A kind of variety identification of okra, okra variety fingerprint and identification method of SSR primer combination

Abstract

The present application relates to a kind of SSR primer combination of identification of variety of Viterica sinensis, variety fingerprint of Viterica sinensis and identification method, belong to biological detection field.The present application provides a kind of SSR primer combination for the identification of variety of Viterica sinensis, it includes HmSSR160 primer pair as SEQ ID NO.1-2, HmSSR147 primer pair of SEQ ID NO.3-4, HmSSR041 of SEQ ID NO.5-6 and HmSSR106 primer pair of SEQ ID NO.7-8.The present application further provides a kind of construction method of variety fingerprint database of Viterica sinensis.The present application further provides a kind of identification detection kit of variety of Viterica sinensis.The present application further provides a kind of identification method of variety of Viterica sinensis, realizes the identification of 40 kinds of Viterica sinensis, and it is significant to new variety right protection of Viterica sinensis, safeguard breeder legal rights and interests, promote high-quality development of Viterica sinensis industry.

Description

Technical Field

[0001] This invention relates to a fingerprint spectrum, SSR primer combination, detection kit, and identification method for Hibiscus mutabilis varieties, belonging to the field of biological detection. Background Technology

[0002] Plant variety identification is a core aspect of botanical research and application, involving the accurate identification and classification of different plant species or varieties through multi-dimensional methods such as morphological characteristics, genetic information, and physiological and biochemical indicators. With the increasing demands for agricultural modernization, the refinement of the horticultural industry, and biodiversity conservation, accurate plant variety identification has become fundamental to ensuring germplasm resource security, protecting varietal rights, and guiding scientific cultivation and breeding. Traditional variety identification primarily relies on morphological observation, including phenotypic traits such as plant type, leaf shape, flower color, and fruit characteristics. This method is intuitive and simple but easily affected by environmental factors. The development of modern molecular biology techniques has provided more reliable means for variety identification. For example, DNA molecular marker technology, by analyzing genomic differences, can achieve precise identification at the gene level, offering advantages such as high throughput, high stability, and immunity to environmental influences. Furthermore, combined with emerging technologies such as spectral analysis and artificial intelligence image recognition, plant variety identification is continuously evolving towards faster, more intelligent, and standardized processes.

[0003] Hibiscus mutabilis L., a traditional famous flower in my country and the city flower of Chengdu, not only has a profound cultural heritage but is also an important tree species for urban landscaping and ecological construction. For a long time, domestic research institutions, especially the Chengdu Botanical Garden, have made a series of advances in the collection of germplasm resources, breeding of new varieties, cultivation and propagation techniques, and adaptability research of Hibiscus mutabilis (Shi Xiaoqing et al. 2021. Adaptability study of Hibiscus mutabilis germplasm resources in Chengdu. Sichuan Forestry Science and Technology, 42(4): 90–93.), successfully cultivating many new varieties with excellent ornamental value and resistance, such as 'Jinxiu Zi' and 'Bai Ri Hua Cai', and successfully introducing them to many parts of China. With the increasing demand for distinctive landscape plants, the industrial application prospects of Hibiscus mutabilis are broad, and the need for accurate identification, efficient management, and intellectual property protection of its varietal resources is becoming increasingly urgent.

[0004] However, the identification and classification of Hibiscus mutabilis varieties still face significant challenges. Long-term artificial hybridization and breeding have led to complex genetic relationships and blurred kinship among varieties, and some varieties exhibit highly similar phenotypes, making accurate and reproducible identification difficult relying solely on traditional morphological indicators such as flower shape and color. Although palynology and quantitative taxonomy studies have provided useful references for the differentiation of Hibiscus mutabilis varieties, these methods still have limitations such as insufficient stability, significant influence from selected indicators, or a limited range of tested varieties.

