SSR molecular markers for distinguishing the morphological variants of Phyllostachys edulis and their application

Abstract

The present application belongs to the technical field of plant breeding, and particularly relates to SSR molecular markers for distinguishing Phyllostachys edulis varieties and application thereof. 209 SSR molecular markers of the genome of Phyllostachys edulis varieties are screened. The 209 SSR molecular markers of the genome of Phyllostachys edulis varieties and the polymorphic primers based on the molecular markers can be applied in the development of SSR molecular markers of Phyllostachys edulis varieties and the construction of DNA fingerprint maps, and can be used for the difference analysis of SSR marker sites of Phyllostachys edulis varieties, thereby providing a theoretical basis for the protection and utilization of Phyllostachys edulis resources and identification. In addition, the SSR molecular markers have the characteristics of good stability, high polymorphism and strong universality, and the fingerprint analysis of Phyllostachys edulis varieties by using the SSR marker sites has the advantages of convenience, rapidness and good sharing.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically relating to SSR molecular markers for distinguishing bamboo variants and their applications. Background Technology

[0002] bamboo( Phyllostachys edulis As one of the most representative bamboo species in my country, bamboo possesses ornamental, edible, economic, and ecological value, with wide applications and enormous development potential. Accurate identification of bamboo germplasm resources is a prerequisite for their efficient development and utilization. However, current variety identification techniques still have significant shortcomings: traditional morphological identification methods rely on phenotypic characteristics, resulting in long identification cycles, results easily affected by environmental factors, and large human judgment biases, especially making it difficult to distinguish closely related varieties; while existing low-throughput molecular marker-assisted methods (such as RAPD, AFLP, ISSR, etc.) generally suffer from low specificity, poor repeatability, and low information utilization efficiency, failing to meet the needs of accurate identification of subspecies-level bamboo germplasm resources, severely hindering the advancement of bamboo resource protection and variety intellectual property protection.

[0003] With the development of high-throughput sequencing technology, SSR (simple sequence repeat) molecular marker technology has become an important tool for crop variety identification due to its high stability, rich polymorphism, and strong versatility. SSR markers are developed based on short tandem repeat sequences in the genome and, combined with whole-genome sequencing and capillary electrophoresis, effectively overcome the shortcomings of traditional molecular markers, such as high development costs and low resolution in gel electrophoresis. Currently, they have been successfully applied to variety identification and genetic diversity analysis in crops, fruit trees, and forest trees, including rice, peach, tomato, peony, and poplar. However, in the field of moso bamboo, reports on using SSR marker technology for variant differentiation, variety identification, and genetic diversity research are still lacking, and a molecular marker system suitable for precise identification of moso bamboo subspecies grades has not yet been established.

[0004] Accurate identification of bamboo varieties and cultivars is fundamental to their resource conservation, rational development, and intellectual property protection. Currently, whole-genome data of bamboo and its varieties can be obtained using next-generation genome sequencing technology. However, significant technological gaps remain in the development of SSR molecular markers for bamboo varieties, the construction of DNA fingerprinting, and infraspecific classification techniques. Therefore, there is an urgent need to develop a stable, highly polymorphic, and universally applicable SSR molecular marker system to achieve rapid and accurate differentiation of bamboo varieties. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide an SSR molecular marker for distinguishing bamboo variants and related applications.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides an SSR molecular marker for distinguishing bamboo varieties, wherein the SSR molecular marker is selected from... One or more combinations of the molecular markers SSR1, SSR2, SSR4, SSR5, SSR15, SSR22, SSR28, SSR30, SSR49, SSR55, SSR57, SSR58, SSR77, SSR81, SSR104, SSR118, SSR120, SSR133, SSR139, SSR151, SSR155, SSR168, SSR177, or SSR209 are used, and the position information of the above molecular markers is shown in Table 4 below (see Table 4 in Example 2).

[0007] Preferably, the above-mentioned SSR molecular markers further include one or more combinations of SSR3, SSR6 to SSR14, SSR16 to SSR21, SSR23 to SSR27, SSR29, SSR31 to SSR48, SSR50 to SSR54, SSR56, SSR59 to SSR76, SSR78 to SSR80, SSR82 to SSR103, SSR105 to SSR117, SSR119, SSR121 to SSR132, SSR134 to SSR138, SSR140 to SSR150, SSR152 to SSR154, SSR156 to SSR167, SSR169 to SSR176, or SSR178 to SSR208 molecular markers, and the information of the above molecular markers is shown in Table 4 below (see Table 4 in Example 2).

[0008] Preferably, the repeating motif information of the SSR molecular markers SSR1, SSR2, SSR4, SSR5, SSR15, SSR22, SSR28, SSR30, SSR49, SSR55, SSR57, SSR58, SSR77, SSR81, SSR104, SSR118, SSR120, SSR133, SSR139, SSR151, SSR155, SSR168, SSR177 and SSR209 are shown in Table 4 below (see Table 4 in Example 2).

