Primer combination for identifying peony resources and application thereof

Abstract

The present application belongs to the technical field of molecular biology, and particularly relates to a primer combination for identifying peony resources and application thereof. The present application aims to provide a new method for identifying peony resources. The technical scheme of the present application is a primer combination for identifying peony resources. The present application takes the progeny germplasm resources obtained by natural pollination of Central Plains peony and Northwest peony as materials, and uses 57 pairs of SSR marker primers for screening. Finally, 9 core SSR markers are screened for genotype analysis, and a molecular identity card system is successfully constructed for 190 peony germplasms by using a coding mode combining numbers and uppercase and lowercase English letters.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to primer combinations for the identification of peony resources and their applications. Background Technology

[0002] Peony (Paeonia sect. Moutan) is a perennial woody plant belonging to the genus Paeonia in the family Paeoniaceae. It is a traditional Chinese flower with a long history, often referred to as the "King of Flowers." Widely loved not only as an ornamental plant but also for its significant medicinal value, it is commonly cultivated in temperate regions. There are approximately 2,000 cultivars of peonies worldwide, which can be divided into about 17 major cultivar groups. China, as the origin of peonies, boasts over 1,600 years of cultivation and breeding history, accumulating rich germplasm resources and cultivation experience through long-term practice, and has now cultivated over 1,000 excellent varieties. Based on geographical distribution and cultivation characteristics, Chinese peonies can be mainly divided into four major cultivar groups: Central Plains, Northwest, Jiangnan, and Southwest. Each cultivar group has its own unique characteristics in flower shape, color, and ecological adaptability, collectively forming the rich and diverse horticultural landscape and cultural connotations of Chinese peonies.

[0003] Among the four major peony cultivar groups, the Central Plains peony cultivar group occupies a core position. It is considered the group with the longest cultivation history, the richest variety, and the most profound horticultural influence, serving as the "main thread" of China's peony cultivation system. After more than a thousand years of cultivation, the Central Plains peony has developed over 500 varieties. The Northwest peony cultivar group, also known as the Purple Spotted Peony cultivar group, is most notably characterized by distinct purple spots at the base of its petals. This group comprises approximately 300 varieties, making it the second largest peony cultivar group in China, second only to the Central Plains peony. Since peony seed oil was approved as a new resource food by the Chinese Ministry of Health in 2011, the economic value of Northwest peony, as an important woody oil crop, has received widespread attention nationwide. The healthy development of the peony industry depends on the accurate identification and stable propagation of varieties. However, currently, both the Central Plains and Northwest peonies suffer from widespread confusion in cultivar names and serious issues of homonyms and variants, which pose significant obstacles to cultivar identification, market transactions, and industry promotion, hindering the effective utilization of peony resources and the sustainable development of the industry. Therefore, establishing a scientific and unified identification system has become a key prerequisite for promoting the standardization and specialization of the peony industry.

[0004] DNA molecular markers (Deoxyribonucleic acid molecular markers) are a class of genetic markers based on variations in the nucleotide sequence of genetic material. They directly reflect the genetic differences of individuals at the DNA level and can be stably inherited. Compared with traditional morphological and biochemical markers, DNA molecular markers have significant advantages: First, their detection is not affected by the location of the organism's tissue, developmental stage, or external environmental conditions; second, they exhibit rich variability and high polymorphism among individuals; third, there are diverse and numerous marker types; fourth, most markers show co-dominance, enabling accurate identification of homozygous and heterozygous genotypes. Currently, DNA fingerprinting and molecular identification based on molecular marker technology are common methods for the standardized, digital, and precise management of germplasm resources, and have been widely used in the protection of ornamental plant germplasm resources and the evaluation of new varieties. For example, researchers have successfully identified varieties of ornamental plants such as Spiraea japonica, Phalaenopsis orchids, lilies, orchids, peonies, lotus, chrysanthemums, bougainvillea, osmanthus, cherry blossoms, and crape myrtles using various markers such as SRAP, RAPD, CDDP, SSR, and SNP. For peonies, while morphological characteristics are the most direct basis for variety identification, this method is usually limited to observation during flowering and is easily influenced by subjective judgment, leading to errors. Therefore, constructing unique molecular identifiers for peony germplasm with similar morphology has become a necessary approach to achieve accurate identification. Among numerous molecular markers, SSR (Simple Sequence Repeats) molecular markers are the preferred method for constructing plant DNA fingerprints designated by the International Union for the Protection of New Varieties of Plants (IUPAC). SSR fluorescent markers developed using fluorescence sequencing technology are considered ideal tools for peony germplasm resource identification due to their high sensitivity, rich polymorphism, co-dominant inheritance, good repeatability, and relatively simple operation. Developing molecular identifiers for peonies using this technology can effectively overcome the limitations of traditional morphological identification, providing reliable technical support for variety resource management, intellectual property protection, and industrial applications. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a new method for identifying peony resources.

