SNP (Single Nucleotide Polymorphism) molecular marker combination for constructing fructus aurantii fingerprint spectrum and application of SNP molecular marker combination

Abstract

The invention discloses an SNP molecular marker combination for constructing a fructus aurantii fingerprint spectrum and application of the SNP molecular marker combination, SNP molecular markers in the SNP molecular marker combination have the advantages of being single in copy, high in polymorphism, homozygous in site and high in universality, and fructus aurantii varieties in four major production areas can be efficiently identified. The DNA fingerprint spectrum established by using the set of SNP molecular marker combination can be used for database contrast of unknown fructus aurantii varieties to query the fructus aurantii source, can also be used for contrast differentiation of known varieties, and can assist variety authenticity identification and market supervision.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural molecular biology, and in particular relates to a set of SNP molecular marker combinations for constructing a fingerprint map of Fructus Aurantii Immaturus and applications thereof. Background Art

[0002] Citrus aurantium (Fructus Aurantium) is the dried, immature fruit of Citrus aurantium L., a plant of the Rutaceae family (Citrus) and its cultivars. It is a commonly used Traditional Chinese Medicine (TCM) with benefits such as regulating qi, relieving fullness, relieving stagnation and distension, and resolving phlegm. Numerous cultivars have been continuously selected through long-term artificial cultivation. Traditional identification of medicinal herb varieties relies primarily on morphological characteristics, which are susceptible to environmental influences. Consequently, the market often suffers from a mixture of superior and inferior varieties, resulting in a chaotic variety mix and inconsistent quality. This seriously impacts the quality of the medicinal herb and is detrimental to the healthy and sustainable development of the Fructus Aurantium (Fructus Aurantium) industry. Establishing a fingerprint of Fructus Aurantium and classifying Fructus Aurantium from different regions based on this fingerprint is urgently needed in the Fructus Aurantium (Fructus Aurantium) industry to provide a reference for quality control of Fructus Aurantium (Fructus Aurantium) in different major producing areas.

[0003] Currently, mainstream Citrus aurantium products are categorized into four main production areas based on their origin. Citrus aurantium is primarily produced in the Sichuan Basin, commonly known as "Chuan Zhi Ke"; Citrus aurantium is primarily produced in the Dongting Lake Plain, commonly known as "Xiang Zhi Ke"; Citrus aurantium is primarily produced in the Poyang Lake Plain, commonly known as "Jiang Zhi Ke"; and Citrus aurantium dulcis is primarily produced in the Jinqu Basin, commonly known as "Qu Zhi Ke." Existing techniques for distinguishing Citrus aurantium from different origins involve measuring the content of naringin, neohesperidin, and luteolin in Citrus aurantium using liquid chromatography. Cluster analysis and principal component analysis are then used to generate coefficient variables to construct similarity fingerprints, thereby distinguishing Citrus aurantium varieties from different regions. With cross-breeding across different regions, the limitations of liquid chromatography have become increasingly apparent. Therefore, developing an economical, efficient, and accurate method for regional cultivar identification is crucial for improving the management and conservation of Citrus aurantium germplasm resources, as well as the quality of the medicinal material. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a set of SNP molecular marker combinations for constructing a fingerprint of Fructus Aurantii Immaturus.

[0005] The present invention also provides a primer set and / or probe for detecting the above-mentioned SNP molecular marker combination.

[0006] The present invention also provides a kit.

[0007] The invention also provides a gene chip.

[0008] The present invention also proposes the application of the above molecular marker combination, primer set, kit and / or gene chip.

[0009] The invention also provides a method for constructing a DNA fingerprint of a Fructus Aurantii Immaturus variety.

[0010] The present invention also provides a DNA fingerprint of the Fructus Aurantii Immaturus varieties constructed by the above construction method.

[0011] The invention also provides a method for identifying the varieties of Fructus Aurantii Immaturus.