[0009] Preferably, the repeating motif information of the SSR molecular markers SSR3, SSR6 to SSR14, SSR16 to SSR21, SSR23 to SSR27, SSR29, SSR31 to SSR48, SSR50 to SSR54, SSR56, SSR59 to SSR76, SSR78 to SSR80, SSR82 to SSR103, SSR105 to SSR117, SSR119, SSR121 to SSR132, SSR134 to SSR138, SSR140 to SSR150, SSR152 to SSR154, SSR156 to SSR167, SSR169 to SSR176 and SSR178 to SSR208 are shown in Table 4 below (see Table 4 in Example 2).

[0010] In another aspect, the present invention provides a primer pair for PCR amplification of the above-mentioned SSR molecular markers, wherein the primer pairs for PCR amplification of SSR1 to SSR209 molecular markers are shown in Table 1 below (see Table 1 in Example 1).

[0011] In another aspect, the present invention provides a reagent or kit for distinguishing different varieties of moso bamboo, comprising the primer pairs described above.

[0012] In another aspect, the present invention provides an application of the above-described SSR molecular marker, primer pair, reagent, or kit, the application including any one or more of the following: (i) Application in constructing DNA fingerprinting of moso bamboo; (ii) Application in molecular marker-assisted breeding of moso bamboo; (iii) Application in the identification of bamboo variants; (iv) Application in the genetic diversity analysis of moso bamboo variants; (v) Application in the identification of kinship of moso bamboo germplasm resources.

[0013] In another aspect, the present invention provides a method for distinguishing bamboo variants, the method comprising: detecting the polymorphism of SSR molecular markers as described above in the bamboo to be tested, and distinguishing the bamboo variants to be tested based on the detection results.

[0014] Preferably, the above method includes: extracting genomic DNA from the bamboo to be tested, performing PCR amplification using the above primer pair, and distinguishing the bamboo variants to be tested based on the amplification results.

[0015] Preferably, the above method includes: comparing the detection results of the bamboo to be tested with the DNA fingerprint, and distinguishing the mutation of the bamboo to be tested based on the comparison results; wherein, the DNA fingerprint is obtained by constructing the polymorphism of the above-mentioned SSR molecular marker in bamboo.

[0016] In another aspect, the present invention provides a device for distinguishing bamboo variants, the device comprising: The DNA fingerprint acquisition module is configured to acquire the DNA fingerprint of the bamboo to be tested. The DNA fingerprinting comparison module is configured to traverse, compare, and identify the DNA fingerprint of the bamboo to be tested that has the highest similarity to the DNA fingerprint in the standard library. The identification module is configured to output the bamboo mutation information to be detected based on the DNA fingerprint pattern with the highest similarity. The DNA fingerprint was obtained by constructing the polymorphism of the aforementioned SSR molecular markers in moso bamboo.

[0017] The beneficial effects of this invention include: the 209 SSR molecular markers for moso bamboo varieties and the polymorphic primers based on these markers provided by this invention can be applied to the development of SSR molecular markers for moso bamboo varieties and the construction of DNA fingerprint maps. This allows for differential analysis of SSR marker sites among moso bamboo varieties, providing a theoretical basis for the protection, utilization, and identification of moso bamboo resources. It also enables the differentiation of different moso bamboo varieties and the distinction of infraspecies levels. Furthermore, the SSR molecular markers of this invention are characterized by good stability, high polymorphism, and strong versatility. Using the SSR marker sites of this invention for moso bamboo variety fingerprint analysis offers advantages such as convenience, speed, and good shareability. Attached Figure Description

[0018] Figure 1 To demonstrate the effectiveness of screening SSR primers based on agarose gel electrophoresis in Example 1, the figure shows the electrophoresis diagrams of 24 pairs of SSR primers (numbered 029-052) out of 332 primer pairs for amplification of bamboo and tortoise shell bamboo; wherein, each pair of primers uses two DNA templates, DNA1: bamboo genomic DNA, DNA2: tortoise shell bamboo genomic DNA. Figure 2 This is a cluster diagram of the kinship of 20 bamboo varieties in Example 4. The letters on the right side of the diagram represent the abbreviations of the 20 bamboo varieties (same as Table 2). The genetic similarity coefficient is shown at the bottom of the diagram. Figure 3 This is a capillary electrophoresis image of 20 bamboo varieties amplified by the primer pair SSR177 used in Example 5. Each fluorescence signal peak represents an allele. The top of the image shows the size of the nucleic acid fragment (bp), and the left side of the image shows RFU (relative fluorescence unit), indicating the fluorescence intensity. Figure 4The DNA fingerprinting of 20 bamboo varieties constructed based on Example 2 in Example 5 is used to detect the bamboo variety. The left side of the figure is abbreviated as 20 bamboo varieties (same as Table 2). The top of the figure shows the SSR primers used to construct the DNA fingerprinting. The numbers at the bottom of the figure are the allele sizes (bp) amplified by each primer. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0021] In a first aspect, embodiments of the present invention provide an SSR molecular marker for distinguishing bamboo variants. The SSR molecular marker is selected from one or more combinations of SSR1, SSR2, SSR4, SSR5, SSR15, SSR22, SSR28, SSR30, SSR49, SSR55, SSR57, SSR58, SSR77, SSR81, SSR104, SSR118, SSR120, SSR133, SSR139, SSR151, SSR155, SSR168, SSR177, or SSR209 molecular markers. The position information of the above molecular markers is shown in Table 4 below (see Table 4 in Embodiment 2).

