Lily SSR primer and application thereof in population genetic diversity analysis

Abstract

The invention belongs to the technical field of molecular markers, and particularly relates to a lily SSR primer and application thereof in population genetic diversity analysis. The lily SSR primer comprises 17 pairs of SSR labeled primers, namely, IdS1-IdS17, and 17 pairs of SSR labeled primers, namely, IdS1-IdS17. Each pair of SSR labeled primers comprises a forward primer and a reverse primer. According to the invention, SSR primers with relatively high polymorphism and relatively strong specificity are screened from lily germplasm by utilizing an SSR molecular marker technology, and the genetic diversity and genetic relationship of 200 parts of lily experimental materials under 20 populations are analyzed through 17 pairs of core primers obtained by screening. According to the method, the genetic diversity of the lilies is analyzed from the molecular level, the main source of genetic differentiation of the lilies is disclosed, clustering analysis is carried out on 200 lily germplasms, and the relation between geographic positions and genetic differentiation of lily populations is explored.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and particularly relates to lily SSR primers and applications thereof in population genetic diversity analysis. Background Art

[0002] Lily is a member of the Liliaceae family ( Lilicaeae ) Lilium ( Lilium Lily is a herbaceous plant with medicinal, edible, and ornamental uses. my country is rich in lily resources, with approximately 47 species and 18 varieties, of which 36 species and 15 varieties are unique to my country. The 2020 edition of the Chinese Pharmacopoeia specifies three lily strains as medicinal: Lilium spp., Lilium dasyphyllum, and Lilium tenuifolium. Their underground bulbs are used as medicine and have anti-tumor, antioxidant, anti-inflammatory, antidepressant, and antiviral properties.

[0003] Due to its significant medicinal, edible, and ornamental value, lilies have seen increasing market demand in recent years. Limited wild lily resources are struggling to meet this demand, further stimulating the development of the cultivated lily industry. However, this expansion in lily cultivation has not been based on quality. Most growers lack professional knowledge in lily cultivation and management, leading to confusion in breeding practices. Furthermore, issues such as long production cycles, high production costs, susceptibility to toxins during asexual reproduction, and inherent varietal degeneration are becoming increasingly prominent. This results in inconsistent lily quality on the market, hindering the sustainable and healthy development of Hunan's lily production system.

[0004] Genetic diversity research aims to quantify the genetic variation characteristics between individuals and populations, and plays an important role in the collection, protection, evaluation and utilization of lily germplasm resources. Molecular markers are powerful tools for genetic diversity analysis. They can be used for genetic diversity assessment, kinship identification, gene mapping, conservation genetics and breeding, and can provide data support for future species genetics and evolutionary studies. Genetic diversity analysis based on molecular markers has the advantages of high stability, comprehensive information and little influence from external factors. It can better understand the genetic richness and kinship of species. At present, the analysis of genetic information and kinship of Hunan lilies is relatively limited, and there is an urgent need to develop new molecular markers to meet the research on genetic information and kinship analysis of Hunan lilies. Summary of the Invention

[0005] The present invention aims to develop a molecular marker for analyzing the genetic information and kinship of Hunan lilies, and provides a lily SSR primer and its application in population genetic diversity analysis. To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] The present invention provides a lily SSR primer, which comprises 17 pairs of SSR marker primers: IdS1-IdS17.

[0007] The above 17 pairs of SSR marker primers are shown in Table 1.

[0008] Table 1 Lily-specific SSR primers Based on CNKI documents, the present invention selected 100 SSR primer pairs. PCR amplification was then performed using genomic DNA from three lily germplasms as templates. The products were screened on a 2.0% agarose gel to identify 40 SSR primer pairs. PCR amplification was then performed using genomic DNA from 10 lily germplasms from different populations as templates using the 40 pre-screened SSR primer pairs. The products were then electrophoresed on 8.0% denaturing polyacrylamide gels and then silver-stained. Ultimately, 17 SSR primer pairs were identified, designated IdS1 to IdS17.

[0009] The 17 pairs of SSR markers provided by the present invention all showed target bands with clear bands and high specificity, and can be used for subsequent SSR genetic diversity analysis and phylogenetic analysis. The 17 pairs of SSR markers provided by the present invention can be used for research on the genetic information and phylogenetic analysis of Hunan lilies, which can solve the problem that the current genetic information and phylogenetic analysis based on Hunan lilies is relatively limited.

[0010] The present invention also provides the application of the lily SSR primer in lily genetic diversity analysis and kinship analysis.

[0011] Preferably, the method for analyzing lily genetic diversity and kinship using the lily SSR primers comprises the following steps: The genomic DNA of the lily to be tested is extracted as a template, and each pair of SSR marker primers in the lily SSR primers is used for PCR amplification respectively. Then, the PCR amplification products are subjected to electrophoresis, and the genetic diversity analysis and kinship analysis of the lily to be tested are performed according to the electrophoresis results.

