Molecular marker related to high citric acid content of plum fruits and application of molecular marker

Abstract

The invention discloses a molecular marker related to high citric acid content of plum fruits and application of the molecular marker, and belongs to the technical field of fruit tree molecular marker assisted breeding. Based on comparative genomics analysis of plum and sibling apricot, in combination with weighted gene co-expression network analysis, a key gene significantly related to the high accumulation character of fruit citric acid is identified, a specific structural variation exists in a promoter region of the gene, and the gene can be used for early screening of germplasm with high content of citric acid in plum fruits. The molecular marker provided by the invention has important application value, and provides an efficient and reliable technical means for molecular marker assisted breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology for fruit trees, specifically relating to a molecular marker related to the high citric acid content of plum (Prunus mume) fruit and its application. Background Technology

[0002] Plum (Prunus mume Sieb. et Zucc.) is a woody drupe fruit tree belonging to the genus Prunus in the family Rosaceae. Its fruit is known for its sourness, hence the saying "looking at plums quenches thirst." It has a high organic acid content, reaching 4-7%, with a rich variety of acids, primarily citric acid, followed by malic acid, and small amounts of tartaric acid, acetic acid, lactic acid, and succinic acid. The organic acid content of the fruit directly affects its fresh consumption and processing value. Acidity is a crucial factor in shaping the flavor of fresh fruits and an important quality indicator for processed fruits. High-acid fruits have irreplaceable advantages in brewing, processing, and other fields. Since my country achieved a moderately prosperous society and proposed the concept of a "big food" perspective, people have increasingly focused on healthy eating and the research and development of functional foods. Sour foods and safe and convenient processed fruits are gradually becoming a new consumer trend. Analyzing the molecular mechanisms of organic acid accumulation in fruits and further applying them to breeding and production is beneficial to promoting industrial development.

[0003] However, in traditional breeding, the screening of high acidity traits faces significant bottlenecks: First, phenotypic identification depends on the peak fruiting period, and fruit acidity can only be measured after the plant has entered the peak fruiting period, which is time-consuming and costly; Second, the accumulation of organic acids is easily affected by environmental factors, such as light, temperature, and soil, which significantly affect the accuracy of phenotypic selection; Third, the genotype-phenotype relationship is complex, and acidity, as a quantitative trait controlled by multiple genes, exhibits severe segregation in offspring in conventional hybridization breeding, resulting in low efficiency in selecting superior plants.

[0004] Currently, marker-assisted selection (MAS) technology provides a new pathway for fruit tree breeding, but research on the high acidity trait in plums still faces challenges: the reported acidity-related QTL sites have low resolution, making it difficult to accurately locate related variations; the development of conventional markers based on SSRs and SNPs largely relies on genetic linkage analysis, lacking support from molecular mechanisms at the functional gene level, resulting in insufficient marker universality and stability. In recent years, comparative genomics and co-expression network analysis (such as WGCNA) have provided new ideas for discovering candidate genes for important traits. Studies have shown that the PH4 gene regulates vacuolar proton pump activity in fruit trees such as citrus and apples, directly participating in the transport and accumulation of organic acids, but the relationship between its allelic variations and acidity in plums remains unclear, and no highly efficient molecular markers based on functional gene structural variations have been found.

[0005] Therefore, it is urgent to discover molecular markers that co-segregate with the high acidity trait of plums and have functional interpretability, so as to achieve early and accurate identification of germplasm resources and accelerate the breeding process of varieties with high citric acid content in fruits. Summary of the Invention

[0006] This invention aims to solve the technical problems of long identification cycle, low accuracy and lack of functional association markers in the existing technology of identifying the acidic trait of maltodextrin, and to provide a molecular marker based on key gene structural variation and its efficient detection method.

[0007] To achieve the above objectives, the present invention provides a molecular marker associated with high citric acid content in plum fruit. The molecular marker is a 377bp sequence inserted 365bp upstream of the transcription start site of the key gene for citric acid accumulation in plum, PmPH4. The sequence of the molecular marker SV is shown in SEQ ID NO.1.