[0022] It should be noted that the embodiments of the present invention are based on the resequencing data of 20 moso bamboo variant genomes (see the literature "Liu, Y., Zhu, C., Yue, X., et al., 2024. Evolutionary relationship of moso bambooforms and a multihormone regulatory cascade involving culm shape variation. Plant Biotechnol. J. 22, 2578-2592.") and moso bamboo reference genome data (see the literature "Zhao, H., Gao, Z., Wang, L., et al., 2018. Chromosome-level reference genome and alternative splicing atlas of moso bamboo"). Phyllostachys edulis (GigaScience 7, giy115). Using MISA and BWA-MEM2 software, genome data of *Phyllostachys edulis* variants were compared with reference genome data, and genome-wide SSR polymorphism loci were detected, resulting in the identification of 27,801 potential polymorphic SSR loci. Primers were then designed in batches for all SSR candidate loci using Primer 3.0 software to amplify products of 100bp-300bp with a GC content of 40%–60%; primer lengths ranged from 18bp to 27bp, with an optimal length of 22bp; and the temperature range (Tm) was 55℃–65℃. A total of 26,169 effective primer sites were obtained. Based on the SSR site location annotation file on the genome, 760 SSR sites located in gene regions were screened. A total of 332 pairs of SSR primers were synthesized for primer effectiveness amplification detection. Primers with clear and single target bands were selected for capillary electrophoresis detection, and 209 pairs of polymorphic SSR primers were finally obtained. The location information is shown in Table 4 below (see Table 4 in Example 2). Among them, the SSR29 to SSR52 molecular markers have higher polymorphism and can more specifically distinguish bamboo varieties. Therefore, one or more combinations of SSR29 to SSR52 molecular markers are preferred in this invention.

[0023] It should also be noted that the gene version referenced in this invention is the second version of the moso bamboo reference genome (Zhao et al., 2018). Assembly version number: gigadb.HIC.

[0024] In some specific examples, the above-mentioned SSR molecular markers also include one or more combinations of SSR3, SSR6 to SSR14, SSR16 to SSR21, SSR23 to SSR27, SSR29, SSR31 to SSR48, SSR50 to SSR54, SSR56, SSR59 to SSR76, SSR78 to SSR80, SSR82 to SSR103, SSR105 to SSR117, SSR119, SSR121 to SSR132, SSR134 to SSR138, SSR140 to SSR150, SSR152 to SSR154, SSR156 to SSR167, SSR169 to SSR176, or SSR178 to SSR208 molecular markers. The information of the above molecular markers is shown in Table 4 below (see Table 4 in Example 2).

[0025] It should be noted that this invention used Popgene 1.32 software to calculate genetic diversity indicators such as allele count, effective allele count, Shannon information index, observed heterozygosity, and expected heterozygosity for 209 SSR molecular markers, collectively reflecting the total genetic variation of the *Phyllostachys edulis* variant. The allele count ranged from 2 to 6, with an average of 3; the effective allele count ranged from 1.051 to 2.996, with an average of 1.864, indicating that these SSR markers exhibit a certain degree of polymorphism, suggesting the existence of detectable genetic variation within the *Phyllostachys edulis* variant, indicating that it is not a population with a highly homogeneous genetic background. Furthermore, the Shannon information index ranged from 0.117 to 1.305, with an average of 0.677, indicating relatively weak genetic differentiation within the *Phyllostachys edulis* variant. In addition, the observed heterozygosity ranged from 0 to 1, with an average of 0.641; the expected heterozygosity value was between 0.050 and 0.683, with an average of 0.428; 143 pairs of primers had expected heterozygosity less than observed heterozygosity, accounting for more than half, indicating that there is a large number of heterozygous excesses in the moso bamboo variant.

[0026] In some specific examples, the repeating motif information of the above SSR molecular markers SSR1, SSR2, SSR4, SSR5, SSR15, SSR22, SSR28, SSR30, SSR49, SSR55, SSR57, SSR58, SSR77, SSR81, SSR104, SSR118, SSR120, SSR133, SSR139, SSR151, SSR155, SSR168, SSR177 and SSR209 are shown in Table 4 below (see Table 4 in Example 2).

