SSR molecular marker for identification of hainan zhu leaves and application thereof

Abstract

The application provides a primer pair, which is SSR1, SSR2, SSR3, SSR5, SSR6, SSR7, SSR8, SSR9, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19 or SSR21. The application successfully designs and screens 19 primer pairs, which can verify the DNA of Heichun leaf and Baichun leaf materials, can effectively distinguish Heichun leaf and Baichun leaf, realizes rapid, stable and accurate molecular identification of Chunan leaf germplasm, and has good repeatability, stability and reliability and rapid detection.

Description

Technical Field

[0001] This invention relates to an SSR molecular marker, and more particularly to an SSR molecular marker for germplasm identification of *Hymenochloa hainanensis* and its application. Background Technology

[0002] *Phrynium hainanense* TL Wu et Senjen is a perennial herbaceous plant belonging to the genus *Phrynium* in the Marantaceae family. It is a highly distinctive economic plant in South my country, serving as the core ingredient in traditional zongzi (sticky rice dumpling) leaves. Its leaves, also known as Hainan zongzi leaves or Hainan winter leaves, possess multiple values ​​including medicinal, ornamental, and ecological soil-stabilizing properties. It is commonly intercropped in rubber plantations and areca nut orchards, showing broad prospects for industrial application. *Phrynium hainanense* has abundant wild resources and cultivated germplasm types, exhibiting significant genetic variation in traits such as leaf size, leaf color, plant type, and yield. Accurate identification of germplasm resources, evaluation of genetic diversity, and selection of superior individual plants are crucial for the conservation, variety identification, and breeding utilization of *Phrynium hainanense*.

[0003] Currently, the identification and evaluation of Hainan holly leaves mainly rely on morphological traits, such as plant height, leaf shape, number of tillers, and leaf color depth. This method is easily affected by factors such as plant growth period, cultivation conditions, and environmental climate, resulting in problems such as long identification cycles, unstable accuracy, and easy phenotypic confusion. It is difficult to achieve early, rapid, and accurate germplasm identification, and cannot meet the needs of large-scale germplasm resource screening and molecular breeding. Hainan holly leaves are mainly divided into two types: black holly leaves and white holly leaves. Black holly leaves have darker and wider leaves, excellent commercial quality, higher market price, and outstanding medicinal value. They prefer shady, moist, and organic-rich soil, have poor cold resistance, and slow plant growth; they are mostly reusable for use as zongzi leaves. White holly leaves have relatively lighter and narrower leaves, obvious high yield advantage, lower market price, and relatively mild medicinal effects. This germplasm has higher light tolerance, drought and poor soil tolerance, and vigorous growth; it is mostly single-use for zongzi leaves. Because the two germplasms are very similar in appearance and difficult to distinguish with the naked eye, they are often confused, making it difficult to select the appropriate germplasm during cultivation and hindering normal sales later. Therefore, accurately distinguishing the two germplasms is of great significance for the cultivation and application of Hainan holly leaves.

[0004] Simple repeat sequence (SSR) molecular markers have advantages such as high polymorphism, good stability, co-dominant inheritance, strong reproducibility, and convenient detection, making them the preferred marker method for plant germplasm identification, genetic diversity analysis, DNA fingerprinting, and molecular-assisted breeding. Compared with markers such as RAPD, AFLD, and SNP, SSR markers are simpler to operate, produce clearer amplified bands, and are less expensive, making them more suitable for promotion and application in grassroots research and breeding units.

[0005] Current research on the molecular biology of *Hylocereus undatus* is weak, with a lack of genomic information. Specific SSR molecular markers developed based on transcriptomics and stably used for germplasm identification are still scarce. An efficient, unified, and scalable SSR marker detection system has not yet been established, severely hindering the accurate evaluation of *Hylocereus undatus* germplasm resources, the identification of genuine varieties, and the development of molecular breeding work. Therefore, developing highly polymorphic, specific, and amplified stable *Hylocereus undatus* SSR molecular markers and establishing rapid and accurate germplasm molecular identification methods has significant theoretical value and practical application implications. Summary of the Invention

[0006] To address the problems of phenotypic dependence, low accuracy, long cycle, and lack of specific SSR markers in the identification of *Hylocereus undatus* germplasm in existing technologies, this invention provides an SSR molecular marker for the identification of *Hylocereus undatus* germplasm in Hainan and its application, enabling rapid, stable, and accurate molecular identification of *Hylocereus undatus* germplasm.