[0008] The inventors, through comparative genomics analysis of plum and apricot (Prunus armeniaca) combined with WGCNA co-expression network analysis, identified the key gene PmPH4 regulating citric acid accumulation; a specific insertion variant (SV) of 377 bp was identified in the promoter region of the PmPH4 gene (approximately 365 bp upstream of the transcription start site). Figure 7 (a) This SV showed a significant co-segregation relationship with the high acidity trait of plum fruit and was defined as a molecular marker of high citric acid germplasm in plum fruit.

[0009] The present invention also provides a primer pair for specifically detecting the molecular marker, comprising primer PH4SV-F1 and primer PH4SV-R1, wherein the nucleotide sequence of primer PH4SV-F1 is AGC AGG AAT TCC ACT GGT ATG AAA G, and the nucleotide sequence of primer PH4SV-R1 is GGG GAT GGA AGA AGA TGG TAA TTG G.

[0010] This primer pair can amplify target regions containing SV. The amplification product is 686 bp (including a 377 bp insertion) in high-citrate germplasm and 309 bp (without insertion) in low-citrate germplasm.

[0011] This invention also provides the application of the above-mentioned molecular markers or primer pairs in plum breeding, including:

[0012] (a) Screening of high-citric acid germplasm from plum germplasm resource bank;

[0013] (b) Early marker-assisted selection of high-citrate individuals in hybrid progeny populations;

[0014] (c) Identification of plum variety purity.

[0015] This invention also provides a method for identifying malachite citric acid germplasm, comprising the following steps:

[0016] Step 1: Extract genomic DNA from the plum plant to be tested using the CTAB method or a DNA extraction kit;

[0017] Step 2: Perform PCR amplification using the primer pairs described above;

[0018] Step 3: Detect the size of the amplified product bands by agarose gel electrophoresis;

[0019] Step 4: If the amplification product shows a band at 686bp (homozygous genotype: single band; heterozygous genotype: double bands at 686bp and 309bp), it indicates that the plant being tested carries the high-citric acid associated SV; if only a single band at 309bp is present, it indicates that the plant being tested does not carry the high-citric acid associated SV.

[0020] Based on comparative genomics analysis of plum and its closely related species apricot, combined with weighted co-expression network analysis (WGCNA), this invention identified a key gene, PmPH4 (LOC103320403), significantly associated with the high citric acid accumulation trait in plum fruit. A specific structural variation (SV) of 377 bp exists in its promoter region (approximately 365 bp upstream of the transcription start site). This SV shows a significant co-segregation relationship with the high acidity trait in plum fruit, serving as a molecular marker for high citric acid germplasm in plum fruit. Based on this structural variation, specific primers were designed to amplify the corresponding target fragment in the plum genomic DNA via PCR. The difference in band size of the amplified products was then detected by agarose gel electrophoresis, allowing for rapid and accurate determination of whether the tested plant carries the allele associated with the high citric acid trait.

[0021] This invention enables rapid and accurate early identification of high-citric acid traits through DNA testing at the seedling stage, effectively shortening the breeding cycle by 4-6 years. Furthermore, the identification method boasts high accuracy and stability, based on structural variations of functional genes, unaffected by environmental factors, and exhibits high detection accuracy. It is also simple and low-cost to operate, requiring only conventional PCR and agarose gel electrophoresis equipment, with detection completed within 2 hours, without the need for sequencing or specialized instruments. The marker has broad applicability: suitable for screening high-acid germplasm in all cultivated and wild relatives of the genus *Prunus*.

[0022] The molecular markers of the present invention can be applied to: (1) high-throughput screening of germplasm resource banks: rapid identification of high-acid germplasm and construction of core breeding populations; (2) early selection of hybrid offspring: selection of high-acid single plants at the seedling stage to reduce field management costs; (3) identification of variety purity: distinguishing between high-citric acid varieties and low-citric acid varieties to ensure seedling quality.