[0027] In some specific examples, the repeating motif information of the SSR molecular markers SSR3, SSR6 to SSR14, SSR16 to SSR21, SSR23 to SSR27, SSR29, SSR31 to SSR48, SSR50 to SSR54, SSR56, SSR59 to SSR76, SSR78 to SSR80, SSR82 to SSR103, SSR105 to SSR117, SSR119, SSR121 to SSR132, SSR134 to SSR138, SSR140 to SSR150, SSR152 to SSR154, SSR156 to SSR167, SSR169 to SSR176, and SSR178 to SSR208 are shown in Table 4 below (see Table 4 in Example 2).

[0028] It should be noted that once the gene version, molecular marker location, and repetitive sequence are determined, the flanking conserved sequence can be obtained, so the flanking conserved sequence will not be described again in this application.

[0029] Secondly, embodiments of the present invention provide a primer pair for PCR amplification of the above-mentioned SSR molecular markers. The primer pairs used for PCR amplification of SSR1 to SSR209 molecular markers are shown in Table 1 below (see Table 1 in Example 1).

[0030] It should be noted that the 209 primer pairs in Table 1 of this invention were selected by capillary electrophoresis of 332 SSR primer pairs designed based on the location, functional annotation, and potential polymorphism of SSR sites on the genome. Polymorphic SSR primers were identified through this process. Variations in the number of repeats of SSRs located in gene regions are more likely to affect gene expression and protein structure through multiple mechanisms, thereby being associated with phenotypic variations. Furthermore, this invention analyzed the 209 primer pairs using Popmarker 3.25 software. PIC (Polymorphism Information Index), among all primer pairs PIC The average value of the (polymorphic information index) is 0.339. PIC Primers with a strength ≥0.25 accounted for 72%; indicating that the primers have the ability to distinguish different genotypes and can be used for genetic diversity analysis of moso bamboo variants.

[0031] It should also be noted that a three-primer method is commonly used in PCR amplification. Specifically, the first primer is a primer with an M13 tail formed by linking the 5' end of the SSR forward primer to the M13 sequence; the second primer is the normal SSR reverse primer; and the third primer is an M13 primer with a fluorescent label (5-FAM, 5-ROX, 5-HEX, or 5-TAMRA) at its 5' end. In this way, only the universal M13 primer needs to be fluorescently labeled to achieve fluorescence detection of the SSR amplification product, while also reducing primer synthesis costs.

[0032] Thirdly, embodiments of the present invention provide a reagent or kit for distinguishing different varieties of moso bamboo, including the primer pairs described above.

[0033] It should be noted that the primer pairs in this invention can be used to prepare reagents or kits for distinguishing bamboo varieties. The form of the reagents or kits for distinguishing bamboo varieties is well-known in the art; for example, the reagents for distinguishing bamboo varieties may also include auxiliary reagents used in PCR amplification (such as Taq PCR mix or ddH2O). Preparing the reagents into kit form is for easier storage and transportation; the structure of the kits is well-known in the art.

[0034] Fourthly, embodiments of the present invention provide an application of the above-described SSR molecular marker, primer pair, reagent, or kit, the application including any one or more of the following: (i) Application in constructing a DNA fingerprint map of moso bamboo; specifically, this invention can extract the genomes of 20 moso bamboo variants using the CTAB method, and perform PCR amplification and capillary electrophoresis detection on all DNA samples using the aforementioned 209 primer pairs. The position of the fluorescence signal peak in the capillary electrophoresis results indicates the size of the amplified product fragment. Data comparison analysis is performed to record the allele size of each marker site in the 20 samples; finally, SSR molecular marker combinations that can distinguish the 20 moso bamboo variants are screened out, and the recorded data is transformed into 0 / 1 matrix data (with a 9-bit deletion site) using the SSR processing macro program DataTrans (version 2003). Finally, the fingerprint map is drawn using Tbtools software; in addition, the DNA fingerprint map of moso bamboo variants replaces traditional morphological identification with precise identification at the molecular level, and it does not change due to changes in growth stage and environmental factors; its application spans multiple aspects such as germplasm resource collection, breeding, and variety protection. If the differences in the shoot stage or morphology of moso bamboo variants are small, the DNA fingerprint map can be compared with the primer combination amplification, which can be used for faster and more accurate identification; (ii) Application in molecular marker-assisted breeding of moso bamboo; (iii) Application in the identification of bamboo variants; (iv) Application in the genetic diversity analysis of moso bamboo variants; (v) Application in the identification of kinship among bamboo germplasm resources; Specifically, in this invention, genotypic data of 209 loci from 20 bamboo varieties were analyzed using NTSYS software and the UPGMA (Universal Genetic Distance) algorithm for Euclidean genetic distance, and a clustering dendrogram was drawn. The 20 bamboo varieties were divided into different groups, and the genetic background between them was analyzed. The clustering analysis results showed that the 20 bamboo varieties were divided into two groups (I and II), with golden bamboo clustered separately into group I, and the other 19 varieties clustered into group II, indicating that golden bamboo has a relatively distant genetic background from other varieties. Group II was further divided into two subgroups, with Anji purple bamboo clustered separately into II-1, and the other 18 bamboo varieties clustered into II-2, indicating that the 18 bamboo varieties have a relatively close genetic background. This demonstrates that SSR molecular markers can be applied to the identification of kinship among bamboo germplasm resources.

