Chrysanthemum DNA Fingerprint Map Based on SNP Markers, Its Construction Method and Application

Abstract

The present invention discloses a DNA fingerprint of chrysanthemum based on SNP markers, a construction method, and an application thereof. The DNA fingerprint includes 179 specific SNP sites. DNA fingerprints for chrysanthemums of different color systems can be used for accurate and specific identification of chrysanthemum varieties of the same color system or different flower colors. The method for constructing a DNA fingerprint is based on the specificity of DNA of chrysanthemums of the same color system or different color systems, providing a general strategy for constructing fingerprints of chrysanthemum varieties. In combination with population structure analysis, DNA fingerprints of chrysanthemum varieties of the same color system or different color systems are constructed. This method has the advantages of low application cost, short identification time, high accuracy, and better results in identifying chrysanthemum varieties of the same color system or different flower colors.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a chrysanthemum DNA fingerprint map and a construction method and application thereof. Background Art

[0002] Chrysanthemum (Chrysanthemum morifolium Ramat.), a perennial herbaceous flower in the genus Chrysanthemum of the family Asteraceae, is a traditional famous flower with a long history of cultivation in my country and one of the four major cut flowers in the world. It has a wide variety of varieties, vibrant colors, and is widely cultivated and applied, possessing high ornamental and commercial value. Variety identification is a key component of chrysanthemum germplasm resource evaluation. Flower color, as the primary ornamental characteristic of chrysanthemum, is a key trait for variety identification. However, traditional morphological identification methods, such as flower color, are significantly affected by the external cultivation environment (light, temperature, etc.), and have certain limitations in identifying varieties within the same color family. Therefore, constructing DNA fingerprints based on different color families is of great significance for accurately distinguishing and identifying varieties within the same color family.

[0003] In the existing technology, DNA fingerprinting based on molecular markers such as SSR, SRAP, RAPD, AFLP, and RFLP has been widely used for plant variety identification. In recent years, with the rapid development of sequencing technology, SNP markers, as a third-generation molecular marker technology, have been considered to be a molecular marker technology with great application prospects due to their advantages such as high stability, good polymorphism, and large number. They have been identified as recommended markers for the construction of DNA fingerprint databases in the BMT testing guidelines of the International Union for the Protection of Plant Varieties' Rights (UPOV). Currently, the main methods for obtaining SNP data include SNP chips and genome sequencing. Among them, SNP chips have higher accuracy and reliability and have a more mature data analysis process. However, their customization is expensive and they mainly target known variant sites. As sequencing costs continue to decrease, genome sequencing can detect all types of variants at a sufficient depth. DNA fingerprinting has been sporadically reported in the identification of chrysanthemum varieties, primarily using ISSR, SSR, SRAP, and other molecular markers for variety identification and DNA fingerprinting. Compared to SNP markers, these markers are more difficult to detect at high throughput, and the construction of DNA fingerprints for chrysanthemum varieties based on SNP marker technology has not yet been reported. Therefore, by analyzing SNP loci and selecting SNP loci with high polymorphic information content as core SNP markers, and constructing fingerprints for chrysanthemum varieties of different color systems, this can provide a scientific basis for accurate and specific identification of chrysanthemum varieties. Summary of the Invention

[0004] Purpose of the invention: In response to the deficiencies in the prior art, the present invention provides a chrysanthemum DNA fingerprint based on SNP markers that can be used for accurate and specific identification of chrysanthemum varieties with the same or different flower colors.

[0005] The present invention provides a method for constructing a chrysanthemum DNA fingerprint based on SNP markers.

[0006] The present invention also provides the application of a chrysanthemum DNA fingerprint map based on SNP markers or a chrysanthemum DNA fingerprint map construction method in identifying chrysanthemum varieties.

