Lithocarpus litseifolius leaf sweet substance significance-related SNP (Single Nucleotide Polymorphism) site

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) site obviously related to sweet substances of Lithocarpus litseifolius leaves and application of the SNP site. The sweet substances comprise trilobatin, phlorizin, 3-hydroxy phlorizin, neohesperidin dihydrochalcone, phloretin and general flavone. The total number of the SNP loci obviously related to different sweet substances provided by the invention is 84, and the physical positions of the SNP loci are compared and determined according to a reference genome with the login number of GCA040182985.1. The specific SNP site physical position and variation information are shown in a table 5. The loci can be further utilized to establish a molecular marker-assisted selection technology to help breeders to quickly screen high-sweetness Lithocarpus litseifolius strains, so that the traditional breeding period is shortened, and the breeding efficiency is improved. The resource guarantee is provided for the development of a novel Lithocarpus litseifolius sweetener, and the reference is also provided for germplasm innovation, variety specificity identification, quality improvement and genetic evolution relationship discussion of excellent Lithocarpus litseifolius.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to SNP loci related to the significant sweetness of Lithocarpus litseifolius leaves. Background Art

[0002] Lithocarpus litseifolius [Hance] Chun is an evergreen tree of the genus Lithocarpus in the Fagaceae family. It is a characteristic plant resource of new food raw materials that widely distributes in the Hunan Xuefeng Mountain range area and uses young leaves as tea beverages (commonly known as sweet tea and Yao tea). "Xupu Yao tea" has been listed as a national geographical indication protection product. Lithocarpus litseifolius is rich in flavonoids and has functions such as blood sugar lowering, blood pressure lowering, and antioxidant.

[0003] Lithocarpus litseifolius has strong adaptability, a large biomass, and strong plant germination ability. New leaves germinate in spring, summer, and autumn, and mature leaves can be picked throughout the year. After being picked, new branches can grow from the leaf axils. Even if the whole plant is cut down, multiple new branches can germinate from the roots. It is a very characteristic resource. It is mainly distributed in mountain forests at an altitude of 200 - 2,000 m south of the Yangtze River, and likes sunlight and is drought-tolerant. The resources in Hunan, Jiangxi and other provinces are particularly rich. According to incomplete statistics, the distribution area of Lithocarpus litseifolius in Hunan Province is about 133,000 hm 2 2, and the area of Lithocarpus litseifolius in Xupu County is about 0.53 hm 2 2. The annual output of fresh young leaves of wild Lithocarpus litseifolius in the whole country is about 8,000 tons, and the annual output of fresh young leaves in the Xuefeng Mountain area of Hunan is 1,600 tons.

[0004] At the present stage, the collection of Lithocarpus litseifolius mainly depends on wild plants, and the artificial cultivation is relatively less. With the development of the Lithocarpus litseifolius industry, the demand for its leaves has increased significantly. If only continuously cutting a large number of wild resources to obtain young leaves, along with the continuous reduction of the wild distribution area, the population and its individual numbers will gradually decrease, and then the loss of genetic diversity within the species will be caused. Therefore, it is particularly important to establish a germplasm resource nursery for Lithocarpus litseifolius to protect its genetic diversity, especially to screen excellent Lithocarpus litseifolius germplasms. Germplasm resource evaluation, from traditional morphological characteristics, cytological structure, physiological and biochemical indexes to modern molecular marker techniques, is committed to revealing the diversity and variation of genetic materials.

[0005] Currently, Lithocarpus litseifolius is mainly developed into tea products with low added value. Considering the chemical components contained in Lithocarpus litseifolius, its potential for in-depth development is relatively large. One is to prepare beverages or instant teas with different flavors based on the characteristics of Lithocarpus litseifolius as a tea drink. The other is to develop it into a new type of sweetener to completely or partially replace sucrose and produce low-sugar or sugar-free foods based on the sweet taste characteristics of Lithocarpus litseifolius.

