Molecular marker for controlling vitamin c content of pepper fruits and use thereof

Abstract

The application provides a molecular marker for controlling vitamin C content of pepper fruits and application thereof, and belongs to the technical field of nucleic acid determination methods. The molecular marker is a combination of two Indel molecular markers, nucleotide sequences of the two Indel molecular markers are respectively shown in SEQ ID NO. 2 and SEQ ID NO. 3 in the sequence table; the molecular marker shown in SEQ ID NO. 2 is located at 260909281-260909295 of the full-length sequence of the 3rd chromosome of a pepper reference genome, and the molecular marker shown in SEQ ID NO. 3 is located at 210356054-210356077 of the full-length sequence of the 11th chromosome of the pepper reference genome. The Indel molecular marker significantly related to the vitamin C content of the pepper fruits can be used for assisted selection breeding and rapid directional breeding of pepper varieties with high vitamin C content.

Description

TECHNICAL FIELD

[0001] The present application relates to a molecular marker for controlling vitamin C content of pepper fruits and application thereof, and belongs to the technical field of nucleic acid detection methods. BACKGROUND

[0002] Pepper is a widely cultivated vegetable crop in China, and the vitamin C content of its fruits varies among different varieties. There are significant differences in vitamin C content among different pepper varieties. For example, the vitamin C content of the fruits of some pepper varieties can reach more than 120 mg per 100 g, while the vitamin C content of some sharp pepper varieties is slightly lower, but still significantly higher than that of most fruits, which can meet the daily nutritional needs.

[0003] The traditional detection method for the vitamin C content of pepper fruits needs to be completed by chemical analysis at the fruit ripening stage, which is a destructive detection method and needs to be processed sample by sample, time-consuming and labor-intensive, and the results are easily affected by the operating environment and experimental conditions, with poor stability. Using molecular marker detection, the potential of vitamin C content can be non-destructively screened at an early stage of pepper growth, significantly shortening the breeding cycle, reducing labor and time costs, while avoiding damage to the plants, maintaining the complete growth state, and improving the preservation and utilization efficiency of breeding materials.

[0004] In molecular markers, Indel (insertion-deletion) markers are the preferred tool for molecular identification of vitamin C content in peppers due to their characteristics of being based on PCR technology, rich polymorphism, high stability, and simple operation. This marker can accurately locate the key gene region related to vitamin C synthesis and accumulation by detecting the insertion or deletion polymorphism of DNA fragments in the genome, providing a reliable technical path for molecular marker development.

[0005] In the prior art, although there have been reports on QTL positioning and candidate gene research related to vitamin C content in peppers, there is still no related report on Indel molecular markers closely related to vitamin C content. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides a molecular marker for controlling vitamin C content of pepper fruits and application thereof, which realizes non-destructive and high-accuracy vitamin C content detection, and meets the demand for rapid screening of high-vitamin C varieties in pepper breeding.

[0007] To solve the above technical problems, the present application adopts the following technical solutions:

[0008] A molecular marker for controlling vitamin C content of pepper fruits, which is a combination of two Indel molecular markers, the nucleotide sequences of which are shown as SEQ ID NO. 2 and SEQ ID NO. 3 in the sequence listing, respectively; the molecular marker shown as SEQ ID NO. 2 is located at 260909281-260909295 of the full-length sequence of chromosome 3 of pepper reference genome, and the molecular marker shown as SEQ ID NO. 3 is located at 210356054-210356077 of the full-length sequence of chromosome 11 of pepper reference genome.

[0009] The primers for amplifying the molecular marker shown as SEQ ID NO. 2 include forward primer a and reverse primer a, the nucleotide sequence of the forward primer a is shown as SEQ ID NO. 6 in the sequence listing, and the nucleotide sequence of the reverse primer a is shown as SEQ ID NO. 7 in the sequence listing; the primers for amplifying the molecular marker shown as SEQ ID NO. 3 include forward primer b and reverse primer b, the nucleotide sequence of the forward primer b is shown as SEQ ID NO. 10 in the sequence listing, and the nucleotide sequence of the reverse primer b is shown as SEQ ID NO. 11 in the sequence listing.

