SNP molecular markers closely linked to the red xylem trait of plum blossoms and their applications

Abstract

This invention relates to the fields of plant genetic engineering, molecular biology, and genetic breeding, and discloses a SNP molecular marker closely linked to the red xylem trait in plum blossoms and its application. Through genome-wide association analysis, this invention discovered an SNP locus located at 2669885 bp on chromosome 3 of the plum blossom genome that is significantly associated with the red xylem trait. Plum trees with the CC genotype at this locus all exhibited the cinnabar trait. This SNP is located at... PmbHLH163 The gene promoter (1037 bp upstream of ATG) was located, and gene expression pattern analysis clarified that... PmbHLH163 A significant association was found between the gene and the red xylem trait in plum blossoms; this gene is primarily expressed in the stems and xylem of plum trees. The molecular marker and candidate gene screening method of this invention can be widely applied in the molecular breeding of plum blossoms for early prediction of Cinnabar Plum varieties, thereby improving 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 red xylem 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 regarded 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 Cinnabar Plum is a unique group of plum blossom varieties, with purplish-red flowers, a strong fragrance, and pale purplish-red xylem in its new shoots, commonly known as "red in the bone," giving it high ornamental value. Currently, although metabolomics analysis has clarified that cyanidin rutin and cyanidin-3-glucoside are the main causes of the red xylem in the Cinnabar Plum variety, and transcriptomics analysis has identified a series of transcription factors and enzyme genes regulating its coloration (Qiu, Like, TangchunZheng, Weichao Liu, et al. Integration of Transcriptome and Metabolome Reveals the Formation Mechanism of Red Stem in Prunus Mume). Frontiers in Plant Science (13 2022, 884883.), however, no molecular markers have been developed for the identification of Cinnabar Plum, and research on the molecular mechanism of the formation of the red xylem trait in Cinnabar Plum remains relatively limited. Therefore, screening and identifying regulatory genes related to the formation of the red xylem trait in Cinnabar Plum and developing corresponding molecular markers are of great significance for the genetic improvement and molecular breeding of Cinnabar Plum varieties. Summary of the Invention

[0003] The purpose of this invention is to provide SNP molecular markers closely linked to the red xylem characteristics of plum blossoms and their applications.

[0004] Another object of the present invention is to provide genes related to the formation of red xylem traits in *Prunus cerasifera*. PmbHLH163 And its applications.

[0005] This invention is based on whole-genome sequencing 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 three traits: xylem color, petal color, and filament color. A single SNP locus, chr3:2669885 (A>C), was identified as strongly associated with all three traits. A significant association was found between the genotype at this locus and xylem color, petal color, and filament color; the CC genotype was almost exclusively found in the cinnabar variety. This locus is located on the gene encoding a basic helical-circular-helical protein. bHLH163 The promoter region of the gene (1037 bp upstream of ATG) is named PmbHLH163 Further, by integrating transcriptome data from 22 different tissues of plum blossom, it was discovered that... PmbHLH163 The gene is expressed only in the one-year-old branches of plum blossoms, and its expression level is highest in the xylem of mature branches. Its expression in the xylem and phloem of Prunus cerasifera is significantly higher than that in Prunus mume, indicating that it may play an important regulatory role in the xylem coloration of Prunus cerasifera.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides an SNP molecular marker closely linked to the red xylem trait of plum blossom (i.e., an SNP molecular marker for identifying cinnabar plum), wherein the SNP molecular marker contains a nucleotide sequence with a polymorphism of A / C at 2669885 bp on chromosome 3 of plum blossom, denoted as marker Pm3_2669885; The physical location of the SNP molecular markers was determined based on the Meihua reference genome, which is located at https: / / ngdc.cncb.ac.cn / gwh and has the accession number PRJCA032427.

[0007] Furthermore, plum blossom materials with the genotype CC at the aforementioned polymorphic locus exhibit red xylem traits (i.e., plum blossom materials with the genotype CC at the aforementioned polymorphic locus are Cinnabar Plum).

[0008] Secondly, the present invention provides primer pairs for amplifying the SNP molecular marker, the primer pairs comprising an upstream primer as shown in SEQ ID NO:7 and a downstream primer as shown in SEQ ID NO:8.

