A CAPS molecular marker for Amaryllis and its application in variety identification

Abstract

The present invention belongs to the field of gene engineering technology and discloses a SNP molecular marker for constructing a molecular identity card of an amaryllis variety. The SNP molecular marker for constructing a molecular identity card of an amaryllis variety provided by the present invention can accurately identify an amaryllis variety at the molecular level. Theoretically, a total of 3 amaryllis varieties can be identified based on the 20 SNP sites. 20 = 3,486,784,401 genotypes, which can meet the identification of complex and diverse amaryllis germplasm resources. Further providing an amaryllis molecular ID card based on SNP molecular markers can identify the identity and genetic relationship of different amaryllis germplasms, providing technical support for the management and development and utilization of amaryllis germplasm resources, variety protection and evaluation.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and particularly relates to a CAPS molecular marker of Amaryllis and application thereof in variety identification. Background Art

[0002] Hippeastrum spp. is a bulbous flowering plant in the Amaryllidaceae family. It has peculiar flower shapes and is of great ornamental value. Hippeastrum originated in the subtropical regions of America and is widely distributed in eastern Brazil, Peru, Argentina, Bolivia and other countries or regions. Most species in this genus and their hybrids have huge, spectacular colorful flowers and are of high decorative and ornamental value. The Netherlands, the United States, South Africa and other countries have conducted breeding research and bred a number of varieties for cut flowers, potted plants and garden viewing. To date, breeders have bred more than 600 varieties of commercial value through hybrid breeding and other means, and the number is increasing year by year. For example Figure 1 As shown in the figure, the identification of varieties of Amaryllis plants mainly relies on the specificity of flower organs, and it is difficult to identify varieties during the non-flowering period. With the increase in the number of new varieties cultivated, it has become more difficult to identify Amaryllis cultivars, hybrid offspring and original species. This has also brought great difficulties to the collection and protection of Amaryllis germplasm resources, commercial variety trading and identification, and variety rights protection.

[0003] Molecular markers developed based on DNA sequence differences between different varieties are an effective method for identifying amaryllis varieties. Compared with identification through floral organ traits, identification of amaryllis varieties using molecular markers is not affected by growth conditions and growth periods, and is stable and repeatable. Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. SNP has the characteristics of high density, strong representativeness, and good genetic stability in the whole genome; SNP markers are diallelic genes, representing the smallest genetic variation unit in the genome, and data statistics are simple and accurate; SNP detection methods are flexible and diverse, and easy to automate. SNP-CAPS is essentially a molecular marker technology based on SNP PCR technology combined with restriction endonuclease (RE) technology. In simple terms, a certain SNP happens to be located at the restriction site. By designing primers for the SNP site, the corresponding product fragments obtained after PCR are subjected to enzyme cutting, electrophoresis and other steps, and finally the polymorphism of the sample is judged by observing different bands. Currently, there is no SNP-CAPS molecular marker suitable for identifying Amaryllis species. Therefore, there is an urgent need to develop a molecular marker that can quickly, economically and stably identify species of Amaryllis plants. Summary of the invention

[0004] In this study, a set of SNP-CAPS molecular markers with EcoRI restriction sites were developed to identify the varieties of Amaryllis based on the differences in the molecular fingerprints of enzyme cleavage of different markers, and a unique molecular ID card was produced for each variety.

[0005] The purpose of the first aspect of the present invention is to provide a SNP molecular marker for constructing a molecular identity card of amaryllis varieties.

[0006] The purpose of the second aspect of the present invention is to provide a primer pair for amplifying the SNP molecular marker of the first aspect of the present invention.

[0007] The third aspect of the present invention aims to provide a detection reagent, a gene chip or a kit.

[0008] The purpose of the fourth aspect of the present invention is to provide an application of the SNP site of the first aspect of the present invention, the primer pair of the second aspect of the present invention, or the detection reagent, gene chip or kit of the third aspect of the present invention.

