SSR markers for identification of rhizome aroma germplasm resources of zhusending and their application
By constructing a fingerprint map of amaryllis aroma germplasm resources using primer combinations of HiSSR10, HiSSR22, and HiSSR37, the problem of low identification efficiency of amaryllis germplasm resources was solved, achieving efficient, simple, and accurate identification of germplasm resources and analysis of phylogenetic relationships.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2026-06-19
AI Technical Summary
In the existing technology, there are not many SSR marker primers for amaryllis, and most of them have unclear backgrounds. There is a lack of effective methods for germplasm resource identification, which makes the identification of amaryllis germplasm resources inefficient, inconvenient and inaccurate.
Three primer pairs, HiSSR10, HiSSR22, and HiSSR37, are provided to construct aroma fingerprints of amaryllis germplasm resources. Varieties are distinguished by PCR amplification and fluorescent capillary electrophoresis, and combined with UPGMA cluster analysis, achieving efficient, simple, and accurate germplasm resource identification.
It enables efficient differentiation and accurate identification of nine amaryllis varieties, provides a reference for phylogenetic analysis and breeding of specific traits, and improves the efficiency and accuracy of amaryllis germplasm resource identification.
Smart Images
Figure CN121204291B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular marker technology, and in particular to SSR markers for the identification of amaryllis aroma germplasm resources and their applications. Background Technology
[0002] Amaryllis (Hippeastrum vittatum) is a general term for perennial herbaceous plants belonging to the genus Hippeastrum in the family Amaryllidaceae. They prefer warm, humid climates, are somewhat cold-hardy, and currently comprise about 75 species with over 600 horticultural varieties. With their beautiful flowers, vibrant colors, and long, slender leaves, amaryllis are ideal for potted plants, landscaping, and cut flowers, possessing both ornamental and economic value. Amaryllis is widely loved and commonly used as a potted plant, cut flower, and for landscaping during holidays.
[0003] Molecular marker technology is widely used in plant phylogenetic analysis and genetic diversity analysis, variety identification, genetic diversity assessment, and assisted breeding. Simple sequence repeats (SSR) markers, also known as microsatellite DNA markers, belong to the second generation of molecular marker technology and are widely distributed in the genome sequence. Currently, there is limited research on the development of SSR markers for amaryllis, and the background of most SSR primers is unclear. SSR molecular marker technology has been successfully applied in ornamental flowers such as roses, anthuriums, and saffron; in crops, it has been used for genetic diversity analysis of sesame, wheat, and melons; for DNA fingerprinting and variety identification of rice; and for authenticity and purity identification of cabbage. However, there are few reports on SSR primers used for amaryllis germplasm resource identification. Summary of the Invention
[0004] The purpose of this invention is to provide SSR markers for the identification of amaryllis aroma germplasm resources and their applications, in order to solve the problems existing in the prior art. The SSR marker primer combination of this invention has the advantages of high efficiency, simplicity and accuracy in constructing fingerprint maps of amaryllis aroma germplasm resources and identifying germplasm resources, and provides a reference for research in the fields of amaryllis hybrid offspring kinship analysis, true and false hybrid identification, and amaryllis germplasm breeding for specific traits.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides a primer combination for amplifying the aroma germplasm resources of Amaryllis using SSR markers, including HiSSR10 primers, HiSSR22 primers, and HiSSR37 primers;
[0007] The nucleotide sequences of the forward and reverse primers of the HiSSR10 primers are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.
[0008] The nucleotide sequences of the forward and reverse primers of the HiSSR22 primers are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.
[0009] The nucleotide sequences of the forward and reverse primers of the HiSSR37 primers are shown in SEQ ID NO.19 and SEQ ID NO.20.
[0010] This invention also provides the use of the primer combination in any of the following:
[0011] (1) Analysis of genetic diversity in amaryllis;
[0012] (2) Construction of the aroma germplasm fingerprint spectrum of amaryllis;
[0013] (3) Identification of amaryllis aroma germplasm resources.
[0014] This invention provides a method for constructing an aroma germplasm fingerprint of amaryllis, comprising the following steps:
[0015] Using the aforementioned primer combination as the core primers, fluorescently labeled primers were synthesized, and PCR amplification was performed using amaryllis DNA as a template.