[0023] The molecular markers of this invention have significant application value, providing an efficient and reliable technical means for molecular marker-assisted breeding. Attached Figure Description

[0024] Figure 1 The genome structural variation analysis diagrams for 'Zhejiang Pearl Plum' and 'Yinxiang White Apricot' include: (a) genome structural variation maps of 'Zhejiang Pearl Plum' and 'Yinxiang White Apricot'; (b) variation types and frequency statistics in the genomes of both; (c) annotation results of genome structural variations in 'Yinxiang White Apricot' and statistics of the number of variant genes; and (d) GO enrichment analysis results of promoter variation, exon variation, and splice site variation genes.

[0025] Figure 2 The weighted gene co-expression network analysis diagram for plum fruit includes: (a) hierarchical clustering tree of WGCNA module; (b) dendrogram of adjacent genes; (c) heatmap of correlation analysis between each module and phenotype; and (d) Venn diagram of 4 candidate genes obtained from the intersection of 256 core genes and structural variation genes in the green module.

[0026] Figure 3 This is a phylogenetic tree of MYB transcription factors in plum, among which Pmu000042 is most closely related to the PH4 gene in citrus and petunia.

[0027] Figure 4 The results of spatiotemporal expression analysis of the PmPH4 gene include its expression in tissues such as roots, stems, leaves, flowers, and fruits.

[0028] Figure 5 The graph shows the expression analysis of PmPH4 in plum fruits, including: (a) the changes in the relative expression level of PmPH4 during the fruit development of 'Zhejiang Pearl Plum', including S1 (33 DAF), S3 (48 DAF), S5 (62 DAF), S7 (77 DAF), and S9 (92 DAF); (b) the comparison of the relative expression level of PmPH4 in mature fruits of 'Zhejiang Pearl Plum' (ZJZZM), 'Fenghou' (FH), and 'Jintaiyang' (JTY).

[0029] Figure 6 The results of transient overexpression analysis of PmPH4 in tobacco and plum fruit include: (a) the relative expression level of PmPH4 in tobacco leaves; (b) the change in citric acid content in tobacco leaves; (c) the relative expression level of PmPH4 in plum fruit; and (d) the change in citric acid content in plum fruit.

[0030] Figure 7The diagram shows the transcriptional activity analysis of the PmPH4 promoter, including: (a) analysis of cis-acting elements in the PmPH4 promoter region; (b) detection of the transcriptional activity of the PmPH4 promoter in tobacco leaves using a dual-luciferase (LUC) reporter system; and (c) assessment of the effect of structural variations on the transcriptional activity of the PmPH4 promoter by the LUC / REN ratio.

[0031] Figure 8 The image shows the sequence alignment of the PH4 gene promoter in plum and apricot. The materials involved include: Prunus armeniaca'Marouch_n14' (MAR), 'Yinxiang Baixing' (YXB), 'Fenghou' (FH), 'Zhejiang Zhenzhumei' (ZJZZM), 'Nanhong' (NH), and 'Zangmei' (ZM).

[0032] Figure 9 PCR identification results of PH4 promoter structural variants: (a) PCR identification diagram of SV in plum, apricot and apricot populations; (b) PCR identification diagram of SV in peach, plum and sweet cherry populations.

[0033] Figure 10 The results of citric acid content determination in the fruits of different varieties of plum, apricot, and apricot included the following varieties: Soft-striped Red Plum (RTHM), Pinzi Plum (PZM), Zhejiang Pearl Plum (ZJZZM), Longan (LY), Nan Hong (NH), Yanglao (YL), Ina Bungo (YNFH), Takada Bungo (GTFH), Bungo (FH), Setsuda Plum (JTM), JAM Bungo (JAMFH), Changli Apricot Plum (CLXM), Golden Sun (JTY), and Luntai Small White Apricot (XBX). Detailed Implementation

[0034] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. It should be noted that these descriptions of embodiments are intended to aid in understanding the present invention and are not intended to limit it. Furthermore, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0035] Example 1