[0010] The application of the primers for amplifying the molecular marker in identifying the vitamin C content of pepper fruits.

[0011] The application comprises the following steps:

[0012] (1) extracting the genomic DNA of the pepper leaves to be detected;

[0013] (2) using the genomic DNA of the pepper leaves as a template, performing PCR amplification by using the primers for amplifying the molecular marker, and detecting the PCR amplification product;

[0014] (3) if the molecular marker shown as SEQ ID NO. 2 amplifies an amplification product with a length of 264 bp and the molecular marker shown as SEQ ID NO. 3 amplifies an amplification product with a length of 313 bp, it is predicted that the vitamin C content of the pepper germplasm is ≥ 110 mg / 100 g, which is a pepper germplasm material with high vitamin C content, and if the molecular marker shown as SEQ ID NO. 2 amplifies an amplification product with a length of 249 bp and the molecular marker shown as SEQ ID NO. 3 amplifies an amplification product with a length of 289 bp, it is predicted that the vitamin C content of the pepper germplasm is < 110 mg / 100 g, which is a pepper germplasm material with low vitamin C content.

[0015] The nucleotide sequence of the 264bp amplification product is shown in SEQ ID NO. 14 in the sequence listing, the nucleotide sequence of the 249bp amplification product is shown in SEQ ID NO. 15 in the sequence listing; the nucleotide sequence of the 313bp amplification product is shown in SEQ ID NO. 18 in the sequence listing, and the nucleotide sequence of the 289bp amplification product is shown in SEQ ID NO. 19 in the sequence listing.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The Indel molecular marker developed by the present application is significantly associated with the vitamin C content of pepper fruits, and can be used for marker-assisted selection (MAS) breeding to achieve early non-destructive screening. High / low vitamin C content genotypes can be quickly distinguished by PCR amplification at an early generation (such as seedling stage) of pepper, and high-vitamin C content pepper varieties can be quickly selected and bred, so as to realize precise improvement of nutritional quality. The marker can be applied at an early generation of pepper breeding, compared with traditional phenotype screening, through the marker-assisted screening, the breeding cost can be significantly reduced, the destructive operation of traditional chemical detection can be avoided, the breeding cycle can be shortened, and the selection efficiency of pepper molecular marker-assisted breeding can be improved.

[0018] Through large sample verification (94 pepper materials), the combination of the two molecular markers Indel-2 and Indel-4 of the present application can effectively distinguish high-vitamin C pepper and low-vitamin C content pepper germplasm, and the high-vitamin C content pepper fruits screened by the combination of the above-mentioned molecular markers have a vitamin C content increased by 19.43% compared with the low-vitamin C content pepper fruits, which has a significant statistical significance. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a column chart of vitamin C content in four periods of P241098 and P241135 pepper materials;

[0020] Figure 2 It is a cluster heat map of gene expression amount in four periods of P241098 and P241135 pepper materials;

[0021] Figure 3 It is a principal component scatter plot of gene expression amount in four periods of P241098 and P241135 pepper materials;

[0022] Figure 4 It is a volcano plot of DeSeq2 differential expression analysis;

[0023] Figure 5 It is a Wayne plot of DeSeq2 differential expression genes;

[0024] Figure 6Soft threshold scatter plot for WGCNA analysis of differentially expressed genes;

[0025] Figure 7 Module clustering plot for WGCNA analysis of differentially expressed genes;

[0026] Figure 8 Module-trait correlation coefficient heatmap for WGCNA analysis of differentially expressed genes;

[0027] Figure 9 Distribution density plot of Indel variants in chromosomes;

[0028] Figure 10 PCR amplification results of Indel-2 marker in 20 pepper germplasms;

[0029] Figure 11 PCR amplification results of Indel-4 marker in 20 pepper germplasms;