[0007] Technical solution: To achieve the above objectives, the present invention provides a DNA fingerprint of chrysanthemum based on SNP markers, wherein the DNA fingerprint includes 179 SNP sites, and the sites and their specific nucleotides are as follows:

[0008]

[0009]

[0010]

[0011] The method for constructing a DNA fingerprint of chrysanthemum based on SNP markers according to the present invention comprises the following steps:

[0012] (1) The genomic DNA of 141 chrysanthemum materials of different color systems was extracted using the CTAB method. After the quality of the DNA samples was tested, the genomic DNA was digested with the restriction endonuclease (MseI);

[0013] (2) The enzyme-digested fragments obtained in step (1) were connected to sequencing adapters, and then PCR amplified, sample pooled, and fragments recovered to construct a GBS library. After the GBS library passed the quality inspection, paired-end 150 sequencing was performed using the Illumina HiSeq sequencing platform;

[0014] (3) The high-quality clean data obtained from GBS genome sequencing in step (2) were aligned to the chrysanthemum reference genome using BWA software (parameters: mem-t 4-k 32-M). SNPs were detected and filtered using SAMTOOLS software and VCFtools software, respectively, to screen out 370,562 high-quality SNP sites in different color groups of chrysanthemum;

[0015] (4) The SNP sites screened in step (3) were further screened for different color groups based on the missing rate, MAF value, heterozygosity, and PIC value, and one of the two sites with the higher LD value was removed. A total of 293 SNP sites in different color groups of chrysanthemum were screened. Finally, the minimum marker set that can distinguish all varieties was screened using a Python program, and a total of 179 core SNP sites were finally obtained for each color group.

[0016] (5) The DNA fingerprint of different color chrysanthemums based on SNP markers was successfully constructed.

[0017] In step (3), SNP typing data were processed based on high-quality clean data obtained by filtering GBS (Genotyping-by-sequencing) genome sequencing data. High-quality clean reads were aligned to the chrysanthemum reference genome using BWA software (parameters: mem-t 4-k 32-M), and SNP calling was performed using SAMTOOLS software. GBS genome sequencing and SNP marker development were completed by Beijing Novogene Bioinformatics Technology Co., Ltd. Using VCFtools software, 370,562 high-quality SNP sites were obtained by filtering under the conditions of "--minDP 3 --min-alleles 2 --max-alleles2 --max-missing 0.80 --maf 0.05" for subsequent analysis.

[0018] Preferably, the criteria for further screening the SNP sites of different color groups in step (4) are as follows: the deletion rate is 0, the MAF value is greater than 0.1, the heterozygosity is less than 0.2, and the PIC value of the white group is greater than 0.20, the PIC value of the orange group is greater than 0.35, the PIC value of the pink-purple group is greater than 0.20, the PIC value of the red group is greater than 0.35, the PIC value of the yellow group is greater than 0.20, and the PIC value of the green group is greater than 0.25.

[0019] Furthermore, in step (4), core SNP site screening was performed by calculating the minimum allele frequency (MAF), observed heterozygosity (Ho), polymorphism information content (PIC), and linkage disequilibrium (LD) in different color populations using VCFtools and Plink1.9 software based on the genotyping results. The SNP sites in different color populations were screened according to the following criteria: (1) missing data rate of 0; (2) MAF value > 0.1; (3) heterozygosity < 0.2; (4) PIC value of white > 0.20, PIC value of orange > 0.35, PIC value of pink-purple > 0.20, PIC value of red > 0.35, PIC value of yellow > 0.20, and PIC value of green > 0.25. For the SNP sites obtained by screening, one of the two sites with higher LD values was removed using Plink1.9 software according to the condition --indep-pairwise 50 10 0.3, and the Python (https: / / github.com / wan230114 / Get-least-diff-feature) program was used to further screen the minimum markers that can distinguish all varieties to form the core SNP marker set.

[0020] Among them, the chrysanthemum DNA fingerprint map based on SNP markers is used to identify chrysanthemum varieties of the same color or different colors.

[0021] Furthermore, the chrysanthemum DNA fingerprint map based on SNP markers is used to identify chrysanthemum varieties of the same color system.

[0022] Furthermore, the chrysanthemum DNA fingerprint based on SNP markers is used to identify chrysanthemum varieties with different colors.

[0023] Among them, the method for constructing a chrysanthemum DNA fingerprint based on SNP markers is used in identifying chrysanthemum varieties of the same color system or different color systems.

[0024] Furthermore, the SNP marker-based chrysanthemum DNA fingerprint construction method is used to identify chrysanthemum varieties of the same color system.

[0025] Furthermore, the SNP marker-based chrysanthemum DNA fingerprint map construction method is applied in identifying chrysanthemum varieties with different colors.

[0026] The specific process of applying the chrysanthemum DNA fingerprint based on SNP markers in identifying chrysanthemum varieties of the same color or different colors is as follows:

[0027] (1) The DNA of the species to be identified was extracted using the CTAB method.