[0006] The technical solution of this invention is a primer pair for identifying peony resources, including at least one pair of primers selected from PR73, PR75, PR96, PR49, PR147, PR163, PR103, PR25, and PR90. The PR73 primer pair is shown as Seq ID No. 57 and Seq ID No. 58; the PR75 primer pair is shown as Seq ID No. 59 and Seq ID No. 60; the PR96 primer pair is shown as Seq ID No. 71 and Seq ID No. 72; the PR49 primer pair is shown as Seq ID No. 37 and Seq ID No. 38; the PR147 primer pair is shown as Seq ID No. 105 and Seq ID No. 106; the PR163 primer pair is shown as Seq ID No. 113 and Seq ID No. 114; and the PR103 primer pair is shown as Seq ID No. 79 and Seq ID No. 60. As shown in No. 80, the PR25 primer pair is shown in Seq ID No. 19 and Seq ID No. 20, and the PR90 primer pair is shown in Seq ID No. 63 and Seq ID No. 64.

[0007] This invention also provides the application of SSR markers amplified by the primer combination or the primer combination thereof in identifying the cultivar group to which peony germplasm belongs, obtaining the genotype of peony germplasm, or constructing molecular identity cards for peony germplasm.

[0008] Specifically, the cultivar group refers to either Central Plains Peony or Northwest Peony.

[0009] Specifically, the peony germplasm is the Central Plains peony variety 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu', or 'Guifeichacuicui'.

[0010] Specifically, the parent plants of the peony germplasm are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu', and / or 'Guifeichacuicui'.

[0011] Among them, the peony germplasm is the Northwest peony variety 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao', or 'Hanhai Bingxin'.

[0012] Among them, the parent plants of the peony germplasm are Northwest peony varieties 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

[0013] This invention also provides the application of SSR markers amplified by the primer combination or the primer combination thereof in marker-assisted breeding of peony or identification of new peony varieties.

[0014] The present invention also provides a method for identifying the genotype of peony germplasm, comprising the following steps: performing PCR amplification on the genomic DNA of the peony germplasm to be tested using the primer set, obtaining PCR products, detecting the size of the PCR products of each primer pair, and determining the genotype of the peony germplasm to be tested.

[0015] Specifically, the germplasm of the variety is either Central Plains Peony or Northwest Peony.

[0016] Specifically, the peony germplasm is the Central Plains peony variety 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu', or 'Guifeichacuicui'.

[0017] Specifically, the parent plants of the peony germplasm are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu', and / or 'Guifeichacuicui'.

[0018] Among them, the peony germplasm is the Northwest peony variety 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao', or 'Hanhai Bingxin'.

[0019] Among them, the parent plants of the peony germplasm are Northwest peony varieties 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

[0020] The present invention also provides a method for obtaining molecular identification of peony germplasm, comprising the following steps: performing PCR amplification on the genomic DNA of the peony to be tested using the primer combination to obtain PCR products, detecting the size of the PCR products of each primer pair, labeling each PCR product of each primer pair with different numbers or letters, and then arranging the number or letter labels according to the primer order to obtain the molecular identification of the peony germplasm to be tested.

[0021] This invention also provides a method for determining whether peony germplasm is a known variety, comprising the following steps: performing PCR amplification on the genomic DNA of the peony to be tested using the primer combination to obtain PCR products; detecting the size of the PCR products of each primer pair; labeling each PCR product of each primer pair with different numbers or letters; arranging the numbers or letters according to the primer order to obtain the molecular identification card of the peony germplasm to be tested; comparing the molecular identification card of the peony germplasm to be tested with the molecular identification card of a known variety; if they are the same, it is a known variety; if they are different, it is not a known variety.