[0009] The purpose of the fifth aspect of the present invention is to provide a method for constructing a molecular identity card of Amaryllis varieties.

[0010] The sixth aspect of the present invention aims to provide a molecular identity card for Amaryllis varieties.

[0011] The seventh aspect of the present invention aims to provide a method for identifying Amaryllis germplasm and varieties.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] In a first aspect of the present invention, a SNP molecular marker for constructing a molecular identity card of an amaryllis variety is provided, wherein the SNP molecular marker comprises Hcaps1, Hcaps16, Hcaps20, Hcaps27, Hcaps49, Hcaps92, Hcaps93, Hcaps106, Hcaps114, Hcaps117, Hcaps136, Hcaps137, Hcaps139, Hcaps148, Hcaps152, Hcaps156, Hcaps178, Hcaps185, Hcaps198 and Hcaps203, wherein the Hcaps1 is located at the 312th base of the sequence shown in SEQ ID NO:1, and the base is T / A; the Hcaps16 is located at the 225th base of the sequence shown in SEQ ID NO:2, and the base is A / G; the Hcaps20 is located at the 312th base of the sequence shown in SEQ ID NO:3, and the base is A / G; NO:3 is located at the 306th base of the sequence shown in SEQ ID NO:3, and the base is A / G; the Hcaps27 is located at the 226th base of the sequence shown in SEQ ID NO:4, and the base is G / C; the Hcaps49 is located at the 259th base of the sequence shown in SEQ ID NO:5, and the base is G / A; the Hcaps92 is located at the 294th base of the sequence shown in SEQ ID NO:6, and the base is C / T; the Hcaps93 is located at the 223rd base of the sequence shown in SEQ ID NO:7, and the base is G / A; the Hcaps106 is located at the 255th base of the sequence shown in SEQ ID NO:8, and the base is C / T; the Hcaps114 is located at the 392nd base of the sequence shown in SEQ ID NO:9, and the base is T / A; the Hcaps117 is located at the 257th base of the sequence shown in SEQ ID NO:10, and the base is A / G; the Hcaps136 is located at the 255th base of the sequence shown in SEQ ID NO:8, and the base is C / T; NO: 11 is located at the 317th base of the sequence shown in SEQ ID NO: 11, and the base is G / A; the Hcaps137 is located at the 345th base of the sequence shown in SEQ ID NO: 12, and the base is G / A; the Hcaps139 is located at the 222nd base of the sequence shown in SEQ ID NO: 13, and the base is C / T; the Hcaps148 is located at the 265th base of the sequence shown in SEQ ID NO: 14, and the base is C / T; the Hcaps152 is located at the 331st base of the sequence shown in SEQ ID NO: 15, and the base is A / G; the Hcaps156 is located at the 263rd base of the sequence shown in SEQ ID NO: 16, and the base is T / C; the Hcaps178 is located at the 260th base of the sequence shown in SEQ ID NO: 17, and the base is G / A; the Hcaps185 is located at the 278th base of the sequence shown in SEQ ID NO: 18, and the base is G / A;The Hcaps981 is located at the 211th base of the sequence shown in SEQ ID NO: 19, and the base is T / C; the Hcaps203 is located at the 231st base of the sequence shown in SEQ ID NO: 20, and the base is T / G. ;

[0014] The second aspect of the present invention provides a primer pair for amplifying the SNP molecular marker of the first aspect of the present invention, wherein the primer pair is SEQ ID NO: 21 to SEQ ID NO: 60.

[0015] In some embodiments of the present invention, every two nucleic acid sequences of SEQ ID NO: 21 to SEQ ID NO: 60 constitute a primer pair, which correspond to Hcaps1 to Hcaps203 respectively. By using the primer pairs provided by the present invention to prepare molecular markers, the rapid construction of the molecular ID card of Amaryllis can be achieved.