[0005] The introduction of molecular biology techniques has provided a new solution for the identification of Hibiscus mutabilis varieties. In recent years, DNA molecular marker technology has shown great potential in the identification and phylogenetic analysis of Hibiscus mutabilis varieties due to its advantages of being unaffected by environmental conditions and being able to directly reflect the genetic essence. The literature (Li Zhenbing. Taxonomy, chloroplast genome comparison and phylogenetic study of Hibiscus mutabilis varieties and their closely related species [D].: Sichuan University, 2022. DOI:10.27342 / d.cnki.gscdu.2022.004188.) used eight Hibiscus mutabilis varieties as research objects and found that the chloroplast genome structure was conserved, but there were differences in the length of large / small single copy regions (LSC / SSC), the boundary of inverted repeat regions (IR), simple repeat sequences (SSR), and repeat sequences. However, chloroplast genomes are mostly maternally inherited, with conserved sequences and low recombination rates. Literature also mentions that the whole chloroplast genome sequences of the varieties "Single-Petal Red" and "Peony Pink" are completely identical, and the structures of the sequences of the four varieties "Colorful Clouds," "Hundred Days of Splendor," "Brocade Purple," and "Brocade Buds" are also completely identical. Therefore, these varieties cannot be distinguished by chloroplast genomes. Patent application number: CN202410425844, invention title: A Hibiscus mutabilis ISSR-PCR reaction system and molecular marker method and application, discloses: an ISSR-PCR reaction system and molecular marker method suitable for Hibiscus mutabilis, used for variety identification, genetic diversity analysis, and molecular marker-assisted breeding of Hibiscus mutabilis. This method can distinguish 16 varieties of Hibiscus mutabilis.

[0006] Among numerous molecular markers, SSRs have been widely used for fingerprinting and genetic diversity analysis of ornamental plants, fruit trees, and vegetables due to their advantages such as high polymorphism, good reproducibility, and codominant inheritance. However, significant gaps and deficiencies remain in SSR molecular identification research for Hibiscus mutabilis. Therefore, developing primers for Hibiscus mutabilis-specific SSR molecular markers, constructing a standardized variety identification technology system, and establishing an authoritative variety DNA fingerprint database have become key technological needs for overcoming current industry bottlenecks, protecting the legitimate rights and interests of breeders, and promoting the high-quality development of the Hibiscus mutabilis industry. Summary of the Invention

[0007] This invention develops SSR markers to construct DNA fingerprint profiles and analyze genetic diversity of 40 Hibiscus mutabilis varietal resources. Four pairs of core primers were selected, which can completely distinguish the 40 Hibiscus mutabilis varietals, and fingerprint codes for these 40 varietal resources were constructed based on these primers. This invention provides molecular technical support for the protection, precise identification, and breeding innovation of new Hibiscus mutabilis varieties, and also provides important technical support for the promotion of improved varieties.

[0008] This invention provides an SSR primer combination for identifying Hibiscus mutabilis varieties, comprising the following primer pairs:

[0009] The forward primer of the HmSSR160 primer pair is shown in SEQ ID NO.1, and the reverse primer is shown in SEQ ID NO.2;

[0010] The forward primer of the HmSSR147 primer pair is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4;

[0011] The forward primer of the HmSSR041 primer pair is shown in SEQ ID NO.5, and the reverse primer is shown in SEQ ID NO.6;

[0012] The forward primer of the HmSSR106 primer pair is shown in SEQ ID NO.7, and the reverse primer is shown in SEQ ID NO.8.

[0013] Preferably, the 5' end of the above-mentioned forward primer has a fluorescent modification, and the fluorescent modification includes one or more of FAM, HEX, ROX, and TAMRA.

[0014] Further preferred embodiments include: the forward primer of the HmSSR160 primer pair has a fluorescent modification group of FAM at the 5' end; the forward primer of the HmSSR147 primer pair has a fluorescent modification group of TAMRA at the 5' end; the forward primer of the HmSSR041 primer pair has a fluorescent modification group of ROX at the 5' end; and the forward primer of the HmSSR106 primer pair has a fluorescent modification group of HEX at the 5' end.

