SNP molecular marker closely linked to heavy petal trait of prunus mume and application thereof

Abstract

The application relates to the field of plant genetic engineering, molecular biology and genetic breeding technology, and discloses an SNP molecular marker closely linked to plum blossom double-petal traits and application thereof. Through genome-wide association analysis (GWAS), the application finds that single nucleotide polymorphisms (SNPs) located at positions 7963262 and 8053162 of a plum blossom genome are significantly related to petal number. Plum blossom plants with genotypes TT at the positions 7963262 and 8053162 both exhibit double-petal flower types. Based on the SNP molecular marker, further screening of candidate genes related to flower type development is carried out, and through gene expression pattern analysis, it is determined that PmZFP-like a gene is significantly related to plum blossom double-petal traits. The molecular marker and the candidate gene screening method can be widely applied to molecular breeding of plum blossoms, are used for early prediction of flower type traits, and improve breeding efficiency.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering, molecular biology and genetic breeding technology, and more specifically, to SNP molecular markers closely linked to the double-petal trait of plum blossoms and their applications. Background Technology

[0002] plum bossom( Prunus mume Sieb. et Zucc.) is a member of the genus *Prunus* in the family Rosaceae. Prunus Plum blossoms are important ornamental plants, widely admired for their rich flower colors, strong fragrance, and diverse flower shapes (Chen Junyu. Illustrated Catalogue of Chinese Plum Blossom Varieties [M]. China Forestry Publishing House, 2010). The number of petals and the morphology of floral organs are important traits that determine the ornamental value and horticultural applications of plum blossoms. Among them, the double-petal trait not only affects the appearance of the flower but is also closely related to reproductive development and breeding direction. Double-petaled varieties are usually formed due to abnormal differentiation of floral organ development or differences in gene expression regulation, and their molecular basis has long been the focus of research on plum blossom genetic improvement and molecular breeding.

[0003] Transcription factors play a central role in the molecular regulatory network of plant flower development. Zinc finger proteins are an important class of transcriptional regulators that play a crucial role in plant flower development. C2H2-type zinc finger proteins influence flower formation by regulating cell division and proliferation of floral organ primordia and interacting with hormone signaling pathways. (Lyu T, Cao J. Cys2 / His2 zinc-finger proteins in transcriptional regulation of flower development[J]) International Journal of Molecular Sciences, 2018, 19(9):2589). In Arabidopsis thaliana, C2H2 type zinc finger proteins ZFP1 and ZFP8 are expressed in leaves and undeveloped floral organs, and can inhibit type B ( APETALA3 , PISTILLATA ) and Class C ( AGAMOUS Transcription of homologous genes for floral organs prevents premature activation of these genes at inappropriate locations. In the floral primordium, transcription factors... LEAFY ( LFY )and APETALA1 ( AP1 Downward adjustment ZFP1 and ZFP8The expression of these genes relieves their repression on class B and C genes, allowing organs such as petals, stamens, and carpels to form in the correct space and time (Lei L, Lin S, Wang H, et al. LEAFY and APETALA1 down-regulate ZINCFINGER PROTEIN 1 and 8 to release their repression on class B and C floralhomeotic genes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119(42): e2221181120). In addition, Zhang et al. used 328 plum blossom varieties to locate the quantitative trait of plum blossom petals to the interval 4058003-7693997 on chromosome 1 of plum blossom through genome-wide association analysis (Zhang Q, Zhang H, Sun L, et al. The genetic architecture of floral traits in the woody plant). Prunus mume [J]. Nature Communications, 2018,9(1):1702).

[0004] However, current research on the molecular mechanisms underlying the formation of double-petaled traits in plum blossoms remains relatively limited. The diversity of plum blossom flower shapes and the complexity of its genome increase the difficulty of identifying key genes. Therefore, screening and identifying regulatory genes related to the specific expression of floral organs and the formation of double-petaled traits is of great significance for elucidating the molecular basis of plum blossom flower shape development, establishing a molecular marker system, and accelerating molecular breeding. Summary of the Invention

[0005] The purpose of this invention is to provide SNP molecular markers closely linked to the double-petal trait of plum blossoms and their applications.

