A molecular detection method for identifying prunus plants as true mei, apricot or aprumei hybrids

Abstract

This invention relates to the fields of plant genetic engineering and molecular biology, and discloses a molecular detection method for identifying plum species as true plum, apricot, or apricot-plum hybrids. This invention also provides plum blossom... PmBBX24 The first intron of the gene relative to apricot PaBBX24 Structural variations in the first intron of a gene, specifically manifested as plum blossom patterns. PmBBX24 The gene has a 1554bp insertion in its first intron, which is heterozygous in apricot-plum. This site provides a basis and molecular tool for the identification of true plum / apricot-plum / apricot. PmBBX24 Gene expression is involved in the response of plum blossoms to seasonal climate change. There are significant differences in the expression patterns of seasonal climate change in winter between the true plum and apricot plum varieties. This can be used to identify the true plum / apricot plum varieties, enabling rapid and accurate identification of plum blossom varieties during the seedling stage, shortening the breeding cycle and greatly reducing the workload of breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular biology, and more specifically, to a molecular detection method for identifying plum plants as true plum, apricot, or apricot-plum hybrids. Background Technology

[0002] plum bossom( Prunus mume ) belongs to the genus *Prunus* of the family Rosaceae. Prunus Plum blossom (Prunus armeniaca) is one of China's ten traditional famous flowers, possessing significant ornamental and economic value. Originating in southwestern China, it has a cultivation history of over 3000 years (Chen Junyu. Illustrated Catalogue of Chinese Plum Blossom Varieties [M]. China Forestry Publishing House, 2010). Plum blossoms are characterized by early flowering, diverse flower colors, and rich flower / plant shapes; however, they have relatively weak cold tolerance, and their suitable habitat is in the Yangtze River basin and areas south of it. Apricot (… P. arminiaca Plum (Prunus apricot) is one of the oldest cultivated fruit trees in my country, with strong adaptability and a wide distribution, widely planted in northern my country. Under natural conditions, due to the similar geographical distribution and flowering period of apricot and plum, there is a certain degree of natural hybridization between the two. In recent decades, in order to improve the cold resistance of plum blossoms, a series of apricot-plum varieties have been bred by hybridizing plum with the more cold-resistant apricot (Chen Junyu. Illustrated Catalogue of Chinese Plum Varieties [M]. China Forestry Publishing House, 2010). There are currently more than 400 internationally registered varieties of true plum and apricot-plum, some of which have small differences in traits, making them even more difficult to distinguish, especially in the seedling stage. The conventional identification method for true plum and apricot-plum is based on the plant shape of perennial plants, the color of branches after leaf fall in autumn, or the morphology of flowers after flowering, which is highly subjective and time-consuming. With the widespread application of plum blossoms in landscaping, according to the principle of planting the right tree in the right place, it is necessary to accurately assess and identify the genetic background of plum blossoms.

[0003] Plants respond to seasonal climate change by exhibiting phenotypic adaptations. Apricot and apricot-plum blossoms shed their leaves earlier than true plum blossoms in late autumn and early winter. Along with leaf fall, the color of one-year-old branches changes from green to reddish-brown, while the one-year-old branches of true plum blossoms remain green. In recent years, although high-quality genome sequencing and resequencing of plum blossoms have been completed, a large number of genes and their functions remain to be studied, especially those related to plum blossoms' response to seasonal climate change and those related to plum blossom varieties. Research on the function and structural variation molecular markers of genes related to plum blossoms' response to seasonal climate change is of great significance for breeding, variety identification, and landscaping. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular detection method for identifying plum species as true plum, apricot, or apricot-plum hybrids. Specifically, it involves the plum blossom B-box protein PmBBX24, molecular markers with structural variations associated with the plum blossom B-box protein PmBBX24, and a molecular detection method for rapidly identifying true plum / apricot-plum / apricot varieties using molecular markers with structural variations associated with the B-box protein PmBBX24.

[0005] To achieve the objective of this invention, in a first aspect, this invention provides a molecular detection method for identifying plum plants as true plum, apricot, or apricot-plum hybrids, comprising the following steps:

[0006] (1) Extract genomic DNA from the Prunus species to be tested;

[0007] (2) Using the genomic DNA from (1) as a template, PCR amplification was performed using the primers shown in SEQ ID NO:11-12;

[0008] (3) Analyze the amplification products: If the amplification product is only a single characteristic band of 1837bp, the plant to be tested is true plum; if the amplification product is only a single characteristic band of 283bp, the plant to be tested is apricot; if the amplification product shows two characteristic bands of 1837bp and 283bp, the plant to be tested is an apricot-plum hybrid.