[0022] Specifically, the known varieties are Central Plains Peony or Northwest Peony.

[0023] Specifically, the known varieties are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu', or 'Guifeichacuicui'.

[0024] Specifically, the parent plants of the known varieties are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshimoyu', 'Baihuacongxiao', 'Xiangyu', and / or 'Guifeichacuicui'.

[0025] Among them, the known varieties are Northwest peony varieties such as 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiangmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao', or 'Hanhai Bingxin'.

[0026] Among them, the parent plants of the known varieties are the Northwest peony varieties 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

[0027] This invention also provides a method for distinguishing whether two peony germplasms are of the same variety, comprising the following steps: performing PCR amplification on the genomic DNA of the two peony germplasms using primer sets to obtain PCR products; comparing the size of the PCR products of each primer pair in the two peony germplasms; if the size of the PCR amplification products of each primer pair in the primer set is the same for the two peony samples, they are either the same variety or candidates for the same variety; if the size of the PCR amplification products of at least one primer pair in the primer set is different for the two peony samples, they are either different varieties or candidates for different varieties.

[0028] Specifically, the germplasm of the variety is either Central Plains Peony or Northwest Peony.

[0029] Specifically, the peony germplasm is the Central Plains peony variety 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu', or 'Guifeichacuicui'.

[0030] Specifically, the parent plants of the peony germplasm are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu', and / or 'Guifeichacuicui'.

[0031] Among them, the peony germplasm is the Northwest peony variety 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao', or 'Hanhai Bingxin'.

[0032] Among them, the parent plants of the peony germplasm are Northwest peony varieties 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

[0033] The beneficial effects of this invention are as follows: This invention uses offspring germplasm resources obtained through natural pollination of peony varieties from Central China and Northwest China as materials, and employs primers with 57 pairs of SSR markers for screening. Among the markers used, in addition to PR17, PR21, PR53, PR91, PR105, and PR131, 51 markers possess the ability to independently distinguish different germplasms. The number of germplasms that a single marker can distinguish ranges from 1 to 14, with an average of 4 germplasms per marker. This indicates that these SSR markers have high resolution in peony germplasm identification and can serve as an effective molecular tool for the identification, classification, and identification of peony germplasm resources. This invention ultimately screened out 9 core SSR markers for genotypic analysis, and successfully constructed a molecular identification system for 190 peony germplasms using a coding method combining numbers and uppercase and lowercase English letters. This successfully achieved the differentiation between peony populations (especially between Central China and Northwest China peonies, and can also be used to distinguish between different germplasms). The SSR marker-based molecular identification system for peony germplasm developed in this invention has clear application potential and promotional value in peony research and related industries, mainly reflected in the following aspects: 1. Germplasm Resource Management and Intellectual Property Protection: The constructed "molecular ID card" system provides a reliable technical means for the unique identification, standardized archiving, and dynamic management of peony germplasm resources. This system is particularly suitable for resolving the problem of "different names for the same species" or "different species with the same name" caused by similar morphology and confusion in naming of some peony varieties, laying the foundation for the standardized management of germplasm resource banks. At the same time, the molecular ID card can serve as a key basis for the identification and protection of variety rights, providing molecular-level proof of breeders' rights.

[0034] 2. Parental Selection and Offspring Identification in Breeding Practice: The highly efficient SSR markers screened in this invention (especially the 51 markers that can independently distinguish germplasm) can be directly applied to the genetic background analysis of parents in hybridization breeding, helping breeders to more accurately assess the genetic distance between parents and optimize hybrid combination design. Furthermore, this technology can quickly and accurately identify the authenticity of hybrid offspring, distinguishing between true hybrids and self-pollinated seedlings, thereby improving breeding efficiency.

[0035] 3. Variety purity testing and market regulation: This molecular identification system can be used to test the variety purity and authenticity of commercial peony seedlings, which helps to regulate the seedling market, curb the phenomenon of counterfeiting and selling inferior products, protect the legitimate rights and interests of producers and consumers, and promote the healthy development of the peony industry.