[0006] Another objective of this invention is to provide genes associated with the formation of double-petaled plum blossom traits. PmZFP-like And its applications.

[0007] This invention is based on whole-genome sequencing (GWAS) of 328 plum blossom varieties, with data available from the GenBank database (accession number: PRJNA352648). Genome-wide association analysis (GWAS) was used to screen for gene loci significantly associated with the double-petal trait in plum blossoms. Nucleotide diversity analysis was performed using single-petal and double-petal populations. Combining GWAS and nucleotide diversity analysis across different populations, the locus was successfully located within the 7.40 Mb to 8.01 Mb region of chromosome 1 in plum blossoms. Within this region, the SNP loci chr1:7963262 and chr1:8053162 showed a strong correlation with the number of petals, and a significant association existed between the genotype at these loci and the number of petals. Varieties with the TT genotype at both loci exhibited the double-petal trait. A new candidate gene was also discovered within this candidate region. Pm1AG1294 Named PmZFP-like This gene encodes a zinc finger protein. PmZFP-like The expression level was higher in double-petaled plum blossom varieties and lower in single-petaled varieties. Further analysis using transcriptome data from the entire plum blossom tissue revealed... PmZFP-like The gene is specifically expressed only in petals and stamens, suggesting that it may play an important regulatory role in flower development. Figure 6 ).

[0008] To achieve the objectives of this invention, in a first aspect, this invention provides a SNP molecular marker closely linked to the double-petal trait of plum blossoms, wherein the SNP molecular marker contains a nucleotide sequence with a C / T polymorphism at position 7963262 bp on chromosome 1 of the plum blossom, denoted as marker Pm1_7963262; and / or,

[0009] The SNP molecular marker contains a C / T polymorphic nucleotide sequence at position 8053162 bp on chromosome 1, denoted as marker Pm1_8053162;

[0010] The above physical location is based on the Prunus mume reference genome, which is located at https: / / ngdc.cncb.ac.cn / gwh and has the accession number PRJCA032427.

[0011] Furthermore, plum blossom materials with the TT genotype at the aforementioned polymorphic locus exhibit the double-petal trait. Plum blossom materials with the heterozygous or CC genotype at the aforementioned polymorphic locus can have both single and double petals, with the TT genotype having a significantly higher number of petals than the other two genotypes.

[0012] Secondly, the present invention provides primer pairs for amplifying the SNP molecular marker, corresponding to the marker Pm1_7963262, wherein the primer pairs include an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:3;

[0013] Corresponding to the marker Pm1_8053162, the primer pair includes an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5.

[0014] Thirdly, the present invention provides detection reagents or kits containing the primer pairs.

[0015] Fourthly, the present invention provides a method for identifying the single / double petal trait of plum blossoms. Using the genome of the plum blossom to be tested as a template, PCR amplification is performed using the primer pair or the detection reagent or kit, the PCR amplification product is analyzed, the genotype of the site with the polymorphism is obtained, and the single / double petal trait of the plum blossom to be tested is determined based on the genotype.

[0016] Furthermore, the plum blossom material with the TT genotype exhibits the double-petal trait.

[0017] Fifthly, the present invention provides any of the following applications of the SNP molecular marker or its detection reagent:

[0018] (1) Used for the identification or early prediction of single or double petal characteristics of plum blossoms;

[0019] (2) Molecular marker-assisted breeding for single or double petal traits of plum blossoms.

[0020] Sixthly, this invention provides genes related to the formation of double-petaled plum blossom traits. PmZFP-like , PmZFP-like The genes are:

[0021] i) The nucleotide sequence shown in SEQ ID NO:1;

[0022] ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:1 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function;

[0023] iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or,

[0024] iv) Nucleotide sequences that have more than 90% homology with i), ii) or iii) and express the same functional protein.

[0025] In a seventh aspect, the present invention provides PmZFP-like Application of genes or biological materials containing these genes in the regulation of double-petaled traits in plum blossoms.

[0026] Furthermore, the regulation described is a positive regulation.