[0009] Further, the PCR amplification reaction system consisted of: 100-150 ng genomic DNA, 25 μL Planta Mix, 0.5 μM of the upstream primer shown in SEQ ID NO:11, 0.5 μM of the downstream primer shown in SEQ ID NO:12, and ddH2O to a final volume of 50 μL.

[0010] Furthermore, the PCR amplification reaction conditions were as follows: 98℃ for 3 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 30 s, for 30 cycles; 72℃ for 5 min; and stored at 4℃.

[0011] Secondly, this invention provides molecular markers for identifying Prunus species as true plum, apricot, or apricot-plum hybrids, located in the 12470029bp~12471865bp region on chromosome 4 of the plum tree (reference genome: Prunus longyouensis: https: / / www.rosaceae.org / Analysis / 13114608), which are used to identify true plum varieties relative to apricot varieties. BBX24 The first intron of a gene contains a 1554 bp DNA insertion fragment.

[0012] Thirdly, the present invention provides primers for the said molecular marker, including the upstream primer shown in SEQ ID NO:11 and the downstream primer shown in SEQ ID NO:12.

[0013] Fourthly, the present invention provides detection reagents or kits containing primers SEQ ID NO:11-12.

[0014] Fifthly, the present invention provides any of the following applications of the molecular marker, the primer, or the detection reagent or kit:

[0015] 1) Used for early identification of plum species as true plum, apricot, or apricot-plum hybrids;

[0016] 2) Used for identifying plum blossom stem color, leaf deciduousness, or traits that respond to seasonal climate change;

[0017] 3) Used for molecular marker-assisted breeding of true plum, apricot, or apricot-plum hybrids of plum species.

[0018] Sixthly, this invention provides a plum blossom gene. PmBBX24 It is a gene that encodes either protein (a) or (b) as follows:

[0019] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or

[0020] (b) A protein derived from (a) with the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function.

[0021] Gene PmBBX24 The nucleotide sequence is as follows:

[0022] i) The nucleotide sequence shown in SEQ ID NO:2;

[0023] ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function;

[0024] iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:2 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,

[0025] iv) Nucleotide sequences that have more than 90% homology with i), ii) or iii) and express the same functional protein.

[0026] In a seventh aspect, the present invention provides biological materials containing the said gene, including but not limited to expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0027] Eighthly, the present invention provides any of the following applications of the gene or biological materials containing the gene:

[0028] i. Used to regulate the plum blossom's response to seasonal climate change traits;

[0029] ii. Used for plum blossom genetic breeding.

[0030] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0031] (I) The present invention provides PmBBX24 The length of the first intron of a gene differs significantly between apricot and plum; therefore, the present invention provides... PmBBX24 The corresponding structural variation molecular markers and their detection primers can be used to identify plum blossom varieties in natural hybrid populations, enabling rapid and accurate identification of true plum / apricot / apricot varieties during the seedling stage of plum blossoms. This shortens the breeding cycle, greatly reduces the breeding workload, lays the foundation for accelerating the screening and propagation of plum blossom varieties, and has guiding significance for the selection of plum blossom varieties for landscaping.

[0032] (II) The invention provides PmBBX24 Genes in the *Prunus mume* and *Prunus armeniaca* varieties respond differently to seasonal climate change. PmBBX24 The relative expression level of genes can quickly distinguish plum blossom varieties, providing a basis for the rapid selection and breeding of superior plum blossom varieties.

[0033] (III) The molecular markers related to plum stem color provided by this invention have extremely high accuracy in identifying traits such as plum stem color and apricot / true plum / apricot varieties, enabling rapid early identification of plum and apricot varieties. Attached Figure Description

[0034] Figure 1 This is a local collinearity diagram comparing the plum blossom genome and the apricot genome in Example 1 of the present invention.

[0035] Figure 2 The plum blossom in Embodiment 1 of the present invention PmBBX24 Genes and Apricots PaBBX24 A comparative diagram of gene structures.

[0036] Figure 3 The plum blossom in Embodiment 2 of the present invention PmBBX24 Phylogenetic tree of the protein and B-box proteins in other species.

[0037] Figure 4In Example 4 of this invention, different plum blossom varieties during the wintering stage... PmBBX24 Changes in gene expression levels.