[0036] 4. Genetic Diversity Conservation and Ecological Application: By clearly distinguishing between the Central Plains peony and Northwest peony groups, this study provides tools for genetic structure analysis and the mining of distinctive gene resources in these two ecotypes. This will help to more scientifically select materials and configure populations in resource conservation, ecological restoration, or landscaping applications, promoting the effective protection and sustainable utilization of peony genetic diversity.

[0037] 5. Prospects for Technological Expansion and System Integration: The molecular identification scheme using numerical and alphanumeric encoding established in this invention features a unified format, large information capacity, and ease of digital management, making it readily integrateable into germplasm resource information databases. In the future, this SSR marker set can be further correlated with gene markers of important traits (such as flower color, flowering period, and resistance), promoting peony breeding towards a genotype-phenotype synergistic selection approach.

[0038] In summary, the SSR marker system developed in this invention not only enables effective identification between Central Plains peony and Northwest peony, but also has practical application prospects in multiple aspects such as resource management, breeding innovation, industry standardization and biodiversity conservation, providing effective molecular technology support for the high-quality development of the peony seed industry. Detailed Implementation

[0039] Studies have shown that SSR marker technology has good applicability and distinguishing ability in the molecular identification of peony varieties. Previous studies have shown that this technology can effectively identify peony varieties with specific economic traits or uses, such as accurately distinguishing between peony tea-specific varieties and oil-producing peony varieties with relatively homogeneous genetic backgrounds. Of particular note is the stable distinguishing power of SSR markers when facing mixed populations with greater genetic diversity. For example, one study used 10 SSR markers to effectively distinguish 48 varieties from diverse sources, including Chinese, Japanese, and European and American peony varieties, confirming the reliable potential of this technology in large-scale, cross-regional genetic assessment and identification of peony germplasm resources.

[0040] Currently, the application of molecular marker technology in the field of plant variety protection in my country is mainly concentrated in the implementation and protection of variety rights. Its application in the early stages of variety distinctiveness, uniformity, and stability (DUS) testing is relatively limited, appearing only in crops with a large number of varieties, such as maize. To expand the application of this technology in peony DUS testing, future research could focus on the nine SSR core loci selected in this study, combined with representative peony varieties in terms of morphological characteristics, for joint analysis. Specifically, according to the "Peony DUS Testing Guidelines," key group traits such as plant shape, leaf type, flower type, flower color, fruit, stamens, pistils, and coating could be selected and graded according to quantitative and qualitative traits, respectively, to calculate the phenotypic distance and SSR molecular distance between varieties. Through correlation analysis, the correspondence between molecular difference values ​​and morphological difference values ​​can be clarified, especially defining the range of morphological difference values ​​when a single difference locus (i.e., molecular difference value ≥ 1) is used as the judgment threshold. This research will promote the integration of molecular marker technology with traditional DUS testing, providing a new method for screening similar peony varieties and for the precise management of variety resource banks. It will also lay a key technical foundation for establishing industry standards for peony SSR identification technology and improving the efficiency and accuracy of new variety rights examination.

[0041] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with preferred embodiments, is provided below.

[0042] Example 1: Distribution of Specific Allelic Genotypes and Screening of Highly Efficient Identification Primers The peony materials used were collected during the flowering period from the Peony Germplasm Resource Bank of Henan Agricultural University in Zhengzhou, Henan Province (34°51′N, 113°35′E) and the Peony Garden of Beijing Guose Peony Technology Co., Ltd. in Yanqing District, Beijing (40°46′N, 116°07′E), totaling 190 germplasms (Tables 2 and 4). Among them, the peony materials with germplasm numbers 1 to 80 in Tables 2 and 4 were taken from Zhengzhou. They were plants obtained by naturally pollinating the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshimoyu', 'Baihuacongxiao', 'Xiangyu', and 'Guifeichacuicui' through mixed planting and seedling sowing. The materials with germplasm numbers 81 to 190 in Tables 2 and 4 were taken from Beijing. They were plants obtained by naturally pollinating the Northwest peony varieties 'Shushengpengmo', 'Zidiyingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihaiyinbo', 'Xiongmao', 'Dabanlan', 'Saihuanghou', 'Gaoyuanshenghuo', 'Yeguangbei', 'Hongloucangjiao', and 'Hanhaibingxin' through mixed planting and seedling sowing. Take tender leaves from each germplasm, wrap them in paper towels, and then place them in a silicone resealable bag to dry and preserve them.