[0016] The PCR products were subjected to fluorescent capillary electrophoresis to obtain alleles. The obtained alleles were arranged in tandem to construct the aroma germplasm fingerprint of amaryllis.
[0017] Optionally, the fluorescent label includes a FAM label.
[0018] Optionally, the PCR amplification reaction system consists of 2.5 μL of Mg-containing Taq Buffer, 1 μL of template DNA, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of dNTP mix, 0.2 μL of Taq enzyme, and double-distilled water to a final volume of 25 μL.
[0019] Optionally, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 10 cycles, with the annealing temperature decreasing by 0.5℃ per cycle; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ repair extension for 10 min.
[0020] This invention provides the application of the fingerprint spectrum of amaryllis aroma germplasm resources constructed according to the described construction method in the identification of amaryllis aroma germplasm resources.
[0021] Optionally, the amaryllis aroma germplasm resources include Qingxiu, Xiangyi, Ningxiang, Xiyangyang, Rosalie, Hua Kongque, Hongchun, Minawa, and Dajiang.
[0022] This invention provides a method for identifying amaryllis aroma germplasm resources, comprising the following steps:
[0023] Using the primer combination described above as the core primers, fluorescently labeled primers were synthesized, and PCR amplification was performed using the DNA of the amaryllis to be tested as a template.
[0024] The PCR products were subjected to fluorescent capillary electrophoresis to obtain allelic loci. The amaryllis aroma germplasm fingerprint was compared with the above-mentioned fingerprint to determine the variety of the amaryllis to be tested.
[0025] Optionally, the PCR amplification reaction system consists of 2.5 μL of Mg-containing Taq Buffer, 1 μL of template DNA, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of dNTP mix, 0.2 μL of Taq enzyme, and double-distilled water to a final volume of 25 μL.
[0026] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 10 cycles, with the annealing temperature decreasing by 0.5℃ per cycle; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ repair extension for 10 min.
[0027] The present invention discloses the following technical effects:
[0028] This invention selected three primer pairs (HiSSR10, HiSSR22, and HiSSR37) from 14 primer pairs to form primer combinations. These primer combinations can completely distinguish nine varieties of Amaryllis, and fingerprint profiles of these nine varieties were successfully constructed based on these primer combinations. This provides reliable SSR marker primers for the identification and innovative utilization of Amaryllis aroma germplasm resources. UPGMA cluster analysis results show that when the genetic coefficient is 0.3, the nine Amaryllis varieties can be divided into five groups: Group I contains one variety: Grand Prize; Group II contains one variety: Rosalie; Group III contains one variety: Minawa; Group IV contains three varieties: Qingxiu, Hongchun, and Xiangyi; and Group V contains three varieties: Ningxiang, Xiyangyang, and Huakongque. The SSR marker primer combination of this invention has the advantages of high efficiency, simplicity and accuracy in constructing fingerprint maps of amaryllis varieties and identifying varieties, and provides a reference for research in the fields of kinship analysis of amaryllis hybrid offspring, identification of true and false hybrids, and breeding of amaryllis germplasm with specific traits. Attached Figure Description
[0029] 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.
[0030] Figure 1 Front views of flowers from nine varieties of amaryllis; a: Minawa; b: Peacock; c: Rosalie; d: Red Lips; e: Ningxiang; f: Xiangyi; g: Xiyangyang; h: Qingxiu; i: Grand Prize;
[0031] Figure 2 The image shows the electrophoresis results of primers HiSSR1-HiSSR25 and HiSSR50; where H1-H25 and H50 represent primers HiSSR1-HiSSR25 and HiSSR50, respectively, M is the marker, and 1, 2, 3, and 4 represent 4 replicates.
[0032] Figure 3 This is the electrophoresis result of primers HiSSR26-HiSSR49; where H26-H29 represent primers HiSSR26-HiSSR29 respectively, M is the marker, and 1, 2, 3, and 4 represent four replicates.