[0038] Figure 5 The figures show the PCR cloning results of structural variation molecular markers in different varieties of plum blossom, closely related species, and hybrids in Example 5 of this invention. In the figure: JM-'Jiangmei', SLYD-'Sanlun Yudie', BJYD-'Beijing Yudie', ZLE-'Zao Lü'e', GF-'Gongfen', WYY-'Wuyuyu', FHZS-'Fenhong Zhusha', XRB-'Xiangrui Bai', MRM-'Meirenmei', SC-'Songchun', FH-'Fenghou', DFH-'Dan Fenghou'. Detailed Implementation

[0039] This invention aims to provide the B-box protein PmBBX24, which represents the response of plum blossom to seasonal climate change, along with its encoding gene and related structural variation molecular markers, and to utilize... PmBBX24 Methods for identifying true plum / apricot / apricot varieties using genes or their related molecular markers.

[0040] This invention utilizes high-quality genomes of plum and apricot, performing alignment and filtering analysis, and employing Syri software to analyze and visualize structural variations. Genome sequence annotation results show that some genes associated with structural variations are involved in abiotic stress processes in plum. One gene located on chromosome 4 was identified among these genes. PmBBX24 The gene. In plum blossoms, a 1554 bp insertion exists within an intron of this gene, but this insertion is absent in the intron of its homologous gene in apricots. Further PCR cloning confirmed that this insertion exists only in plum blossoms and not in apricots, and that apricot and plum blossoms exhibit heterozygous behavior at this locus. Considering... BBX24 The key role of homologous genes in anthocyanin synthesis, coupled with the fact that apricot and apricot-plum branches accumulate anthocyanins during winter while plum branches remain green, suggests that the insertion in this intron affects plum blossoms. PmBBX24 The expression of this gene limits its anthocyanin synthesis capacity, thus affecting the color of the branches. In-depth research on the gene's sequence, bioinformatics prediction, and expression pattern revealed significant differences in how this gene responds to seasonal climate change between apricot plum and true plum. This characteristic and its related structural variation molecular markers can be used for screening and identification of true plum / apricot plum / apricot varieties, significantly shortening the breeding cycle and possessing practical application value.

[0041] The present invention adopts the following technical solution:

[0042] This invention provides a plum blossom PmBBX24 protein, which has any one of the following amino acid sequences (1) to (3):

[0043] (1) The amino acid sequence as shown in SEQ ID NO:1;

[0044] (2) The amino acid sequence of a protein with the same function obtained by deleting, substituting and inserting one or more amino acids as shown in SEQ ID NO:1;

[0045] (3) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO:1 and yields a protein with the same function.

[0046] This invention, through functional domain prediction analysis, found that the above-mentioned plum blossom PmBBX24 protein belongs to the B-box protein family. This family plays a key role in abiotic stress and anthocyanin synthesis. Therefore, it is speculated that this protein may be involved in cold stress and anthocyanin synthesis in plum blossom branches.

[0047] In (2) above, this can be achieved by deleting, replacing or inserting amino acids in the inactive region that will not affect the protein function, or by replacing conserved amino acids in the active region that will not affect the protein function.

[0048] In (3) above, the homology is preferably at least 95%, more preferably at least 99%.

[0049] The present invention also provides a gene encoding the plum blossom PmBBX24 protein.

[0050] Specifically, the gene encoding the plum blossom PmBBX24 protein has any one of the following nucleotide sequences (1) to (3):

[0051] (1) The nucleotide sequence shown in SEQ ID NO:2;

[0052] (2) A nucleotide sequence encoding a protein with the same function obtained by substituting, deleting or inserting one or more nucleotides as shown in SEQ ID NO:2;

[0053] (3) A nucleotide sequence that, under strict conditions, can hybridize with a nucleotide sequence as described in (1) or (2) and encode a protein with the same function.

[0054] Those skilled in the art should understand that, based on the principle of codon degeneracy, different gene sequences encoding the PmBBX24 protein can be designed according to the codon preferences of different species, and all genes capable of encoding the PmBBX24 protein are within the protection scope of this invention.

[0055] The present invention also provides the plum blossom PmBBX24 The first intron of a gene.

[0056] Specifically, plum blossoms PmBBX24 The first intron of the gene has any of the following nucleotide sequences (1) to (2):

[0057] (1) The nucleotide sequence shown in SEQ ID NO:3;

[0058] (2) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO:3.