[0027] The biological materials include, but are not limited to, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0028] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0029] (I) Through genome-wide association analysis (GWAS) and nucleotide diversity analysis, this invention successfully screened SNP loci closely related to the double-petal trait of plum blossoms and located them in key regions of chromosome 1 of the plum blossom. In particular, the SNP loci Pm1_7963262 and Pm1_8053162 are closely related to variations in the number of petals and can accurately distinguish between double-petal and single-petal plum blossoms through genotyping. This method not only improves the efficiency of molecular identification of plum blossom flower types but also provides reliable molecular markers for early breeding screening, accelerating the improvement process of plum blossom varieties.

[0030] (II) This invention successfully screened out PmZFP-like The gene encodes a C2H2 type zinc finger protein and is specifically expressed in the petals and stamens of plum blossoms. Transcriptome analysis revealed that... PmZFP-like The expression level of the gene was significantly higher in double-petaled plum blossoms than in single-petaled plum blossoms, indicating that this gene may play an important regulatory role in plum blossom flower shape development. This finding provides a new perspective on the molecular mechanism of the double-petaled trait in plum blossoms and lays the foundation for further research on plum blossom flower shape development.

[0031] (III) The SNP molecular markers and related genes provided by this invention ( PmZFP-like The application of SNP markers provides strong technical support for molecular-assisted breeding of plum blossoms. Using the developed molecular markers, breeders can perform precise flower type screening in the early stages of plum blossom breeding, not only improving breeding efficiency but also accelerating the selection process for double-flowered traits. Furthermore, combining SNP markers and... PmZFP-like Genetic testing can more effectively improve plum blossom varieties, promote the innovation and optimization of plum blossom germplasm resources, and has significant economic and ecological benefits. Attached Figure Description

[0032] Figure 1The GWAS analysis results in Embodiment 1 of the present invention include (A) Manhattan plot; (B) QQ plot.

[0033] Figure 2 This is a nucleotide diversity diagram from the genome selection analysis in Example 1 of the present invention.

[0034] Figure 3 This is a haplotype block diagram of plum blossom trait-related genes in Example 1 of the present invention.

[0035] Figure 4 Box plots showing the number of plum petals for different genotypes in Example 1 of this invention.

[0036] Figure 5 The plum blossom in Embodiment 2 of the present invention PmZFP-like Phylogenetic tree of the protein and ZAT-type zinc finger proteins of other species.

[0037] Figure 6 Different tissues of plum blossom in Example 3 of the present invention PmZFP-like Gene expression heatmap.

[0038] Figure 7 This invention relates to single and double-petaled plum blossoms in Embodiment 4 of the present invention. PmZFP-like Differences in gene expression levels. express P <0.0001. Detailed Implementation

[0039] This invention aims to provide a gene related to the formation of double-petaled plum blossom traits. PmZFP-like Its molecular markers and applications.

[0040] The present invention adopts the following technical solution:

[0041] This invention provides two SNP molecular markers closely linked to the double-petal trait of plum blossoms: one located at 7963262 bp on chromosome 1 of plum blossoms, with a polymorphism of C / T (named Pm1_7963262); and the other located at 8053162 bp on chromosome 1 of plum blossoms, with a polymorphism of C / T (named Pm1_8053162).

[0042] The SNPs (T / C) located at 7963262bp and 8053162bp on chromosome 1 of plum blossom are closely linked to the double / single petal trait of plum blossom. The genotype of the above-mentioned Pm1_7963262 SNP locus corresponding to the double petal trait of plum blossom is TT, and the genotype of the above-mentioned Pm1_8053162 SNP locus is TT.

[0043] Furthermore, the two SNP molecular markers, Pm1_7963262 and Pm1_8053162, are located at position 301 bp of the sequences shown in SEQ ID NO:6 and SEQ ID NO:7, respectively (n is c or t).

[0044] The two SNP molecular markers mentioned above can be used individually for the identification of double / single petal traits in plum blossoms, or they can be used in combination for the identification of double / single petal traits in plum blossoms.

[0045] It should be noted that within a 5 kb radius of the SNP site, there are sites that are completely linked or highly co-segregated with that site (r 2 Equivalent polymorphic sites with a value ≥ 0.8 are all within the scope of protection of this invention.