[0035] Fifthly, embodiments of the present invention provide a method for distinguishing bamboo variants, the method comprising: detecting the polymorphism of SSR molecular markers as described above in the bamboo to be tested, and distinguishing the bamboo variants to be tested based on the detection results.

[0036] In some specific examples, the above method includes: extracting genomic DNA from the bamboo to be tested, performing PCR amplification using the primer pairs described above, and distinguishing the bamboo variants to be tested based on the amplification results.

[0037] It should be noted that in this invention, the differences in fluorescence signal peaks presented by the capillary electrophoresis results of the PCR amplification products can be used to determine whether they are the same variant. Specifically, when interpreting the capillary electrophoresis results, determining the target peak shape is crucial, including: (1) the size of the PCR amplification product of the bamboo to be tested is correct; (2) the fluorescence signal peak shapes of the bamboo to be tested are the same; (3) there is a difference in the fold of repetitive sequences. First, it is determined that the size of the SSR primer amplification products of the bamboo and the bamboo to be tested DNA is similar. Further analysis shows that the fluorescence signal peak shapes of the bamboo variants to be tested are the same but the positions are different, and there is a difference in the fold of repetitive sequences, indicating that there are differences between the bamboo variants to be tested and they are not the same variant; all the fluorescence signal peak shapes of the bamboo to be tested are the same and the positions are the same, indicating that they are the same variant. For example, capillary electrophoresis was performed on the DNA amplification products of 20 bamboo varieties using primer pair SSR177. Two peaks were detected in *Phyllostachys edulis* (yellow-skinned bamboo), at 176 bp and 180 bp respectively, while one peak (156 bp) was detected in *Phyllostachys edulis* (Anji purple bamboo), indicating that they are not the same variety. However, the fluorescence signal peaks of *Phyllostachys edulis* (yellow-skinned bamboo) and *Phyllostachys edulis* (flowering bamboo) were the same, and in this case, primer pair SSR177 alone could not distinguish between them. Therefore, further amplification and identification using the aforementioned primer pair combinations are necessary.

[0038] In some specific examples, the above method includes: comparing the detection results of the bamboo to be tested with the DNA fingerprint, and distinguishing the mutation of the bamboo to be tested based on the comparison results; wherein, the DNA fingerprint is obtained by constructing the polymorphism of the above-mentioned SSR molecular marker in bamboo.

[0039] It should be noted that in this invention, a DNA fingerprint can be constructed first, and then bamboo variants can be distinguished based on the DNA fingerprint. For example, the 24 pairs of SSR primers (primer pairs corresponding to SSR29 to SSR52 molecular markers) used to construct the fingerprint can be used to perform capillary electrophoresis on the DNA of the bamboo variant to be detected. The capillary electrophoresis results are then interpreted and transferred to an Excel spreadsheet. The recorded data is then converted into a 0 / 1 matrix using the SSR processing macro program DataTrans (version 2003). The results of the 24 primer combinations are then compared with the constructed fingerprint of 20 bamboo variants. If the fingerprint information is exactly the same as that of the control bamboo variant, it indicates that the bamboo variant to be detected is the control bamboo variant; otherwise, it is not the control bamboo variant.

[0040] Sixthly, embodiments of the present invention provide a device for distinguishing bamboo variants, the device comprising: The DNA fingerprint acquisition module is configured to acquire the DNA fingerprint of the bamboo to be tested. The DNA fingerprinting comparison module is configured to traverse, compare, and identify the DNA fingerprint of the bamboo to be tested that has the highest similarity to the DNA fingerprint in the standard library. The identification module is configured to output the bamboo mutation information to be detected based on the DNA fingerprint pattern with the highest similarity. The DNA fingerprint was obtained by constructing the polymorphism of the aforementioned SSR molecular markers in moso bamboo.

[0041] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0042] Example 1 This invention provides a screening process for SSR molecular markers and a screening and verification process for primer polymorphisms of SSR molecular markers.