[0006] Molecular markers are a type of genetic markers that are based on DNA sequence differences and show genetic diversity at the nucleic acid molecular level. They have the advantages of a large number, a large amount of information, high diversity, and being unaffected by the environment. Moreover, the results are highly stable and reliable, making them a powerful tool in the study of genetic diversity of plant germplasm resources. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide SNP loci significantly related to the sweet substances in the leaves of Lithocarpus litseifolius.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] 1. SNP loci significantly related to the sweet substances in the leaves of Lithocarpus litseifolius, and the SNP loci include the following position information:

[0010]

[0011] The sweet substance is trilobatin.

[0012] Further, for the SNP loci significantly related to the sweet substances in the leaves of Lithocarpus litseifolius, when the sweet substance is 3-hydroxyphloridzin, the SNP loci further include the following position information:

[0013]

[0014] Further, for the SNP loci significantly related to the sweet substances in the leaves of Lithocarpus litseifolius, when the sweet substance is phloretin, the SNP loci further include the following position information:

[0015]

[0016]

[0017] Further, for the SNP loci significantly related to the sweet substances in the leaves of Lithocarpus litseifolius, when the sweet substance is phloridzin, the SNP loci further include the following position information:

[0018]

[0019] Further, for the SNP loci significantly related to the sweet substances in the leaves of Lithocarpus litseifolius, when the sweet substance is neohesperidin dihydrochalcone, the SNP loci further include the following position information:

[0020]

[0021] Further, for the SNP loci significantly related to the sweet substances in the leaves of Lithocarpus litseifolius, when the sweet substance is total flavonoids, the SNP loci further include the following position information:

[0022]

[0023] Furthermore, the physical positions of the above SNP loci were determined by alignment with the reference genome with the accession number GCA_040182985.1.

[0024] Specifically, the corresponding variation information for each SNP locus is as follows:

[0025]

[0026]

[0027] Properties and characteristics of several sweet substances in Lithocarpus litseifolius:

[0028] Phloridzin: The molecular formula is C 21 H 24 O 10 , with a molecular weight of 436.41, is a light yellow crystalline powder, non-hygroscopic, easily soluble in hot water and ethanol, slightly soluble in cold water, and has a sweetness about 300 times that of sucrose. It has been included by the Joint FAO / WHO Expert Committee on Food Additives (#2171) and listed as a generally recognized as safe substance (#4674).

[0029] Phloretin: The molecular formula is C 21 H 28 O 12 , with a molecular weight of 472.44, is a slightly yellow needle-shaped crystal. It has good water solubility at low content and is hardly soluble in water at high content. Phloretin is a glycoside formed by the combination of phloretin and the glycoside body as glucoside, and its sweetness is about 300 times that of sucrose.

[0030] 3-Hydroxyphloretin: The molecular formula is C 21 H 24 O 11 , with a molecular weight of 452.41, and its sweetness is about 300 times that of sucrose.

[0031] Neohesperidin dihydrochalcone: The molecular formula is C 28 H 36 O 15 , with a molecular weight of 612.58, is a white needle-shaped crystalline powder, has a very strong sweetness, is non-hygroscopic, has a melting point of 152 - 154 °C, is insoluble in ether, soluble in water, and soluble in dilute alkali solution. It has been included in the Hygienic Standard for the Use of Food Additives GB2760-2007 (I1297) and listed as a generally recognized non-toxic compound. It has high sweetness, low calorie, slow sweetness, long duration, good stability, and no toxicity, and is widely used in the food industry. Its sweetness is about 1500 times that of sucrose.

[0032] Phloretin: The molecular formula is C 15 H 14O5, with a molecular weight of 274.27, is a light yellow to pure white powder. It is soluble in methanol, ethanol, and acetone and is almost insoluble in water. It has been included in the National Food Safety Standard for the Use of Food Additives (GB1886.261-2016) in China, and its sweetness is about 300 times that of sucrose.

[0033] 2. A reagent for detecting the SNP locus described in any one of the above-mentioned schemes.

[0034] Furthermore, the reagent is a molecular probe or a liquid-phase chip.

[0035] 3. Application of the reagent for detecting the SNP locus described in any one of the above-mentioned schemes in identifying or screening the leaf sweet substance traits of Litsea cubeba.

[0036] 4. Application of the reagent for detecting the SNP locus described in any one of the above-mentioned schemes in screening Litsea cubeba germplasm resources or artificial assisted breeding.