[0012] According to a first aspect of the present invention, a set of SNP molecular marker combinations for constructing a fingerprint of Fructus Aurantii Immaturus is proposed, wherein the SNP molecular marker combination includes a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, a fourth SNP molecular marker, a fifth SNP molecular marker, a sixth SNP molecular marker, and a seventh SNP molecular marker;

[0013] The first SNP molecular marker is located at nucleotide 5059088 of chromosome scaffold85909_cov90 of the reference genome, and the polymorphism is A or T;

[0014] The second SNP molecular marker is located at nucleotide 844624 of chromosome scaffold85962_cov92 of the reference genome, and the polymorphism is T or C;

[0015] The third SNP molecular marker is located at nucleotide 3327 of chromosome scaffold86470_cov3269 of the reference genome, and the polymorphism is C or A;

[0016] The fourth SNP molecular marker is located at nucleotide 377175 of chromosome scaffold85934_cov88 of the reference genome, and the polymorphism is C or A;

[0017] The fifth SNP molecular marker is located at nucleotide 522217 of chromosome scaffold132_cov94 of the reference genome, and the polymorphism is C or T;

[0018] The sixth SNP molecular marker is located at nucleotide 1237093 of chromosome scaffold85919_cov74 of the reference genome, and the polymorphism is A or G;

[0019] The seventh SNP molecular marker is located at nucleotide 1427222 of chromosome scaffold4070_cov90 of the reference genome, and the polymorphism is A or T;

[0020] The reference genome is the citrus reticulate reference genome, scaffold level, and the link to the reference genome location is http: / / citrus.hzau.edu.cn / orange / download / index.php.

[0021] According to a second aspect of the present invention, a primer set and / or probe for detecting the above-mentioned SNP molecular marker combination is proposed.

[0022] According to a third aspect of the present invention, a kit is provided, comprising the above-mentioned primer set and / or probe.

[0023] According to a fourth aspect of the present invention, a gene chip is provided, wherein the gene chip comprises the above-mentioned primer set and / or probe.

[0024] According to a fifth aspect of the present invention, the use of the above-mentioned SNP molecular marker combination, primer set, kit and / or gene chip in any one of the following 1) to 5) is proposed:

[0025] 1) Identify or assist in identifying varieties of Fructus Aurantii Immaturus;

[0026] 2) Analysis of genetic structure of Fructus Aurantii Immaturus cultivars;

[0027] 3) Analysis of genetic diversity of Fructus Aurantii Immaturus resources;

[0028] 4) Construction of DNA fingerprint of Fructus Aurantii Immaturus;

[0029] 5) Genotyping of Fructus Aurantii Immaturus varieties.

[0030] In some embodiments of the present invention, the varieties of the fructus citron include Zhejiang Pan'an Daidaihua, Zhejiang Huyou, Fujian Xingtian Huyou, Zhejiang Quzhou Daidaihua, Sichuan Shugeng Agriculture Daidaihua grafted seedlings, Guangdong Xinhui tangerine peel, Hunan Yuanjiang yellow peel sour orange, Jiangxi Xingan stinky orange, Jiangxi Xingan County Xinxiang No. 1, Nancheng stinky orange, Nancheng fragrant orange, Zhejiang Zhuluan, Zhejiang small red orange, wide peel orange, and pomelo.

[0031] According to a sixth aspect of the present invention, a method for constructing a DNA fingerprint of Fructus Aurantii Immaturus varieties is proposed, the method comprising the following steps: extracting total DNA from different Fructus Aurantii Immaturus varieties; performing SNP site polymorphism detection using the above-mentioned SNP molecular marker combination, and determining the genotyping of the SNP molecular marker based on the detection results of each SNP site in the combination, thereby constructing a DNA fingerprint of the Fructus Aurantii Immaturus varieties;

[0032] Among them, the varieties of Citrus aurantium include Zhejiang Pan'an Daidaihua, Zhejiang Huyou, Fujian Xingtian Huyou, Zhejiang Quzhou Daidaihua, Sichuan Shugeng Agriculture Daidaihua grafted seedlings, Guangdong Xinhui tangerine peel, Hunan Yuanjiang yellow peel sour orange, Jiangxi Xingan stinky orange, Jiangxi Xingan County Xinxiang No. 1, Nancheng stinky orange, Nancheng fragrant orange, Zhejiang Zhuluan, Zhejiang small red orange, wide-peeled orange, and pomelo.