[0016] The third aspect of the present invention provides a detection reagent, a gene chip or a kit comprising the primer pair of the second aspect of the present invention.

[0017] In some embodiments of the present invention, the kit further comprises one or more of dNTPs, DNA polymerase, PCR reaction buffer, restriction endonuclease and standard positive template. In some embodiments of the present invention, the restriction endonuclease is EcoRI endonuclease.

[0018] The fourth aspect of the present invention provides the use of the SNP site of the first aspect of the present invention, the primer pair of the second aspect of the present invention, or the detection reagent, gene chip or kit of the third aspect of the present invention in at least one of (1) to (6): (1) identifying amaryllis varieties; (2) preparing and identifying amaryllis products; (3) constructing a molecular identity card of amaryllis varieties; (4) identifying the kinship of amaryllis germplasm; (5) managing, developing and utilizing amaryllis germplasm resources; and (6) protecting and evaluating amaryllis varieties.

[0019] The fifth aspect of the present invention provides a method for constructing a molecular identity card of amaryllis varieties, comprising the step of using the primer pair of the second aspect of the present invention or the detection reagent, gene chip or kit of the third aspect of the present invention to detect the SNP molecular marker of the first aspect of the present invention on the test sample.

[0020] In some embodiments of the present invention, the construction method comprises the following steps: 1) using the primer pair of the second aspect of the present invention or the detection reagent, gene chip or kit of the third aspect of the present invention to perform PCR amplification on the DNA of the sample to be tested; 2) analyzing the SNP site genotype of the PCR amplification product, digitizing each molecular marker according to the SNP site genotype, arranging them in sequence in the order of Hcaps1 to Hcaps203, and concatenating them into a molecular ID code.

[0021] In some embodiments of the present invention, the analyzing of the genotype of the SNP site of the PCR amplification product comprises performing restriction digestion and electrophoresis on the PCR amplification product; or, the analyzing of the genotype of the SNP site of the PCR amplification product comprises sequencing the PCR amplification product.

[0022] In some embodiments of the present invention, after the PCR amplification product is digested with a restriction endonuclease, the digestion product is subjected to electrophoresis detection of characteristic bands to determine the genotype of the SNP site, and A, T, C, and G are converted to 1, 2, 3, and 4 in sequence according to the genotype, and the deletion site is converted to 0. The sequences are arranged in the order of Hcaps1 to Hcaps203 and connected in series to form a molecular ID code.

[0023] Those skilled in the art should understand that there are many methods for analyzing the base type of a SNP site, including sequencing analysis, allele-specific PCR analysis, enzyme digestion product polymorphism analysis, and the like.

[0024] In some embodiments of the present invention, the germplasm resource category (the first digit of the ID card, such as 1 represents wild resources (populations), 2 represents wild resources (family), 3 represents wild resources (asexual lines), 4 represents local varieties, 5 represents selected varieties, 6 represents selected strains, and 7 represents genetic data) and the resource source area code (the second to fifth digits of the ID card, such as 0031 represents the origin of the Netherlands) are digitized and placed before the molecular ID card code (the sixth to forty-fifth digits of the ID card) to form a 45-digit molecular ID card number.

[0025] In some embodiments of the present invention, the restriction endonuclease is EcoRI endonuclease.

[0026] In some embodiments of the present invention, the PCR reaction system includes: 2-3 ng / μL of the sample DNA to be tested, 0.2-0.3 μM of the upstream primer, 0.2-0.3 μM of the downstream primer, 0.3-0.7 μL / μL of 2×Taq PCR StarMix, and the rest is water.

[0027] In some embodiments of the present invention, the PCR reaction conditions are: pre-denaturation at 94-96°C for 4-6 min in the first stage; denaturation at 93-95°C for 18-22 s, annealing at 53-55°C for 18-22 s, extension at 71-73°C for 28-32 s, and 36-42 cycles in the second stage; and extension at 71-73°C for another 4-6 min in the third stage.