[0033] Figure 4 The results of cluster analysis for nine varieties of amaryllis are shown. Detailed Implementation
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] Example 1
[0040] 1. Experimental materials
[0041] The nine amaryllis varieties used in this embodiment are all fragrant and are preserved in the amaryllis germplasm resource nursery of the Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences. The variety names are Qingxiu, Xiangyi, Ningxiang, Xiyangyang, Rosalie, Huakongque, Hongchun, Minawa, and Dajiang. Germplasm resource information is shown in Table 1, and flower characteristics are shown in Table 2 and... Figure 1 .
[0042] Table 1 Information on Amaryllis Varieties
[0043] serial number variety source 1 delicate and pretty Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 2 Xiangyi Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 3 Ningxiang Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 4 Happy Sheep Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 5 Luo Lisa Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 6 Peacock Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 7 red lips Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 8 Minawa Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences 9 Grand Prize Amaryllis Germplasm Resource Nursery, Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences
[0044] Table 2. Flower characteristics of 9 amaryllis varieties
[0045] variety Main features Minawa Single-petaled, red, with star-shaped petal patterns, 15.7 cm in diameter, fragrant, and each flower lasts for 4 days. Peacock Double-petaled, red, with flame-shaped petal patterns, 16.5 cm in diameter, fragrant, and each flower lasts for 8 days. Rosalie Single-petaled, red with single-colored petals, 16.7 cm in diameter, fragrant, and each flower lasts for 6 days. red lips Double-petaled, red, with striped and spotted petals, 16.2 cm in diameter, fragrant, and each flower lasts for 8 days. Ningxiang Double-petaled, deep red, with striped petals; flower diameter 14.6 cm; fragrant; single flower blooms for 12 days. Xiangyi Double-petaled, orange-red, with striped patterns on the petals, 15.1 cm in diameter, fragrant, and each flower lasts for 12 days. Happy Sheep Double-petaled, red, with flame-shaped petal patterns, 16.7 cm in diameter, fragrant, and each flower lasts for 8 days. delicate and pretty Double-petaled, predominantly white with secondary red hues, petals with a flame-like pattern, flower diameter 16.7 cm, fragrant, single flower bloom lasts 8 days. Grand Prize Single-petaled, red with single-colored petals, 15.5 cm in diameter, fragrant, and each flower lasts for 6 days.
[0046] Major instruments: The major instruments are PCR instrument (Veriti™ 96well, ABI, USA), gel imaging system (FR-980A, Shanghai Furi Technology Co., Ltd.), sequencer (3730XL, ABI, USA), benchtop high-speed centrifuge (TD5A-WS, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), electrophoresis apparatus (DYY-6C, Beijing Liuyi Instrument Factory), electrophoresis tank (DYCP-32B, Beijing Liuyi Instrument Factory), and UV-Vis Spectrophotometer (SMA4000, Merinton).
[0047] 2. Experimental Methods
[0048] 2.1 DNA Extraction and Electrophoresis
[0049] DNA was extracted using the Ezup column-based plant tissue genomic DNA extraction kit.
[0050] Fifty SSR markers were randomly selected from the *Amaryllis* transcriptome, primers were designed, and electrophoresis was performed. The electrophoresis results of the 50 primer pairs are shown below. Figure 2 and Figure 3 (Each primer pair was tested using 4 samples numbered 1-4). Fourteen primer pairs with clear bands and good polymorphism (HiSSR4, HiSSR10, HiSSR13, HiSSR17, HiSSR18, HiSSR22, HiSSR30, HiSSR32, HiSSR34, HiSSR37, HiSSR41, HiSSR42, HiSSR49, and HiSSR50) were selected for subsequent experiments. Primer Premier 5 was used for primer design.
[0051] 2.2 PCR reaction system and procedure
[0052] The 14 primer pairs selected are shown in Table 5. PCR amplification was performed using these 14 primer pairs. The PCR reaction system and reaction conditions are shown in Tables 3 and 4.
