A set of SNP molecular marker combination for constructing a fingerprint of fructus aurantii and application thereof

Abstract

The application discloses a set of SNP molecular marker combination for constructing a fingerprint of fructus aurantii and application thereof. The SNP molecular marker in the SNP molecular marker combination has the advantages of single copy, high polymorphism, site homozygosity and high universality, and can efficiently identify fructus aurantii varieties in four large production areas. The set of SNP molecular marker combination is used to establish a DNA fingerprint, which can be used for database comparison of unknown fructus aurantii varieties to query the source of fructus aurantii, and can also be used for comparison and distinction of known varieties, and can help to identify the authenticity of the varieties and market supervision.

Description

Technical Field

[0001] This invention belongs to the field of agricultural molecular biology, specifically involving a set of SNP molecular marker combinations for constructing the fingerprint spectrum of Citrus aurantium and their applications. Background Technology

[0002] Citrus aurantium L., a plant belonging to the Rutaceae family and its cultivated varieties, is the dried, immature fruit of the orange tree. It is a commonly used traditional Chinese medicine with functions such as regulating qi, relieving chest congestion, promoting digestion, and resolving phlegm. Through long-term artificial cultivation, numerous cultivars of Citrus aurantium have been developed. However, traditional identification of medicinal materials relies primarily on morphological characteristics, which are highly susceptible to environmental factors. Consequently, the market often experiences a mix of superior and inferior varieties, leading to confusion and inconsistent quality, severely impacting the quality of the medicinal materials and hindering the healthy and sustainable development of the Citrus aurantium industry. Therefore, establishing a fingerprint spectrum for Citrus aurantium and classifying it based on this spectrum to provide a reference for quality control in different major producing areas is urgently needed by the Citrus aurantium industry market.

[0003] Currently, the mainstream commercial products of Citrus aurantium (Zhi Ke) are mainly divided into four major producing areas: sour orange, mainly produced in the Sichuan Basin, commonly known as "Sichuan Zhi Ke"; yellow-skinned sour orange, mainly produced in the Dongting Lake Plain, commonly known as "Xiang Zhi Ke"; stinky orange, mainly produced in the Poyang Lake Plain, commonly known as "Jiang Zhi Ke"; and Changshan pomelo, mainly produced in the Jinhua-Quzhou Basin, commonly known as "Quzhou Zhi Ke". Existing techniques for distinguishing Zhi Ke from different producing areas involve measuring the content of naringin, neohesperidin, and luteolin in the medicinal material using liquid chromatography (LC), and then using cluster analysis and principal component analysis to construct a similarity fingerprint spectrum based on the content coefficients, thereby differentiating the regional differences in Zhi Ke varieties. However, with cross-breeding in different regions, the limitations of LC measurement are becoming increasingly apparent. Therefore, finding an economical, efficient, and accurate method for regional variety identification is of great significance for improving the efficiency of Zhi Ke germplasm resource management, germplasm resource protection capabilities, and 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 prior art. To this end, the present invention proposes a set of SNP molecular marker combinations for constructing the fingerprint spectrum of Citrus aurantium.

[0005] The present invention also proposes primer sets and / or probes for detecting the above-mentioned SNP molecular marker combinations.

[0006] The present invention also proposes a reagent kit.

[0007] This invention also proposes a gene chip.

[0008] This invention also proposes applications of the above-mentioned molecular marker combinations, primer sets, kits, and / or gene chips.

[0009] This invention also proposes a method for constructing a DNA fingerprint of a Citrus aurantium variety.

[0010] This invention also proposes a DNA fingerprint of the Citrus aurantium variety constructed using the above method.

[0011] This invention also proposes a method for identifying varieties of Citrus aurantium.

[0012] According to a first aspect of the present invention, a set of SNP molecular marker combinations for constructing fingerprint patterns of Citrus aurantium is proposed, the SNP molecular marker combination including 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 on 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 on chromosome scaffold85962_cov92 of the reference genome, with a polymorphism of T or C;

[0015] The third SNP molecular marker is located at nucleotide 3327 on 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 on 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 on 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 on 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 on chromosome scaffold4070_cov90 of the reference genome, and the polymorphism is A or T;

[0020] The reference genome is the Citrus reticulate scaffold reference genome of the mandarin orange, 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 invention, primer sets and / or probes for detecting the above-described SNP molecular marker combinations are provided.

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

[0023] According to a fourth aspect of the present invention, a gene chip is provided, the gene chip comprising the aforementioned primer set and / or probes.