[0009] Thirdly, the present invention provides detection reagents or kits containing the primer pairs.

[0010] Furthermore, the kit is a kit for identifying Cinnabarberry.

[0011] Fourthly, the present invention provides a method for identifying Cinnabar Plum, using the genomic DNA of the plum blossom to be tested as a template, performing PCR amplification using the primer pair or the detection reagent or kit, analyzing the PCR amplification product, obtaining the genotype of the site with the polymorphism, and determining whether the plum blossom to be tested is Cinnabar Plum based on the genotype.

[0012] Furthermore, the plum blossom material with genotype CC is *Prunus cerasifera*.

[0013] Fifthly, the present invention provides any of the following applications of the SNP molecular marker or its detection reagent: (1) Used for the identification or early prediction of the color of the xylem, petals and filaments of plum blossoms; (2) Used for the identification or early prediction of Cinnabar Plum; (3) Used for molecular marker-assisted breeding of Cinnamon Plum.

[0014] Sixthly, the present invention provides plum blossom PmbHLH163 Genes, which are: i) The nucleotide sequence shown in SEQ ID NO:1; 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; iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses the same functional protein, 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 solution; iv) Nucleotide sequences that have more than 90% homology with nucleotide sequences i), ii), or iii) and express the same functional protein; or, v) A nucleotide sequence that is completely complementary to the nucleotide sequences of i), ii), iii) or iv).

[0015] Seventhly, the present invention provides plum blossom PmbHLH163 Application of the gene or biological material containing the gene in the regulation of xylem color in plum blossoms.

[0016] Furthermore, PmbHLH163 The gene is positively correlated with the red xylem trait of plum blossoms.

[0017] The biological materials include, but are not limited to, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0018] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) Through genome-wide association analysis (GWAS) and nucleotide diversity analysis, this invention successfully screened SNP loci closely related to the xylem color, petal color, and filament color traits of plum blossoms, and located them in key regions of chromosome 3 of the plum blossom. In particular, the SNP locus Pm3_2669885 is closely related to differences in xylem color, petal color, and filament color, and can accurately distinguish *Prunus cerasifera* var. *crimsonii* through genotyping. This method not only improves the efficiency of molecular identification of plum blossom varieties but also provides reliable molecular markers for early breeding screening, accelerating the improvement process of plum blossom varieties.

[0019] (II) This invention successfully screened out PmbHLH163 The gene encodes a basic helical-loop-helical protein, which is expressed in the xylem of one-year-old and mature branches of plum blossoms. Transcriptome analysis revealed that... PmbHLH163 The expression levels of the gene in the xylem and phloem of *Prunus cerasifera* were significantly higher than those in *Prunus mume*, indicating that this gene may play an important regulatory role in the xylem coloration of *Prunus cerasifera*. This finding provides new insights into the molecular mechanism of red xylem formation in *Prunus cerasifera*.

[0020] (III) The SNP molecular markers and related genes provided by this invention ( PmbHLH163 The application of molecular markers provides strong technical support for molecular-assisted breeding of plum blossoms. Using the developed molecular markers, breeders can accurately identify *Prunus cerasifera* (red plum) in the early stages of plum breeding, not only improving breeding efficiency but also accelerating the selection process. Furthermore, combining SNP markers and... PmbHLH163 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

[0021] Figure 1 The results of GWAS analysis for three different phenotypes in Example 1 of this invention include (A) Manhattan and QQ plots of xylem color association analysis; (B) Manhattan and QQ plots of petal color; and (C) Manhattan and QQ plots of filament color.

[0022] Figure 2 This is a haplotype block diagram of genes related to the red xylem trait of plum blossom in Example 1 of the present invention.

[0023] Figure 3 In Embodiment 1 of the present invention PmbHLH163 Haplotype heatmap of the promoter and its downstream genes.

[0024] Figure 4 Different tissues of plum blossom in Example 3 of the present invention PmbHLH163Gene expression heatmap.

[0025] Figure 5 The phloem and xylem of Prunus cerasifera and Prunus cerasifera in Example 4 of this invention PmbHLH163 Differences in gene expression levels. express P <0.05, express P <0.001.