[0036] Identification, expression analysis and functional verification of key genes for citric acid accumulation in plum fruit

[0037] (1) Comparative genomic analysis of genomic differences between 'Zhejiang Pearl Plum' and 'Ginkgo biloba'

[0038] The inventors initially conducted comparative genomic analysis of 'Zhejiang Pearl Plum' and 'Yinxiang White Apricot' using nucmer2 and SyRI. The results showed that 'Yinxiang White Apricot' exhibited richer genomic structural variations compared to 'Zhejiang Pearl Plum', including duplications, translocations, and inversions. Figure 1(a). In addition, it contains smaller variations including 3,811,704 SNPs, 668,493 Indels, and 5,382 SVs. Figure 1 (b) SVs were annotated, and genes located at the promoter (1210), exon (178), and splice site (4) were selected for analysis. Figure 1 (c). GO enrichment analysis revealed multiple molecular functions related to organic acids, including vacuole transport, cellular pH regulation, and bioregulation. Figure 1 (d).

[0039] (2) Analysis of organic acids in plum fruit and WGCNA identification of key correlations in citric acid accumulation

[0040] Based on the organic acid content during the developmental stages of 'Zhejiang Pearl Bush', transcriptome sequencing was performed at stages S1 (33 DAF), S3 (48 DAF), S5 (62 DAF), S6 (70 DAF), S8 (84 DAF), and S9 (92 DAF) to more accurately screen core candidate genes for citric acid accumulation. Transcriptome analysis was performed using Hisat2 combined with StringTie, and the resulting FPKM will be directly used for subsequent WGCNA (Weighted Gene Co-expression Network Analysis). Genes were divided into 47 modules based on their expression patterns. Figure 2 Among the components (a and b), the green module showed the highest correlation with citric acid at 0.96. Figure 2 (c). Combining the previously screened structural variation genes related to organic acids with the green module, there are four overlapping genes: Pmu000042, Pmu004537, Pmu013038, and Pmu002391. Figure 2 (d).

[0041] Candidate genes were annotated using eggNOG-mapper, revealing Pmu000042 and Pmu002391 to be MYB transcription factors. While the regulation of organic acids by MYB transcription factors has been extensively reported, the annotation and function of these specific MYB transcription factors remain unclear. Therefore, MYB transcription factors were identified genome-wide using the HMM model in the high-quality genome of *Pteris vittata* (Zhejiang Pearl Bush), and a phylogenetic tree was constructed using IQTREE. The results showed that the *Pteris vittata* genome contains 134 MYB transcription factors, and Pmu000042 belongs to the same phylogenetic group as PetuPH4 (petunia) and CsPH4 (citrus). Therefore, it was named PmPH4. Figure 3 ).

[0042] (3) Spatiotemporal expression pattern analysis of PmPH4 gene

[0043] Spatiotemporal expression analysis of PmPH4 using transcriptome (PRJNA172987) showed that PmPH4 expression was highest in fruit and lowest in flower. Figure 4 The expression of the PmPH4 gene was analyzed using RT-qPCR to validate the assay. Figure 5 (a). The expression level of PmPH4 in fruit gradually increases with fruit development and is significantly correlated with citric acid accumulation. Figure 2 In addition, the relative expression level of PH4 in pure plum was significantly higher than that in apricot plum, while the relative expression level in apricot plum was significantly higher than that in apricot (c). Figure 5 (b). Therefore, it is speculated that it is a key gene that promotes citric acid accumulation.

[0044] (4) An expression vector for the PmPH4 gene was constructed, and preliminary studies on gene function were conducted.

[0045] Construct a 35S:PmPH4 overexpression vector and transiently transform tobacco and 'Zhejiang Pearl Plum' fruit, respectively. Figure 6 The results showed that PmPH4 significantly promoted citric acid accumulation in both tobacco and plum fruits. Therefore, PmPH4 was identified as a key gene promoting citric acid accumulation.