[0007] SSR1 TCTAAGATCGGGGGTGCAT TGCATTTGGATTGGTGGCAA SSR2 TGAGCAAGACGACGATCAAG TGGTGCAGATCAATTACGCA SSR3 CTTCGGCGACTCCAATGTG TGGTGACCCTTCATCACAAG SSR5 GCCATACACGGGAAAGGTAA GAGGAGCACCGTAAAATGG SSR6 AAAGAAGCTCCAGCTCAAAATAG GTGGGAAGGAGTTGATG SSR7 CCTTCACCGACTTCGTCGT TCCGCAAATCGAGGATCAGT SSR8 CGTCTCCATTCTCCGAACC TGGGAGGATCTTGACGGA SSR9 GAAACCCGACTGGGTTACTG ATCTCCGGTATCCACACCC SSR10 GTCATCCACTCCGATTCCAG TTATGCCCGAATCGCTTCAC SSR11 CTGCTCATCATCTCTGGCTC CCGAAAGCTATCCCAACA SSR12 TATTAACCTGTTGAGCTCGTCG AAATTGCCAAGCTCCGACAAC SSR13 CAAGCGAAAAGTTGAGGTGC TGGGAAAGTGAGATCCAGGT SSR14 ATCGATCAGCGGATCACGAA CCACGCATCASUMMERGT SSR15 TGTTGTAGATCCTCTTCAGGTG GGCAAAGGTGAAGATGTCG SSR16 CCATGATCACCATCCAGTCC QUESTIONQUESTION SSR17 GCAGATCTTAGCAGAAACG GAATGCGGGAAAAGGAACCTG SSR18 TCGATGCCACAACATTCACA HELPGAGTCGTTTGGGA SSR19 AGGTACCCTGCTCAACTCTT TACCCATAAGACTCACCGGC SSR21 AGAGAACACAGGGAC GGAATTATTGACGAACCAGGAG .

[0008] SSR1 TCTAAGATCGGGGGTGCAT TGCATTTGGATTGGTGGCAA SSR2 TGAGCAAGACGACGATCAAG TGGTGCAGATCAATTACGCA SSR3 CTTCGGCGACTCCAATGTG TGGTGACCCTTCATCACAAG SSR5 GCCATACACGGGAAAGGTAA GAGGAGCACAGCGTAAATGG SSR6 AAAGAAGCTCCAGCTCAAAATAG GTGGGAAGGAAGAGAGTTGATG SSR7 CCTTCACCGACTTCGTCGT TCCGCAAATCGAGGATCAGT SSR8 CGTCTCCATTCTCCGAACC TGGGAGGAGATCTTGACGGA SSR9 GAAACCCGACTGGGTTACTG ATCTCCGGTATCCACACCC SSR10 GTCATCCACTCCGATTCCAG TTATGCCCGAATCGCTTCAC SSR11 CTGCTCATCATCTCTGGCTC CCGAAAGCTATCCCACAACA SSR12 TATTAACCTGTTGAGCTCGTCG AAATTGCAAGCTCCGACAAC SSR13 CAAGCGAAAAGTTGAGGTGC TGGGAAAGTGAGATCCAGGT SSR14 ATCGATCAGCGGATCACGAA CCACACGCATCAAGAACAGT SSR15 TGTTGTAGATCCTCTTCAGGTG GGCAAAGGTGAAGAATGTCG SSR16 CCATGATCACCATCCAGTCC AAGCATGAGACAAGAACATGAAC SSR17 GCAGATCTTAGCAGAAGAACG GAATGCGGGAAAGGAACCTG SSR18 TCGATGCCACAACATTCACA CATCAAGGAGTCGTTTGGGA SSR19 AGGTACCCTGCTCAACTCTT TACCCATAAGACTCACCGGC SSR21 AAGAGACACACACAGAGGGAC GGAATTATTGACGAAGCAGAGAG .

[0009] Preferably, the primer set includes primer pair SSR3 and / or primer pair SSR16.