[0026] Figure 6 This is a sequence alignment diagram of the 300bp upstream and downstream regions of Pm3_2669885 in different plum blossoms in Example 4 of this invention. Wherein, WYY represents the cinnabar plum variety 'Wuyuyu', FHZS represents the cinnabar plum variety 'Fenhongzhusha', HXZS represents the cinnabar plum variety 'Hongxuzhusha', ZLE represents the green calyx plum variety 'Zaolv', SLYD represents the jade disc plum variety 'Sanlunyudie', and JM represents the plum variety Jiangmei. Detailed Implementation

[0027] This invention aims to provide a gene associated with the formation of red xylem traits in Prunus cerasifera. PmbHLH163 Its molecular markers and applications.

[0028] The present invention adopts the following technical solution: This invention provides a tightly linked SNP molecular marker for the red xylem trait of Prunus cerasifera, located at 2669885 bp on chromosome 3 of Prunus cerasifera, with a polymorphism of A / C (named Pm3_2669885).

[0029] The SNP (polymorphism A / C) located at 2669885 bp on chromosome 3 of plum blossom is closely linked to the xylem color, petal color, and filament color traits of plum blossoms. Among these, 54 varieties of *Prunus cerasifera* have the genotype CC at the aforementioned Pm3_2669885 SNP locus, 8 varieties have the genotype AC at the same locus, and the remaining 266 non-*Prunus cerasifera* varieties have either the genotype AC or AA at the same Pm3_2669885 SNP locus. All varieties with the CC genotype at this locus are *Prunus cerasifera*.

[0030] Furthermore, the SNP molecular marker Pm3_2669885 is located at the 300th bp of the sequence shown in SEQ ID NO:6 (n is a or c).

[0031] The SNP molecular markers of this invention can be used for the identification of cinnabar plum varieties.

[0032] 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.

[0033] The present invention also provides detection primers for the SNP molecular marker, wherein the detection primer for the Pm3_2669885 molecular marker is primer pair 1, the sequence of which is shown in SEQ ID NO:7-8.

[0034] Forward primer F: 5'-CCAACTTTAGTTTCTCTTGAGGGC-3' (SEQ ID NO:7); Reverse primer R: 5'-AACAACAAACTATTTGGTAAGTA-3' (SEQ ID NO:8).

[0035] The present invention also provides a kit containing detection primers for the SNP molecular marker. The kit can be used for the detection of *Prunus armeniaca* varieties.

[0036] 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 Cinnamon plum varieties.

[0037] 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.

[0038] This invention also provides a method for identifying varieties of Cinnabar Plum, comprising the following steps: 1) Extract genomic DNA from the sample to be tested (extract genomic DNA from the plum blossom to be tested); 2) Using the genomic DNA described above as a template, perform PCR amplification using the primer pair or the kit described above (using genomic DNA as a template, perform PCR amplification using the primers shown in SEQ ID NO:7-8). 3) Analyze the amplification product: If the amplification product matches the sequence corresponding to the SNP site at position 2669885 on chromosome 3 of the plum blossom genome and the genotype is CC, then it is identified as Cinnabar Plum.

[0039] Preferably, the PCR amplification reaction system consists of 100 ng template (DNA), 25 μL Planta Mix, 0.5 μM upstream primer (SEQ ID NO:7), 0.5 μM downstream primer (SEQ ID NO:8), and ddH2O to a final volume of 50 μL. Reaction conditions: 98℃ for 3 min; 98℃ for 10 s, 58℃ for 15 s, 72℃ for 30 s (30 cycles); 72℃ for 5 min; and storage at 4℃.

[0040] Further, sequencing was used for identification, and the identification criteria were as follows: when the 300th base in the amplification product was all C, the variety to be tested was Cinnabar Plum.

[0041] This invention also provides that, in SNP molecular markers, the Pm3_2669885 molecular marker is located in PmbHLH163 1037 bp upstream of the ATG gene. PmbHLH163 The gene encodes a basic helical-circular-helical protein. This invention obtains the expression pattern of this gene through transcriptome analysis. PmbHLH163 The gene is expressed only in the xylem of branches and mature branches, therefore PmbHLH163 Genes can serve as important candidate genes involved in the regulation of the red xylem traits of Prunus cerasifera.