[0012] Preferably, the electrophoresis results are converted into "1,0" gene matrix data, and then subjected to genetic diversity analysis to obtain the genetic diversity analysis results of the lily to be tested.

[0013] Preferably, genetic diversity analysis software is used in the genetic diversity analysis to calculate the matrix data to obtain observation indicators, and the genetic diversity information of the lily to be tested is obtained by analyzing the above observation indicators.

[0014] The observation indicators include the number of alleles, the effective number of alleles, the expected heterozygosity, the Shannon information index, the genetic differentiation coefficient, gene flow, the variation rate between and within populations, the genetic similarity and the genetic distance.

[0015] Preferably, the electrophoresis result is converted into "1,0" gene matrix data, and then subjected to kinship analysis to obtain the kinship analysis result of the lily to be tested.

[0016] Preferably, cluster analysis software is used in the kinship analysis, and the hierarchical cluster analysis method of the statistical model is used to analyze the genotype matrix data to obtain the kinship analysis results of the lily to be tested.

[0017] Preferably, the PCR amplification system is: 0.7 μL of 40 ng / μL template DNA, 10 μL of 2×Taq MasterMix, 0.2 μL of each pair of SSR marker primers, 100 μM forward primer, and 100 μM reverse primer of each pair of SSR marker primers, and sterile water is added to 20 μL.

[0018] 10 μL of 2×Taq MasterMix consists of Taq DNA polymerase, Mg 2+ , dNTPs, PCR stabilizers and enhancers.

[0019] Preferably, the procedure of the PCR amplification is: Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 53°C-58°C for 30 s, extension at 72°C for 1 min 30 s, 35 cycles; final extension at 72°C for 10 min.

[0020] The present invention has the following beneficial effects: The present invention provides a lily SSR primer, which fills a gap in the research on the SSR genetic diversity of Hunan lilies and efficiently obtains the genetic information of Hunan lilies. The lily SSR primer developed by the present invention has been screened and verified multiple times and has good stability and high polymorphism. It can be applied to lily genetic diversity analysis and phylogenetic relationship analysis, and can also be used for genetic identification of lily germplasm resources and assisted breeding. The lily SSR primer provided by the present invention can be used for research on the genetic information and phylogenetic relationship analysis of Hunan lilies, which can solve the problem that the current genetic information and phylogenetic relationship analysis based on Hunan lilies is relatively limited.

[0021] The 17 pairs of lily SSR primers showed high polymorphism and were able to provide reasonable genetic information. Genetic diversity analysis showed that there was little gene exchange among the 20 lily populations, resulting in a high degree of genetic differentiation, with greater inter-population variation than within populations. Phylogenetic relationship analysis completely separated the three lily varieties, clustered all lily germplasm from the same population into a single group, and divided closely related lily populations into a single category, providing a strong basis for future lily genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows the electrophoresis patterns of PCR amplification of some primers in three lily experimental materials in the present invention; among them, BH-SSR8, BH-SSR7, BH-SSR6, BH-SSR5, BH-SSR4, BH-SSR3, BH-SSR2 and BH-SSR1 are tested primers, and labels 1 to 3 are No. 196, Jinbaili, Dingcheng District, Changde, No. 116, Nanjiangqiao Town, Pingjiang County, Yueyang, and No. 146, Zhouwang Town, Longhui County, Shaoyang, respectively; M is a 2000bp marker.

[0023] Figure 2 The figure shows the electrophoresis patterns of PCR amplification of some primers in 10 lily experimental materials; among them, BH-SSR-80, BH-SSR86, BH-SSR-87, BH-SSR88 and BH-SSR90 are tested primers, and labels 1 to 10 are No. 176 Maoping Village, Longhui County, Shaoyang, No. 126 Xiafeng Village, Shuiling Township, Dong'an County, Yongzhou, No. 66 Longdong Village, Shipai Town, Longshan County, Xiangxi, No. 46 Hongxing Village, Longshan County, Xiangxi, No. 196 Golden Lily, Dingcheng District, Changde, No. 96 Wild Lily, Pingjiang County, Yueyang, No. 136 Tantou Town, Longhui County, Shaoyang, No. 86 Longya Lily, Dingcheng District, Changde, No. 36 Shangmu Village, Shipai Town, Longshan County, Xiangxi, and No. 16 Taoxing Village, Shipai Town, Longshan County, Xiangxi; M is a 500bp marker.

[0024] Figure 3 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS1 primer in the present invention. From left to right, the samples are No. 21-30 in Dajing Village, Xiluo Town, Longshan, Xiangxi, No. 91-100 in Pingjiang, Yueyang, No. 101-110 in Yanmen Town, Mayang Miao Autonomous County, Huaihua, and No. 111-120 in Nanjiang Bridge, Pingjiang, Yueyang.

[0025] Figure 4 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS2 primer in the present invention. From left to right, the samples are No. 21-30 in Dajing Village, Xiluo Town, Longshan, Xiangxi, No. 91-100 in Pingjiang, Yueyang, No. 101-110 in Yanmen Town, Mayang Miao Autonomous County, Huaihua, and No. 111-120 in Nanjiang Bridge, Pingjiang, Yueyang.