[0010] A third aspect of the invention is to provide the use of primer pairs as described in the first aspect of the invention, or primer sets as described in the second aspect of the invention, in distinguishing between black holly leaves and white holly leaves.

[0011] A fourth aspect of the present invention is to provide the application of primer pairs as described in the first aspect of the present invention, or primer sets as described in the second aspect of the present invention, in molecular marker-assisted breeding of *Hymenochloa hainanensis*.

[0012] The fifth aspect of the present invention is to provide a method for distinguishing between black holly leaves and white holly leaves, using the primer pairs described in the first aspect of the present invention, or the primer sets described in the second aspect of the present invention.

[0013] Preferably, the method includes the following steps: extracting DNA from the leaves of the *Hylocereus undatus* to be tested, using this DNA as a template, performing PCR amplification with primer pairs as described in the first aspect of the present invention, or performing PCR amplification with primer sets as described in the second aspect or 3 of the present invention, and detecting and analyzing the amplification products using agarose gel electrophoresis.

[0014] If the primer pair used for SSR1 amplifies a black holly leaf (SSR1), and a bright band appears around 313 bp, then it is a white holly leaf; if the primer pair used for SSR2 amplifies a black holly leaf (SSR2), and a bright band appears around 316 bp, then it is a black holly leaf (SSR2); if there is no bright band, then it is a white holly leaf (SSR2). If the primer pair used for SSR3 amplifies a black holly leaf (SSR3), and a bright band appears around 240 bp, then it is a black holly leaf (SSR3); if there is a bright band around 250 bp, then it is a white holly leaf (SSR3); if the primer pair used for SSR5 amplifies a black holly leaf (SSR5), and a bright band appears around 245 bp, then it is a white holly leaf (SSR5); if the primer pair used for SSR6 amplifies a black holly leaf (SSR6), and a bright band appears around 255 bp, then it is a black holly leaf (SSR5). If a bright band is visible around 271 bp, it indicates a white holly leaf; if no bright band is visible when using primer pair SSR7, it indicates a black holly leaf; if a bright band is visible around 271 bp, it indicates a white holly leaf; if a bright band is visible around 263 bp when using primer pair SSR8, it indicates a black holly leaf; if no bright band is visible, it indicates a white holly leaf; if no bright band is visible when using primer pair SSR9, it indicates a black holly leaf; if no bright band is visible when using primer pair SSR10, it indicates a black holly leaf; if no bright band is visible when using primer pair SSR1 ... If a bright band is visible around 239 bp, it indicates a white holly leaf; if no bright band is visible when using primer pair SSR12, it indicates a black holly leaf; if a bright band is visible around 244 bp, it indicates a white holly leaf; if no bright band is visible when using primer pair SSR13, it indicates a black holly leaf; if a bright band is visible around 240 bp, it indicates a white holly leaf; if no bright band is visible when using primer pair SSR14, it indicates a black holly leaf; if a bright band is visible around 240 bp, it indicates a white holly leaf; if a bright band is visible around 250 bp when using primer pair SSR15, it indicates a black holly leaf; if no bright band is visible when using primer pair SSR16, it indicates a black holly leaf; if a bright band is visible around 245 bp, it indicates a white holly leaf. If there is a distinct bright band around 248 bp, it is a white holly leaf; if amplified using primer pair SSR17, there is a distinct bright band around 248 bp, it is a black holly leaf; if there is no distinct bright band, it is a white holly leaf; if amplified using primer pair SSR18, there is no distinct bright band, it is a black holly leaf; if there is a distinct bright band around 230 bp, it is a white holly leaf; if amplified using primer pair SSR19, there is no distinct bright band, it is a black holly leaf; if there is a distinct bright band around 331 bp, it is a white holly leaf.If the SSR21 amplification using primers produces no obvious bright band, it indicates a black holly leaf; if there is a distinct bright band around 219 bp, it indicates a white holly leaf.

[0015] This invention successfully designed and screened 19 primer pairs, all of which can verify the DNA of black and white holly leaves, effectively distinguishing between them, and achieving rapid, stable, and accurate molecular identification of holly leaf germplasm with good repeatability, stability, reliability, and rapid detection. Attached Figure Description

[0016] Figure 1 The germplasm phenotypes are black-leaved and white-leaved.