[0028] In some embodiments of the present invention, the enzyme digestion reaction system includes: 5-15 μL / μL of PCR amplification product, 0.05-0.1 μL / μL of EcoRI endonuclease, and 0.5-1.5 μL / μL of Buffer.

[0029] In some embodiments of the present invention, the enzyme digestion reaction conditions are: 33-40° C., 2-4 h.

[0030] The sixth aspect of the present invention provides a molecular identity card of Amaryllis varieties, which is constructed by the construction method of the fifth aspect of the present invention.

[0031] In some embodiments of the present invention, the molecular ID card of the Amaryllis variety also includes the following information: germplasm resource category, resource source area code and / or germplasm resource information.

[0032] In some embodiments of the present invention, the presentation form of the molecular ID card of the Amaryllis variety includes a barcode and / or a QR code.

[0033] The seventh aspect of the present invention provides a method for identifying amaryllis germplasm and varieties, which utilizes the uniqueness of the amaryllis variety molecular identity card of the sixth aspect of the present invention to determine the corresponding amaryllis germplasm or variety.

[0034] The beneficial effects of the present invention are:

[0035] The present invention provides a set of SNP molecular markers that can be used to construct molecular ID cards for Amaryllis varieties, which can accurately identify Amaryllis at the molecular level. Theoretically, a total of 3 20 =3,486,784,401 genotypes, which can meet the identification of complex and diverse amaryllis germplasm resources.

[0036] The SNP molecular marker-based amaryllis molecular identity card developed in the present invention can identify the identities and genetic relationships of different amaryllis germplasms, and provide technical support for the management and development and utilization of amaryllis germplasm resources, and the protection and evaluation of varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the plants of 'Haku' Amaryllis and 'Fairy' Amaryllis.

[0038] Figure 2 Phylogenetic tree of different Hippeastrum cultivars constructed using molecular markers.

[0039] Figure 3 The results of electrophoresis were verified by enzyme digestion of some SNP molecular markers of 24 Hippeastrum varieties (Hcaps1, Hcaps16, Hcaps20, Hcaps27, Hcaps49 and Hcaps92, respectively); in the figure, CK represents the electrophoresis band of the PCR product of the corresponding species, D represents the electrophoresis band of the PCR product after adding EcoRI enzyme, and the middle band is the 5000bp Marker.

[0040] Figure 4 The electrophoresis results of enzyme digestion verification of some SNP molecular markers of 24 Hippeastrum varieties (Hcaps93, Hcaps106, Hcaps114, Hcaps117, Hcaps136 and Hcaps137, respectively); in the figure, CK represents the electrophoresis band of the PCR product of the corresponding species, D represents the electrophoresis band of the PCR product after adding EcoRI enzyme, and the middle band is the 5000bp Marker.

[0041] Figure 5 The electrophoresis results of enzyme digestion verification of some SNP molecular markers of 24 Hippeastrum varieties (Hcaps139, Hcaps148, Hcaps152, Hcaps156, Hcaps178 and Hcaps185, respectively); in the figure, CK represents the electrophoresis bands of PCR products of the corresponding species, D represents the electrophoresis bands after the PCR product is added with EcoRI enzyme, and the middle band is the 5000bp Marker.

[0042] Figure 6 The results of electrophoresis for the restriction digestion verification of some SNP molecular markers (Hcaps198 and Hcaps203, respectively) of 24 Hippeastrum varieties; in the figure, CK represents the electrophoresis bands of the PCR products of the corresponding species, D represents the electrophoresis bands after the PCR products were treated with EcoRI enzyme, and the middle band is the 5000bp Marker.

[0043] Figure 7 This is the molecular ID card of 'Flower Peacock' Amaryllis.

[0044] Figure 8 These are the molecular identity cards for 'Minawa', 'Double Emperor', 'Lemon Star', 'Red Heaven', 'Haku' and 'Biyi' amaryllis.