[0033] In some embodiments of the present invention, sequencing, chip, KASP or QPCR methods are used to detect SNP site polymorphism.

[0034] According to a seventh aspect of the present invention, a DNA fingerprint of a Fructus Aurantii Immaturus variety constructed by using the above-mentioned method for constructing a DNA fingerprint of a Fructus Aurantii Immaturus variety is proposed.

[0035] According to an eighth aspect of the present invention, a method for identifying a Fructus Aurantii Immaturus variety is proposed, wherein the total DNA of the sample to be tested is extracted, and the SNP site polymorphism is detected using the above-mentioned SNP molecular marker combination. The genotyping of the SNP molecular marker is determined based on the detection results of each SNP site in the combination, and then compared with the genotyping in the Fructus Aurantii Immaturus variety DNA fingerprint constructed by the above-mentioned method for constructing a DNA fingerprint of the Fructus Aurantii Immaturus variety.

[0036] In some embodiments of the present invention, sequencing, chip, KASP or QPCR methods are used to detect SNP site polymorphism.

[0037] According to an embodiment of the present invention, a set of SNP molecular marker combinations for constructing a fingerprint map of Citrus aurantium has at least the following beneficial effects: the SNP molecular markers in the SNP molecular marker combination screened by the present invention have the advantages of single copy, high polymorphism, site homozygosity, and high universality, and can efficiently identify Citrus aurantium varieties from four major producing areas. Using this set of SNP molecular marker combinations to establish a DNA fingerprint map can, on the one hand, be used for database comparison of unknown Citrus aurantium varieties to query their origin, and can also be used for comparison and differentiation of known varieties, which can assist in variety authenticity identification and market supervision.

[0038] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0041] In the description of the present invention, if there are descriptions of first, second, third, etc., they are only used to distinguish technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0042] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0043] Example 1 A set of SNP molecular marker combinations for detecting Fructus Aurantii Immaturus varieties

[0044] This example provides a set of SNP molecular marker combinations for detecting Fructus Aurantii Immaturus varieties, and the method for obtaining the combination is as follows:

[0045] (1) Collection of Citrus aurantium resources

[0046] Through research on various planting bases and markets, we obtained more than 30 market varieties and selected a list of 15 representative Citrus aurantium resources as shown in Table 1 below.

[0047] Table 1

[0048]

[0049] (2) Genome sequencing

[0050] The leaves of Citrus aurantium were collected and DNA was extracted and sequenced at the Huazhi National Key Laboratory. The reference genome used was the citrus reticulate reference genome at the scaffold level. The link for the reference genome location was http: / / citrus.hzau.edu.cn / orange / download / index.php, and SNP differential sites were mined.

[0051] (3) SNP screening

[0052] SNP mining was performed on the SNP site data obtained through sequencing based on the following parameters: 1) there were no missing SNPs in all samples; 2) the base type was homozygous; 3) the frequency of the lowest genotype was greater than 0.2.

[0053] Finally, 7899 effective SNP sites were obtained. Further site simplification screening was performed, and the sites with the highest frequency of the lowest genotype were selected and superimposed with sites that efficiently distinguished different samples for replacement. The minimum number of sites was selected to distinguish 15 Fructus Aurantii Immaturus resources. Finally, 7 SNP sites were obtained, which were able to distinguish the 15 Fructus Aurantii Immaturus varieties shown in Table 1. The SNP site information is shown in Table 2 below.

[0054] Table 2

[0055] ID Chromosome Pos Ref Alt SNP1 scaffold85909_cov90 5059088 A T SNP2 scaffold85962_cov92 844624 T C SNP3 scaffold86470_cov3269 3327 C A SNP4 scaffold85934_cov88 377175 C A SNP5 scaffold132_cov94 522217 C T SNP6 scaffold85919_cov74 1237093 A G SNP7 scaffold4070_cov90 1427222 A T

[0056] The sequence information of the specific molecular markers is shown in Table 3 below, SNP1 is located at 100 bp of the sequence shown in SEQ ID NO: 1; SNP2 is located at 100 bp of the sequence shown in SEQ ID NO: 2; SNP3 is located at 100 bp of the sequence shown in SEQ ID NO: 3; SNP4 is located at 100 bp of the sequence shown in SEQ ID NO: 4; SNP5 is located at 100 bp of the sequence shown in SEQ ID NO: 5; SNP6 is located at 100 bp of the sequence shown in SEQ ID NO: 6; SNP7 is located at 100 bp of the sequence shown in SEQ ID NO: 7.