[0017] Figure 2 This is an electrophoresis pattern of SSR marker PCR amplification for candidate primers. The left side of the primers shows a black holly leaf, and the right side shows a white holly leaf. M: 2000 DNA marker.

[0018] Figure 3 Electrophoresis diagram of PCR amplification of 12 samples using primers SSR3 and SSR16. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0020] Example 1: Development of SSR molecular markers in *Hymenoptera* leaves (1) Two different germplasms unique to Hainan (black holly leaves and white holly leaves, the two germplasms cannot be distinguished by the naked eye) were selected. Figure 1 Six samples of fresh, disease-free, young holly leaves were collected and stored in an ultra-low temperature freezer at -80℃ for later use.

[0021] (2) Total RNA was extracted from *Hylocereus undatus* leaves using a polysaccharide and polyphenol plant total RNA extraction kit, following the instructions. RNA integrity and concentration were assessed using agarose gel electrophoresis and Nanodrop to ensure the RNA sample quality met library construction requirements. Qualified RNA samples were sent to BioMed Sequencing for transcriptome library construction. Parameter-free transcriptome sequencing was performed using the Illumina high-throughput sequencing platform. The sequenced data underwent quality control, filtering, and assembly to obtain a high-quality Unigene sequence set for subsequent SSR site mining.

[0022] (3) The Unigene sequences obtained by transcriptome splicing were used to search for SSR sites across the entire genome using MISA software. The screening criteria were set as follows: dinucleotide repeats ≥ 5 times; trinucleotide repeats ≥ 5 times; tetranucleotide repeats ≥ 3 times; single nucleotide repeats (A / T, G / C) were removed; and the length of flanking sequences on both sides of the SSR site was ≥ 150 bp. High-quality SSR sequences were obtained according to the above criteria. Sequences that were too close to the end of the sequence, had continuous single base repeats on the flanking sequences, or had abnormal GC content were further removed. Finally, 22 candidate SSR sites were obtained (Table 1) for primer design.

[0023] (4) Based on the screened SSR loci and their flanking conserved sequences, primers were designed using Primer 3.0 online software with the following parameters: primer length: 18-22 bp; amplification product length: 100–300 bp; annealing temperature (Tm): 55-62 ℃, with a Tm difference of ≤2 ℃ between upstream and downstream primers; GC content: 40%-60%; hairpin structures, primer dimers, mismatches, and consecutive bases were avoided. One pair of candidate primers was designed for each SSR locus. The primer sequences were compared with the entire transcriptome of *Phyllostachys pubescens* using BLAST alignment to ensure that the primers specifically matched the target locus and had no other homologous sequences. The final candidate specific primer pairs were obtained (Table 2).