[0053] Table 3 PCR reaction system
[0054] Components concentration volume Template DNA 30 ng / μL 1 μL forward primer 10 μM 1 μL reverse primer 10 μM 1 μL dNTP (mix) 10 mM 1 μL <![CDATA[Taq Buffer(with MgCl2)]]> 10X 2.5 μL Taq enzyme 5 U / μL 0.2 μL <![CDATA[Add ddH2O to]]> 25 μL
[0055] Table 4 PCR reaction conditions
[0056] Serial Number program temperature time 1 Pre-variation 95℃ 5 min 2 transsexual 94℃ 30 sec 3 annealing 63 (decreases by 0.5℃ per cycle) 30 sec 4 extend 72℃ 30 sec 5 Cycle 2-4 10 cycles 6 transsexual 95℃ 30 sec 7 annealing 58℃ 30 sec 8 extend 72℃ 30 sec 9 Cycle 6-8 30 cycles 10 Repair and extension 72℃ 10 min
[0057] Table 5 SSR primer sequences (5'-3')
[0058]
[0059] 2.3 Capillary electrophoresis
[0060] Based on the 14 selected SSR markers, FAM markers (for blue channel detection) were added to 14 forward primers to synthesize fluorescently labeled primers for fluorescent capillary electrophoresis. The size of the separated fragments determined the genotype and allele frequencies were calculated.
[0061] 2.4 Data Analysis
[0062] SSR data were analyzed using Genemapper software, and routine analysis was performed using Excel. Samples were tested using an ABI sequencer. Values were assigned based on the presence or absence of fluorescence signals at the target gene peaks; a selected peak of the same size was recorded as "1", and the absence as "0", thus establishing a database. The sequential combination of "1"s and "0"s generated by the selected primer pairs at these polymorphic fragment sites constitutes the fingerprint encoding of the material.
[0063] 3. Results and Analysis
[0064] 3.1 Capillary electrophoresis results of PCR amplification of 9 amaryllis varieties using 14 primer pairs
[0065] SSR data were analyzed using Genemapper software, and electrophoresis images were obtained and analyzed using Excel. A total of 53 polymorphic allelic fragments were amplified from 14 primer pairs in 9 amaryllis varieties (Table 6), with an average of 3.79 polymorphic allelic fragments amplified per primer pair.
[0066] Table 6. Capillary electrophoresis results of PCR amplification of 9 amaryllis varieties using 14 primer pairs
[0067] Primers Allele size (bp) Number of alleles (NA) HiSSR4 136，140 2 HiSSR10 136，137，138，140，142，144 6 HiSSR13 289，291 2 HiSSR17 282，322 2 HiSSR18 191，194，195 3 HiSSR22 256，258，262，266，270，272，275，277 8 HiSSR30 286，287 2 HiSSR32 258，261，266，267 4 HiSSR34 206，209，235，236 4 HiSSR37 253，259，261，262，264，265，268，271 8 HiSSR41 342，348，351 3 HiSSR42 192，193，195，198 4 HiSSR49 279，295，359，365 4 HiSSR50 243 1
[0068] As shown in Table 6, a total of 53 alleles were amplified by 14 primer pairs. Among them, HiSSR10, HiSSR22 and HiSSR37 had the highest polymorphism (6, 8 and 8 alleles, respectively), while HiSSR-50 had the lowest polymorphism (1 allele). Therefore, HiSSR10, HiSSR22 and HiSSR37 were selected as the 3 core primer pairs.
[0069] 3.2 Construction of fingerprint profiles for 9 Amaryllis varieties
[0070] The alleles amplified by the three core primer pairs (HiSSR10, HiSSR22, and HiSSR37) obtained from screening in nine amaryllis varieties were tandemly arranged to obtain the corresponding fingerprint codes (Table 7). The results showed that the codes of the nine amaryllis varieties were all different. For example, the genotype combination of Xiangyi was HiSSR10-001000-HiSSR22-10001000-HiSSR37-01010000, which was different from the combinations of other amaryllis varieties. This confirmed the high efficiency and accuracy of these markers for variety identification and that they can effectively distinguish different amaryllis varieties.