[0015] This invention also provides a method for constructing a fingerprint database of Hibiscus mutabilis varieties, which includes the following steps:

[0016] a. Collect fresh leaves from 40 varieties of Hibiscus mutabilis and extract DNA;

[0017] b. Perform PCR amplification on the extracted DNA using primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106, respectively. For the blank control, ddH2O was used instead of DNA template.

[0018] c. Perform capillary electrophoresis on the PCR products from step b to obtain the length of the PCR products;

[0019] d. Based on the capillary electrophoresis results, the amplification results of the sample DNA of each variety of Hibiscus mutabilis were used to form primer letter numbers plus PCR product length numerical or letter banding numbers, resulting in a fingerprint database of 40 Hibiscus mutabilis varieties.

[0020] Preferably, the specific information of the 40 Hibiscus mutabilis varieties in step a of the above-mentioned method for constructing the Hibiscus mutabilis varietal fingerprint database is shown in the table below:

[0021] Table 40 Hibiscus mutabilis Varieties Catalogue

[0022]

[0023] The PCR amplification in step b consists of the following PCR reaction system: a total volume of 20 μL, including 17 μL of DNA polymerase premix, 1 μL each of 10 μM upstream and downstream primers, and 1 μL of template DNA; the PCR amplification program is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing for 10 s according to the Tm value of each primer pair, extension at 72℃ for 10 s, for a total of 35 cycles, and a final extension at 72℃ for 5 min;

[0024] The encoding method in step d is as follows: Primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106 are encoded as A, B, C, and D, respectively. The amplified sites from each primer pair are encoded according to fragment size, starting from 1 to 9. Sites exceeding 9 sites are encoded using English letters. Combining the primer numbers with the corresponding site codes yields the fingerprint database shown in the table below:

[0025] Table 40 fingerprint codes of Hibiscus mutabilis varieties

[0026] .

[0027] The present invention also provides a kit for identifying Hibiscus mutabilis varieties, which includes the above-mentioned SSR primer combination for identifying Hibiscus mutabilis varieties, and the above-mentioned Hibiscus mutabilis variety fingerprint database.

[0028] The aforementioned kit for identifying Hibiscus mutabilis varieties also includes a DNA polymerase premix.

[0029] This invention also provides a method for identifying Hibiscus mutabilis varieties, which uses the above-mentioned Hibiscus mutabilis variety identification kit and includes the following steps:

[0030] a. Collect fresh leaves of Hibiscus mutabilis and extract genomic DNA from Hibiscus mutabilis;

[0031] b. Perform PCR amplification on the extracted DNA using primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106, respectively. For the blank control, ddH2O was used instead of DNA template.

[0032] c. Take the PCR product from step b and perform capillary electrophoresis detection;

[0033] d. Encode the capillary electrophoresis results according to the above coding method, and compare the coding results with the Hibiscus mutabilis varietal fingerprint database to obtain the Hibiscus mutabilis varietal identification results.

[0034] The identification method for the above-mentioned Hibiscus mutabilis varieties, in step (b), the PCR reaction system is as follows: total system 20 μL, including 17 μL DNA polymerase premix, 1 μL each of upstream and downstream 10 μM primers, and 1 μL template DNA; the PCR amplification program is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing at TM value for 10 s, extension at 72℃ for 10 s, for a total of 35 cycles, and finally extension at 72℃ for 5 min.

[0035] The present invention also provides the application of the above-mentioned SSR primer combination for identifying Hibiscus mutabilis varieties, the reagent kit for identifying Hibiscus mutabilis varieties, and the identification method for Hibiscus mutabilis varieties in the identification of Hibiscus mutabilis varieties.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] Current methods for identifying Hibiscus mutabilis varieties rely on traditional phenotypic identification. However, some varieties are very similar in appearance, leading to potential errors in phenotypic identification. Furthermore, phenotypic identification requires the flowering period. This invention, however, allows for identification through leaf analysis, eliminating the need to wait for flowering. Moreover, identification based on the plant's own DNA offers high accuracy, reducing the difficulty of identification. Compared to the literature (CN202410425844 - A Hibiscus mutabilis ISSR-PCR reaction system and molecular marker method and application - application disclosure) which distinguishes 16 varieties, this invention expands the scope of variety identification to 40 varieties. This is of great significance for the protection of new Hibiscus mutabilis variety rights, safeguarding the legitimate rights and interests of breeders, and promoting the high-quality development of the Hibiscus mutabilis industry. Attached Figure Description