[0046] The present invention also provides detection primers for the SNP molecular markers, wherein the detection primers for the Pm1_7963262 molecular marker include primer pair 1, the sequence of which is shown in SEQ ID NO:2-3; and the detection primers for the Pm1_8053162 molecular marker include primer pair 2, the sequence of which is shown in SEQ ID NO:4-5.

[0047] SEQ ID NO:2: Forward primer F:

[0048] 5'-CCTCTCAATGTAAAATTGCGT-3';

[0049] SEQ ID NO:3: Reverse primer R:

[0050] 5'-TTCAATTCAAAACAGAACCCG-3'.

[0051] SEQ ID NO:4: Forward primer F:

[0052] 5'-CTTTGACCACTCAAACTAGAG-3';

[0053] SEQ ID NO:5: Reverse primer R:

[0054] 5'-TTTCTTACCTAAGCACAACAC-3'.

[0055] This invention also provides a kit containing detection primers for the aforementioned SNP molecular marker. The kit can be used for the detection of double / single petal traits in plum blossoms.

[0056] The present invention also provides the application of the SNP molecular marker or the detection primer of the SNP molecular marker or the kit containing the detection primer in the identification of double / single petal traits of plum blossom.

[0057] The present invention also provides the application of the SNP molecular marker, or the detection primer of the SNP molecular marker, or the kit containing the detection primer in the molecular marker-assisted breeding of plum blossom.

[0058] This invention also provides a method for identifying double-petaled / single-petaled plum blossom varieties, comprising the following steps:

[0059] 1) Extract genomic DNA from the sample to be tested (extract genomic DNA from the plum blossom to be tested);

[0060] 2) Using the genomic DNA described above as a template, perform PCR amplification using the primer pairs or the kit described above (using genomic DNA as a template, perform PCR amplification using the primers shown in SEQ ID NO:2-3 or SEQ ID NO:4-5).

[0061] 3) Analyze the amplification products: If the amplification products match the sequences corresponding to the SNP sites at positions 7963262 and 8053162 on chromosome 1 of the plum blossom genome and the genotype is TT, then it is determined to be a double-flowered trait.

[0062] Preferably, the PCR amplification reaction system consists of 100 ng template (DNA), 25 μL Planta Mix, 0.5 μM upstream primer (SEQ ID NO:2), 0.5 μM downstream primer (SEQ ID NO:3), and ddH2O to a final volume of 50 μL. Reaction conditions: 98℃ for 3 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 30 s (30 cycles); 72℃ for 5 min; stored at 4℃.

[0063] Preferably, the reaction procedure is as follows: 100 ng template (DNA), 25 μL Planta Mix, 0.5 μM upstream primer (SEQ ID NO:4), 0.5 μM downstream primer (SEQ ID NO:5), and ddH2O to a final volume of 50 μL. Reaction conditions: 98℃ for 3 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 30 s (30 cycles); 72℃ for 5 min; store at 4℃.

[0064] Further, sequencing was used for identification, and the identification criteria were as follows: when the 301st base in the amplification product was all T, the variety to be tested was a double-flowered plum blossom.

[0065] The Pm1_7963262 molecular marker of the present invention is located at PmZFP-like The marker Pm1_8053162 is located 4110 bp upstream of the ATG of the gene, 94010 bp upstream of the ATG. PmZFP-like The gene encodes a C2H2 type protein. This invention obtains the expression pattern of this gene through transcriptome analysis.PmZFP-like The gene is expressed only in the petals and stamens, therefore PmZFP-like Genes can serve as important candidate genes involved in the regulation of double-petaled plum blossom traits.

[0066] This invention also provides PmZFP-like Application of genes in the regulation of double-petaled plum blossom traits; the aforementioned PmZFP-like The gene has a sequence as shown in SEQ ID NO:1 or a sequence as shown in SEQ ID NO:1 obtained by deletion, substitution or insertion of one or more nucleotides to encode a protein with the same function.

[0067] This invention also provides PmZFP-like Application of genes in the genetic breeding of double-petaled plum blossoms; PmZFP- like The gene has a sequence as shown in SEQ ID NO:1 or a sequence as shown in SEQ ID NO:1 obtained by deletion, substitution or insertion of one or more nucleotides to encode a protein with the same function.