[0059] The present invention also provides the apricot PaBBX24 The first intron of a gene.

[0060] Specifically, apricot PaBBX24 The first intron of the gene has any of the following nucleotide sequences (1) to (2):

[0061] (1) The nucleotide sequence shown in SEQ ID NO:4;

[0062] (2) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO:4.

[0063] The present invention also provides biological materials containing a gene encoding the plum blossom PmBBX24 protein, said biological materials including expression cassettes, vectors, host cells, engineered bacteria or transgenic plant cell lines.

[0064] The aforementioned vectors include, but are not limited to, cloning vectors, expression vectors, and plasmid vectors. All vectors containing the gene encoding the plum blossom PmBBX24 protein are within the scope of protection of this invention.

[0065] The host cell or engineered bacteria can be derived from microorganisms, plants, or animals. All host cells or engineered bacteria containing the gene encoding the PmBBX24 protein are within the scope of protection of this invention.

[0066] Furthermore, the present invention also provides specific amplification primers for the gene encoding the plum blossom PmBBX24 protein, having the sequence shown in SEQ ID NO:5-6.

[0067] On the other hand, the present invention also provides a molecular marker for structural variation related to plum blossom stem color, which is located on chromosome 4 of plum blossom, PmuVar_Chr4_0218 ( PmBBX24 In the first intron of the gene, there is a 1554bp insertion in the plum blossom, while in the apricot... PaBBX24 The insertion segment does not exist in the first intron.

[0068] Furthermore, the present invention also provides detection primers for the structurally variable molecular markers, having sequences as shown in SEQ ID NO:11-12.

[0069] Among them, the primers SEQ ID NO:11 and SEQ ID NO:12 are used in PmuVar_Chr4_0218 ( PmBBX24 The first intron structural variation of the gene is detected. When the primer pair is used to amplify plum, apricot, and apricot, products of different lengths can be obtained. The plum, apricot, and apricot can be identified based on the length of the PCR product.

[0070] This invention demonstrates through experiments that, during the onset of winter, true plum and apricot plum... BBX24 Gene expression levels change, and BBX24 Genes in *Prunus mume* and *Prunus apricot* are induced or suppressed by different environmental factors, resulting in significantly different expression patterns. These differential expression patterns can be used for rapid identification of *Prunus mume* and *Prunus apricot* strains and their hybrid offspring, or for further analysis. BBX24 Gene regulation of plum blossom's response to seasonal climate change traits.

[0071] On the other hand, the present invention provides any of the following applications of the gene encoding the BBX24 protein or biological materials containing the gene:

[0072] (1) Application in regulating plum stem color, leaf fall traits or traits that respond to seasonal climate change;

[0073] (2) Application in plum blossom genetic breeding.

[0074] This invention demonstrates through experiments that the structural variation molecular markers related to plum stem color can be used for the identification of true plum / apricot plum / apricot varieties, and this locus can completely separate different varieties.

[0075] On the other hand, the present invention provides any of the following applications of the gene encoding the PmBBX24 protein or its specific amplification primers, or the structural variation molecular marker or its detection primers:

[0076] (1) Application in the identification of apricot plum / true plum blossom varieties;

[0077] (2) Application in the identification of seedlings of apricot-plum hybrid offspring;

[0078] (3) Application in the identification of plum stem color, leaf fall characteristics or seasonal climate change response characteristics.

[0079] Specifically, the above applications include: detecting the presence of certain substances in plum blossoms. PmBBX24 The regulation of genes by seasonal climate change, according to PmBBX24Differential gene expression under different climatic conditions was used to identify apricot / true plum flower varieties; or...

[0080] The above applications include: using plum genomic DNA as a template, detecting the genotype of the structural variation molecular marker associated with the first intron of PmBBX24, and identifying the true plum / apricot plum / apricot strains.

[0081] This invention provides a method for identifying true plum / apricot plum / apricot varieties. The method includes: culturing the plum blossoms to be tested under different seasonal climatic conditions, extracting RNA from the plum blossoms to be tested, analyzing the differential expression of the gene encoding the PmBBX24 protein in the plum blossoms to be tested cultured under different seasonal climatic conditions, and determining the variety of the plum blossoms to be tested.

[0082] The protein encoded by the PmBBX24 gene has any one of the following amino acid sequences (1) to (3):

[0083] (1) The amino acid sequence as shown in SEQ ID NO:1;

[0084] (2) The amino acid sequence of a protein with the same function obtained by deleting, substituting and inserting one or more amino acids as shown in SEQ ID NO:1;

[0085] (3) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO:1 and yields a protein with the same function.