[0024] According to a fifth aspect of the present invention, the application of the above-described SNP molecular marker combinations, primer sets, kits, and / or gene chips in any of the following 1)-5):

[0025] 1) To identify or assist in the identification of varieties of Citrus aurantium;

[0026] 2) Genetic structure analysis of *Citrus aurantium* cultivars;

[0027] 3) Genetic diversity analysis of Citrus aurantium resources;

[0028] 4) Construct a DNA fingerprint of Citrus aurantium;

[0029] 5) Genotyping of trifoliate orange varieties.

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

[0031] According to a sixth aspect of the present invention, a method for constructing a DNA fingerprint of a bitter orange variety is provided. The method includes the following steps: extracting total DNA from different bitter orange varieties; using the above-mentioned SNP molecular marker combination to detect SNP site polymorphism; determining the genotype of the SNP molecular marker based on the detection results of each SNP site in the combination; thereby constructing a DNA fingerprint of a bitter orange variety.

[0032] Among them, the varieties of bitter orange include Pan'an bitter orange from Zhejiang, Zhejiang pomelo, Xingtian pomelo from Fujian, bitter orange from Quzhou, Zhejiang, grafted bitter orange seedlings from Shugeng Agriculture in Sichuan, Chenpi from Xinhui, Guangdong, Huangpi sour orange from Yuanjiang, Hunan, stinky orange from Xingan, Jiangxi, Xinxiang No. 1 from Xingan County, Jiangxi, stinky orange from Nancheng, fragrant orange from Nancheng, Zhuluan from Zhejiang, small red orange from Zhejiang, loose-skinned mandarin orange, and pomelo.

[0033] In some embodiments of the present invention, sequencing, microarray, KASP, or qPCR methods are used to detect SNP site polymorphisms.

[0034] According to a seventh aspect of the present invention, a DNA fingerprint of a bitter orange variety is constructed using the above-described method for constructing a DNA fingerprint of a bitter orange variety.

[0035] According to an eighth aspect of the present invention, a method for identifying varieties of Citrus aurantium is proposed, wherein total DNA is extracted from the sample to be tested, SNP site polymorphism is detected using the above-mentioned SNP molecular marker combination, 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 Citrus aurantium constructed by the above-mentioned method for constructing DNA fingerprint of Citrus aurantium varieties.

[0036] In some embodiments of the present invention, sequencing, microarray, KASP, or qPCR methods are used to detect SNP site polymorphisms.

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

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

[0039] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort 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; unless otherwise specified, the materials and reagents used are commercially available.

[0041] In the description of this invention, the use of terms such as first, second, third, etc., is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0042] "And / or" is used to indicate that one or both of the described 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 markers for detecting varieties of Citrus aurantium

[0044] This embodiment provides a set of SNP molecular marker combinations for detecting varieties of Citrus aurantium, obtained as follows:

[0045] (1) Collection of Citrus aurantium resources

[0046] Through research at various planting bases and markets, more than 30 varieties were identified, and 15 representative and high-quality bitter orange peel resources were selected, as shown in Table 1 below.

[0047] Table 1

[0048]

[0049] (2) Genome sequencing

[0050] Leaves of *Citrus trifoliata* were collected, and DNA extraction and sequencing were performed at the Huazhi National Key Laboratory. The reference genome used was the *Citrus reticulate* citrus reference genome at the scaffold level. The link to the reference genome location is http: / / citrus.hzau.edu.cn / orange / download / index.php. SNP differential sites were mined.

[0051] (3) SNP screening

[0052] SNP mining was performed using SNP locus data obtained from sequencing, based on the following parameters: 1) no SNP deletions in all samples; 2) homozygous base types; 3) frequency of the lowest genotype greater than 0.2.

[0053] A total of 7899 valid SNP loci were obtained. Further simplification screening was adopted, and the loci with the highest frequency of the lowest genotype were selected and superimposed with loci that could efficiently distinguish different samples for replacement. The minimum number of loci was selected to distinguish 15 bitter orange resources, and finally 7 SNP loci were obtained, which can distinguish 15 bitter orange varieties as shown in Table 1. The SNP locus information is shown in Table 2 below.

[0054] Table 2

[0055] 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 specific sequence information of the 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; and 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 Citrus aurantium

[0061] 1. Obtaining the DNA fingerprint of Citrus aurantium varieties

[0062] Based on the SNP molecular marker combinations obtained in Example 1 for detecting the varieties of Citrus aurantium, the position information and base information of the corresponding SNP molecular marker combinations of the standard varieties of Citrus aurantium were obtained, including Zhejiang Pan'an Daidaihua, Zhejiang Huyou, Fujian Xingtian Huyou, Zhejiang Quzhou Daidaihua, Sichuan Shugeng Agriculture Daidaihua grafted seedlings, Guangdong Xinhui Chenpi, Hunan Yuanjiang Huangpi Suancheng, Jiangxi Xingan Choucheng, Jiangxi Xingan County Xinxiang No. 1, Nancheng Choucheng, Nancheng Xiangcheng, Zhejiang Zhuluan, Zhejiang Xiaohongcheng, Kuanpiju, and Youzi. The DNA fingerprint of the Citrus aurantium varieties was obtained, as shown in Table 4.