[0043] (1) Using 20 varieties of moso bamboo, including butterfly moso bamboo, Anji purple moso bamboo, green groove moso bamboo, oil moso bamboo, hemp-skin moso bamboo, yellow-skin moso bamboo, yellow-skin flower moso bamboo, green-skin moso bamboo, strong bamboo, thick-walled moso bamboo, spotted moso bamboo, Anji purple moso bamboo, Anji brocade moso bamboo, golden silk moso bamboo, sacred bamboo, flower-stem golden silk moso bamboo, tortoise-shell bamboo, flower tortoise bamboo, square moso bamboo and Buddha belly moso bamboo, as samples, genome resequencing data were obtained; for specific operations, refer to "Liu Y, Xiao X, Li G, et al., 2022. Comprehensive analyses of simple sequence repeat (SSR) in bamboo genomes and development of SSR markers with peroxidase genes. Genes 9 (13), 1518".

[0044] (2) Based on the resequencing data of moso bamboo variant genomes (see the literature "Liu, Y., Zhu, C., Yue, X., et al., 2024. Evolutionary relationship of moso bamboo forms and a multihormoneregulatory cascade involving culm shape variation. Plant Biotechnol. J. 22,2578-2592.") and the data of the moso bamboo reference genome (Zhao et al., 2018) (see the literature "Zhao, H., Gao, Z., Wang, L., et al., 2018. Chromosome-level reference genome and alternativesplicing atlas of moso bamboo"), the resequencing data were compared with the data of the moso bamboo reference genome (Zhao et al., 2018). Phyllostachys edulis (GigaScience 7,giy115.) was used for comparative analysis and detection of whole-genome polymorphic sites.

[0045] (3) Screening of SSR molecular marker sites The basic principles for SSR marker site selection are as follows: high site sequencing quality; high site sequencing depth; appropriate repeat unit length; strong site sequence integrity; selection of sites with high polymorphic potential; clear chromosomal location of the site; high conservation of flanking regions of repeat sequences; high sequence specificity of amplified fragments; uniform GC content distribution of amplified fragments; and uniform distribution of sites on the genome.

[0046] The SSR molecular marker screening method is as follows: (3-1) SSR locus identification Identification was performed using the microsatellite identification tool (MISA), with the reference genome in FASTA format. The search criteria were set as follows: the minimum number of repetitions for single nucleotide motifs was 12, for dinucleotide motifs 6, for trinucleotide motifs 5, for tetranucleotide and pentanucleotide motifs 4, and for hexanucleotide, heptanucleotide, octanucleotide, nonanucleotide, and decanucleotide motifs 3. (3-2) Sequencing data alignment The quality-controlled sequences were aligned to the bamboo reference genome using BWA-MEM2 software, and the BAM files with redundant sequences removed were subjected to SSR polymorphism detection. (3-3) SSR primer design SSR markers were designed from 27,801 potential polymorphic SSR sites generated and filtered by resequencing. Primers were designed in batches for all SSR candidate sites using Primer 3.0 software to amplify products of 100-300 bp with a GC content of 40%–60%. Primer lengths ranged from 18-27 bp, with an optimal length of 22 bp. The temperature range (Tm) was 55-65℃. A total of 26,169 effective primer sites were obtained. (3-4) Screening for SSR loci located in the gene region Based on the SSR locus location annotation file on the genome, a total of 760 SSR loci located in gene regions were screened; based on potential SSR polymorphisms and gene function annotation, a large number of SSR primers were synthesized; after routine PCR amplification using the genomes of *Phyllostachys pubescens* and *Phyllostachys tortoise-shell bamboo* as templates, the results were detected by 2% agarose gel electrophoresis. Figure 1 As shown in Table 1, SSR primers with clear bands that could effectively amplify were selected, resulting in 332 pairs of SSR primers. Then, the 332 pairs of SSR primers were used to perform capillary electrophoresis on moso bamboo and 20 varieties, respectively. The samples with consistent amplification bands in 21 samples were removed, and finally 209 pairs of SSR primers were selected. The size of the amplification products varied in the samples tested. The nucleotide sequences of the 209 pairs of SSR primers are shown in Table 1.

[0047] Table 1. Primer pair nucleotide sequences corresponding to 209 SSR molecular markers Example 2 (1) Genomic DNA was extracted from 20 bamboo variant samples (as shown in Table 2) for constructing fingerprint maps using the CTAB method. PCR amplification was performed using primer pairs of 209 SSR molecular markers shown in Table 2 to obtain capillary electrophoresis images. The fluorescently labeled TP-M13-SSR system and procedure are shown in Table 3 below.

[0048] Table 2. Relevant information for 20 bamboo variant samples. Table 3 PCR amplification reaction system and reaction procedure (2) Compare and analyze the capillary electrophoresis data; import the compared genotype loci into Excel software and record the genotypes of 209 loci for each sample, as shown in Table 4.

