C in the primer mixture were 12 μM, 12 μM, and 30 μM, respectively. The PCR detection procedure was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s; annealing and extension at 61~55℃ for 40 s, 10 cycles, with the temperature decreasing by 0.6℃ per cycle; 95℃ denaturation for 20 s, amplification at 55℃ for 40 s, 30~34 cycles, and storage at 4℃.