[0057] Table 3

[0058]

[0059]

[0060] Example 2 Construction of DNA fingerprint library and its application in identifying varieties of Fructus Aurantii Immaturus

[0061] 1. Obtaining DNA fingerprints of Fructus Aurantii Immaturus varieties

[0062] Based on the SNP molecular marker combination for detecting the varieties of Citrus aurantium obtained in Example 1, the position information and base information of the corresponding SNP molecular marker combination of the standard varieties of Citrus aurantium Zhejiang Pan'an Daidaihua, Zhejiang Huyou, Fujian Xingtian Huyou, Zhejiang Quzhou Daidaihua, Sichuan Shugeng Agricultural Daidaihua grafted seedlings, Guangdong Xinhui Chenpi, Hunan Yuanjiang Huangpi Sour Orange, Jiangxi Xingan Bitter Orange, Jiangxi Xingan County Xinxiang No. 1, Nancheng Bitter Orange, Nancheng Fragrant Orange, Zhejiang Zhuluan, Zhejiang Xiaohongcheng, Kuanpiju, and Pomelos were obtained, and the DNA fingerprints of the Citrus aurantium varieties were obtained, as shown in Table 4.

[0063] Table 4

[0064]

[0065] 2. Verification of fingerprint data

[0066] DNA was re-extracted from another 15 samples of Fructus Aurantii Immaturus resources, identified as Daidaihua from Pan'an, Zhejiang, Huyou from Zhejiang, Huyou from Xingtian, Fujian, Daidaihua from Quzhou, Zhejiang, grafted Daidaihua seedlings from Shugeng Agriculture in Sichuan, dried tangerine peel from Xinhui, Guangdong, yellow-peeled sour orange from Yuanjiang, Jiangxi, Xingan bitter orange, Jiangxi, Xinxiang No. 1 from Xingan County, Nancheng bitter orange, Nancheng fragrant orange, Zhuluan from Zhejiang, Xiaohongcheng from Zhejiang, Kuanpi orange, and pomelo. Each variety was replicated three times. The 200 bp upper and lower flanking sequences of the corresponding sites were obtained using the reference genome and sent to Shanghai Bioengineering for first-generation sequencing. The fingerprint loci data of the 15 Fructus Aurantii Immaturus resources were verified by first-generation sequencing and are shown in Table 5 below. At the same time, the whole genome of the samples was sequenced to confirm the Fructus Aurantii Immaturus varieties. The loci data were compared with the sequencing data, and the obtained data were consistent and could be used in the fingerprint reference database.

[0067] Table 5

[0068]

[0069]

[0070] 3. Fingerprint and database applications

[0071] Based on the varieties sold on the market, 5 varieties were randomly selected from the Internet for identification. The process is as follows

[0072] 1) Purchase five varieties of Citrus aurantium from the market;

[0073] 2) Perform first-generation sequencing on the loci corresponding to the variety and extract the corresponding 7 SNP loci information;

[0074] 3) The corresponding site information was compared with the established fingerprint database. At the same time, the whole genome of the sample was sequenced to confirm the variety of Fructus Aurantii Immaturus. The results are shown in Table 6 below.

[0075] Table 6

[0076]

[0077] The results are shown in Table 6. It can be seen from the table that the fingerprint of the present invention can be used to accurately identify the varieties of Fructus Aurantii Immaturus, which is consistent with the actual results obtained by sequencing.