[0037] The beneficial effects of the present invention are as follows: This study systematically explores the variation law of the content of sweet substances in Litsea cubeba, conducts reduced-representation genome sequencing, further performs GWAS analysis based on a large number of high-quality SNPs, and finally obtains SNP loci that are significantly correlated with trilobatin, phloridzin, 3-hydroxyphloridzin, neohesperidin dihydrochalcone, phloretin, and total flavonoids. Further utilization of these loci can establish a molecular marker-assisted selection technology to help breeders quickly screen high-sweetness Litsea cubeba strains, shorten the traditional breeding cycle, improve breeding efficiency, while the traditional method requires many years of field trials. It can accelerate the quality improvement breeding of Litsea cubeba. Or combined with gene editing technology (such as CRISPR), directly target and regulate the key genes for the synthesis of sweet substances to achieve the directional improvement of the leaf sweet substances of Litsea cubeba and cultivate high-quality varieties that meet market demands. Further solve the problems that the current industrial development of Litsea cubeba mainly focuses on traditional tea products, with low added value, can only utilize young and tender leaves, low yield, and a taste that is too sweet to be accepted by consumers. At the same time, developing Litsea cubeba into a new type of natural sweetener can not only broaden the source of sweeteners but also make full use of Litsea cubeba resources to achieve industrial upgrading and transformation, which has very important theoretical significance and practical value. The information of these SNP loci not only provides resource guarantee for the development of new sweeteners from Litsea cubeba but also provides reference for the innovation of excellent Litsea cubeba germplasm, the identification of variety specificity, quality improvement, and the exploration of genetic relationship and evolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0039] Figure 1 It is a DNA agarose gel electrophoresis diagram.

[0040] Figure 2 It is the GWAS result of the trait of trilobatin in Lithocarpus litseifolius.

[0041] Figure 3 It is the GWAS result of the trait of 3-hydroxyphloridzin in Lithocarpus litseifolius.

[0042] Figure 4 It is the GWAS result of the trait of phloretin in Lithocarpus litseifolius.

[0043] Figure 5 It is the GWAS result of the trait of total flavonoids in Lithocarpus litseifolius.

[0044] Figure 6 It is the GWAS result of the trait of phloridzin in Lithocarpus litseifolius.

[0045] Figure 7 It is the GWAS result of the trait of neohesperidin dihydrochalcone in Lithocarpus litseifolius. Detailed implementation manners

[0046] Next, the technical solutions of the preferred embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. For the experimental methods without specific conditions noted in the embodiments, they are usually in accordance with conventional conditions or the conditions recommended by the manufacturers.

[0047] Regarding the use of "including", "comprising", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are intended to include but not limited to.

[0048] Example 1

[0049] Materials: The standardized planting base of Lithocarpus litseifolius and the surrounding wild Lithocarpus litseifolius of Hunan Aokang Biotechnology Co., Ltd. were used as test materials, and it was identified as Lithocarpus litseifolius [Hance] Chun of the genus Lithocarpus in the Fagaceae family. The standardized planting base of Lithocarpus litseifolius is located in Heiniutian Village, Yanxi Township, Liangyaping Town, Xupu County, Hunan Province (110.58°E, 27.74°N), with an altitude of about 676m, an average annual temperature of 16.9°C, an average annual rainfall of 1539.1mm, and an average annual frost-free period of 286 days.

[0050] In the early stage of the present invention, the content change rules of five sweet substances, namely trilobatin, phloridzin, 3-hydroxyphloridzin, neohesperidin dihydrochalcone and phloretin, in Lithocarpus litseifolius with different parts (the results are shown in Table 1), tree ages, leaf ages, harvest periods and processing technologies were systematically studied. The results show that trilobatin and phloridzin are the two sweet substances with the highest proportions.

[0051] Table 1 Changes in the Contents of Different Sweet Substances in Different Parts of Lithocarpus litseifolius

[0052]

[0053]

[0054] Note: Different lowercase letters in the same column indicate significant differences in the same substance among different treatments (P<0.05). The same applies hereinafter.