[0024] Table 1 Candidate SSR sites SSR1 TRINITY_DN801_c1_g2 210 231 TTCTTCTTCTTCTTCTTCTTC 3 7 SSR2 TRINITY_DN20121_c0_g1 217 238 GAAGAAGAAGAAGAAGAAGAA 3 7 SSR3 TRINITY_DN13811_c0_g1 224 245 GAAGAAGAAGAAGAAGAAGAA 3 7 SSR4 TRINITY_DN1526_c0_g1 231 252 TTCTTCTTCTTCTTCTTCTTC 3 7 SSR5 TRINITY_DN48466_c0_g2 236 257 GAAGAAGAAGAAGAAGAAGAA 3 7 SSR6 TRINITY_DN34301_c0_g1 244 265 TTCTTCTTCTTCTTCTTCTTTCTT 3 7 SSR7 TRINITY_DN10205_c0_g1 275 296 GAGAGAGAGAGAGAGAAA 3 7 SSR8 TRINITY_DN6359_c0_g1 296 317 GAGAGAGAGAGAGAGAAA 3 7 SSR9 TRINITY_DN666_c0_g1 387 408 GAGAGAGAGAGAGAGAAA 3 7 SSR10 TRINITY_DN70_c0_g1 424 445 GAGAGAGAGAGAGAGAAA 3 7 SSR11 TRINITY_DN20924_c0_g1 443 464 TTCTTCTTCTTCTTCTTCTTTCTT 3 7 SSR12 TRINITY_DN10837_c0_g1 520 538 AGTAGTAGTAGTAGTAGT 3 6 SSR13 TRINITY_DN4408_c0_g1 537 558 GAGAGAGAGAGAGAGAAA 3 7 SSR14 TRINITY_DN2461_c2_g1 591 612 GAGAGAGAGAGAGAGAAA 3 7 SSR15 TRINITY_DN8144_c0_g1 619 640 ATCATCATCATCATCATCATC 3 7 SSR16 TRINITY_DN100508_c1_g1 793 814 GAGAGAGAGAGAGAGAAA 3 7 SSR17 TRINITY_DN1019_c0_g1 848 869 GAGAGAGAGAGAGAGAAA 3 7 SSR18 TRINITY_DN3959_c0_g1 907 928 GAGAGAGAGAGAGAGAAA 3 7 SSR19 TRINITY_DN16558_c1_g1 1044 1065 GAGAGAGAGAGAGAGAAA 3 7 SSR20 TRINITY_DN998_c0_g2 1823 1844 ATCATCATCATCATCATCATC 3 7 SSR21 TRINITY_DN10015_c0_g3 2046 2067 TTCTTCTTCTTCTTCTTCTTTCTT 3 7 SSR22 TRINITY_DN8894_c2_g1 3003 3024 GAGAGAGAGAGAGAGAAA 3 7 Table 2 Candidate Specific SSR Primer Pairs SSR1 TTCTTCTTCTTCTTCTTCTTTCTT TCTAAGATCGGGGGTGCAT TGCATTTTGGATTGGTGGCAA SSR2 GAGAGAGAGAGAGAGAAA TGAGCAAGACGACGACGATCAAG TGGTGCAGATCAAATTACGCA SSR3 GAGAGAGAGAGAGAGAAA CTTCGGCGACTCCAATGTG TGGTGACCCTTCATCACAAG SSR4 TTCTTCTTCTTCTTCTTCTTTCTT ACGACAGATCACGGTTTTGG GATTGAGGCGAGGAGAGAAG SSR5 GAGAGAGAGAGAGAGAAA GCCATACACGGGAAGGTAA GAGGAGCACAGCGTAAATGG SSR6 TTCTTCTTCTTCTTCTTCTTTCTT AAAGAAGCTCCAGCTCAAAATAG GTGGGAAGGAAGAGTTGATG SSR7 GAGAGAGAGAGAGAGAAA CCTTCACCGACTTCGTCGT TCCGCAAATCGAGGATCAGT SSR8 GAGAGAGAGAGAGAGAAA CGTCTCCATTCTCCGAACC TGGGAGGAGAGATCTTGACGGGA SSR9 GAGAGAGAGAGAGAGAAA GAAACCCGACTGGGTTACTG ATTCCCGGTATCCACACCCC SSR10 GAGAGAGAGAGAGAGAAA GTCATCCACTCCGATTCCAG TTATGCCCGAATCGCTTCAC SSR11 TTCTTCTTCTTCTTCTTCTTTCTT CTGCTCATCATCTCTGGCTC CCGAAAGCTATCCCACAACA SSR12 AGTAGTAGTAGTAGTAGT TATTAACCTGTTGAGCTCGTCG AAATTGCAAGCTCCGACAAC SSR13 GAGAGAGAGAGAGAGAAA CAAGCGAAAAGTTGAGGTGC TGGGAAAGTGAGATCCAGGT SSR14 GAGAGAGAGAGAGAGAAA ATCGATCAGCGGATCACGAA CCACACGCATCAAGAACAGT SSR15 ATCATCATCATCATCATCATC TGTTGTAGATCCTCTTCAGGTG GGCAAAGGTGAAGATGTCG SSR16 GAGAGAGAGAGAGAGAAA CCATGATCACCATCCAGTCC AAGCATGAGACAAGAACATGAAC SSR17 GAGAGAGAGAGAGAGAAA GCAGATCTTAGCAGAAGAACG GAATGCGGGAAGGAACCTG SSR18 GAGAGAGAGAGAGAGAAA TCGATGCCCAACATTCCACA CATCAAGGAGTCGTTTGGGA SSR19 GAGAGAGAGAGAGAGAAA AGGTACCCTGCTCAACTCTT TACCCATAAGACTCACCGGC SSR20 ATCATCATCATCATCATCATC ATCATGGATTCCACACTCGG GTTCAAATGGCAGCATTGGC SSR21 TTCTTCTTCTTCTTCTTCTTC AAGAGACACACACAGAGGGAC GGAATTATTGACGAAGCAGAGAG SSR22 GAAGAAGAAGAAGAAGAAGAA CTGAGACTGGGCAAGTGTG CTGGGAAGATATTGTGGTGGG