[0026] Figure 5 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS3 primer in the present invention. The samples from left to right are No. 1-10 in Xiaohe Village, Xiluo Town, Longshan District, Xiangxi, No. 71-80 in Xiaoshui Village, Yushan Town, Longhui District, Shaoyang, No. 81-90 in Longya Lily, Dingcheng District, Changde, and No. 11-20 in Taoxing Village, Shipai Town, Longshan District, Xiangxi.

[0027] Figure 6This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS4 primer in the present invention. The samples from left to right in the figure are No. 1-10 in Xiaohe Village, Xiluo Town, Longshan District, Xiangxi, No. 71-80 in Xiaoshui Village, Yushan Town, Longhui District, Shaoyang, No. 81-90 in Longya Lily, Dingcheng District, Changde, and No. 11-20 in Taoxing Village, Shipai Town, Longshan District, Xiangxi.

[0028] Figure 7 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS5 primer in the present invention. The samples from left to right are No. 1-10 in Xiaohe Village, Xiluo Town, Longshan District, Xiangxi, No. 71-80 in Xiaoshui Village, Yushan Town, Longhui District, Shaoyang, No. 81-90 in Longya Lily, Dingcheng District, Changde, and No. 11-20 in Taoxing Village, Shipai Town, Longshan District, Xiangxi.

[0029] Figure 8 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS6 primer in the present invention. The samples from left to right are No. 1-10 in Xiaohe Village, Xiluo Town, Longshan District, Xiangxi, No. 71-80 in Xiaoshui Village, Yushan Town, Longhui District, Shaoyang, No. 81-90 in Longya Lily, Dingcheng District, Changde, and No. 11-20 in Taoxing Village, Shipai Town, Longshan District, Xiangxi.

[0030] Figure 9 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS7 primer in the present invention. The samples from left to right are No. 1-10 in Xiaohe Village, Xiluo Town, Longshan District, Xiangxi, No. 71-80 in Xiaoshui Village, Yushan Town, Longhui District, Shaoyang, No. 81-90 in Longya Lily, Dingcheng District, Changde, and No. 11-20 in Taoxing Village, Shipai Town, Longshan District, Xiangxi.

[0031] Figure 10 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS8 primer in the present invention. The samples from left to right are No. 141-150 Zhouwang Town, Longhui District, Shaoyang, No. 151-160 Mojia Village, Longhui District, Shaoyang, No. 61-70 Longdong Village, Shipai Town, Longshan District, Xiangxi, and No. 161-170 Zhongfang Town, Huaihua City.

[0032] Figure 11 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS9 primer in the present invention. From left to right, the samples are No. 21-30 in Dajing Village, Xiluo Town, Longshan, Xiangxi, No. 91-100 in Pingjiang, Yueyang, No. 101-110 in Yanmen Town, Mayang Miao Autonomous County, Huaihua, and No. 111-120 in Nanjiang Bridge, Pingjiang, Yueyang.

[0033] Figure 12This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS10 primer in the present invention. Among them, the samples from left to right are No. 1-10 in Xiaohe Village, Xiluo Town, Longshan, Xiangxi, No. 71-80 in Xiaoshui Village, Yushan Town, Longhui, Shaoyang, No. 81-90 in Longya Lily, Dingcheng District, Changde, and No. 11-20 in Taoxing Village, Shipai Town, Longshan, Xiangxi.

[0034] Figure 13 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS11 primer in the present invention. The samples from left to right are No. 191-200 Jinbaili in Dingcheng District, Changde, No. 51-60 Xiangao Village in Pingjiang, Yueyang, No. 141-150 Zhouwang Town in Longhui, Shaoyang, and No. 151-160 Mojia Village in Longhui, Shaoyang.

[0035] Figure 14 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS12 primer in the present invention. From left to right, the samples are No. 21-30 in Dajing Village, Xiluo Town, Longshan, Xiangxi, No. 91-100 in Pingjiang, Yueyang, No. 101-110 in Yanmen Town, Mayang Miao Autonomous County, Huaihua, and No. 111-120 in Nanjiang Bridge, Pingjiang, Yueyang.

[0036] Figure 15 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS13 primer in the present invention. From left to right, the samples are No. 21-30 in Dajing Village, Xiluo Town, Longshan, Xiangxi, No. 91-100 in Pingjiang, Yueyang, No. 101-110 in Yanmen Town, Mayang Miao Autonomous County, Huaihua, and No. 111-120 in Nanjiang Bridge, Pingjiang, Yueyang.

[0037] Figure 16 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS14 primer in the present invention. The samples from left to right are No. 191-200 Jinbaili in Dingcheng District, Changde, No. 51-60 Xiangao Village in Pingjiang, Yueyang, No. 141-150 Zhouwang Town in Longhui, Shaoyang, and No. 151-160 Mojia Village in Longhui, Shaoyang.