[0049] Table 4 Information on 209 SSR molecular markers (3) Finally, 24 pairs of SSR molecular marker combinations (SSR1, SSR2, SSR4, SSR5, SSR15, SSR22, SSR28, SSR30, SSR49, SSR55, SSR57, SSR58, SSR77, SSR81, SSR104, SSR118, SSR120, SSR133, SSR139, SSR151, SSR155, SSR168, SSR177 and SSR209) with high polymorphism or ability to specifically distinguish moso bamboo varieties were selected and used to construct DNA fingerprint maps of 20 moso bamboo varieties.

[0050] Example 3 This invention utilizes 209 pairs of SSR primers to analyze the genetic diversity of 20 bamboo varieties. Specifically, Popmarker 3.25 and Popgene 1.32 software were used to perform SSR locus polymorphism analysis on the 20 bamboo varieties using 209 pairs of SSR primers, and the genetic diversity parameters were obtained (as shown in Table 5). The average number of alleles for the 209 pairs of SSR primers was 3, ranging from 2 to 6; the effective number of alleles varied from 1.051 to 2.996, with an average of 1.864. This indicates that these SSR markers have a certain degree of polymorphism, and there is a detectable genetic variation basis within the bamboo varieties, indicating that they are not a population with a highly homogeneous genetic background. The Shannon information index ranged from 0.117 to 1.305, with an average of 0.677, indicating weak genetic differentiation within the *Phyllostachys pubescens* variant. The observed heterozygosity ranged from 0 to 1, with an average of 0.641. The expected heterozygosity value ranged from 0.050 to 0.683, with an average of 0.428. 146 primer pairs had expected heterozygosity < observed heterozygosity, accounting for more than half, indicating a significant excess of heterozygotes within the *Phyllostachys pubescens* variant. PIC The value ranged from 0.048 to 0.607, among all primers. PIC The average value of the (polymorphic information index) is 0.339. PIC Primers with a strength ≥0.25 accounted for 72%; these primers can be used for genetic diversity analysis of moso bamboo variants.

[0051] Table 5 Genetic diversity parameters Example 4 The 209 pairs of SSR primers provided in this invention can be used to construct subspecies fingerprint maps of moso bamboo. Considering the genotype loci that distinguish moso bamboo varieties, they can also be used for the identification of phylogenetic relationships in moso bamboo germplasm resources. Specifically, the genotype data of 209 loci from 20 moso bamboo varieties are used in NTSYS software, and the UPGMA (Universal Genetic Distance) algorithm is selected for cluster analysis to draw a cluster dendrogram. The results are as follows: Figure 2 As shown, the results indicate that 20 bamboo varieties were divided into two groups (I and II). Golden bamboo clustered alone in group I, while the other 19 varieties clustered in group II, indicating that golden bamboo has a relatively distant genetic background from the other varieties. Group II was further divided into two subgroups. Anji purple bamboo was assigned to subgroup II-1, while the other 18 bamboo varieties clustered in subgroup II-2, indicating that the 18 bamboo varieties have a relatively similar genetic background.

[0052] The above data demonstrates that the 209 SSR primer pairs provided by this invention can successfully detect genetic differences in moso bamboo and can also distinguish kinship through cluster analysis, proving that this "tool" can accurately perform "genotyping" of moso bamboo and has strong practicality. Furthermore, this method can be used to draw a "genetic family tree" of moso bamboo, indicating that it is feasible to use it to establish a "variety-specific genetic fingerprint" for moso bamboo, enabling rapid identification of different moso bamboo varieties.

[0053] Example 5 In this embodiment of the invention, to determine that the SSR primer amplification products of bamboo and the tested bamboo variant DNA are similar in size, the peak shape and position of the fluorescence signal between the tested bamboo variants are further analyzed. Specifically, if the peak shape of the fluorescence signal is the same but the position of occurrence is different, indicating a difference in the fold change of the repetitive sequence, it indicates that there is a difference between the tested bamboo variants and they are not the same variant; if all the fluorescence signal peak shapes are the same and the positions of occurrence are the same, they belong to the same variant.

[0054] The primer pair SSR177 was used to detect the DNA amplification products of 20 bamboo variants by capillary electrophoresis. The results are as follows: Figure 3 As shown, the results indicate that two peaks were detected in *Phyllostachys pubescens*, at 176 bp and 180 bp respectively, while one peak was detected in *Phyllostachys anjiensis*, at 156 bp, indicating that the two are not the same variant. However, the fluorescence signal peaks of *Phyllostachys pubescens* and *Phyllostachys pubescens* are the same, and at this point, the SSR177 primer pair alone cannot distinguish between the two. Therefore, the above-mentioned primer pair combination is needed for amplification and further identification.