[0068] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0069] Example 1: Mining SNP molecular markers related to double-petaled plum blossom traits based on GWAS analysis

[0070] 1. Experimental materials

[0071] This embodiment, based on a high-quality T2T genome, aligned the resequencing data of 328 plum blossom varieties from the previous study to a reference genome, and screened SNP loci associated with ornamental traits of plum blossoms using genome-wide association analysis (GWAS). A total of 1,995,841 SNP loci were identified, and 6 SNPs significantly associated with the number of petals were identified. The QTL controlling the number of petals in plum blossoms was preliminarily located in the chr1 range of 6.50 Mb–8.10 Mb. Figure 1 ).

[0072] Nucleotide diversity was calculated using a sliding window for a population of double-flowered varieties (n=35) and a population of single-flowered varieties (n=22), respectively. Comparison of the nucleotide diversity between the two populations revealed a significant differentiation interval on chromosome 1 (7.40 Mb–8.10 Mb). Figure 2 This interval contains two SNPs that are significantly correlated with the number of petals (chr1:7963262 and chr1:8053162).

[0073] 2. Candidate interval screening

[0074] GWAS analysis revealed that the 6.53 Mb to 8.01 Mb region of chromosome 1 (Plum Blossom) contains multiple SNP loci significantly associated with petal number. Haplotype block division of the candidate region's SNP loci showed that multiple SNP loci were highly linked, forming significant haplotype blocks. Figure 3 The analysis results showed that the cosegregation pattern of these haplotype blocks was significantly associated with the number of petals in different plum blossom varieties. Among them, the SNP loci chr1:7963262 and chr1:8053162 showed a strong correlation. There were significant phenotypic differences between the genotypes at these two loci and the number of petals. Genotypes with both loci being T were denoted as 1 / 1, those with both being C as 0 / 0, and heterozygotes as 0 / 1. Varieties with the 1 / 1 genotype exhibited the double-petal trait. Figure 4 ).

[0075] 3. Mining of SNP molecular markers

[0076] Within this candidate interval, two SNP molecular markers associated with the double-petaled trait of plum blossoms were identified by combining GWAS with phenotypic data:

[0077] Pm1_7963262 (chr1:7963262): The TT genotype showed double petals, and the TT genotype had a significantly higher number of petals than the TC and CC genotypes.

[0078] Pm1_8053162 (chr1:8053162): The TT genotype was significantly associated with the double-petal trait, and the material with the TT genotype had a significantly higher number of petals than the materials with the TC and CC genotypes.

[0079] Example 2: GWAS Related Region PmZFP-like Gene cloning and analysis

[0080] Total RNA extraction from the plum blossom variety ('Green Calyx') was performed according to the instructions of the plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0081] Total RNA was reverse transcribed into cDNA using a kit, and the cDNA was used as a template for... PmZFP-like The gene's ORF was cloned by PCR. The reaction mixture consisted of 100-150 ng template (cDNA), 25 μL PrimeSTAR HS (Premix), and upstream primer (…). PmZFP- like _F, SEQ ID NO:8) 1μL, downstream primer ( PmZFP-like_R (SEQ ID NO:9) 1 μL, ddH2O added to 50 μL. Reaction conditions: 98℃ for 2 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 60 s (35 cycles); 72℃ for 5 min; store at 4℃.

[0082] The target band was recovered by gel electrophoresis, ligated, transformed, and sequenced to obtain the target sequence. DNAMAN software was then used for further analysis. ZFP-like The gene's ORF sequence was compared, and the sequences matched perfectly. Therefore, this... ZFP-like Gene naming PmZFP-like .

[0083] With plum blossoms PmZFP-like The amino acid sequence of the protein was used as the target sequence. ZAT gene family sequences were obtained from the Arabidopsis genome database (https: / / www.arabidopsis.org / ), and sequence alignment and phylogenetic tree construction were performed using MEGA 7.0 software. The alignment results showed that the PmZFP-like protein from *P. truncatula* has high homology with the ZFP4 protein from *Arabidopsis*, indicating a closer phylogenetic relationship. Figure 5 ).