[0038] Figure 17 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS15 primer in the present invention. The samples from left to right are No. 1-10 in Xiaohe Village, Xiluo Town, Longshan District, Xiangxi, No. 71-80 in Xiaoshui Village, Yushan Town, Longhui District, Shaoyang, No. 81-90 in Longya Lily, Dingcheng District, Changde, and No. 11-20 in Taoxing Village, Shipai Town, Longshan District, Xiangxi.

[0039] Figure 18This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS16 primer in the present invention. From left to right, the samples are No. 21-30 in Dajing Village, Xiluo Town, Longshan, Xiangxi, No. 91-100 in Pingjiang, Yueyang, No. 101-110 in Yanmen Town, Mayang Miao Autonomous County, Huaihua, and No. 111-120 in Nanjiangqiao, Pingjiang, Yueyang.

[0040] Figure 19 This is the electrophoresis pattern of partial PCR amplification of 200 lily germplasms using the IdS17 primer in the present invention. From left to right, the samples are No. 21-30 in Dajing Village, Xiluo Town, Longshan, Xiangxi, No. 91-100 in Pingjiang, Yueyang, No. 101-110 in Yanmen Town, Mayang Miao Autonomous County, Huaihua, and No. 111-120 in Nanjiang Bridge, Pingjiang, Yueyang.

[0041] Figure 20 This is a phylogenetic cluster diagram of 200 lily germplasm resources based on SSR markers in the present invention. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] Example 1: Development of primer sets based on SSR markers 1.1. Synthesis of SSR marker primers The China National Knowledge Infrastructure (CNKI) literature (https: / / www.cnki.net / ) was consulted to find 100 pairs of SSR primers related to lily (see Table 2 for details). They were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai) and purified using HAP.

[0044] Table 2 100 pairs of SSR marker primers 1.2 Primer validity and polymorphism analysis 1.2.1 Lily experimental materials and sampling Lily resources were collected from Xiangxi, Shaoyang, Yueyang, Changde, Huaihua, and Yongzhou in Hunan Province and uniformly transplanted to a Chinese medicinal material planting base (see Table 3 for details). Tender green leaves of lily plants were collected from April to May of the following year and stored in a container filled with dry ice. After being transported back to the laboratory, they were rinsed with clean water to remove dirt and other impurities and wiped clean with paper towels. Finally, the lily samples (experimental materials) were placed in sealed bags, marked, filled with liquid nitrogen, and stored in a -80°C refrigerator for later use.

[0045] Table 3 Basic information of lily experimental materials 1.2.2 Lily DNA extraction and quality control A plant genomic DNA rapid extraction kit (Tiangen Biotechnology Co., Ltd.) was used to extract genomic DNA from 200 lily germplasm leaves (lily samples). The extracted DNA concentration was detected using an ultraviolet spectrophotometer (Nanodrop2000 / 2000C) and 1.2% agarose gel electrophoresis.

[0046] All 200 samples of lily genomic DNA were able to amplify fragments of approximately >5000bp; a microspectrophotometer was used to detect the purity (OD value: A260 / A280) and concentration of each DNA sample. The lowest concentration was sample number 161 (20.6ng / μL), and the highest concentration was sample number 99 (61.3ng / μL). The OD values ​​ranged from 1.56 to 1.95. The genomic DNA of Polygonatum sibiricum was minimally damaged and had good purity. Impurities such as proteins, RNA, and phenols were relatively completely removed, making it suitable for subsequent lily PCR amplification.

[0047] 1.2.3 PCR amplification of SSR marker primers DNA extracts from ZW-146, DC-JBH-196, and NJQ-116 were used as genomic DNA templates, and 100 pairs of synthesized SSR marker primers (Table 2) were used for PCR amplification. PCR products were detected by 1.2% agarose gel electrophoresis. Primers with clear bands and PCR product sizes within the expected range were considered valid. Figure 1 shown.

[0048] Depend on Figure 1 It can be seen that the bands amplified by the four primers BH-SSR1, BH-SSR4, BH-SSR5 and BH-SSR6 are clear and do not contain any other bands, and can be used for subsequent primer rescreening.

[0049] 40 pairs of primers were initially screened from 100 pairs of SSR primers, as shown in Table 4.

[0050] Table 4 40 pairs of SSR primers for primary screening Among them, PCR amplification uses a 20 μL reaction system, which contains 2 μL of template DNA (20 ng / μL), 2× TaqMasterMix (including: Taq DNA polymerase, Mg2 ＋ , dNTPs, PCR stabilizer and enhancer) 10 μL, forward primer (100 μM) and reverse primer (100 μM) 2 μL each, and make up to 20 μL with sterile water.

[0051] The reaction program for amplification was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 1 min 30 s, 35 cycles; and finally extension at 72°C for 10 min.