[0038] Figure 1 The image shows the amplification results of SSR160 in some varieties;

[0039] Figure 2 This image shows the amplification results of SSR032 in some varieties. Detailed Implementation

[0040] Example 1: Establishing a fingerprint database of Hibiscus mutabilis varieties using the SSR primer combinations of the present invention.

[0041] The selection of core primers is crucial in the construction of a fingerprint database. The principle is to achieve the maximum number of species differentiations with the fewest primer combinations. Generally, primers with higher polymorphism information content (PIC) have stronger species differentiation capabilities. Therefore, core primers are selected based on their PIC values ​​and actual differentiation abilities. This invention selects HmSSR160, HmSSR147, HmSSR041, and HmSSR106 as core primers for fingerprint database construction. The primers used are shown in the table below.

[0042] Table 1. SSR primer combinations for constructing fingerprint patterns

[0043]

[0044] 1. Sample collection and DNA extraction

[0045] Fresh leaves from 40 varieties of Hibiscus mutabilis were collected. The variety information of Hibiscus mutabilis is shown in Table 2. DNA was extracted from Hibiscus mutabilis using the TSINGKE Plant DNA Extraction Kit (General Type).

[0046] Table 2. Catalogue of 40 Hibiscus mutabilis varieties

[0047]

[0048] 2. PCR amplification

[0049] Using the above-mentioned Hibiscus mutabilis DNA as a template, PCR amplification was performed using fluorescently modified primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106, respectively. For the blank control, ddH2O was used instead of DNA template.

[0050] The total PCR reaction volume was 20 μL, including 17 μL of Chinco Gold MixVer.2, 1 μL each of forward and reverse primers, and 1 μL of template DNA.

[0051] The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing at TM value for 10 s, extension at 72℃ for 10 s, for a total of 35 cycles, and a final extension at 72℃ for 5 min.

[0052] 3. Capillary electrophoresis analysis

[0053] Take the PCR amplification products and perform capillary electrophoresis on an ABI 3730xl genetic analyzer. If the blank control has no band, the sample has a bright band near 93-197 bp and can be used for subsequent experiments; if the blank control has no band and the sample has no band, PCR amplification needs to be repeated; if the blank control has a band and the sample has a bright band at 93-197 bp, the detection test needs to be repeated.

[0054] 4. Fingerprint encoding method for Hibiscus mutabilis

[0055] The primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106 were coded as A, B, C, and D, respectively. The sites amplified by each primer pair were coded from 1 to 9 according to the fragment size. Sites with more than 9 sites were coded with English letters (Table 3). The fingerprint pattern of the variety was obtained by combining the primer number with the corresponding site code. Different primers were separated by "-". For example, DBB was coded as A6-B1256-C2359-D5 according to this method. Finally, fingerprint pattern codes for 40 varieties were established (Table 4).

[0056] Table 3. Band size and coding criteria for 4 primer pairs

[0057]

[0058] Table 4. Fingerprint codes of 40 Hibiscus mutabilis varieties

[0059]

[0060] Example 2: Method for identifying Hibiscus mutabilis varieties according to the present invention

[0061] 1. Sample collection and DNA extraction

[0062] Fresh leaves of Hibiscus mutabilis were collected, and DNA was extracted from the leaves using the TSINGKE Plant DNA Extraction Kit (General Type).

[0063] 2. PCR amplification

[0064] Using the DNA from Hibiscus mutabilis to be tested as a template, PCR amplification was performed using primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106, respectively. For the blank control, ddH2O was used instead of DNA template.

[0065] The total PCR reaction volume was 20 μL, including 17 μL of Chinco Gold MixVer.2, 1 μL each of forward and reverse primers, and 1 μL of template DNA.