[0030] Figure 12 PCR amplification results of Indel-2 marker in pepper germplasms No.1-36 described in Example 3;

[0031] Figure 13 PCR amplification results of Indel-2 marker in pepper germplasms No.37-72 described in Example 3;

[0032] Figure 14 PCR amplification results of Indel-2 marker in pepper germplasms No.73-94 described in Example 3;

[0033] Figure 15 PCR amplification results of Indel-4 marker in pepper germplasms No.1-94 described in Example 3;

[0034] In the figures, CK1 is the band corresponding to P241098 germplasm, and CK2 is the band corresponding to P241135 germplasm. DETAILED DESCRIPTION

[0035] Example 1: Mining of key genes for vitamin C in pepper fruits

[0036] 1. Pepper fruit transcriptome sequencing and bioinformatics analysis

[0037] The pepper materials used in the present application are two pepper germplasms with significant differences in vitamin C content, with germplasm numbers P241098 and P241135. The above materials are provided by Shandong Yongsheng Agricultural Development Co., Ltd.

[0038] At four stages of fruit development, the pulp of P241098 and P241135 was cut for transcriptome sequencing and vitamin C content determination (2,6-dichlorophenol indophenol colorimetry).

[0039] The four periods of fruit development and their characteristics are shown in Table 1, and the vitamin C content is shown in Figure 1 .

[0040] Table 1 Characteristics of P241098 and P241135 fruits at four periods of fruit development

[0041]

[0042] The transcriptome of the pulp of P241098 and P241135 pepper fruits at four periods of development was sequenced using the Illumina Novaseq 6000 platform, and a total of 185.94 Gb of high-quality base data was obtained (results shown in Table 2).

[0043] Table 2 Transcriptome sequencing results

[0044]

[0045] The sequencing data of P241098 and P241135 at each of the four periods was aligned to the pepper reference genome (ASM51225 v2, https: / / plants.ensembl.org / info / data / ftp / index.html) using STAR (v2.7.10a) software (alignment results shown in Table 3). The results showed that the overall alignment rate ranged from 90.6% to 94.04%, and the number of paired uniquely aligned reads accounted for 79.6-86.8%, indicating that the quality of the sequencing data alignment was good and suitable for downstream differential expression analysis.

[0046] Table 3 Transcriptome alignment results

[0047]

[0048] FeatureCounts (v2.0.3) software was used for expression statistics, and Pheatmap program was used to draw gene expression clustering map (results shown in Figure 2 ), and ggplot2 was used to draw principal component scatter plot of expression (results shown in Figure 3 ). The analysis results showed that the pepper samples at the same period were clustered together, indicating that the sample selection and experimental design were reasonable.

[0049] Differential expression analysis was performed using R / DESeq2 (v1.38.3) software, and the screening criteria were the expression difference fold |log2FoldChange|≥1 and the multiple test correction P value Padj<0.05. Finally, 4491, 3471, 6171, and 3903 significant differentially expressed genes were detected in S1, S2, S3, and S4 periods, respectively, of which 1510 genes were significantly differentially expressed in the four periods. The volcano plot of differentially expressed genes is shown in Figure 4 , and the Venn diagram is shown in Figure 5 .

[0050] 2. Key gene mining for vitamin C content in pepper fruits

[0051] The expression data of 1510 significantly differentially expressed genes and the vitamin C content data of two varieties in four periods were analyzed by WGCNA (weighted gene co-expression network analysis) Figure 6 ). The soft threshold was set to 13, and the results divided the 1510 genes into 6 modules (ME1-ME6) Figure 7 , Figure 8 ). Among them, the ME1 and ME4 modules were significantly associated with the vitamin C content in pepper fruits, with correlation coefficients of 0.74 and -0.73, respectively.

[0052] For the genes in ME1 and ME4 modules, the key genes associated with the vitamin C content in pepper fruits were mined according to the two statistical quantities of MM value (module membership) and GS value (gene significance), with the MM threshold set to 0.9 and the GS threshold set to 0.6. Finally, 45 potential key genes controlling the vitamin C content in pepper fruits were obtained (Table 4).