[0052] Then, 10 lily accessions (MP-176, SLXXF-126, LD-66, HX-46, DC-JBH-196, YS-96, TT-136, DC-LY-86, SM-36, and TX-16) from different populations were used as DNA templates. PCR amplification of the primary screening primers was performed using the optimal SSR reaction system. The products were subjected to 8.0% denaturing polyacrylamide gel electrophoresis (150V, 1h2min) and silver staining. Primers with clear bands and high polymorphism were selected as core primers. Some of the results are shown in Figure 2. Figure 2 shown.

[0053] Depend on Figure 2 It was found that the bands amplified by the two primers BH-SSR87 and BH-SSR90 were clear and highly polymorphic, and thus could be used as core primers.

[0054] Among them, PCR amplification used a 20 μL reaction system, which contained 0.7 μL of template DNA (20 ng / μL), 10 μL of 2×TaqMasterMix, 0.2 μL each of forward primer (100 μM) and reverse primer (100 μM), and sterile water was added to 20 μL.

[0055] Among them, the components of 2×Taq MasterMix 10μL include: Taq DNA polymerase, Mg 2＋ , dNTPs, PCR stabilizers and enhancers.

[0056] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing for 30 s, extension at 72°C for 1 min 30 s, 35 cycles; final extension at 72°C for 10 min.

[0057] The PCR products were subjected to 8.0% denaturing polyacrylamide gel electrophoresis (150 V, 1 h 2 min). The polyacrylamide gel was then rinsed twice with clean water and placed in the fixative for 13 min. Subsequently, it was rinsed with purified water for 30 s and placed in silver staining solution (mass fraction 0.1% AgNO3) for 8 min. It was rinsed with purified water for 30 s three times and finally placed in the developer and shaken continuously until bands appeared. The gel was then photographed and preserved.

[0058] The fixative was made of 400 mL of purified water, 40 mL of anhydrous ethanol, and 2 mL of glacial acetic acid.

[0059] The developer was prepared from 400 mL of pure water, 2 mL of 37% formaldehyde solution and 5 g of NaOH.

[0060] Finally, 17 pairs of core SSR marker primers (BH-SSR16, BH-SSR23, BH-SSR24, BH-SSR35, BH-SSR37, BH-SSR38, BH-SSR40, BH-SSR57, BH-SSR58, BH-SSR59, BH-SSR64, BH-SSR71, BH-SSR77, BH-SSR78, BH-SSR The above 17 pairs of SSR marker primers were renamed as IdS1, IdS2, IdS3, IdS4, IdS5, IdS6, IdS7, IdS8, IdS9, IdS10, IdS11, IdS12, IdS13, IdS14, IdS15, IdS16 and IdS17) and used for genetic diversity analysis of lily germplasm resources.

[0061] The nucleotide sequences of the above 17 pairs of SSR marker primers are shown in Table 1.

[0062] Wherein, IdS1: the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2.

[0063] IdS2: The nucleotide sequence of the forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0064] IdS3: The nucleotide sequence of the forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6.

[0065] IdS4: The nucleotide sequence of the forward primer is shown in SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.8.

[0066] IdS5: The nucleotide sequence of the forward primer is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.10.

[0067] IdS6: The nucleotide sequence of the forward primer is shown in SEQ ID NO.11, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.12.

[0068] IdS7: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 14.

[0069] IdS8: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 15, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 16.

[0070] IdS9: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 18.

[0071] IdS10: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 19, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 20.

[0072] IdS11: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 21, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 22.

[0073] IdS12: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 23, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 24.

[0074] IdS13: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 25, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 26.

[0075] IdS14: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 27, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 28.

[0076] IdS15: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 29, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 30.

[0077] IdS16: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 31, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 32.

[0078] IdS17: The nucleotide sequence of the forward primer is shown in SEQ ID NO. 33, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 34.

[0079] Example 2: Application of Lily SSR Markers 2.1 Lily Experimental Materials and Sampling Lily resources were collected from Xiangxi, Shaoyang, Yueyang, Changde, Yongzhou, and Huaihua in Hunan Province and uniformly transplanted to a Chinese medicinal material planting base (see Table 3). Tender green leaves of lily plants were collected from April to May of the following year and stored in a container filled with dry ice. After being transported back to the laboratory, soil impurities were rinsed with clean water and wiped clean with paper towels. Finally, the lily samples were placed in sealed bags, marked, filled with liquid nitrogen, and stored in a -80°C refrigerator for later use.

[0080] 2.2 Lily DNA Extraction and Quality Control Genomic DNA was extracted from 200 lily germplasm leaves using a plant genomic DNA rapid extraction kit (Tiangen Biochemical Technology Co., Ltd.). DNA concentration was determined using a UV spectrophotometer (Nanodrop 2000 / 2000C) and 1.2% agarose gel electrophoresis.