[0045] Fig. 9 The molecular identity cards for the amaryllis species 'Afre', 'Fen Dai', 'Shuang Meng', 'Amigo', 'Wuhua Tianbao', 'Orange Sevi', 'Nanhai Girl', 'Moonlight', 'Mandela', 'Red Peacock', 'Fairy', 'Sakura' and 'Ocean Star'. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below through specific examples.

[0047] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0048] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0049] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0050] Example 1 Development of molecular markers

[0051] Fresh leaves of native Amaryllis, White Rib and Amaryllis superior variety 'Flower Peacock' were collected for RNA extraction. After the RNA library was constructed, high-throughput sequencing was performed by Illumina to obtain three transcriptome Raw data. Trinity was used to assemble the paired 150bp sequences of the three transcriptome data sets into Scalfold sequences with a length greater than 1000bp. BioEdit (v.7.2.5) software was used to search for homologous sequences of the three transcriptome data, and used as reference sequences for the next step to search for SNP sites. The three transcriptome Raw data were aligned with the reference sequences using Burrows-Wheeler Aligner (BWA) to obtain SNP sites. SAMtools and VarScan software were used to analyze and filter SNP information. SNP2CAPS or EditPlus software was used to detect the SNP sites located in the EcoRI region (GAATTC, i.e., the restriction site), and finally 20 Scaffold sequences with SNP sites in the EcoRI region (GAATTC) were obtained, including TRINITY_DN12563_c0_g1_i3, TRINITY_DN16367_c0_g1_i12, TRINITY_DN10054_c0_g1_i2, TRINITY_DN4347_c0_g1_i13, TRINITY_DN1601_c0_g1_i3, TRINITY_DN7232_c0_g1_i3, TRINITY_DN8448_c0_g1_i9, TRINITY_DN4110_c0_g1_i3, and TRINITY_DN10054_c0_g1_i4. ITY_DN3838_c0_g1_i3, TRINITY_DN5903_c0_g1_i2, TRINITY_DN23219_c0_g1_i3, TRINI TY_DN2934_c0_g1_i5, TRINITY_DN5583_c0_g1_i1, TRINITY_DN5764_c0_g1_i1, TRINITY_ DN15239_c0_g1_i10, TRINITY_DN6465_c0_g1_i7, TRINITY_DN12540_c0_g1_i7, TRINITY _DN815_c0_g1_i3, TRINITY_DN530_c0_g1_i4, TRINITY_DN1888_c1_g1_i13, the specific sequences are as follows: SEQ ID NO:1~20. Primers were designed using NCBI and Primer Premier (v.5) for the 100-300 bp sequence before and after the SNP. After screening, 20 pairs of polymorphic and universal SNP site primers were finally obtained. The primer sequence information is shown in Table 1.

[0052] Table 1 SNP site primer sequence information

[0053]

[0054]

[0055] Example 2 SNP primer amplification and enzyme digestion verification

[0056] (1) Tender leaf samples were cut from 24 tested Hippeastrum varieties ('Minawa', 'Double Emperor', 'Lemon Star', 'Red Heaven', 'Haku', 'Biyi', 'Afre', 'Flower Peacock', 'Fen Dai', 'Shuang Meng', 'Amigo', 'Wuhua Tianbao', 'Orange Saiwei', 'Nanhai Girl', 'Moonlight', 'Mandela', 'Red Peacock', 'Fairy', 'Sakura', 'Ocean Star', 'Princess Tikka', 'Yang Gaozhao', 'Dancing Queen', 'Daisy') and DNA was extracted using the Plant Genomic DNA Extraction Kit (Tiangen, China) according to the instructions.