[0055] Example 2

[0056] Thirty-eight germplasm resources of Lithocarpus litseifolius in this study were collected from 12 provinces such as Yunnan, Sichuan, Hunan, and Guizhou, and transplanted in the germplasm resource nursery of Lithocarpus litseifolius in Hunan Yaocha Engineering Technology Research Center (110.58°E, 27.74°N). The specific germplasm resource information is shown in Table 2.

[0057] In April 2022, in the germplasm resource nursery of Lithocarpus litseifolius, three plants were randomly selected from each germplasm, and young leaves (one bud with three leaves) on the current year's new shoots were collected in the east, south, west, and north directions. Mature leaves were collected in the same way in September of the same year, blanched at 105°C for 5 min, dried at 65°C, and pulverized with a universal pulverizer for the determination of the contents of phloridzin, phloretin, 3-hydroxyphloretin, neohesperidin dihydrochalcone, and phloretin.

[0058] Table 2 Germplasm Resource Information

[0059]

[0060]

[0061] Evaluation of Lithocarpus litseifolius Germplasm Resources Based on the Contents of Sweet Substances

[0062] The results of the contents of sweet substances in the young leaves of different Lithocarpus litseifolius germplasm resources are shown in Table 3. The content of phloridzin in the young leaves is between 58.80 mg / g and 144.29 mg / g. The content of phloridzin in SP2 is the lowest (P<0.05), and the content of phloridzin in HX4 is the highest (P<0.05). The content of phloretin is between 1.75 mg / g and 16.87 mg / g, the content of 3-hydroxyphloretin is between 0.32 mg / g and 4.08 mg / g, the content of neohesperidin dihydrochalcone is between 0.18 mg / g and 2.51 mg / g, and the content of phloretin is between 0.24 mg / g and 4.84 mg / g. The contents of the five sweet substances vary greatly.

[0063] Table 3 Contents of Sweet Substances in Young Leaves of Different Lithocarpus litseifolius Germplasms

[0064]

[0065]

[0066] Example 3

[0067] Reduced-representation genome sequencing

[0068] (1) DNA extraction and detection

[0069] CTAB method was used for DNA extraction. Preliminary DNA detection: quantitative analysis by agarose gel electrophoresis, One drop; concentration: Onedrop; purity: One drop, agarose gel electrophoresis. Detection parameters for DNA agarose gel electrophoresis: gel concentration: 1%; voltage: 120v; electrophoresis time: 25min. The DNA quality inspection results are as Figure 1 shown.

[0070] (2) Reduced-representation genome sequencing and genetic diversity analysis

[0071] First, a double-digestion experiment was carried out to construct a pair-end library for the DNA samples with qualified quality inspection, and then Illumina HiSeq PE150 sequencing was used to search for SNP molecular markers. Filter the SNPs to obtain high-quality SNPs for PCA, phylogenetic tree, population structure and other analyses.

[0072] After sequencing on the Illumina platform, a total of 61.19 Gb of clean reads were obtained from 38 Litsea elongata germplasm samples, and Q30 reached over 91.83%. The number of reads for each sample was between 3,761,362 and 8,036,446, and the GC content was between 35.05% and 36.34%. Among them, HH3 obtained the largest amount of data (8,036,446 reads), and GQ1 obtained the smallest amount of data (3,761,362 reads). The number of assembled sequences was between 363,163 and 491,389, the total depth of sequence coverage was between 5,843,978 and 13,285,337, and the average depth of the sequences was between 14.95 and 28.18, indicating high sequencing quality.

[0073] Genetic diversity analysis of the Litsea elongata population

[0074] Population genetic diversity analysis: The population-level variation information obtained by the populations program shows that the number of SNPs ranges from 311,516 to 439,710, the number of SNPs of the transition type ranges from 215,167 to 302,767, the number of SNPs of the transversion type ranges from 97,836 to 136,943, the ratio of transition to transversion types ranges from 2.09 to 2.22, the number of heterozygous SNPs ranges from 175,601 to 253,461, the number of homozygous SNPs ranges from 109,674 to 202,912, the average depth ranges from 13.77 to 25.60, the number of SNPs with genotype missing ranges from 1,837,029 to 2,278,818, and the number of SNPs identical to the reference ranges from 1,788,219 to 2,163,084. The genomic data can be obtained from GenBank with the accession number PRJNA1055704.