[0066] The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing at TM value for 10 s, extension at 72℃ for 10 s, for a total of 35 cycles, and a final extension at 72℃ for 5 min.

[0067] 3. Capillary electrophoresis analysis

[0068] Same as Part 3 of Example 1.

[0069] 4. Result Determination

[0070] The electrophoresis results were encoded according to the encoding method in Part 4 of Example 1. The encoded results were compared with the Hibiscus mutabilis varietal fingerprint database of the present invention to obtain the varietal information of the Hibiscus mutabilis to be identified.

[0071] Example 3: Screening experiment of SSR primer combinations of the present invention

[0072] 1 Experimental Methods

[0073] 1.1 SSR Primer Source and Screening

[0074] Thirty-five pairs of primary screening primers (SEQ ID NO. 1-70) were designed based on the Hibiscus mutabilis genome and transcriptome, and fluorescent modifications were added. For screening, DNA extracted from 16 samples (DBB, DBH, JR, JQS, MDF, CX, YR, ZY, MX, HY, FD, HS004, BRHC, A14, B03, and C05) were used as amplification templates. A total PCR reaction volume of 20 μL was established, containing 217 μL of Qingke Gold Mix Ver., 1 μL each of forward and reverse primers (10 μM), and 1 μL of template DNA. The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; annealing at different TM values ​​for 10 s; extension at 72℃ for 10 s; for a total of 35 cycles; and a final extension at 72℃ for 5 min. Finally, the well-amplified fluorescent PCR products were detected using an ABI 3730xl genetic analyzer, and the obtained data were analyzed using Genemapper software. Based on the analysis results, it was determined whether different primers exhibited fragment polymorphism.

[0075] 1.2 DNA Extraction and Detection

[0076] In July 2025, fresh leaves of 40 Hibiscus mutabilis varieties (as shown in Table 1) were collected from the research nursery of Chengdu Botanical Garden. DNA was extracted using the TSINGKE Plant DNA Extraction Kit (General Type).

[0077] 1.3 Amplification and Detection of All Samples

[0078] The selected polymorphic primers were synthesized into fluorescently modified primers, and all 40 samples were amplified and detected by capillary electrophoresis. The amplification system was 25 μL, containing 22 μL of Gold Mix Ver.2, 1 μL each of forward and reverse primers, and 1 μL of template DNA. The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; annealing at different TM values ​​for 10 s; 72℃ extension for 10 s, for a total of 40 cycles; and a final extension at 72℃ for 5 min.

[0079] The amplified products were subjected to capillary electrophoresis on an ABI 3730xl genetic analyzer to detect SSR polymorphisms in 40 varietal resources, and the locus information was read using Gene Mapper 4.1 software.

[0080] 1.4 Genetic diversity analysis

[0081] In GenAlEx version 6.501 and Popgen32 software, various genetic diversity indicators of SSR loci were calculated, including observed alleles (Na), effective alleles (Ne), gene diversity (H), Shannon index (I), and polymorphism content (PIC).

[0082] 2 Results and Analysis

[0083] 2.1 SSR primer screening and genetic diversity analysis

[0084] Using 16 varieties of Hibiscus mutabilis as templates, 20 pairs of polymorphic primers with high specificity, high resolution, and good sample repeatability were screened from 35 primer pairs. Figure 1 and Figure 2 Primers with high specificity and good polymorphism are listed separately, as well as primers without polymorphism (due to space limitations, only the results of some varieties are shown). Figure 1 The results show the amplification of SSR160 in some varieties. Different varieties amplified different band sizes, indicating that the primer has high specificity and good polymorphism, and can be selected for further experiments. Figure 2 The amplification results of SSR032 in some varieties are given as an example. Primer SSR032 only amplified the band pattern 203 in all the screened varieties, indicating that this primer does not have polymorphism and was eliminated in the initial screening process.