[0057] (2) PCR amplification was performed using the extracted DNA as template and the 20 pairs of SNP primers in Table 1 as primers. The reaction system for 20 μL was as follows: DNA template (50 ng / μL) 1 μL, upstream / downstream primers (5 μM) 1 μL, 2×Taq PCR StarMix 10 μL, ddH2O 7 μL. The reaction procedure was as follows: 95°C for 5 min; 94°C for 20 s, 54°C for 20 s, 72°C for 30 s, 40 cycles; 72°C for 5 min; and storage at 4°C.

[0058] The reaction yielded the primary PCR product;

[0059] (3) The SNP primer amplification product was digested with enzymes. The digestion system for 10 μL was as follows: 5 μL of PCR product, 1 μL of EcoRI endonuclease, 1 μL of buffer, and 3 μL of ddH2O. The digestion conditions were 37°C water bath for 3 h.

[0060] (4) Using the non-enzyme-digested PCR product as a blank control and the enzyme-digested product as a treatment control, perform 1% agarose gel electrophoresis for 25 minutes and then observe the electrophoresis results in a gel imager.

[0061] According to the enzyme electrophoresis bands, the enzyme cleavage of different markers in different varieties was typed. In the genotype, the type with enzyme cleavage site was typed as A, and the type without enzyme cleavage site was typed as a. Each variety was typed as diploid, that is, homozygous AA and aa, and heterozygous Aa. According to the genotype of each marker in 24 varieties, the genotype matrix was constructed, and the phylogenetic tree was constructed using NTSYS software.

[0062] Experimental results: The electrophoresis results are as follows Figure 3 to Figure 6 The following table shows the amplification results of 20 marker primers in 24 varieties of Amaryllis. The phylogenetic tree constructed using NTSYS software showed that all 24 varieties could be significantly distinguished by the 20 markers ( Figure 2 ). The genetic distance between different varieties is between 0.0303 and 0.2395, and the genetic distance is greater than 0, indicating that the 20 markers developed can well distinguish these 24 varieties. Each marker can distinguish 3 genotypes (AA, Aa, aa), so the 20 pairs of primers provided by the invention can theoretically identify 3 20 =3,486,784,401 genotypes.

[0063] Example 3 Preparation of molecular ID card of Amaryllis varieties

[0064] According to the fingerprint information of the designed molecular markers in all collected amaryllis varieties, a specific molecular ID card for each variety was developed. The first digit of the ID card is the germplasm resource category, with reference to the "Technical Regulations for Survey and Cataloging of Camellia Oil Genetic Resources" (LY / T 2247-2014) and "Construction of Molecular ID Cards of Camellia Oil Main Variety Resources Based on Transcriptome SNP" (Lin Ping and Wang Kailiang et al. (2023). "Construction of Molecular ID Cards of Camellia Oil Main Variety Resources Based on Transcriptome SNP." Chinese Agricultural Science 56(2):217-235.), with slight modifications. 1 represents wild resources (population), 2 represents wild resources (family), 3 represents wild resources (clonal line), 4 represents local varieties, 5 represents selected varieties, 6 represents selected lines, and 7 represents genetic data; the second to fifth digits are the resource source area code; starting from the sixth digit, the SNP site genotype of each variety after verification, A, T, C, G are converted to 1, 2, 3, 4 in sequence, and missing sites are replaced with 0 to construct a specific molecular fingerprint map for each variety. Combine germplasm resource information and molecular fingerprints, and use barcode generators and QR code generators to construct molecular ID cards specific to different varieties. Figure 7 Molecular ID template of the peacock flower shown.

[0065] Take the flower peacock amaryllis as an example ( Figure 7), its molecular ID number is 500312144444341321132224441413222113341412322, indicating that it is a selected variety (5), and its origin is the Netherlands (0031). After PCR amplification, according to the results of restriction enzyme electrophoresis, the SNP variant site bases of the 20 core markers are TA in sequence (Note: the genotype is heterozygous, and the bases of the restriction enzyme site are in front and the bases of the non-restriction site are in the back; if the base The 45-digit molecular ID number of each variety was used to make variety-specific barcodes and QR codes (http: / / qr-batch.com / ). The molecular ID numbers of other amaryllis varieties are as follows: Figure 8-Figure 9 shown.