[0046] (5) PmPH4 promoter activity analysis

[0047] Analysis of the PmPH4 promoter using PlantCARE ( Figure 7 (a) The results showed that the SV region contained a large number of transcriptional activating elements, suggesting that the SV could enhance the transcriptional capacity of the promoter. Therefore, the PmPH4 promoter was inserted into the pGreenII 0800-LUC vector using homologous recombination to drive firefly luciferase expression for transcriptional activity detection. Simultaneously, the PmPH4 promoter was mutated using overlap PCR to remove the SV sequence as a control. The results showed that the transcriptional activity of the PmPH4 promoter containing the SV was significantly higher than that of the promoter lacking the SV. Figure 7 (b, c). Therefore, it is further clarified that this is the key structural variation that promotes citric acid accumulation.

[0048] Example 2

[0049] Identification of SV variants in the PmPH4 promoter region

[0050] First, the promoter sequence of PmPH4 was extracted from the genomes of five varieties, including plum, apricot, and apricot-plum, for analysis. Sequence alignment was performed using DNAMAN. The results showed that only the plum PmPH4 promoter had a 377bp sequence (5`-3`) located 365bp upstream of the promoter. ATGTCAAATGAATATTTTATTTTTTTTAAAGACAGCTAGTTGTAACTTTCATTTATTATTTTCCTTTTTTTTTTTTTCTTTATCTTAAATGATTTGACTGTTATAATAATTATTTGACATATAGGGTGGAGTAAAAAACTGTGTAAATGTCAAATTGCCACATCAGCCATCAAATTTAAATTTGATGTTTTGATATTTGACATTTCTCTTGGAGATGCTCTTGG, SEQ ID NO.1) Insert ( Figure 8 Therefore, stable identification primers (PH4SV-F1: AGC AGGAAT TCC ACT GGT ATG AAAG and PH4SV-R1: GGG GAT GGA AGAAGA TGG TAATTG G) were developed for PCR in the conserved regions upstream and downstream of the SV. In addition, SV was extracted from 339 plum resequencing data. The results showed that all pure plum varieties contained this SV. Notably, the apricot-plum population contained both homozygous and heterozygous mutations (Table 1). Among them, the SV deletion genotypes of Gaotian plum, Jam Fenghou, and Changli apricot plum are consistent with those of apricot, thus suggesting that these three varieties are low-acid varieties.

[0051] Table 1. Resequencing of the PmPH4 promoter SV structural variants identified in the Mei population

[0052]

[0053] Example 3

[0054] This molecular marker was used to screen high-citric acid and low-citric acid germplasm.

[0055] (1) PCR test for the presence and absence of PH4 promoter SV

[0056] Fifteen accessions of plum (Prunus mume), apricot (P. armeniaca), and apricot-plum (P. mume var. bungo Makino) were randomly selected from the National Plum and Waxberry Germplasm Resource Center for PCR verification. Using the primer pair PH4SV-F1 / PH4SV-R1, amplification of the genomic DNA target region (containing a 377bp SV insertion) was performed using a high-fidelity DNA polymerase system (fidelity ≥100×Taq enzyme). The program settings were: 98℃ for 10s denaturation, 60℃ for 30s annealing, 72℃ for 1min extension, 35 cycles. The PCR system consisted of 20μL of 10μL high-fidelity DNA polymerase premix, 0.2μM each of PH4SV-F1 / R1 primers, and 50ng of DNA template. The amplified products were detected by 1% agarose gel electrophoresis (120V, 30 minutes, GelRed staining). The results showed that all 5 plum samples amplified a 686bp band; all 5 apricot samples cloned a 309bp band. Figure 9 (a) indicates that the SV variant is absent in the PH4 promoter region of apricot; the apricot-plum population (5 accessions) showed polymorphic segregation, with the JAM-rich variety exhibiting a homozygous deletion, consistent with the apricot genotype. PCR verification results were completely consistent with genome sequence alignment and resequencing analysis results, therefore, all three methods can identify this SV. Further verification of marker specificity: representative drupe fruit trees of the Rosaceae family were selected: five accessions each of peach (P. persica), plum (P. salicina), and sweet cherry (P. avium), and the same primers and program were used for detection. Results showed that all control species cloned only a 309bp band, proving that this SV is a variant specific to the genus Prunus (Prunus). Figure 9 (b). Therefore, this molecular marker is only applicable to the identification of high acidity in plum and its hybrid (apricot plum), and shows no cross-reactivity in closely related species such as apricot, peach, and cherry, exhibiting strict species specificity.