[0028] (2) After testing the quality of the DNA sample, the DNA was digested with a restriction endonuclease (MseI), and the resulting digested fragments were connected to sequencing adapters. PCR amplification, sample pooling, and fragment recovery were then performed to construct a GBS library. After the GBS library passed the quality inspection, paired-end 150 sequencing was performed using the Illumina HiSeq sequencing platform.

[0029] (3) Obtain the genotype of the core SNP sites of the corresponding color system of the variety and compare it with the fingerprint map. If it is consistent with the corresponding fingerprint map, it is the variety; if it is inconsistent, it is not the variety.

[0030] The core SNP sites finally obtained in each color system in step (4) are 23 for white, 43 for orange, 24 for pink and purple, 21 for red, 40 for yellow and 28 for green.

[0031] The selection of core SNP markers suitable for the identification of different color chrysanthemum variety populations is the key to constructing DNA fingerprints. The marker screening criteria vary depending on the size of the population and the genotype, and the number of core SNP markers screened is also different. The present invention sets different standards based on the different PIC value distribution ranges of SNP sites in different color variety populations, and finally obtains each color core SNP site to constitute a marker set. The population structure analysis of the core SNP marker set and the 370,562 SNP marker sets shows that the core SNP marker set has strong representativeness and discrimination ability, so fewer markers are used to distinguish as many varieties as possible, which is more convenient for variety identification, reduces application costs, and saves identification time. The present invention utilizes 370,562 high-quality SNP sites identified by simplified genome sequencing, and according to parameters such as specific minimum allele frequency (MAF), observed heterozygosity (Ho), polymorphism information content (PIC) and linkage disequilibrium level (LD), respectively, core SNP sites are screened out from 6 color chrysanthemum variety populations to construct DNA fingerprints of different color chrysanthemum varieties. To provide a scientific basis for promoting the application of SNP molecular markers in the accurate and efficient identification of chrysanthemum germplasm resources.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0033] (1) The method for constructing DNA fingerprints of the present invention is based on the specificity of DNA of chrysanthemums of the same color system or different color systems, providing a general strategy for constructing fingerprints of chrysanthemum varieties. In combination with population structure analysis, DNA fingerprints of chrysanthemum varieties of the same color system or different color systems are constructed. In the identification of chrysanthemum varieties of the same color system and different flower colors, it has the advantages of low application cost, short identification time, high accuracy and better effect.

[0034] (2) The chrysanthemum DNA fingerprint of the present invention has the advantages of low application cost, short identification time, high accuracy and better effect in identifying chrysanthemum varieties of the same color system or different flower colors.

[0035] (3) The chrysanthemum DNA fingerprint of the present invention can be used for accurate and specific identification of chrysanthemum varieties of the same color or different flower colors, and can accurately distinguish all tested chrysanthemum varieties of the same color. At the same time, it plays a guiding role in the protection of new varieties, preliminary identification of varieties, and determination of the authenticity and specificity of varieties.

[0036] (4) The fingerprint map construction of the present invention is based on SNP markers to achieve high-throughput detection, and the data analysis is simple and accurate. The key to the fingerprint map construction is to select core SNP markers that are suitable for the same color or different color chrysanthemum variety groups with good stability, strong identification ability and relatively uniform distribution. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The MAF, observed heterozygosity, and PIC value change distribution diagram of the core SNP sites;

[0038] Figure 2 To analyze the population structure of different color groups using the core SNP marker set and all 370,562 SNP loci, including cross-validation error and population structure diagram, A: white, B: orange, C: pink-purple, D: red, E: yellow, and F: green;

[0039] Figure 3 These are the fingerprints of chrysanthemum varieties with different colors: A: white, B: orange, C: pink and purple, D: red, E: yellow and F: green. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0041] The experimental methods described in this example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0042] Example 1

[0043] (1) Experimental materials: 141 chrysanthemum varieties preserved in the “China Chrysanthemum Germplasm Resource Conservation Center” of Nanjing Agricultural University. The flower colors mainly include white, orange, pink purple, red, yellow and green, as shown in Table 1. The white series are shown in NO.B1-B33, the orange series are shown in NO.O1-O10, the pink purple series are shown in NO.P1-P33, the red series are shown in NO.R1-R13, the yellow series are shown in NO.Y1-Y30, and the green series are shown in NO.G1-G22.