[0086] 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.

[0087] Example 1: Genomic differences between plum and apricot based on comparative genomics analysis

[0088] The Mummer software (https: / / github.com / mummer4 / mummer) was used to compare and analyze the plum blossom genome (https: / / www.rosaceae.org / Analysis / 13114608) and the apricot genome (https: / / www.rosaceae.org / Analysis / 9642068). After filtering, the structural variations were visualized and annotated using the Syri software.

[0089] Comparative genomic analysis revealed a 1554 bp insertion in the first intron of the PmuVar_Chr4_0217 gene on chromosome 4 of the Meihua gene. This gene is annotated as follows: BBX24Homologous genes were further analyzed, and the CDS sequences of this gene were extracted from plum and apricot, respectively. It was found that the CDS sequences of plum and apricot differed by 4 nucleotides, and the protein sequences of plum and apricot differed by only one amino acid, indicating very little difference in their protein sequences. Figure 1 and Figure 2 ).

[0090] Example 2 PmBBX24 Gene cloning and analysis

[0091] Total RNA extraction from the plum blossom variety ('Early Green Calyx') was performed according to the instructions of the plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0092] Total RNA was reverse transcribed into cDNA using a kit, and the cDNA was used as a template for... BBX24 The gene's ORF was cloned by PCR. The reaction mixture consisted of 100 ng template (cDNA), 25 μL Planta Mix, 0.3 μM upstream primer (PmBBX24_F, SEQ ID NO:5), 0.3 μM downstream primer (PmBBX24_R, SEQ ID NO:6), and ddH2O to a final volume of 50 μL. Reaction conditions were: 98℃ for 3 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 30 s (30 cycles); 72℃ for 5 min; and stored at 4℃.

[0093] The target band was recovered by gel electrophoresis, ligated, transformed, and sequenced to obtain the target sequence. This was then combined with DNAMAN software for further analysis. BBX24 The gene's ORF sequence was compared, and the sequences matched perfectly. Therefore, this... BBX24 Gene naming PmBBX24 .

[0094] With plum blossoms PmBBX24 Using the amino acid sequence of the protein as the target sequence, homologous amino acid sequences of 15 species, including Arabidopsis, peach, and plum, were obtained from the NCBI database using Blastp. Phylogenetic trees were constructed using MEGA 7.0 software for alignment. The results showed that the PmBBX24 protein of plum blossom and PaBBX24 of apricot are more closely related to PsBBX24 of wild apricot, PpBBX24 of peach, and the B-box protein of almond. Figure 3 ).

[0095] Example 3: Plum and Apricot BBX24 Cloning of the first intron of a gene

[0096] Total DNA extraction of plum blossom variety ('Early Green Calyx') and apricot was performed according to the instructions of the plant total DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0097] Using DNA as a template BBX24 The first intron of the gene was cloned by PCR. The reaction mixture consisted of 100 ng template (DNA), 25 μL Planta Mix, 0.5 μM upstream primer (BBX24int_F, SEQ ID NO:7), 0.5 μM downstream primer (BBX24int_R, SEQ ID NO:8), and ddH2O to a final volume of 50 μL. Reaction conditions were: 98℃ for 3 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 30 s (30 cycles); 72℃ for 5 min; and stored at 4℃.

[0098] The target band was recovered by gel electrophoresis, ligated, transformed, and sequenced to obtain the target sequence. The DNAMAN software was then used to analyze the two... BBX24 Sequence comparison of the first intron of the gene revealed significant differences between the two, including plum blossoms. PmBBX24 The first intron of the gene is 1837 bp in length, while the apricot... PaBBX24 The first intron of the gene is 284 bp in length.

[0099] Example 4: Seasonal Climate Change PmBBX24 Gene expression level change analysis

[0100] 1. Material selection for plum blossoms during the winter season

[0101] Mature true plum ('Beijing Yudie') and apricot plum ('Danfenghou') with consistent growth were selected from the Beijing Jiufeng Plum Germplasm Resource Nursery as experimental materials. Observations were conducted during the wintering period, with sampling performed every two weeks, specifically on September 30, October 14, October 28, November 12, and November 26, for different varieties of plum branches.

[0102] 2. PmBBX24 Gene expression analysis

[0103] 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 1ststrand 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.