[0025] Example 2: Polymorphism identification of DNA from different germplasm leaves of *Hylocereus undatus* using SSR primers. (1) Select 6 samples of fresh, disease-free young leaves of *Hylocereus undatus* (three samples of black leaves and three samples of white leaves) from different germplasms and store them in an ultra-low temperature freezer at -80℃ for later use.

[0026] (2) Plant DNA extraction reagents were used to extract genomic DNA from the tested *Potamogeton crispus* leaves. DNA integrity was detected by 1% agarose gel electrophoresis, and DNA concentration and purity were detected by NanoDrop. Qualified DNA samples were diluted to 50 ng / μL to ensure OD... 260 / 280 Values ​​should be between 1.8 and 2.0. Store at -20℃ for later use.

[0027] (3) Using genomic DNA from two *Hylocereus undatus* germplasm samples as templates, PCR amplification was performed on 20 candidate primer pairs for screening. PCR reaction system (20 μL): 2×Taq PCR Master Mix: 10 μL; 10 μmol / L upstream primer: 0.8 μL; 10 μmol / L downstream primer: 0.8 μL; template DNA: 2 μL; ddH2O: to 20 μL. PCR amplification program: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55-60 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ final extension for 10 min; storage at 4 ℃. The amplification products were observed using agarose gel electrophoresis to observe the polymorphism of bands among different germplasms. Figure 2 (and Table 3). The results show: The black holly leaves amplified by SSR1 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 313 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR2 primers show a distinct bright band at around 316 bp, while the white holly leaves do not, which can effectively distinguish between black and white holly leaves. The two holly leaf germplasm fragments amplified by the SSR3 primers showed obvious bright bands at around 240 bp and 250 bp, respectively, indicating the genetic polymorphism between black and white holly leaves, which can effectively distinguish between them. The black holly leaves amplified by SSR5 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 245 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR6 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 255 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR7 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 271 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR8 primers show a distinct bright band at around 263 bp, while the white holly leaves do not, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR9 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 239 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by the SSR10 primers show a distinct bright band at around 222 bp, while the white holly leaves do not, which can effectively distinguish between the two. The black holly leaves amplified by SSR11 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 257 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR12 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 239 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR13 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 244 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR14 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 240 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR15 primers show a distinct bright band at around 250 bp, while the white holly leaves do not, which can effectively distinguish between black and white holly leaves. The two holly leaf germplasm fragments amplified by the SSR16 primers showed obvious bright bands at approximately 245 bp and 255 bp, respectively, indicating the genetic polymorphism between black and white holly leaves, which can effectively distinguish between them. The black holly leaves amplified by SSR17 primers show a clear bright band at around 248 bp, while the white holly leaves do not show a clear bright band, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR18 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 230 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by SSR19 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 331 bp, which can effectively distinguish between black and white holly leaves. The black holly leaves amplified by the SSR21 primers showed no obvious bright band, while the white holly leaves showed an obvious bright band at around 219 bp, which can effectively distinguish between the two.

[0028] In summary, the bands amplified by SSR1, SSR2, SSR3, SSR5, SSR6, SSR7, SSR8, SSR9, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, and SSR21 showed significant polymorphism and could effectively distinguish between black and white holly leaves. In particular, SSR3 and SSR16 were effective polymorphic sites.

[0029] Table 3 SSR primer amplification results

[0030] Example 3: Repeatability Application Experiment Six samples each of black and white holly leaves were randomly selected, numbered, and then blindly tested. The specific procedure was as follows: using the method described in Example 2, PCR amplification was performed using genomic DNA from each of the 12 holly leaf germplasm samples as templates, with primers SSR3 and SSR16 listed in Table 3. The amplification results are shown in Table 4 and... Figure 3 Based on the PCR amplification results, it can be determined that leaves 1-6 are black holly leaves, and leaves 7-12 are white holly leaves, consistent with the results of identification using other methods. Following the experimental methods described above, PCR amplification using primers for other effective polymorphic sites listed in Table 3 also successfully identified black and white holly leaves.