[0081] All 200 samples of lily genomic DNA were able to amplify fragments of approximately >5000bp; a microspectrophotometer was used to detect the purity (OD value: A260 / A280) and concentration of each DNA sample. The lowest concentration was sample number 161 (20.6ng / μL), and the highest concentration was sample number 99 (61.3ng / μL). The OD values ​​were between 1.56 and 1.95. The genomic DNA of Polygonatum sibiricum was minimally damaged and had good purity. Impurities such as proteins, RNA, and phenols were relatively completely removed, making it suitable for subsequent PCR amplification of lilies.

[0082] 2.3. SSR marker PCR amplification DNA extracts from ZW-146, DC-JBH-196, and NJQ-116 were used as genomic DNA templates and 100 pairs of synthesized SSR marker primers (Table 2) were used for PCR amplification. PCR products were detected by 1.2% agarose gel electrophoresis. Primers with clear bands and PCR product sizes within the expected range were considered valid. Figure 1 shown.

[0083] Depend on Figure 1 It can be seen that the three lily germplasms can all amplify clear target bands under the four primer pairs of BH-SSR1, BH-SSR4, BH-SSR5 and BH-SSR6.

[0084] Among the 100 pairs of SSR primers, 40 pairs can be used for subsequent primer rescreening, as shown in Table 4.

[0085] The reaction system during amplification is the same as above.

[0086] The reaction program for amplification was pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 1 min 30 s, 35 cycles; and finally extension at 72°C for 10 min.

[0087] Then, 10 lily accessions (MP-176, SLXXF-126, LD-66, HX-46, DC-JBH-196, YS-96, TT-136, DC-LY-86, SM-36, and TX-16) from different populations were used as DNA templates. PCR amplification of the primary screening primers was performed using the optimal SSR reaction system. The products were subjected to 8.0% denaturing polyacrylamide gel electrophoresis (150V, 1h2min) and silver staining. Primers with clear bands and high polymorphism were selected as core primers. Some of the results are shown in Figure 2. Figure 2 shown.

[0088] Depend on Figure 2 It was found that the bands amplified by the two primers BH-SSR87 and BH-SSR90 were clear and highly polymorphic, and thus could be used as core primers.

[0089] The PCR amplification used a 20 μL reaction system and the PCR reaction procedure was the same as above.

[0090] The PCR products were subjected to 8.0% denaturing polyacrylamide gel electrophoresis (150 V, 1 h 2 min). The polyacrylamide gel was then rinsed twice with clean water and placed in fixative for 13 min. Subsequently, the gel was rinsed with purified water for 30 s and placed in silver stain for 8 min. The gel was rinsed with purified water for 30 s three times and then placed in developer and shaken continuously until bands appeared. The gel was then photographed and saved, with DL500 as a DNA marker.

[0091] PCR amplification was performed on 17 pairs of SSR primers (BH-SSR-16, BH-SSR23, BH-SSR-24, BH-SSR-35, BH-SSR-37, BH-SSR-38, BH-SSR-40, BH-SSR-57, BH-SSR-58, BH-SSR-59, BH-SSR-64, BH-SSR-71, BH-SSR-77, BH-SSR-778, BH-SSR-84, BH-SSR-87 and BH-SSR-90) obtained by rescreening.

[0092] The PCR amplification used a 20 μL reaction system and the PCR reaction procedure was the same as above.

[0093] The PCR products were electrophoresed on 8.0% denaturing polyacrylamide gel (150V, 1h2min), and then the polyacrylamide gel was rinsed twice with clean water and placed in fixative for 13min, then rinsed with purified water for 30s and placed in silver staining solution for 8min, rinsed with purified water for 30s three times, and finally placed in developer and shaken continuously until bands appeared, and then photographed and saved. Figures 3 to 19 shown.

[0094] Depend on Figures 3 to 19 It can be seen that all 17 pairs of SSR primers can amplify the target bands, and the bands are clear and complete with high polymorphism, which can be used for subsequent analysis.

[0095] 2.4 Genetic diversity parameters of SSR molecular markers The electrophoretic data from the polyacrylamide gel electrophoresis plot was converted into a "1,0" matrix. The 0,1 type gene data from 200 lily accessions were imported into the GenAIEx 6.5 plug-in. The "Color-brand" - "Color-data" - "Color By PopParameters" option was selected, and the "Binary" option was selected. The remaining options were left as default to obtain genetic diversity information (allele information, Shannon index, and heterozygosity) for the SSR primers. The electrophoretic data from the polyacrylamide gel electrophoresis plot was converted into bp format and imported into Powermarker 3.25 to generate polymorphism information. Information on SSR genetic diversity parameters is shown in Table 5.

[0096] Table 5 Genetic diversity parameters based on 17 SSR markers As shown in Table 5, 62 alleles were detected using 17 SSR primer pairs, with the number of alleles per primer ranging from 2 to 7, with an average of 3.647. SSR-71 was the most polymorphic locus, with 7 alleles. SSR-40, SSR-57, SSR-78, and SSR-84 had fewer alleles, each with 2 alleles. The effective number of alleles (Ne) ranged from 1.201 to 4.334, with an average of 2.340; the Shannon index (I) ranged from 0.075 to 0.693, with an average of 0.532; the observed heterozygosity (Ho) ranged from 0.085 to 1.000, with an average of 0.725; the expected heterozygosity (He) ranged from 0.051 to 0.500, with an average of 0.380; and the polymorphism information content (PIC) ranged from 0.078 to 0.375, with an average of 0.317.