[0075] Using the leaf material of the germplasm resource HX4 (Xupu, Hunan) with a relatively high content of sweet substances, the genetic characteristics of the chloroplast and mitochondrial genomes were analyzed.

[0076] (1) Chloroplast genome assembly and annotation

[0077] The whole-genome DNA sequencing library was constructed using Agilent 2100 and sequenced on the Illumina NovaSeq 6000 platform. The chloroplast genome was assembled using Spades, and with the chloroplast genome of MW375417.1 as a reference, the genomic sequencing data map was drawn using Circos (v0.69-9). The NCBI accession number is OM048987.1.

[0078] (2) Mitochondrial genome assembly and annotation

[0079] The library was constructed and sequenced using the Oxford nanopore sequencing device and the Illumina Novaseq 6000 platform. Perl script fill (v 0.2.1) was used to count and filter the sequence data from three different sources. The third-generation alignment software Minimap2 (v2.1) was used to align the initial third-generation data with the reference gene sequence. Canu (v1.4) software was used to perform de novo assembly on the third-generation sequencing data, and Bowtie2 (v2.3.5.1) was used to align the second-generation sequencing data with the third-generation assembled sequence. Then, Unicycler (v0.4.8) software was used to mix and splice the second-generation and third-generation data, and finally, the complete genome of Lithocarpus litseifolius was obtained. The NCBI accession number is ON462106.

[0080] Genotype detection and GWAS

[0081] Method

[0082] Clean reads were aligned to the reference genome GCA_040182985.1_ASM4018298v1_genomic using the Burrows-Wheeler Aligner (BWA) version 0.7.17 (Li and Durbin, 2009) software. The SAM file input to the BWA software was converted to a bam file and sorted using SAMtools version 1.11 (Li et al., 2009). The Picard's MarkDuplicates utility ( https: / / github.com / broadinstitute / picard / releases ) was used to remove potential PCR biases. The Bcftools (Danecek and McCarthy, 2017) software was used to genotype all loci in the genome based on the BAM file and combine the genotype information of all individuals. Then, the bcftools software was used to retain loci with an individual genotyping rate greater than 90% and a MAF greater than 0.05. The quality-controlled data was processed by linear regression using the generalized linear model (GLM), with six phenotypes (Trefoil_glycoside, 3-hydroxyphloridzin, Phloretin, Total_flavonoids, Phlorizin, Neohesperidin_dihydrochalcone) as the dependent variables and PCA as the covariate for population stratification correction.

[0083] Results

[0084] The GWAS analysis of this example covered six phenotypes, and the results revealed multiple SNP loci significantly associated with these phenotypes. Figures 2 to 7 The GWAS analysis results for each phenotype are shown respectively, while Table 5 details the SNP loci, allelic phenotypes, and their significance levels (p-values) related to each phenotype. Table 4 shows the distribution of SNPs on chromosomes.

[0085] Table 4 Distribution of SNPs on chromosomes

[0086]

[0087]

[0088] Table 5 SNP information related to traits

[0089]

[0090]

[0091]

[0092] The GWAS results of the Trefoil_glycoside trait are as follows Figure 2 As shown, in the Trefoil_glycoside trait, significant SNP loci located on chromosomes 7 and 1 were found. Specifically, two loci on chromosome 7 (Chr7_46794273 and Chr7_46794337) showed relatively high significance (both p-values were 9.04E-06), while the locus on chromosome 1 (Chr1_69977740) had slightly lower significance (p = 1.16E-05).

[0093] Figure 3 The GWAS results for the 3-hydroxyphloridzin trait are as follows. For the 3-hydroxyphloridzin trait, significant SNP loci were distributed on chromosomes 1, 3, 5, and 10. Among them, multiple loci on chromosome 3 (Chr3_11359082, Chr3_11359088, and Chr3_11359109) showed relatively high significance (all p-values were 3.94E-05), and the base substitutions at these loci were all A→G. The locus on chromosome 5 (Chr5_63991475) had a significance of 1.33E-05, with a base substitution of A→G. The locus on chromosome 10 (Chr10_23144902) had a significance of 1.01E-06, with a base substitution of T→C.