[0084] Example 3: Plum Blossom PmZFP-like Tissue-specific expression analysis of genes

[0085] In order to explore PmZFP-like The expression characteristics of genes in different tissues of plum blossoms are illustrated in this example. PmZFP- like Transcriptome data from 22 tissues (including roots, stems, leaves, petals, stamens, pistils, and fruits) were analyzed. PmZFP- like Gene expression patterns in various tissues. The results showed that... PmZFP-like The gene expression level was significantly higher in petals and stamens than in other tissues, while it was almost non-existent in tissues such as roots, stems, leaves, pistils, and fruits. The results of this example indicate that... PmZFP-like The gene exhibits distinct tissue-specific expression characteristics, providing a foundation for its application in the regulation of double-petal traits and the development of molecular markers.

[0086] Example 4: Plum Blossom PmZFP-like Gene expression analysis in flower buds of plum varieties with different numbers of petals

[0087] 1. Sample collection

[0088] Take the single-petal variety 'Jiangmei' ( P. mume 'Jiang Mei', 'Pink Droop' ( P. mume 'Fen Chui', 'Single-petaled cinnabar'P. mume 'Dan Ban Zhu Sha'), double-flowered variety 'San Lun Fen Chui' ( P. mume 'San Lun FenChui', 'Green Calyx' P. mume 'Lve', 'Pyrophyllus' ( P. mume Flower buds of 'Wu Yu Yu' were sampled between 9:00 AM and 11:00 AM and detected using RT-qPCR. PmZFP-like Gene expression levels.

[0089] 2. PmZFP-like Gene expression analysis

[0090] Total RNA was extracted from plum blossoms using the RNAsimple Total RNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd., following the instructions in the kit's manual. cDNA synthesis and real-time quantitative PCR were performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit and TB Green® Premix Ex Taq™ (Tli RNaseH Plus) Kit from Takara Bio Engineering (Dalian) Co., Ltd., following the instructions in the kit's manual.

[0091] Using qPCR method to PmZFP-like Gene quantification was performed using qPCR primers shown in SEQ ID NO: 8-9. The results showed that... PmZFP-like The expression level of the gene in single-petaled flower buds was significantly lower than that in double-petaled flower buds. Figure 7 ).

[0092] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for identifying single / double petal characteristics of plum blossoms, characterized in that, The steps for detecting SNP molecular markers closely linked to the double-petal trait in plum blossoms are as follows: Using the genome of the plum blossom to be tested as a template, PCR amplification was performed using primer pairs as shown in SEQ ID NO:2-3 or primer pairs as shown in SEQ ID NO:4-5. The PCR amplification products were analyzed to obtain the genotype of the SNP molecular marker site. The single-petal / double-petal trait of the plum blossom to be tested was determined based on the genotype. The SNP molecular markers are Pm1_7963262 and Pm1_8053162; Pm1_7963262 is located at position 301 bp of the sequence shown in SEQ ID NO:6 on chromosome 1; Pm1_8053162 is located at position 301 bp of the sequence shown in SEQ ID NO:7 on chromosome 1. Among them, plum blossom materials with genotype TT at Pm1_7963262 site exhibit double-flowered characteristics, as do plum blossom materials with genotype TT at Pm1_8053162 site.

2. Any of the following applications of SNP molecular markers or their detection reagents that are closely linked to the double-petal trait of plum blossoms: (1) Used for the identification or early prediction of single or double petal characteristics of plum blossoms; (2) Molecular marker-assisted breeding for single or double petal traits in plum blossoms; The SNP molecular markers are Pm1_7963262 and Pm1_8053162; Pm1_7963262 is located at position 301 bp of the sequence shown in SEQ ID NO:6 on chromosome 1; Pm1_8053162 is located at position 301 bp of the sequence shown in SEQ ID NO:7 on chromosome 1. in, Plum blossom materials with genotype TT at the Pm1_7963262 locus exhibit double-flowered characteristics, as do plum blossom materials with genotype TT at the Pm1_8053162 locus. The detection reagent is a primer pair as shown in SEQ ID NO:2-3 or a primer pair as shown in SEQ ID NO:4-5.

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