[0097] Analysis of the experimental results showed that the 17 pairs of SSR primers had relatively rich genetic diversity and could be used for subsequent genetic diversity analysis and phylogenetic relationship analysis of 20 Hunan lily populations.

[0098] 2.5 Analysis of Lilium Genetic Diversity The electrophoresis data of the above polyacrylamide gel electrophoresis diagram were converted into a "1, 0" matrix. The 0, 1 type gene data of 200 lily germplasms were imported into the GenAIEx 6.5 plug-in. Click "Color-brand" - "Color-data" - "Color By PopParameters", then select the "Binary" option. The remaining options were set by default to obtain the genetic diversity data. Table 6 shows the genetic diversity results.

[0099] Table 6 Genetic diversity levels of 20 lily populations based on 17 pairs of SSR core primers Table 6 shows that the observed allele number for the 20 lily populations ranged from 1.706 to 1.882; the effective allele number remained between 1.700 and 1.859; the observed heterozygosity ranged from 0.447 to 0.605; the expected heterozygosity ranged from 0.594 to 0.835; and the Shannon diversity index remained between 0.351 and 0.435. The average observed allele number, average effective allele number, average observed heterozygosity, average expected heterozygosity, and average Shannon information index were 1.797, 1.745, 0.532, 0.725, and 0.380, respectively.

[0100] From the above experimental results, it can be seen that the genetic diversity levels of lily populations are as follows: YM, TX, YS, DC-LY, XS, LD, SLXXF, MP, ZF, TT, XH, XYJ, NJQ, ZW, DC-JBH, XG, DJ, MJ, SM, HX, and Yanmen Town (YM) in Mayang Miao Autonomous County, Huaihua City has the highest development potential.

[0101] 2.6 Analysis of Lilium Genetic Differentiation Import the 0,1 type gene data of 200 lily accessions into the GenAIEx 6.5 plug-in. Click “Distance-based” - “AMOVA Data Parameter” - “AMOVA Genetic Distance Options”, then select the “POP” option. Leave the other options as default to obtain the Nei’s analysis results and molecular variance analysis results, as shown in Tables 7 and 8.

[0102] Table 7 Nei's analysis of genetic diversity among 20 lily populations Table 8 Molecular variance analysis among lily populations Nei's analysis results (Table 7) show that the total genetic diversity of the 20 lily populations was 0.1845, the genetic diversity within the population was 0.0776, the genetic differentiation coefficient (Gst) was 0.5796, and the gene flow (Nm) value was 0.3627. The variance analysis results (Table 8) show that the percentage of variation between populations was 56%, and the percentage of variation within populations was 44%.

[0103] From the above experimental results, it can be seen that there is a moderate degree of gene exchange among lily populations, and the degree of variation between populations is higher than that within populations.

[0104] 2.7 Genetic distance and homology analysis The electrophoresis data of the polyacrylamide gel electrophoresis diagram were converted into bp format, and the genetic data of 200 lily germplasms were imported into Popgene32 software. The genetic distance and genetic similarity information were obtained by clicking "Analysis" - "Genetic Distance" - "Nei's Distance". See Tables 9 and 10 for details.

[0105] Table 9 Genetic similarity and genetic distance of 20 lily populations Table 10 Genetic similarity and genetic distance among 20 lily populations (Continued from Table 9) Genetic distance and homology analysis among the 20 lily populations (Table 9) showed that the average genetic distance among the 20 Hunan lily populations was 0.0704, and the average genetic similarity was 0.9324. The genetic distance among the 20 Hunan lily populations ranged from 0.0178 to 0.2366, with the smallest genetic distance between SLXXF and TT at 0.0178, and the largest genetic distance between DC-JBH and HX at 0.2366.

[0106] The above experimental results reveal the genetic differences between lily populations to a certain extent, providing a scientific basis for subsequent breeding optimization.

[0107] 2.8 Analysis of Lily Phylogenetic Relationships The electrophoresis data of the polyacrylamide gel electrophoresis diagram was converted into a "1, 0" matrix. The "Multivariate Analysis" function under the "Statistics" item in Origin 2024 software was used. The genotype data of 200 lily experimental materials were used as variables. The number of clusters was set to "7", the cluster direction was set to "circular", and other options were set to default to generate a cluster diagram. The cluster diagram clustered the 20 lily populations into 6 categories. The results are as follows: Figure 20 shown.