[0044] Table 1 Chrysanthemum varieties tested

[0045]

[0046]

[0047]

[0048] (2) The genomic DNA of 141 different color chrysanthemum materials was extracted using the CTAB method. After the DNA sample quality was tested, the genomic DNA was digested with a restriction endonuclease (MseI). The ends of the digested fragments were connected to sequencing adapters P1 and P2Adapter (which can complement the gaps in the digested DNA). Then, PCR was performed to amplify the tag sequences containing P1 and P2 adapters at both ends. DNA fragments were pooled and the DNA of the required interval was recovered by electrophoresis. The GBS library was successfully constructed. Then, Qubit2.0 was used for preliminary quantification, and the library was diluted to 1 ng / μl. Subsequently, the insert size of the library was detected using Agilent 2100. After the insert size met the expectations, the effective concentration of the library was accurately quantified using the Q-PCR method (library effective concentration>2nM) to ensure the quality of the library. After the GBS library passed the quality inspection, different libraries were pooled according to the effective concentration and the target data volume requirements and then sequenced using the Illumina HiSeq sequencing platform for paired-end 150 sequencing. GBS genome sequencing was completed by Beijing Novogene Bioinformatics Technology Co., Ltd.

[0049] (3) The high-quality clean data obtained by filtering the genome sequencing data in step (2) were aligned to the chrysanthemum reference genome (Song A et al. Analyses of a chromosome-scale genome assembly reveal the origin and evolution of cultivated chrysanthemum. Nat Commun, 2023.14:2021) using BWA software (parameters: mem-t 4-k 32-M). SNP calling was performed using SAMTOOLS software. GBS genome sequencing and SNP marker development were completed by Beijing Novogene Bioinformatics Technology Co., Ltd. 370,562 high-quality SNP sites were obtained using VCFtools software under the conditions of "--minDP 3 --min-alleles 2 --max-alleles 2 --max-missing 0.80 --maf 0.05" for subsequent analysis.

[0050] (4) SNP site screening Based on the genotyping results, VCFtools and Plink1.9 software were used to calculate the minimum allele frequency (MAF), observed heterozygosity (Ho), polymorphism information content (PIC), and linkage disequilibrium (LD) in different color populations. SNP sites in different color populations were screened according to the following criteria: (1) missing data rate of 0; (2) MAF value > 0.1; (3) heterozygosity < 0.2; (4) PIC value of white > 0.20, orange > 0.35, pink-purple > 0.20, red > 0.35, yellow > 0.20, and green > 0.25. For the SNP sites obtained by screening, one of the two sites with higher LD values was removed using Plink1.9 software according to the condition --indep-pairwise50 10 0.3, and the minimum markers that can distinguish all varieties were further screened using the Python program (https: / / github.com / wan230114 / Get-least-diff-feature) to form the core SNP marker set.

[0051] Example 2

[0052] Core SNP site screening

[0053] SNP sites were screened for six different color varieties of white, orange, pink-purple, red, yellow, and green according to the method of step (4) of Example 1. First, sites with a deletion rate greater than 0 were removed, and 39,123, 160,822, 45,134, 148,891, 59,285, and 87,380 sites remained for white, orange, pink-purple, red, yellow, and green, respectively; sites with a MAF value less than 10% were removed, and 21,847, 109,983, 25,260, 91,485, 36,758, and 45,649 sites remained for white, orange, pink-purple, red, yellow, and green, respectively; sites with an observed heterozygosity greater than 0.2 were removed, and 23,000 sites remained for white, orange, pink-purple, red, yellow, and green, respectively. 4, 1,963, 418, 2,224, 554, and 610 loci were removed. After removing loci with low PIC values, 41 loci remained in the white color, 89 loci remained in the orange color, 61 loci remained in the pink color, 43 loci remained in the red color, 113 loci remained in the yellow color, and 85 loci remained in the green color after removing loci with a PIC value of less than 0.25. Using the --indep-pairwise 50 10 0.3 condition, Plink 1.9 software was used to remove one of the two loci with high LD values. This resulted in 32, 52, 46, 28, 77, and 58 loci remaining in the white, orange, pink, red, yellow, and green color, respectively. Finally, the minimum number of markers that can distinguish all varieties was calculated using a Python program, and the chromosome coverage of SNP sites reached more than 70%. The final number of core SNP sites obtained was 23, 43, 24, 21, 40, and 28 for white, orange, pink-purple, red, yellow, and green, respectively, as shown in Table 2.