[0043] DNA was extracted using the TIANGEN (DP121221) kit according to the instructions. The resulting DNA stock solution was first detected by 1.0% agarose gel electrophoresis to confirm that the bands were clear and bright; then its concentration was determined by NanoDrop spectrophotometer and diluted with ddH2O to 20~30 ng / μL, and finally stored at -20℃.

[0044] The specific sequence information of the 57 EST-SSR marker primers used is shown in Table 1. FAM (blue), HEX (green), TAMRA (yellow), or ROX (red) fluorescent labels were added to the corresponding upstream primers. The total volume of the SSR-PCR amplification system (one reaction per site per germplasm resource) was 10 µL, containing 5 µL of 2×Power Taq PCR Master Mix (Aidlab Biotechnologies, Beijing, China), 0.5 µL of 10 µmol / L forward and reverse primers, 1 µL of 20–25 ng / µL genomic DNA, and 3 µL of ddH2O. The amplification program was: 95℃, 5 min; 95℃, 30 sec; Tm, 30 sec; 72℃, 1 min; 35 cycles; 72℃, 10 min; 4℃, hold. The amplified products were separated by capillary electrophoresis using an ABI 3730xl DNA analyzer (Applied Biosystems, Carlsbad, CA, USA), and fragment analysis was performed using a GeneScan-500LIZ as an internal standard.

[0045] Table 1 Information on 57 polymorphic primers .

[0046] GeneMarker 2.2.0 software was used to analyze the raw data and obtain the banding patterns of each peony sample at different SSR sites. All banding patterns amplified by each primer were sorted in ascending order of fragment size and assigned values ​​according to the rules of 1–9, A–Z, and a–z. Based on the principle of "distinguishing the most types with the fewest primers," the simplest primer combination capable of completely distinguishing all tested materials was selected according to the number of banding patterns for each SSR primer from highest to lowest.

[0047] SSR amplification analysis using 57 primer pairs across all peony germplasms revealed the detection of specific alleles in 91 germplasms. Germplasm 165 carried the most specific genotypes (26), followed by germplasm 136 (13), germplasm 60 (10), germplasm 112 (9), and germplasm 64 (7). Regarding primer identification efficiency (Table 2), PR96 performed best, independently distinguishing 14 different germplasms; PR49 was second best, independently identifying 11 germplasms.

[0048] Table 2. Germplasm that can be distinguished by one pair of primers

[0049] The selection process was based on the principle of "distinguishing the most germplasm with the fewest primers." Specifically, each SSR primer was ranked from highest to lowest based on the number of bands produced, and then compared and combined step-by-step until the minimum primer set capable of completely distinguishing all tested materials was obtained. The final optimal combination was determined to be PR73, PR75, PR96, PR49, PR147, PR163, PR103, PR25, and PR90, totaling nine primer pairs. This combination can achieve complete identification of all 190 peony germplasm samples with the fewest primers.

[0050] The molecular ID card is composed of the combination of numbers corresponding to the band patterns amplified by each primer. First, the nine best primer pairs were sorted from highest to lowest according to the number of band patterns, in the following order: PR73, PR75, PR96, PR49, PR147, PR163, PR103, PR25, and PR90. Then, the band patterns generated by each primer in all germplasms were converted into corresponding character numbers according to a predetermined rule (the conversion rules and results are detailed in Table 3). Finally, the numbers of each germplasm at each primer site were concatenated in this fixed order to form its unique molecular ID card. Based on this method, the molecular ID cards for all 190 peony germplasms were constructed, and the final coding results are summarized in Table 4.

[0051] Table 3. Amplification band patterns of the 9 primers and their corresponding codes

[0052] Table 4. Molecular identification cards of 190 peony germplasms .

[0053] When using SSR markers for plant variety identification, genetic differences between varieties are often assessed by the similarities and differences in alleles. Generally, the more alleles present, the greater the genetic distance between varieties. The criteria for differentiation differ depending on the crop's reproductive method and genetic characteristics: In sexually reproduced crops, where intravariety is relatively large, two or three alleles are typically used as a threshold for differentiation. For example, in maize identification, if two samples have ≥2 alleles, they are considered different varieties; if only one allele is present, they are considered genetically similar varieties; and if no allele is present, they are classified as potentially the same variety. In crops like wheat, the criteria are more stringent, often requiring ≥3 alleles to classify them as different varieties. In asexually reproduced woody plants, because asexual reproduction maintains genetic stability and intravariety is minimal, only one allele is needed for variety differentiation, such as ash and apricot kernels.