[0042] This invention also provides PmbHLH163 Application of genes in the regulation of red xylem traits in Prunus cerasifera; the aforementioned PmbHLH163 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.

[0043] This invention also provides PmbHLH163 Application of genes in the genetic breeding of red xylem traits in plum blossoms; PmbHLH163 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.

[0044] 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.

[0045] Example 1: GWAS analysis to mine SNP molecular markers related to the red xylem trait of plum blossoms. 1. Experimental materials This embodiment, based on a high-quality plum blossom genome, aligned the resequencing data of 328 plum blossom varieties previously collected by the research group to a reference genome. Genome-wide association analysis (GWAS) was then used to screen for SNP loci associated with ornamental traits in plum blossoms. The resequencing data was obtained from the GenBank database (accession number: PRJNA352648). A total of 55 SNPs significantly associated with xylem color and 2 SNPs associated with flower / filament color were identified out of a total of 847,530 SNP loci. The QTLs controlling xylem color and petal / filament color in plum blossoms were preliminarily located within the chr3 range of 1.02-2.80 Mb. Figure 1 ).

[0046] 2. Candidate interval screening GWAS analysis revealed that the 1.02-2.80 Mb region on chromosome 3 of the plum blossom contained multiple SNP loci significantly associated with the color of different parts of the plum blossom, with three traits all associated with Pm3_2669885. Haplotype block division of neighboring SNP loci showed that this SNP was highly linked to multiple neighboring SNP loci, forming significant haplotype blocks. Figure 2 At the same time, genotypes with all nucleotides C at this locus were denoted as 1 / 1, those with all nucleotides A as 0 / 0, and heterozygotes as 0 / 1. 87.1% of cinnabar varieties (54) had a genotype of 1 / 1 at this locus, and 12.9% of cinnabar varieties (8) had a genotype of 0 / 1 at this locus, showing a very high correlation. Figure 3 ).

[0047] Example 2: GWAS Related Region PmbHLH163 Gene cloning and analysis Total RNA extraction from the plum blossom variety ('Phyllostachys pubescens') was performed according to the instructions of the plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0048] Total RNA was reverse transcribed into cDNA using a kit, and the cDNA was used as a template for... PmbHLH163 The gene's ORF was cloned by PCR. The reaction mixture consisted of 100-150 ng template (cDNA), 25 μL Planta Mix, and upstream primer (…). PmbHLH163 _F, SEQ ID NO:2) 2 μL, downstream primer ( PmbHLH163 _R (SEQ ID NO:3) 2 μL, ddH2O added to 50 μL. Reaction conditions: 98 ℃ 3 min; 98 ℃ 10 s, 60 ℃ 15 s, 72 ℃ 60 s (35 cycles); 72 ℃ 5 min; store at 4 ℃.

[0049] The target band was recovered by gel electrophoresis, ligated, transformed, and sequenced to obtain the target sequence. The ORF of this gene was then compared using DNAMAN software, and a complete sequence match was found. Therefore, the gene was named... PmbHLH163 .

[0050] Example 3 Plum Blossom PmbHLH163 Tissue-specific expression analysis of genes In order to explore PmbHLH163 This example analyzes the expression characteristics of genes in different tissues of plum blossoms, focusing on transcriptome data from 22 tissues (including roots, stems, leaves, petals, stamens, pistils, and fruits). PmbHLH163 Gene expression patterns in various tissues. The results showed that... PmbHLH163 The gene is mainly expressed in branches (including fully lignified, semi-lignified, and non-lignified branches), and its expression level is highest in the xylem of the branches. Figure 4 ).

[0051] Experimental results show that PmbHLH163 The gene exhibits distinct tissue-specific expression characteristics, providing a foundation for its application in the regulation of xylem traits in cinnabar and the development of molecular markers.