[0063] Table 4

[0064]

[0065] 2. Validation of fingerprint data

[0066] DNA was extracted from 15 additional samples of bitter orange (Citrus aurantium) resources, including those from Pan'an, Zhejiang; Zhejiang pomelo; Xingtian, Fujian; Quzhou, Zhejiang; grafted seedlings of Bitter Orange from Shugeng Agriculture, Sichuan; Xinhui, Guangdong; Yuanjiang, Hunan; Xingan, Jiangxi; Xinxiang No. 1, Jiangxi; Nancheng; Nancheng; Zhuluan, Zhejiang; Xiaohongcheng, Zhejiang; Mandarin orange; and pomelo. Three replicates were used for each variety. Using a reference genome, 200 bp sequences were obtained from the upper and lower flanking regions of the corresponding loci and sent to Shanghai Sangon Biotech for first-generation sequencing. The first-generation sequencing verified the fingerprinting locus data of the 15 bitter orange resources, as shown in Table 5 below. Simultaneously, the whole genome of the samples was sequenced to confirm the bitter orange variety. The locus data and sequencing data were compared and found to be consistent, making them suitable for use in the fingerprinting reference database.

[0067] Table 5

[0068]

[0069]

[0070] 3. Application of fingerprinting and database

[0071] Five varieties were randomly selected from those sold in the market and also from online sources for identification. The process is as follows:

[0072] 1) Purchase five varieties of bitter orange peel from the market;

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

[0074] 3) The corresponding locus 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 Citrus aurantium. The results are shown in Table 6 below.

[0075] Table 6

[0076]

[0077] The results are shown in Table 6. As can be seen from the table, the fingerprint spectrum of the present invention can be used to accurately identify the varieties of Citrus aurantium, which is consistent with the actual results obtained from sequencing.

[0078] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of primer sets and / or probes for detecting SNP molecular marker combinations used to construct the fingerprint spectrum of Citrus aurantium in any of the following 1)-4): 1) To identify or assist in the identification of varieties of Citrus aurantium; 2) Genetic diversity analysis of Citrus aurantium resources; 3) Construct a DNA fingerprint of Citrus aurantium; 4) Genotyping of trifoliate orange varieties; 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 100 bp of the sequence shown in SEQ ID NO:1, and has a polymorphism of A or T; The second SNP molecular marker is located at 100 bp of the sequence shown in SEQ ID NO:2, and has a polymorphism of T or C; The third SNP molecular marker is located at 100 bp of the sequence shown in SEQ ID NO:3, and has a polymorphism of C or A; The fourth SNP molecular marker is located at 100 bp of the sequence shown in SEQ ID NO:4, and has a polymorphism of C or A; The fifth SNP molecular marker is located at 100 bp of the sequence shown in SEQ ID NO:5, and has a polymorphism of C or T. The sixth SNP molecular marker is located at 100 bp of the sequence shown in SEQ ID NO:6, and has a polymorphism of A or G; The seventh SNP molecular marker is located at 100 bp of the sequence shown in SEQ ID NO:7, and has a polymorphism of A or T.

2. The use of a reagent kit in any of the following 1)-4): 1) To identify or assist in the identification of varieties of Citrus aurantium; 2) Genetic diversity analysis of Citrus aurantium resources; 3) Construct a DNA fingerprint of Citrus aurantium; 4) Genotyping of trifoliate orange varieties; The kit includes the primer set and / or probe as described in claim 1.

3. An application of a gene chip in any of the following 1)-4): 1) To identify or assist in the identification of varieties of Citrus aurantium; 2) Genetic diversity analysis of Citrus aurantium resources; 3) Construct a DNA fingerprint of Citrus aurantium; 4) Genotyping of trifoliate orange varieties; The gene chip includes the primer set and / or probes as described in claim 1.

4. The use of the SNP molecular marker combination as described in claim 1 in any one of the following 1)-4): 1) To identify or assist in the identification of varieties of Citrus aurantium; 2) Genetic diversity analysis of Citrus aurantium resources; 3) Construct a DNA fingerprint of Citrus aurantium; 4) Genotyping of the varieties of Citrus aurantium.

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