[0054] Peony varieties are primarily propagated asexually (e.g., division, grafting), resulting in a highly consistent genetic background and extremely low inter-individual variation within the same variety. Based on this, the method of this invention allows for the identification of peony varieties using a single differential locus as the criterion: if two compared samples have ≥1 differential locus, they are considered different varieties; if all detected loci are identical (number of differential loci = 0), they are considered potentially identical varieties. In practical applications, if background information such as parental origin and geographical location of the variety being tested is lacking, a core marker combination consisting of nine SSR loci—PR73, PR75, PR96, PR49, PR147, PR163, PR103, PR25, and PR90—can be preferentially used for analysis. This combination represents a highly efficient identification system screened and validated in this study; other markers can be used for supplementation or verification.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A primer combination for identifying peony resources, characterized in that: The primer pair includes at least one pair selected from PR73, PR75, PR96, PR49, PR147, PR163, PR103, PR25, and PR90. The PR73 primer pair is shown as Seq ID No. 57 and Seq ID No. 58; the PR75 primer pair is shown as Seq ID No. 59 and Seq ID No. 60; the PR96 primer pair is shown as Seq ID No. 71 and Seq ID No. 72; the PR49 primer pair is shown as Seq ID No. 37 and Seq ID No. 38; the PR147 primer pair is shown as Seq ID No. 105 and Seq ID No. 106; the PR163 primer pair is shown as Seq ID No. 113 and Seq ID No. 114; the PR103 primer pair is shown as Seq ID No. 79 and Seq ID No. 80; and the PR25 primer pair is shown as Seq ID No. 19 and Seq ID No.

80. As shown in ID No. 20, the PR90 primer pair is shown in Seq ID No. 63 and Seq ID No.

64.

2. The application of the primer combination of claim 1 or the SSR marker amplified by the primer combination of claim 1 in identifying the cultivar group to which peony germplasm belongs, obtaining the genotype of peony germplasm, or constructing a molecular identity card for peony germplasm; Preferably, the variety group is Central Plains Peony or Northwest Peony.

3. The application according to claim 2, characterized in that: Choose one of the following: The peony germplasm is the Central Plains peony varieties 'Luoyang Red', 'Jingyu', 'Wujin Yaohui', 'Heihua Kui', 'Fenlanpan', 'Baihe Woxue', 'Huahudie', 'Mochi Zhenghui', 'Guanshi Moyu', 'Baihua Congxiao', 'Xiangyu', or 'Guifei Chacui'; The parent varieties of the peony germplasm are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshimoyu', 'Baihuacongxiao', 'Xiangyu' and / or 'Guifeichacuicui'; The peony germplasm is the Northwest peony variety 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao', or 'Hanhai Bingxin'; The parent varieties of the peony germplasm are Northwest peony varieties 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

4. The application of the primer combination of claim 1 or the SSR marker amplified by the primer combination of claim 1 in marker-assisted breeding of peony or identification of new peony varieties.

5. A method for identifying the genotype of peony germplasm, characterized in that: The steps include: using the primer set to perform PCR amplification of the genomic DNA of the peony germplasm to be tested, obtaining PCR products, detecting the size of the PCR products of each primer pair, and determining the genotype of the peony germplasm to be tested. Preferably, the germplasm of the variety is Central Plains Peony or Northwest Peony.