[0085] As shown in Table 5, 20 primer pairs detected a total of 147 allele loci in 40 samples, with the smallest number of alleles being 3 (CXSSR019) and the largest being 13 (CXSSR147), resulting in an average of 7.3500 alleles per locus. The total number of effective alleles (Ne) was 25.7559, ranging from 1.0770 (CXSSR008) to 1.4633 (CXSSR031), with an average of 1.2726 effective alleles per locus. The Shannon index (I) ranged from 0.1191 (CXSSR008) to 0.4178 (CXSSR031), with an average of 0.2938. Gene diversity (H) ranged from 0.0632 (CXSSR008) to 0.2734 (CXSSR031), with an average of 0.1786. The polymorphism information content (PIC) of each primer ranged from 0.309 to 0.832, with an average of 0.684, indicating that these primers have high polymorphism and can be used for polymorphism analysis of Hibiscus mutabilis.

[0086] Table 5 Information on 20 SSR primer pairs and their polymorphisms

[0087]

[0088] Continued from Table 5

[0089]

[0090] Note: PP%, percentage of polymorphic bands, Na: observed alleles, Ne: effective alleles, H: gene diversity, I: Shannon index, PIC: polymorphism information content.

[0091] 2.2 Determination of core primers

[0092] Generally, primers with higher polymorphic information content (PIC) have a stronger ability to identify varieties. Therefore, core primers were screened based on their PIC values ​​and actual distinguishing abilities. Among the 20 primer pairs, three pairs had PIC values ​​greater than 0.8: HmSSR160, HmSSR147, and HmSSR041. According to the amplification results, these three primer pairs could distinguish 38 varieties except for HY and B23. However, primer HmSSR095, which ranked fourth in PIC value, could not distinguish between HY and B23, while primer HmSSR106, which ranked fifth in PIC value, could distinguish between HY and B23. Therefore, HmSSR160, HmSSR147, HmSSR041, and HmSSR106 were finally selected as core primers for identifying Hibiscus mutabilis varieties and for constructing fingerprint profiles.

[0093] Example 1: Evaluation of the method for identifying Hibiscus mutabilis varieties using SSR primer combinations of the present invention in practical applications.

[0094] Forty young leaves of Hibiscus mutabilis varieties were collected from the research nursery of Chengdu Botanical Garden as samples to verify the identification of Hibiscus mutabilis varieties using the SSR primer combination of this invention. The samples were numbered 1-40. Genomic DNA was extracted as a template and extracted using the TSINGKE Plant DNA Extraction Kit (Universal).

[0095] Forty samples were amplified using the four pairs of core SSR primers selected in this invention. A total PCR reaction volume of 20 μL was established, containing 17 μL of Qingke Gold Mix Ver.2, 1 μL each of 10 μM forward and reverse primers, and 1 μL of template DNA. The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing at different TM values ​​for 10 s, extension at 72℃ for 10 s, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. Finally, the well-amplified fluorescent PCR products were analyzed using an ABI 3730xl genetic analyzer.

[0096] The amplified products were subjected to capillary electrophoresis on an ABI 3730xl genetic analyzer to detect SSR polymorphisms in 40 varietal resources. Locus information was retrieved using Gene Mapper 4.1 software (Table 6). Based on the fingerprinting principles established according to this invention, fingerprint codes for these 40 samples were obtained. By comparing these codes with the fingerprints of the 40 varietals established according to this invention, the Hibiscus mutabilis varieties corresponding to samples 1-40 were confirmed (Table 7).

[0097] The results showed that the identification results of 40 varieties using the SSR primer combination of the present invention were consistent with the actual varieties, indicating that the SSR primer combination of the present invention can identify the 40 Hibiscus mutabilis varieties in Table 1.

[0098] Table 6. Band information amplified from 40 Hibiscus mutabilis samples using 4 primer pairs.

[0099]

[0100] Continued from Table 6

[0101]

[0102] Continued from Table 6

[0103]

[0104] Continued from Table 6

[0105]

[0106] Continued from Table 6

[0107]

[0108] Table 7. Comparison of variety information obtained from identifying 40 samples using the identification method of this invention with actual variety information.