[0055] In addition, the identification of bamboo varieties was carried out using the 20 bamboo variant fingerprint patterns constructed in Example 2. Specifically, the process included: First, using the 24 pairs of SSR primers used to construct the fingerprint patterns, the amplification products of the bamboo variant DNA to be tested were detected by capillary electrophoresis; then, the capillary electrophoresis results were interpreted and transferred to an Excel spreadsheet. The recorded data was converted into a 0 / 1 matrix data using the SSR processing macro program DataTrans (version 2003). Then, the results of the 24 primer combinations were compared with the constructed 20 bamboo variant fingerprint patterns. If the information was exactly the same as that of the control bamboo variant fingerprint pattern, it indicated that the two were the same variant; otherwise, they were not the same variant.

[0056] Test results as follows Figure 4 As shown in Table 6 below (in Table 6, PE represents moso bamboo, and the others represent different variants). Figure 4In the figure, HPH, HGZ, and DMZ on the left represent 20 bamboo variant samples. The primers at the top of the figure are labeled with the number of alleles amplified by each primer in these samples. The allele length is in bp. Yellow dots indicate the presence of this allele in this sample, and gray dots indicate the absence of this allele in this sample.

[0057] Table 6. Amplification banding of moso bamboo and its 20 variants Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An SSR molecular marker for distinguishing varieties of moso bamboo, characterized in that, SSR molecular markers include combinations of SSR molecular markers as shown in the table below: ； The repeating motif information for the molecular markers SSR1, SSR2, SSR4, SSR5, SSR15, SSR22, SSR28, SSR30, SSR49, SSR55, SSR57, SSR58, SSR77, SSR81, SSR104, SSR118, SSR120, SSR133, SSR139, SSR151, SSR155, SSR168, SSR177, and SSR209 is shown in the table below: ； The reference gene version is the second version of the moso bamboo reference genome, and the assembly version number is gigadb.HIC.

2. The SSR molecular marker according to claim 1, characterized in that, SSR molecular markers also include combinations of SSR molecular markers listed in the table below: ； The repeating motif information for the molecular markers SSR3, SSR6 to SSR14, SSR16 to SSR21, SSR23 to SSR27, SSR29, SSR31 to SSR48, SSR50 to SSR54, SSR56, SSR59 to SSR76, SSR78 to SSR80, SSR82 to SSR103, SSR105 to SSR117, SSR119, SSR121 to SSR132, SSR134 to SSR138, SSR140 to SSR150, SSR152 to SSR154, SSR156 to SSR167, SSR169 to SSR176, and SSR178 to SSR208 is shown in the following table: ； The reference gene version is the second version of the moso bamboo reference genome, and the assembly version number is gigadb.HIC.

3. Primer pairs, characterized in that, Primer pairs are used for PCR amplification of the SSR molecular markers described in claim 1 or 2; wherein the primer pairs used for PCR amplification of the molecular markers SSR1, SSR2, SSR4, SSR5, SSR15, SSR22, SSR28, SSR30, SSR49, SSR55, SSR57, SSR58, SSR77, SSR81, SSR104, SSR118, SSR120, SSR133, SSR139, SSR151, SSR155, SSR168, SSR177 and SSR209 are shown in the table below: ； The primer pairs used for PCR amplification of SSR3, SSR6 to SSR14, SSR16 to SSR21, SSR23 to SSR27, SSR29, SSR31 to SSR48, SSR50 to SSR54, SSR56, SSR59 to SSR76, SSR78 to SSR80, SSR82 to SSR103, SSR105 to SSR117, SSR119, SSR121 to SSR132, SSR134 to SSR138, SSR140 to SSR150, SSR152 to SSR154, SSR156 to SSR167, SSR169 to SSR176, and SSR178 to SSR208 molecular markers are shown in the table below: 。 4. A reagent or kit for distinguishing different varieties of moso bamboo, characterized in that, Includes the primer pair as described in claim 3.

5. The application of the primer pair of claim 3 or the reagent or kit of claim 4 in constructing a DNA fingerprint of moso bamboo.

6. A method for distinguishing bamboo variants, characterized in that, The method includes: detecting the polymorphism of the SSR molecular marker as described in claim 1 or 2 in the bamboo to be tested, and distinguishing the variants of the bamboo to be tested based on the detection results.

7. The method according to claim 6, characterized in that, The methods include: The detection results of the bamboo to be tested are compared with the DNA fingerprint, and the mutation of the bamboo to be tested is distinguished according to the comparison results; wherein, the DNA fingerprint is obtained by constructing the polymorphism of the SSR molecular marker in bamboo as described in claim 1 or 2.

8. A device for distinguishing bamboo variants, characterized in that, The device includes: The DNA fingerprint acquisition module is configured to acquire the DNA fingerprint of the bamboo to be tested. The DNA fingerprinting comparison module is configured to traverse, compare, and distinguish the DNA fingerprint of the bamboo to be tested from the DNA fingerprint of the standard library with the highest similarity. The identification module is configured to output the bamboo mutation information to be detected based on the DNA fingerprint pattern with the highest similarity. The DNA fingerprint is obtained by constructing the polymorphism of the SSR molecular markers described in claim 1 or 2 in moso bamboo.

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