[0108] The results showed that the clustering map divided the 200 lily germplasm resources into six categories. Cluster I (red) contained 40 accessions of Lilium lily, including 10 from Xiaohe Village, Xiluo Town, Longshan, Hunan Province; 10 from Taoxing Village, Shipai Town, Longshan, Hunan Province; 10 from Tantou Town, Longhui County, Shaoyang, Hunan Province; 10 from Dajing Village, Xiluo Town, Longshan, Hunan Province; and 10 from Longdong Village, Shipai Town, Longshan, Hunan Province. Cluster II (blue) contained 30 accessions of Lilium lily, including 10 from Shangmu Village, Shipai Town, Longshan, Hunan Province; 10 from Hongxing Village, Longshan, Hunan Province; and 10 from Xiangao Village, Pingjiang, Yueyang. Cluster III (green) contained 20 accessions of Lilium lily, including 10 from Dingcheng District-Longya Lilium in Changde, Hunan Province; and 10 from Xiaoshui Village, Yushan Town, Longhui County, Shaoyang, Hunan Province. Group IV (purple) contains 50 lily accessions, including 10 wild lily accessions from Pingjiang, Yueyang; 10 from Yanmen Town, Mayang Miao Autonomous County, Huaihua; 10 from Nanjiangqiao, Pingjiang, Yueyang; 10 from Xiafeng Village, Shuiling Township, Dong'an County, Yongzhou; and 10 from Tantou Town, Longhui, Shaoyang. Group V (yellow) contains 50 lily accessions, including 10 from Zhouwang Town, Longhui, Shaoyang; 10 from Mojia Village, Longhui, Shaoyang; 10 from Zhongfang Town, Huaihua; 10 from Maoping Village, Longhui, Shaoyang; and 10 from Xiyangjiang Town, Longhui, Shaoyang. Group VI contains 10 golden lily accessions, including 10 from Dingcheng District, Changde.

[0109] From the above experimental results, it can be seen that the clustering results can objectively reflect the relationship between lily populations and can be verified with the genetic distance to a certain extent.

[0110] In summary, the 17 pairs of SSR marker primer combinations screened by the present invention have good polymorphism, can reflect the rich genetic diversity of Hunan lily, and to a certain extent reveal the phylogenetic relationships and genetic differentiation of 20 Hunan lily populations, laying a theoretical foundation for the future assisted breeding and development and utilization of Hunan lily. The 17 pairs of SSR primers provided by the present invention can be used for research on genetic information and phylogenetic relationship analysis of Hunan lily, which can solve the current problem of the limited genetic information and phylogenetic relationship analysis of Hunan lily.

[0111] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0112] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Lily SSR primer, characterized in that: The lily SSR primers include 17 pairs of SSR marker primers: IdS1 to IdS17; The nucleotide sequences of the forward primers IIdS1 to IdS17 are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, and SEQ ID NO.33, respectively; The nucleotide sequences of the reverse primers of IdS1 to IdS17 are shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32 and SEQ ID NO.34, respectively.

2. Use of the lily SSR primers according to claim 1 in lily genetic diversity analysis and kinship analysis.

3. The use according to claim 2, characterized in that The method for analyzing lily genetic diversity and kinship using the lily SSR primers comprises the following steps: The genomic DNA of the lily to be tested is extracted as a template, and each pair of SSR marker primers in the lily SSR primers is used for PCR amplification respectively. Then, the PCR amplification products are subjected to electrophoresis, and the genetic diversity analysis and kinship analysis of the lily to be tested are performed according to the electrophoresis results.

4. The use according to claim 3, characterized in that The electrophoresis results are converted into matrix data, and then subjected to genetic diversity analysis to obtain the genetic diversity analysis results of the lily to be tested.

5. The use according to claim 4, characterized in that The genetic diversity analysis is performed using genetic diversity analysis software to calculate the matrix data to obtain observation indicators, and the genetic diversity information of the lily to be tested is obtained by analyzing the observation indicators; The observation indicators include the number of alleles, the effective number of alleles, the expected heterozygosity, the Shannon information index, the genetic differentiation coefficient, gene flow, the variation rate between and within populations, the genetic similarity and the genetic distance.

6. The use according to claim 3, characterized in that The electrophoresis results are converted into matrix data, and then subjected to kinship analysis to obtain kinship analysis results of the lily to be tested.

7. The use according to claim 6, characterized in that The kinship analysis is performed using cluster analysis software, and the hierarchical cluster analysis method of the statistical model is used to analyze the matrix data to obtain the kinship analysis results of the lilies to be tested.

8. The use according to claim 3, characterized in that The PCR amplification system is as follows: 0.7 μL of 40 ng / μL template DNA, 10 μL of 2×Taq MasterMix, 0.2 μL of 100 μM forward primer of each pair of SSR marker primers and 100 μM reverse primer of each pair of SSR marker primers, and sterile water is added to 20 μL; 10 μL of 2×Taq MasterMix consists of Taq DNA polymerase, Mg 2+ , dNTPs, PCR stabilizers and enhancers.

9. The use according to claim 3, characterized in that The procedure of the PCR amplification is: Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 53°C-58°C for 30 s, extension at 72°C for 1 min 30 s, 35 cycles; final extension at 72°C for 10 min.

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