[0071] Table 7. DNA fingerprinting of nine amaryllis varieties
[0072] variety Core primer SSR data delicate and pretty HiSSR10-100000-HiSSR22-00001000-HiSSR37-01010000 Xiangyi HiSSR10-001000-HiSSR22-10001000-HiSSR37-01010000 Ningxiang HiSSR10-000101-HiSSR22-00100100-HiSSR37-01000010 Happy Sheep HiSSR10-001100-HiSSR22-00110000-HiSSR37-01000001 Luo Lisa HiSSR10-001010-HiSSR22-01000000-HiSSR37-00010000 Peacock HiSSR10-100010-HiSSR22-10000100-HiSSR37-01000010 red lips HiSSR10-100100-HiSSR22-10001000-HiSSR37-10010000 Minawa HiSSR10-100010-HiSSR22-00110000-HiSSR37-00010100 Grand Prize HiSSR10-010010-HiSSR22-00000011-HiSSR37-00101000
[0073] 3.3 UPGMA cluster analysis of 9 amaryllis varieties
[0074] UPGMA cluster analysis was performed on nine amaryllis cultivars based on the capillary electrophoresis results of the three selected primer pairs. The results are as follows: Figure 3 As shown in the figure. The results showed that when the heritability coefficient was 0.3, the nine amaryllis varieties could be divided into five groups: Group I contained one variety: Grand Prize (DJ); Group II contained one variety: Rosalie (LSL); Group III contained one variety: Minawa (MNW); Group IV contained three varieties: Qingxiu (QX), Hongchun (HC), and Xiangyi (XY); Group V contained three varieties: Ningxiang (NX), Xiyangyang (XYY), and Huakongque (HKQ).
[0075] 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 primer combination for amplifying a SSR marker for identifying a Haematoxylum campechianum germplasm resource, characterized by, Including HiSSR10 primers, HiSSR22 primers, and HiSSR37 primers; The nucleotide sequences of the forward and reverse primers of the HiSSR10 primers are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The nucleotide sequences of the forward and reverse primers of the HiSSR22 primers are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively. The nucleotide sequences of the forward and reverse primers of the HiSSR37 primers are shown in SEQ ID NO.19 and SEQ ID NO.
20.
2. The use of the primer combination of claim 1 in any of the following: (1) Analysis of genetic diversity in amaryllis; (2) Construction of fingerprint map of amaryllis germplasm resources; (3) Identification of amaryllis germplasm resources.
3. A method for constructing a fingerprint map of amaryllis germplasm resources, characterized in that, Includes the following steps: Using the primer combination described in claim 1 as the core primer, fluorescently labeled primers were synthesized, and PCR amplification was performed using amaryllis DNA as a template; The PCR products were subjected to fluorescent capillary electrophoresis to obtain alleles. The obtained alleles were arranged in tandem to construct the fingerprint map of amaryllis germplasm resources.
4. The construction method as described in claim 3, characterized in that, The fluorescent label includes FAM labeling.
5. The construction method as described in claim 3, characterized in that, The PCR amplification reaction system consisted of 2.5 μL of Mg-containing Taq Buffer, 1 μL of template DNA, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of dNTP mix, 0.2 μL of Taq enzyme, and double-distilled water to a final volume of 25 μL.
6. The construction method as described in claim 3, characterized in that, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 10 cycles, with the annealing temperature decreasing by 0.5℃ per cycle; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ repair extension for 10 min.
7. The application of the fingerprint spectrum of amaryllis germplasm resources constructed by the construction method according to any one of claims 3-6 in the identification of amaryllis germplasm resources.
8. The application as described in claim 7, characterized in that, The amaryllis germplasm resources include Qingxiu, Xiangyi, Ningxiang, Xiyangyang, Rosalie, Huakongque, Hongchun, Minawa, and Dajiang.
9. A method for identifying amaryllis germplasm resources, characterized in that, Includes the following steps: Using the primer combination described in claim 1 as the core primer, fluorescently labeled primers were synthesized, and PCR amplification was performed using the DNA of the amaryllis to be tested as a template. The PCR products were subjected to fluorescent capillary electrophoresis to obtain allelic sites. The amaryllis germplasm fingerprint profile described in claim 7 was used to determine the variety of the amaryllis to be tested.
10. The method as described in claim 9, characterized in that, The PCR amplification reaction system consisted of 2.5 μL of Mg-containing Taq Buffer, 1 μL of template DNA, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of dNTP mix, 0.2 μL of Taq enzyme, and double-distilled water to a final volume of 25 μL. The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 10 cycles, with the annealing temperature decreasing by 0.5℃ per cycle; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ repair extension for 10 min.