[0094] Figure 4 The GWAS results for the Phloretin trait are as follows. The analysis of the Phloretin trait showed significant SNP loci on multiple chromosomes, including chromosomes 4, 1, 8, etc. Among them, two loci on chromosome 4 (Chr4_48439054 and Chr4_48439285) showed extremely high significance (both p-values were 4.03E-24), indicating that these loci may be key genetic factors for the Phloretin trait, with base substitutions of C→T and T→C respectively. In addition, Chr1_52024394 on chromosome 1 (p = 2.33E-19) and Chr8_14801901 on chromosome 8 (p = 7.58E-13) also showed significant associations, with base substitutions of C→T and C→T respectively. This trait is related to the phloretin content.

[0095] Figure 5This is the GWAS result of the Total_flavonoids (total flavonoids) trait. The significant SNP loci of the Total_flavonoids trait are mainly concentrated on chromosomes 9 and 6 (see Table 5 for details). Two loci on chromosome 9 (Chr9_31272994 and Chr9_31273095) showed relatively high significance (p-values were both 2.69E-05), and the base substitutions were T→G and G→A respectively. Chr6_11591299 (p = 3.29E-05) on chromosome 6 also showed a significant association, with a base substitution of T→C, and this trait is related to flavonoid content.

[0096] Figure 6 This is the GWAS result of the Phlorizin trait. The significant SNP loci of the Phlorizin trait are distributed on chromosomes 3, 12, 5, and 2. Among them, two loci on chromosome 3 (Chr3_41676062 and Chr3_41676069) showed relatively high significance (p-values were both 1.90E-07), and the base substitutions were G→C and G→A respectively. Chr12_37932912 (p = 6.57E-06) on chromosome 12 and Chr5_88223758 (p = 1.03E-05) on chromosome 5 also showed significant associations, with base substitutions of C→T and C→T respectively. This trait is related to phlorizin content, which is a natural glycoside derivative.

[0097] Finally, the GWAS result of the Neohesperidin_dihydrochalcone trait is as Figure 7 shown. In the Neohesperidin_dihydrochalcone trait, significant SNP loci were found on multiple chromosomes such as chromosomes 4, 1, 11, and 9. Among them, two loci on chromosome 4 (Chr4_48439054 and Chr4_48439285) showed extremely high significance (p-values were both 2.31E-14), indicating that these loci may be key genetic factors for the Neohesperidin_dihydrochalcone trait, and the base substitutions were C→T and T→C respectively. In addition, Chr1_52024394 (p = 8.45E-13) on chromosome 1 and Chr11_17552728 (p = 1.63E-11) on chromosome 11 also showed significant associations, with base substitutions of C→T and T→A respectively. This trait is related to neohesperidin dihydrochalcone content.

[0098] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. The SNP loci significantly associated with sweet substances in the leaves of Ligusticum chuanxiong are characterized by: The SNP site includes the following position information: The sweet substance is clover.

2. The SNP site according to claim 1, characterized in that: The SNP site also includes the following position information: The sweet substance is 3-hydroxyphlorizin.

3. The SNP site according to claim 1, characterized in that: The SNP site also includes the following position information: The sweet substance is phloretin.

4. The SNP site according to claim 1, characterized in that: The SNP site also includes the following position information: The sweet substance is phlorizin.

5. The SNP site according to claim 1, characterized in that: The SNP site also includes the following position information: The sweet substance is neohesperidin dihydrochalcone.

6. The SNP site according to claim 1, characterized in that: The SNP site also includes the following position information: The sweet substance is total flavonoids.

7. A reagent for detecting the SNP site according to any one of claims 1 to 6.

8. The reagent according to claim 7, characterized in that The reagent is a molecular probe or a liquid phase chip.

9. Use of the reagent according to claim 7 in identifying or screening for the properties of sweet substances in the leaves of Ligusticum chuanxiong.

10. Use of the reagent according to claim 7 in screening germplasm resources of Ligusticum chuanxiong or artificial assisted breeding.

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