[0054] Table 2 179 SNP sites in the DNA fingerprint of chrysanthemum based on SNP markers

[0055]

[0056]

[0057]

[0058] Example 3

[0059] Description of core SNP site polymorphism and analysis of its identification ability

[0060] Evaluate the polymorphism information of core SNP sites in different color breed populations, such as Figure 1 As shown, the minimum allele frequency (MAF) of SNP markers in the white population ranged from 0.14 to 0.24, with an average of 0.16; the observed heterozygosity (Ho) ranged from 0 to 0.182, with an average of 0.162; and the polymorphism information content (PIC) ranged from 0.208 to 0.3, with an average of 0.234. In the orange population, the MAF values of SNP markers ranged from 0.35 to 0.45, with an average of 0.41; the Ho ranged from 0 to 0.1, with an average of 0.098; and the PIC values ranged from 0.351 to 0.372, with an average of 0.365. The MAF values of SNP markers in the pink-purple population ranged from 0.14 to 0.28, with an average of 0.17; the Ho values ranged from 0.094 to 0.188, with an average of 0.169; and the PIC values ranged from 0.212 to 0.323, with an average of 0.242. The MAF values of SNP markers in the red population ranged from 0.35 to 0.46, with an average of 0.43; the Ho values ranged from 0 to 0.154, with an average of 0.121; and the PIC values ranged from 0.350 to 0.74, with an average of 0.368. The MAF values of SNP markers in the yellow population ranged from 0.13 to 0.48, with an average of 0.19; the Ho values ranged from 0 to 0.167, with an average of 0.149; and the PIC values ranged from 0.204 to 0.375, with an average of 0.250. The MAF values of the SNP markers in the green population ranged from 0.18 to 0.45, with an average of 0.25; the Ho values ranged from 0 to 0.182, with an average of 0.154; and the PIC values ranged from 0.253 to 0.373, with an average of 0.295. The results showed that the core SNP markers in each color population were highly polymorphic. The core SNP names and polymorphism information for each color population are shown in Table 3.

[0061] Table 3 Core SNP names and polymorphism information of each color line

[0062]

[0063]

[0064]

[0065] The core SNP marker set and all 370,562 high-quality SNP marker sets were used to analyze the population structure of chrysanthemum varieties with different color systems, such as Figure 2The discriminatory power of the core SNP loci was further evaluated. Using the obtained high-quality SNP loci and the core SNP marker set, population structure analysis was performed on different chrysanthemum varieties using Admixture software. The software employed a likelihood nested model, with the K value (number of subpopulations) set between 2 and 5. The cross-validation error (CV error) was derived after the calculation. The K value with the lowest CV error was the optimal number of subpopulations for the population. Data were then visualized in R using a Q file containing the probability that each cultivar's variation originated from a particular subpopulation. A population structure stacking plot was generated. The results showed that the number of subpopulations for each color variety (A: white, B: orange, C: pink-purple, D: red, E: yellow, F: green) was essentially the same using both the core SNP marker set and the full SNP marker set. The population structure analysis results showed little difference. Overall, the core SNP marker sets for the six color varieties exhibited strong representativeness and discriminatory power, and can serve as core SNPs for constructing fingerprints for different color varieties.

[0066] Example 4

[0067] Construction of DNA fingerprint

[0068] Using Microsoft Excel, each row represents a SNP site, and each column represents a variety material number. Based on the obtained core SNP sites of different color systems, DNA fingerprints of 33 white varieties, 10 orange varieties, 33 pink-purple varieties, 13 red varieties, 30 yellow varieties, and 22 green varieties were constructed. Figure 3 As shown (A: white, B: orange, C: pink-purple, D: red, E: yellow, F: green), each row represents a SNP site, each column represents a variety material number, red is homozygous site 00, light blue is heterozygous site 01, and dark blue is homozygous site 11. This fingerprint can be used to identify chrysanthemum varieties with different flower colors.