[0066] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A primer set for amplifying a SNP molecular marker, characterized in that: The primer set is SEQ ID NO: 21 to SEQ ID NO: 60; The SNP molecular markers consist of Hcaps1, Hcaps16, Hcaps20, Hcaps27, Hcaps49, Hcaps92, Hcaps93, Hcaps106, Hcaps114, Hcaps117, Hcaps136, Hcaps137, Hcaps139, Hcaps148, Hcaps152, Hcaps156, Hcaps178, Hcaps185, Hcaps198 and Hcaps203, wherein: The Hcaps1 is located at the 312th base of the sequence shown in SEQ ID NO: 1, and the base is T / A; The Hcaps16 is located at the 225th base of the sequence shown in SEQ ID NO: 2, and the base is A / G; The Hcaps20 is located at the 306th base of the sequence shown in SEQ ID NO: 3, and the base is A / G; The Hcaps27 is located at the 226th base of the sequence shown in SEQ ID NO: 4, and the base is G / C; The Hcaps49 is located at the 259th base of the sequence shown in SEQ ID NO:5, and the base is G / A; The Hcaps92 is located at the 294th base of the sequence shown in SEQ ID NO: 6, and the base is C / T; The Hcaps93 is located at the 226th base of the sequence shown in SEQ ID NO:7, and the base is G / A; The Hcaps106 is located at the 255th base of the sequence shown in SEQ ID NO: 8, and the base is C / T; The Hcaps114 is located at the 392nd base of the sequence shown in SEQ ID NO:9, and the base is T / A; The Hcaps117 is located at the 257th base of the sequence shown in SEQ ID NO: 10, and the base is A / G; The Hcaps136 is located at the 317th base of the sequence shown in SEQ ID NO: 11, and the base is G / A; The Hcaps137 is located at the 345th base of the sequence shown in SEQ ID NO: 12, and the base is G / A; The Hcaps139 is located at the 222nd base of the sequence shown in SEQ ID NO: 13, and the base is C / T; The Hcaps148 is located at the 265th base of the sequence shown in SEQ ID NO: 14, and the base is C / T; The Hcaps152 is located at the 331st base of the sequence shown in SEQ ID NO: 15, and the base is A / G; The Hcaps156 is located at the 263rd base of the sequence shown in SEQ ID NO: 16, and the base is T / C; The Hcaps178 is located at the 260th base of the sequence shown in SEQ ID NO: 17, and the base is G / A; The Hcaps185 is located at the 278th base of the sequence shown in SEQ ID NO: 18, and the base is G / A; The Hcaps198 is located at the 211th base of the sequence shown in SEQ ID NO: 19, and the base is T / C; The Hcaps203 is located at the 231st base of the sequence shown in SEQ ID NO: 20, and the base is T / G.

2. A detection reagent, gene chip or kit, comprising the primer set according to claim 1.

3. Use of the primer set of claim 1 or the detection reagent, gene chip or kit of claim 2 in at least one of (1) to (5): (1) Identify the varieties of Amaryllis; (2) Preparation and identification of amaryllis products; (3) Constructing molecular ID cards for Amaryllis varieties; (4) Identification of the genetic relationship of Amaryllis germplasm; (5) Management, development and utilization of Amaryllis germplasm resources.

4. A method for constructing a molecular identity card of amaryllis varieties, comprising the step of using the primer set described in claim 1 or the detection reagent, gene chip or kit described in claim 2 to detect the SNP molecular marker described in claim 1 on the sample to be tested.

5. A method for identifying amaryllis germplasm and varieties, characterized in that: The uniqueness of the molecular identity card of the Amaryllis variety constructed by the construction method described in claim 4 determines the germplasm or variety to which the corresponding Amaryllis belongs.

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