[0078] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A set of SNP molecular marker combinations for constructing a fingerprint of Fructus Aurantii Immaturus, characterized in that: The SNP molecular marker combination includes a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, a fourth SNP molecular marker, a fifth SNP molecular marker, a sixth SNP molecular marker and a seventh SNP molecular marker; The first SNP molecular marker is located at nucleotide 5059088 of chromosome scaffold85909_cov90 of the reference genome, and the polymorphism is A or T; The second SNP molecular marker is located at nucleotide 844624 of chromosome scaffold85962_cov92 of the reference genome, and the polymorphism is T or C; The third SNP molecular marker is located at nucleotide 3327 of chromosome scaffold86470_cov3269 of the reference genome, and the polymorphism is C or A; The fourth SNP molecular marker is located at nucleotide 377175 of chromosome scaffold85934_cov88 of the reference genome, and the polymorphism is C or A; The fifth SNP molecular marker is located at nucleotide 522217 of chromosome scaffold132_cov94 of the reference genome, and the polymorphism is C or T; The sixth SNP molecular marker is located at nucleotide 1237093 of chromosome scaffold85919_cov74 of the reference genome, and the polymorphism is A or G; The seventh SNP molecular marker is located at nucleotide 1427222 of chromosome scaffold4070_cov90 of the reference genome, and the polymorphism is A or T; The reference genome is the citrus reticulata genome.

2. A primer set and / or probe for detecting the SNP molecular marker combination according to claim 1.

3. A kit, characterized in that The kit comprises the primer set and / or probe according to claim 2.

4. A gene chip, characterized in that The gene chip comprises the primer set and / or probe according to claim 2.

5. Use of at least one of the SNP molecular marker combination according to claim 1, the primer set according to claim 2, the kit according to claim 3, and the gene chip according to claim 4 in any one of the following 1) to 5): 1) Identify or assist in identifying varieties of Fructus Aurantii Immaturus; 2) Analysis of genetic structure of Fructus Aurantii Immaturus cultivars; 3) Analysis of genetic diversity of Fructus Aurantii Immaturus resources; 4) Construction of DNA fingerprint of Fructus Aurantii Immaturus; 5) Genotyping of Fructus Aurantii Immaturus varieties.

6. The use according to claim 5, characterized in that The described varieties of Citrus aurantium include Zhejiang Pan'an Daidaihua, Zhejiang Huyou, Fujian Xingtian Huyou, Zhejiang Quzhou Daidaihua, Sichuan Shugeng Agriculture Daidaihua grafted seedlings, Guangdong Xinhui tangerine peel, Hunan Yuanjiang yellow peel sour orange, Jiangxi Xingan stinky orange, Jiangxi Xingan County Xinxiang No. 1, Nancheng stinky orange, Nancheng fragrant orange, Zhejiang Zhuluan, Zhejiang small red orange, wide-peeled orange, and pomelo.

7. A method for constructing DNA fingerprints of Fructus Aurantii Immaturus varieties, characterized in that: The method comprises the following steps: extracting total DNA from different Fructus Aurantii varieties; performing SNP site polymorphism detection using the SNP molecular marker combination of claim 1, and determining the genotyping of the SNP molecular marker according to the detection results of each SNP site in the combination, thereby constructing a DNA fingerprint of the Fructus Aurantii varieties; The described varieties of Citrus aurantium include Zhejiang Pan'an Daidaihua, Zhejiang Huyou, Fujian Xingtian Huyou, Zhejiang Quzhou Daidaihua, Sichuan Shugeng Agriculture Daidaihua grafted seedlings, Guangdong Xinhui tangerine peel, Hunan Yuanjiang yellow peel sour orange, Jiangxi Xingan stinky orange, Jiangxi Xingan County Xinxiang No. 1, Nancheng stinky orange, Nancheng fragrant orange, Zhejiang Zhuluan, Zhejiang small red orange, wide-peeled orange, and pomelo.

8. The construction method according to claim 7, characterized in that: SNP site polymorphism detection was performed using sequencing, chip, KASP or QPCR methods.

9. The DNA fingerprint of Fructus Aurantii Immaturus obtained by the construction method according to claim 7 or 8.

10. A method for identifying a variety of Fructus Aurantii Immaturus, characterized in that: The total DNA of the sample to be tested is extracted, and the SNP site polymorphism is detected using the SNP molecular marker combination described in claim 1. The genotype of the SNP molecular marker is determined according to the detection results of each SNP site in the combination, and then compared with the genotype in the DNA fingerprint of the Fructus Aurantii Immaturus variety constructed by the method for constructing the DNA fingerprint of the Fructus Aurantii Immaturus variety according to claim 9.

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