[0104] Using qPCR method to PmBBX24 Genes were subjected to relative quantification assays, and the qPCR detection primers are shown in SEQ ID NO:9-10.

[0105] turn out( Figure 4 Different plum blossom varieties PmBBX24 Gene expression levels showed significant differences during the wintering phase, with *Prunus mume* exhibiting these differences before winter. PmBBX24 It exhibits high expression, but its expression gradually decreases during the wintering season, in apricot plums PmBBX24 The expression trend is reversed during the wintering period. Before winter, expression is low, but during the onset of winter, its expression level is continuously induced by the cold, indicating... PmBBX24 The apricot plum responded to the low temperature during its overwintering process.

[0106] Example 5: Application of structural variation markers in the identification of plum blossom varieties

[0107] Eight different varieties of plum blossom, four hybrids, and closely related species (including apricot, wild apricot, and peach) were randomly selected as mature plants, and DNA was extracted from the young leaves according to the instructions of the Plant Genomic DNA Extraction Kit of Tiangen Biotech (Beijing) Co., Ltd.

[0108] Intron insertion regions were cloned by PCR using genomic DNA as a template. The reaction mixture consisted of 100–150 ng template (genomic DNA), 25 μL Planta Mix, 0.5 μM upstream primer (Intron_F, SEQ ID NO:11), 0.5 μM downstream primer (Intron_R, SEQ ID NO:12), and ddH2O to a final volume of 50 μL. The reaction conditions were: 98 °C for 3 min; 98 °C for 10 s, 56 °C for 15 s, 72 °C for 30 s (30 cycles); 72 °C for 5 min; and storage at 4 °C. The PCR products were then subjected to agarose gel electrophoresis.

[0109] Analysis of agarose gel electrophoresis results ( Figure 5 The results showed that a band of approximately 1800 bp could be cloned from the DNA of all plum varieties, while only a 300 bp band could be cloned from closely related species. In the hybrid apricot-plum, both approximately 1800 bp and 300 bp bands could be cloned. These results indicate that the intron insertion exists only in true plum, and is absent in closely related species (apricot and peach). Furthermore, the hybridization of apricot and plum results in this site exhibiting a heterozygous state in apricot-plum.

[0110] This invention establishes a rapid molecular biological method for identifying true plum / apricot plum varieties, utilizing the plum and apricot varieties provided by this invention. BBX24 The detection of structural variation molecular marker sites in gene introns can enable seedling identification and selection of plum blossom varieties, improve the efficiency of plum blossom application in landscaping, and greatly reduce screening costs, thus laying a theoretical foundation for plum blossom selection, breeding and variety identification.

[0111] 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 molecular detection method for identifying plum species as true plum, apricot, or apricot-plum hybrids, characterized in that, Includes the following steps: (1) Extract genomic DNA from the Prunus species to be tested; (2) Using the genomic DNA from (1) as a template, PCR amplification was performed using the primers shown in SEQ ID NO:11-12; (3) Analysis of amplification products: If the amplification product is only a single characteristic band of 1837 bp, the plant to be tested is true plum; if the amplification product is only a single characteristic band of 283 bp, the plant to be tested is apricot; if the amplification product has two characteristic bands of 1837 bp and 283 bp, the plant to be tested is a hybrid of apricot and plum.

2. The method according to claim 1, characterized in that, PCR amplification reaction system: 100-150 ng genomic DNA, 25 μL Planta Mix, 0.5 μM upstream primer shown in SEQ ID NO:11, 0.5 μM downstream primer shown in SEQ ID NO:12, and ddH2O to a final volume of 50 μL.

3. The method according to claim 1 or 2, characterized in that, PCR amplification 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 ℃.

4. Any of the following applications of molecular markers for identifying Prunus species as true plum, apricot, or apricot-plum hybrids, primers for amplifying said molecular markers, or detection reagents or kits containing said primers: 1) Used for early identification of plum species as true plum, apricot, or apricot-plum hybrids; 2) Used for molecular marker-assisted breeding of true plum, apricot, or apricot-plum hybrids of plum species; The molecular marker is located in the 12470029 bp to 12471865 bp range on chromosome 4 of the plum blossom variety. The first intron of the BBX24 gene in the true plum variety, relative to the apricot variety, contains a 1554 bp DNA insertion fragment.

5. The application according to claim 4, characterized in that, The primers used to amplify the molecular marker include the upstream primer shown in SEQ ID NO:11 and the downstream primer shown in SEQ ID NO:12.

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