[0031] Table 4. Sizes of SSR3 and SSR16 amplified fragments (bp) 1 240 245 2 240 245 3 240 245 4 240 245 5 240 245 6 240 245 7 250 255 8 250 255 9 250 255 10 250 255 11 250 255 12 250 255 The above results demonstrate that the detection method described in this invention can accurately identify black holly leaves and white holly leaves, and the detection results have good repeatability and high accuracy.

[0032] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A primer pair, characterized in that, The primer pairs are SSR1, SSR2, SSR3, SSR5, SSR6, SSR7, SSR8, SSR9, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, or SSR21, and the sequences of each primer pair are shown below:

2. A primer set, characterized in that, The primer set consists of two or more primer pairs selected from SSR1, SSR2, SSR3, SSR5, SSR6, SSR7, SSR8, SSR9, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, and SSR21. The sequences of each primer pair are shown below:

3. The primer set according to claim 2, characterized in that, The primer set includes primer pair SSR3 and / or primer pair SSR16.

4. The application of the primer pair as described in claim 1, or the primer set as described in claim 2 or 3, in distinguishing between black holly leaves and white holly leaves.

5. The application of the primer pair as described in claim 1, or the primer set as described in claim 2 or 3, in molecular marker-assisted breeding of *Hymenochloa hainanensis*.

6. A method for distinguishing between black and white holly leaves, characterized in that, The primer pair described in claim 1, or the primer set described in claim 2 or 3, may be used.

7. The method according to claim 6, characterized in that, The process includes the following steps: extracting DNA from the leaves of the holly plant to be tested, using this as a template, performing PCR amplification with the primer pair as described in claim 1, or performing PCR amplification with the primer sets as described in claim 2 or 3 respectively, and detecting and analyzing the amplification products using agarose gel electrophoresis. If the SSR1 amplification using primer pairs produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 313 bp, it is white holly leaf. If the SSR2 amplification using primer pair shows a clear bright band at around 316 bp, it is a black holly leaf; if there is no clear bright band, it is a white holly leaf. If the SSR3 amplification using primer pairs shows a distinct bright band at around 240 bp, it is a black holly leaf; if it shows a distinct bright band at around 250 bp, it is a white holly leaf. If the SSR5 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 245 bp, it is white holly leaf. If the SSR6 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 255 bp, it is white holly leaf. If the SSR7 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 271 bp, it is white holly leaf. If the SSR8 amplification using primer pair shows a distinct bright band at around 263 bp, it is a black holly leaf; if there is no distinct bright band, it is a white holly leaf. If the SSR9 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 239 bp, it is white holly leaf. If the SSR10 amplification using primer pair shows a distinct bright band at around 222 bp, it is a black holly leaf; if there is no distinct bright band, it is a white holly leaf. If the SSR11 primer pair amplifies a product with no obvious bright band, it is a black holly leaf; if there is an obvious bright band at around 257 bp, it is a white holly leaf. If the SSR12 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 239 bp, it is white holly leaf. If the SSR13 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 244 bp, it is white holly leaf. If the SSR14 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 240 bp, it is white holly leaf. If the SSR15 amplification using primer pair shows a distinct bright band at around 250 bp, it is a black holly leaf; if there is no distinct bright band, it is a white holly leaf. If the SSR16 amplified using primer pair has a distinct bright band at around 245 bp, it is black holly leaf; if it has a distinct bright band at around 255 bp, it is white holly leaf. If there is a bright band around 248 bp in the amplified SSR17 using primers, it is a black holly leaf; if there is no bright band, it is a white holly leaf. If the SSR18 primer pair amplifies a product with no obvious bright band, it is a black holly leaf; if there is an obvious bright band at around 230 bp, it is a white holly leaf. If the SSR19 amplification using primer pair produces no obvious bright band, it is black holly leaf; if there is an obvious bright band at around 331 bp, it is white holly leaf. If the primer pair SSR21 amplifies a product with no obvious bright band, it is a black holly leaf; if there is an obvious bright band at around 219 bp, it is a white holly leaf.

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