[0109]

[0110] In recent years, as domestic breeders have continuously cultivated numerous new varieties of ornamental plants, fruit trees, and crops, the importance attached to intellectual property protection has been increasing (Zhang Lu, Zhang Pingping, Xie Weijia, Xu Feng, Peng Luchun, Song Jie, Du Guanghui, Li Shifeng. 2025. Analysis of genetic diversity of evergreen rhododendron germplasm and construction of SSR fingerprint map. Journal of Horticulture, 52(2): 380–394.). Protecting the legitimate rights and interests of variety owners through accurate and efficient variety identification has become an inevitable trend. Against this background, constructing variety DNA fingerprint maps has also become an important research direction in the field of plant breeding.

[0111] This invention screened 20 primer pairs for DNA fingerprinting and genetic diversity analysis of 40 Hibiscus mutabilis varietal resources. The average polymorphism information content (PIC) of the primers was >0.6, indicating that the SSR markers used had high polymorphism, but the overall genetic differences among the varieties were small (average Shannon index I=0.2938).

[0112] SSR markers are codominant markers. In diploid materials, a pair of primers usually amplifies a maximum of two bands (peaks), which can be used to distinguish between homozygous and heterozygous genotypes in genotyping. Since polyploid materials exist in Hibiscus mutabilis, some varieties show multiple bands in their amplification products. This phenomenon also exists in studies of Rosa (Sun Pei, Zhang Hong, Yang Yuan, Wang Hua, Li Maofu, Kang Yanhui, Sun Xiangyi, Jin Wanmei. 2025. Genetic diversity analysis and fingerprinting of Rosa germplasm resources based on SSR markers. Journal of Horticulture, 52(6): 1539–1552.). Therefore, this study converted the data into dominant markers for analysis, without distinguishing between homozygous and heterozygous genotypes, and only distinguished varieties based on the presence or absence of loci. Finally, four pairs of core primers were selected, which can completely distinguish all 40 genus resources. In terms of fingerprint encoding, the research methods of *Lagerstroemia indica* (Tian Shuyan. 2025. Phenotypic evaluation and SSR fingerprint construction of 76 new *Lagerstroemia indica* varieties [Master's thesis]. Changsha: Central South University of Forestry and Technology.) and strawberry (Liu Guoxia, Zhang Quanfang, Yang Xue, Tan Qingqing, Hu Yue, Fan Yangyang, Yao Chuan, Cui Gang, Bu Xun, Chen Xueyan, Liu Bingfu. 2023. Construction and preliminary genetic analysis of DNA fingerprint of octoploid strawberry. Journal of China Agricultural University, 28(2): 35–42.) were referenced. The encoding results can intuitively reflect the amplification of different primers in each variety. The varietal fingerprints constructed in this study will also provide important technical support for the protection of new *Hibiscus mutabilis* variety rights and the promotion of improved varieties.

Claims

1. An SSR primer combination for identifying Hibiscus mutabilis varieties, characterized in that: It includes the following primer pairs: The forward primer of the HmSSR160 primer pair is shown in SEQ ID NO.1, and the reverse primer is shown in SEQ ID NO.2; The forward primer of the HmSSR147 primer pair is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4; The forward primer of the HmSSR041 primer pair is shown in SEQ ID NO.5, and the reverse primer is shown in SEQ ID NO.6; The forward primer of the HmSSR106 primer pair is shown in SEQ ID NO.7, and the reverse primer is shown in SEQ ID NO.

8.

2. The SSR primer combination for identifying Hibiscus mutabilis varieties according to claim 1, characterized in that: The 5' end of the forward primer has a fluorescent modification, which includes one or more of FAM, HEX, ROX, and TAMRA.

3. The SSR primer combination for identifying Hibiscus mutabilis varieties according to claim 1, characterized in that: The forward primer of the HmSSR160 primer pair has a fluorescent modification group of FAM at the 5' end; the forward primer of the HmSSR147 primer pair has a fluorescent modification group of TAMRA at the 5' end; the forward primer of the HmSSR041 primer pair has a fluorescent modification group of ROX at the 5' end; and the forward primer of the HmSSR106 primer pair has a fluorescent modification group of HEX at the 5' end.