6. The method according to claim 5, characterized in that: Choose one of the following: The peony germplasm is the Central Plains peony varieties 'Luoyang Red', 'Jingyu', 'Wujin Yaohui', 'Heihua Kui', 'Fenlanpan', 'Baihe Woxue', 'Huahudie', 'Mochi Zhenghui', 'Guanshi Moyu', 'Baihua Congxiao', 'Xiangyu', or 'Guifei Chacui'; The parent varieties of the peony germplasm are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshimoyu', 'Baihuacongxiao', 'Xiangyu' and / or 'Guifeichacuicui'; The peony germplasm is the Northwest peony variety 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao', or 'Hanhai Bingxin'; The parent varieties of the peony germplasm are Northwest peony varieties 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

7. A method for obtaining molecular identification of peony germplasm, characterized in that: The process includes the following steps: using the primer combination to perform PCR amplification on the genomic DNA of the peony to be tested, obtaining PCR products, detecting the size of the PCR products of each primer pair, labeling each PCR product of each primer pair with different numbers or letters, and then arranging the number or letter labels according to the primer order to obtain the molecular identification of the peony germplasm to be tested.

8. A method for determining whether peony germplasm belongs to a known variety, characterized in that: The process includes the following steps: PCR amplification of the genomic DNA of the peony to be tested using the primer combination to obtain PCR products; detection of the size of the PCR products of each primer pair; labeling each PCR product of each primer pair with different numbers or letters; arranging the numbers or letters according to the primer order to obtain the molecular identification of the peony germplasm to be tested; comparing the molecular identification of the peony germplasm to be tested with the molecular identification of known varieties; if they are the same, it is a known variety; if they are different, it is not a known variety. Preferably, the known varieties are Central Plains Peony or Northwest Peony.

9. The method according to claim 8, characterized in that: Choose one of the following: The known varieties are the Central Plains peony varieties 'Luoyang Red', 'Jingyu', 'Wujin Yaohui', 'Heihua Kui', 'Fenlanpan', 'Baihe Woxue', 'Huahudie', 'Mochi Zhenghui', 'Guanshi Moyu', 'Baihua Congxiao', 'Xiangyu', or 'Guifei Chacui'; The parent plants of the known varieties are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshi Moyu', 'Baihuacongxiao', 'Xiangyu' and / or 'Guifeichacuicui'; The known varieties are Northwest peony varieties such as 'Scholar Holding Ink', 'Purple Butterfly Welcoming the Wind', 'Auspicious Clouds', 'Pink Lotus', 'Blue Lotus', 'Purple Sea Silver Waves', 'Panda', 'Large Petal Blue', 'Queen of the Race', 'Plateau Sacred Fire', 'Night Light Cup', 'Dream House Hiding Beauty', or 'Vast Ocean Ice Heart'; Among them, the parent plants of the known varieties are the Northwest peony varieties 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

10. A method for distinguishing whether two peony germplasms belong to the same variety, characterized in that: The process includes the following steps: using primer sets to perform PCR amplification on the genomic DNA of two peony germplasms to obtain PCR products; comparing the size of the PCR products of each primer pair in the two peony germplasms; if the size of the PCR amplification products of each primer pair in the primer set is the same for the two peony samples, they are considered to be the same variety or candidates for the same variety; if the size of the PCR amplification products of at least one primer pair in the primer set is different for the two peony samples, they are considered to be different varieties or candidates for different varieties. Preferably, the germplasm of the variety is Central Plains peony or Northwest peony; Preferably, the peony germplasm is the Central Plains peony variety 'Luoyang Red', 'Jingyu', 'Wujin Yaohui', 'Heihua Kui', 'Fenlanpan', 'Baihe Woxue', 'Huahudie', 'Mochi Zhenghui', 'Guanshi Moyu', 'Baihua Congxiao', 'Xiangyu', or 'Guifei Chacui'; Preferably, the parent plants of the peony germplasm are the Central Plains peony varieties 'Luoyanghong', 'Jingyu', 'Wujinyaohui', 'Heihuakui', 'Fenlanpan', 'Baihewoxue', 'Huahudie', 'Mochizhenghui', 'Guanshimoyu', 'Baihuacongxiao', 'Xiangyu' and / or 'Guifeichacuicui'; Preferably, the peony germplasm is a Northwest peony variety such as 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao', or 'Hanhai Bingxin'. Preferably, the parent plants of the peony germplasm are Northwest peony varieties such as 'Shusheng Pengmo', 'Zidi Yingfeng', 'Xiangyun', 'Fenhe', 'Lanhe', 'Zihai Yinbo', 'Xiongmao', 'Daban Lan', 'Saihuanghou', 'Gaoyuan Shenghuo', 'Yeguangbei', 'Honglou Cangjiao' and / or 'Hanhai Bingxin'.

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