[0069] Example 5

[0070] DNA fingerprinting applications

[0071] Two chrysanthemum varieties with similar colors but unspecified specific varieties were selected and numbered a and b. First, DNA from the two chrysanthemum varieties was extracted using the CTAB method. After testing the DNA sample quality, the DNA was digested with a restriction endonuclease (MseI). The resulting digested fragments were ligated with sequencing adapters. PCR amplification, sample pooling, and fragment recovery were then performed to construct a GBS library. After the GBS library passed quality inspection, paired-end 150 sequencing was performed using the Illumina HiSeq sequencing platform to obtain the genotype of the core SNP sites of the corresponding color system of the variety. The genotype was compared with the fingerprint map. If the genotype was consistent with the corresponding fingerprint map, the variety was identified as the white variety; otherwise, it was not. If the genotype of the core SNP sites of the white color system for both varieties a and b was obtained and compared with the white color system fingerprint map, variety a was consistent with the B29 fingerprint map and was identified as the variety 'Tehuangrongcai'. B was consistent with the B30 fingerprint map and was identified as the variety 'Huanghua Fuji'. Therefore, the DNA fingerprint of the present invention is applicable to the identification of any chrysanthemum varieties of the same color system or different color systems.

Claims

1. Application of a DNA fingerprint of chrysanthemum based on SNP markers in identifying chrysanthemum varieties, wherein the DNA fingerprint includes 179 SNP sites, and the sites and their specific nucleotides are as follows:

2. The use according to claim 1, characterized in that The application of the chrysanthemum DNA fingerprint map based on SNP markers in identifying chrysanthemum varieties of the same color system or different color systems.

3. The use according to claim 1, characterized in that The invention discloses an application of the method for constructing a chrysanthemum DNA fingerprint based on SNP markers in identifying chrysanthemum varieties of the same color system or different color systems.

4. The use according to claim 1, characterized in that The specific process of the application is: comparing the genotype of the corresponding color SNP site obtained by sequencing with the fingerprint map, if it is consistent with the corresponding fingerprint map, it is the variety, otherwise it is not.

5. The use according to claim 1, characterized in that The specific process of the application is as follows: (1) DNA of the variety to be identified is extracted using the CTAB method; (2) after the quality of the DNA sample is tested, the DNA is digested with the restriction endonuclease MseI, the obtained digested fragments are connected to the sequencing adapter, and then PCR amplification, sample pooling, and fragment recovery are performed to construct a GBS library. After the GBS library passes the quality inspection, paired-end 150 sequencing is performed using the Illumina HiSeq sequencing platform; (3) the genotype of the corresponding color SNP site of the variety is obtained and compared with the fingerprint map. If it is consistent with the corresponding fingerprint map, it is the variety; if it is inconsistent, it is not the variety.

6. The use according to claim 1, characterized in that The method for constructing a chrysanthemum DNA fingerprint based on SNP markers comprises the following steps: (1) The genomic DNA of chrysanthemum materials of different color systems was extracted using the CTAB method. After the quality of the DNA samples was tested, the genomic DNA was digested with the restriction endonuclease MseI; (2) Connecting the enzyme-digested fragments obtained in step (1) to sequencing adapters, followed by PCR amplification, sample pooling, and fragment recovery to construct a GBS library. The library is tested, and sequencing is performed after the library passes the test; (3) The high-quality clean data obtained by GBS genome sequencing in step (2) were aligned to the chrysanthemum reference genome using BWA software, and then SNP detection and filtering were performed to screen out high-quality SNP sites in different color groups of chrysanthemum; (4) The SNP sites screened out in step (3) were screened again, and the SNP sites of different color groups of chrysanthemum were screened and calculated using a Python program to screen the minimum marker set that can distinguish all varieties, and the final core SNP sites of each color group were obtained. The DNA fingerprint map was successfully constructed; In step (3), SNP detection was performed on SNPs of different color groups using SAMTOOLS software, polymorphic sites in the population were detected using a Bayesian model, and SNPs were filtered using VCFtools software under the conditions "--minDP 3--min-alleles 2--max-alleles 2--max-missing 0.80--maf 0.05" to obtain high-quality SNP sites for subsequent analysis.

7. The use according to claim 6, characterized in that The criteria for further screening the SNP sites of different color groups in step (4) are: missing rate 0, MAF value>0.1, heterozygosity<0.2, and PIC value of each color group>0.20-0.

35.

8. The use according to claim 7, characterized in that The PIC values of the color series are: white series PIC value>0.20, orange series PIC value>0.35, pink and purple series PIC value>0.20, red series PIC value>0.35, yellow series PIC value>0.20, and green series PIC value>0.25.

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