[0057] (2) Determination of citric acid content in the selected varieties of fruit

[0058] This invention employs high-performance liquid chromatography (HPLC) to determine the citric acid content in mature fruit samples. The specific method is as follows: 0.5 g of fresh fruit sample is weighed and subjected to two ultrasonic-assisted extractions using a 0.2% (v / v) phosphoric acid aqueous solution at low temperature. The extracts are combined, filtered through a filter membrane, and directly analyzed. Chromatographic analysis was performed using an ACQUITY UPLC HSS T3 C18 column (2.1 × 100 mm, 1.8 μm). The mobile phase consisted of 20 mmol / L sodium dihydrogen phosphate buffer (adjusted to pH = 2.4 with phosphoric acid) and methanol at a ratio of 98:2 (v / v). The flow rate was set at 0.5 mL / min, and the column temperature was 30 °C. Detection was performed using a UV detector at a wavelength of 210 nm, with an injection volume of 2 μL. The citric acid content was calculated based on its peak area using a standard curve plotted using the external standard method. The results showed that the citric acid content of purebred plum varieties was significantly higher than that of apricot and apricot-plum varieties without the molecular marker SV. However, the apricot-plum population exhibited phenotypic segregation, with germplasm containing the molecular marker SV (e.g., Setsuda plum, Bungo, Takada Bungo, Ina Bungo) showing significantly higher citric acid content than apricot and apricot-plum varieties without SV. Conversely, apricot-plum germplasm without this SV (e.g., JAM Bungo) showed significantly lower citric acid content than other plum varieties. Particularly noteworthy is that the citric acid content of Changli apricot-plum was even significantly lower than that of apricot (…). Figure 10 Therefore, this molecular marker (SV marker) is suitable for efficiently and accurately screening germplasm resources of Plum species with high citric acid content, providing a molecular-assisted breeding tool for the quality improvement of Plum crops.

Claims

1. A molecular marker associated with high citric acid content in plum fruit, characterized in that, The molecular marker is a 377bp sequence inserted 365bp upstream of the transcription start site of the key gene for citric acid accumulation. The sequence of the molecular marker SV is shown in SEQ ID NO.

1.

2. A primer pair for detecting the molecular marker of claim 1, characterized in that, It includes primers PH4SV-F1 and PH4SV-R1. The nucleotide sequence of primer PH4SV-F1 is AGC AGG AAT TCC ACT GGT ATG AAA G, and the nucleotide sequence of primer PH4SV-R1 is GGG GAT GGA AGA AGA TGG TAA TTG G.

3. A kit containing the primer pair of claim 2.

4. The application of the molecular marker of claim 1, the primer pair of claim 2, or the kit of claim 3 in plum breeding, comprising: (a) Screening of high-citric acid germplasm from plum germplasm resource bank; (b) Early marker-assisted selection of high-citrate individuals in hybrid progeny populations; (c) Identification of plum variety purity.

5. A method for identifying citric acid germplasm of *Megalocarpus mollissima*, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from the plum plant to be tested using the CTAB method or a DNA extraction kit; Step 2: Perform PCR amplification using the primer pairs described above; Step 3: Detect the size of the amplified product bands by agarose gel electrophoresis; Step 4: If the amplification product shows a single band at 686bp, the plant being tested is a homozygous high-citric acid germplasm; if the amplification product shows a band at both 309bp and 686bp, the plant being tested is a heterozygous high-citric acid germplasm; if the amplification product shows a single band at 309bp, the plant being tested is a low-citric acid germplasm.

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