[0052] Example 4 Plum Blossom PmbHLH163 Analysis of gene expression levels in the xylem and phloem of different plum varieties 1. Sample collection Take the green calyx variety 'Early Green Calyx' ( P. mume 'ZaoLve') and the cinnabar variety 'Black Feather Jade' ( P. mume One-year-old branches of 'WuYu Yu' were carefully scraped with a blade to extract their phloem and xylem, and then tested using RT-qPCR. PmbHLH163 Gene expression levels.

[0053] 2. PmbHLH163 Gene expression analysis Total RNA was extracted 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® PremixEx Taq™ (Tli RNaseH Plus) Kit from Takara Bio Engineering (Dalian) Co., Ltd., following the instructions in the kit's manual.

[0054] Using qPCR method to PmbHLH163Gene quantification was performed using qPCR primers as shown in SEQ ID NO:4-5. The results showed that... PmbHLH163 The expression of the gene in the xylem and phloem of the 'Zhu Sha Mei' variety 'Wu Yu Yu' was significantly higher than its expression in the xylem and phloem of 'Xu Lu Cai'. Figure 5 This suggests that it may play an important role in the coloration of the xylem of Prunus cerasifera.

[0055] Example 5: Application of SNP molecular markers in the identification of plum blossom varieties Three mature plants of Cinnabar Plum and three other plum varieties were randomly selected, 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.

[0056] Genomic sequences of 300 bp before and after the Pm3_2669885 site (SEQ ID NO:6) were extracted, and PCR cloning was performed using genomic DNA as a template. The reaction system consisted of 100-150 ng template (genomic DNA), 25 μL Planta Mix, 0.5 μM upstream primer (SEQ ID NO:7), 0.5 μM downstream primer (SEQ ID NO:8), and ddH2O added to 50 μL. Reaction conditions were: 98 ℃ for 3 min; 98 ℃ for 10 s, 58 ℃ for 15 s, 72 ℃ for 30 s (30 cycles); 72 ℃ for 5 min; and storage at 4 ℃. The PCR products were then subjected to agarose gel electrophoresis, the target band was recovered, ligated, transformed, and sequenced to obtain the target sequence. The DNA sequences were compared using DNAMAN software. Figure 6 It was found that the 300th nucleotide in all Cinnabar Plum blossoms was C, while the 300th nucleotide in other plum blossom varieties was A, indicating that this site can be used to identify Cinnabar Plum varieties.

[0057] 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. Primer pairs for amplifying SNP molecular markers closely linked to the red xylem trait of plum blossoms, characterized in that, The primer pair includes an upstream primer as shown in SEQ ID NO:7 and a downstream primer as shown in SEQ ID NO:8; The SNP molecular marker contains a nucleotide sequence with a polymorphism of A / C at position 2669885 bp on chromosome 3 of the plum blossom; plum blossom materials with the genotype CC at the site of the polymorphism exhibit red xylem traits; The physical location of the SNP molecular markers was determined based on the Meihua reference genome, which is located at https: / / ngdc.cncb.ac.cn / gwh and has the accession number PRJCA032427.

2. A detection reagent or kit containing the primer pair of claim 1.

3. A method for identifying Cinnabar Plum, characterized in that, Using the genome of the plum blossom to be tested as a template, PCR amplification is performed using the primer pair described in claim 1 or the detection reagent or kit described in claim 2. The PCR amplification products are analyzed to obtain the genotype of the site with the polymorphism described. Based on the genotype, it is determined whether the plum blossom to be tested is a Cinnabar Plum.

4. The method according to claim 3, characterized in that, The plum blossom material with genotype CC is Cinnabar Plum.

5. Any of the following applications of SNP molecular markers or their detection reagents closely linked to the red xylem trait of plum blossoms: (1) Used for the identification or early prediction of the color of the xylem, petals and filaments of plum blossoms; (2) Used for the identification or early prediction of Cinnabar Plum; (3) Molecular marker-assisted breeding for Cinnamon Plum; The SNP molecular marker contains a nucleotide sequence with a polymorphism of A / C at position 2669885 bp on chromosome 3 of the plum blossom; plum blossom materials with the genotype CC at the site of the polymorphism exhibit red xylem traits; The physical location of the SNP molecular markers was determined based on the Meihua reference genome, which is located at https: / / ngdc.cncb.ac.cn / gwh and has the accession number PRJCA032427.

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