4. A method for constructing a fingerprint database of Hibiscus mutabilis varieties, characterized in that: It includes the following steps: a. Fresh leaves of 40 varieties of Hibiscus mutabilis were collected and DNA was extracted; the specific information of the 40 varieties of Hibiscus mutabilis is shown in the table below: ； b. Perform PCR amplification on the extracted DNA using the HmSSR160 primer pair, HmSSR147 primer pair, HmSSR041 primer pair, and HmSSR106 primer pair as described in any one of claims 1-3. For the blank control, ddH2O is used instead of DNA template. c. Perform capillary electrophoresis on the PCR amplification product obtained in step b to obtain the length of the amplified fragment; d. Based on the capillary electrophoresis results, the amplification results of the sample DNA of each variety of Hibiscus mutabilis were used to form an encoding method of primer letter code plus amplified fragment length number or letter code, resulting in a fingerprint database of 40 Hibiscus mutabilis varieties.

5. The method for constructing the Hibiscus mutabilis varietal fingerprint database according to claim 4, characterized in that: The PCR amplification in step b consists of the following PCR reaction system: a total volume of 20 μL, including 17 μL of DNA polymerase premix, 1 μL each of 10 μM upstream and downstream primers, and 1 μL of template DNA; the PCR amplification program is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing for 10 s according to the Tm value of each primer pair, extension at 72℃ for 10 s, for a total of 35 cycles, and a final extension at 72℃ for 5 min; The encoding method in step d is as follows: the primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106 are encoded as A, B, C, and D, respectively. The products amplified by each primer pair are encoded according to the length of the amplified fragment, starting from 1 to 9. Fragment lengths exceeding 9 are encoded using English letters. The specific encoding method is shown in the table below: ； The fingerprint database is obtained by combining primer codes with corresponding amplified fragment length codes, as shown in the table below: 。 6. A kit for identifying Hibiscus mutabilis varieties, characterized in that: It includes the SSR primer combination for identifying Hibiscus mutabilis varieties as described in any one of claims 1-3.

7. The kit for identifying Hibiscus mutabilis varieties according to claim 6, characterized in that: It also includes DNA polymerase premix.

8. A method for identifying a variety of Hibiscus mutabilis, characterized in that: It is identified using the hibiscus variety identification kit described in claim 6 or 7, and includes the following steps: a. Collect fresh leaves of Hibiscus mutabilis and extract its genomic DNA; the Hibiscus mutabilis variety is one of the varieties listed in the table below: ； b. Perform PCR amplification on the extracted DNA using primer pairs HmSSR160, HmSSR147, HmSSR041, and HmSSR106, respectively. For the blank control, ddH2O was used instead of DNA template. c. Take the PCR product from step b and perform capillary electrophoresis detection; d. Encode the capillary electrophoresis results according to the encoding method in step d of claim 5, and compare the encoded results with the Hibiscus mutabilis varietal fingerprint database to obtain the Hibiscus mutabilis varietal identification results.

9. The method for identifying Hibiscus mutabilis varieties according to claim 8, characterized in that: The PCR reaction system in step b is as follows: total system 20 μL, including 17 μL DNA polymerase premix, 1 μL each of 10 μM upstream and downstream primers, and 1 μL template DNA; the PCR amplification program is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, annealing at Tm for 10 s, extension at 72℃ for 10 s, for a total of 35 cycles, and a final extension at 72℃ for 5 min.

10. The application of the SSR primer combination for identifying Hibiscus mutabilis varieties according to any one of claims 1-3, the kit for identifying Hibiscus mutabilis varieties according to claim 6 or 7, and the method for identifying Hibiscus mutabilis varieties according to claim 8 or 9 in identifying Hibiscus mutabilis varieties, wherein the Hibiscus mutabilis variety is one of the varieties listed in the table below: 。

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