[0053] Table 4 MM values and GS values of 45 potential key genes

[0054]

[0055] 3. Indel variation detection, primer design, and screening

[0056] Gatk4 program was used to identify Indels in the transcriptome data of P241098 and P241135 in four periods, and finally 122925 Indel sites distributed on 12 chromosomes of the pepper reference genome (ASM51225 v2) were obtained Figure 9 ).

[0057] According to the location information of 45 potential key genes for controlling vitamin C content in pepper fruits obtained by WGCNA analysis on the reference genome, Indel variations located in the gene and the upstream and downstream 1500 bp interval were screened, and finally 468 Indel variations were obtained.

[0058] The above 468 Indel sites were analyzed one by one, and Indel sites meeting the following criteria were selected: ① Indel fragment length ≥6bp; ② Consistency of Indel genotype in P241098 and P241135 in four periods is 100%; ③ Visualizing sequencing data in IGV, Indel sequence and GATK4 results in four periods are 100% consistent. Finally, four Indel sites were screened (Table 5).

[0059] Table 5 Information of four Indel sites associated with vitamin C in pepper fruits

[0060]

[0061] According to the physical location of the Indel site, the DNA sequence of 1000 bp upstream and downstream of the reference genome was extracted, and the primer was designed by using primer3 online program (https: / / ginkgo.zju.edu.cn / genome / tools / primer3 / ). Electronic PCR was performed by using Tbtools software.

[0062] Table 6 Primer sequence designed for four Indel sites

[0063]

[0064] The Indel-1 site is located at 5279013bp-5279024bp of the 3rd chromosome of the pepper reference genome; the nucleotide sequence of the Indel-1 molecular marker is shown in SEQ ID NO. 1 in the sequence listing.

[0065] The primers for amplifying the Indel-1 molecular marker include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO. 4 in the sequence listing; the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 5 in the sequence listing.

[0066] The Indel-2 site is located at 260909281bp-260909295bp of the 3rd chromosome of the pepper reference genome; the nucleotide sequence of the Indel-2 molecular marker is shown in SEQ ID NO. 2 in the sequence listing.

[0067] The primers for amplifying the Indel-2 molecular marker include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 6 in the sequence listing; and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 7 in the sequence listing.

[0068] The Indel-3 site is located at 224709456bp-224709464bp of the 10th chromosome of the pepper reference genome; and the nucleotide sequence of the Indel-3 molecular marker is ATTATATTT.

[0069] The primers for amplifying the Indel-3 molecular marker include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 8 in the sequence listing; and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 9 in the sequence listing.

[0070] The Indel-4 site is located at 210356054bp-210356077bp of the 11th chromosome of the pepper reference genome; and the nucleotide sequence of the Indel-4 molecular marker is shown as SEQ ID NO. 3.

[0071] The primers for amplifying the Indel-4 molecular marker include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 10 in the sequence listing; and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 11 in the sequence listing.

[0072] The electronic PCR and bioinformatics analysis shows that:

[0073] The Indel-1 amplifies a 380bp band in the high-vitamin C strain P241098, and the nucleotide sequence is shown as SEQ ID NO. 12 in the sequence listing; and amplifies a 368bp band in the low-vitamin C strain P241135, and the nucleotide sequence is shown as SEQ ID NO. 13 in the sequence listing;

[0074] The Indel-2 amplifies a 264bp band in the high-vitamin C strain P241098, and the nucleotide sequence is shown as SEQ ID NO. 14 in the sequence listing; and amplifies a 249bp band in the low-vitamin C strain P241135, and the nucleotide sequence is shown as SEQ ID NO. 15 in the sequence listing;

[0075] The Indel-3 amplifies a 253bp band in the high-vitamin C strain P241098, and the nucleotide sequence is shown as SEQ ID NO. 16 in the sequence listing; and amplifies a 244bp band in the low-vitamin C strain P241135, and the nucleotide sequence is shown as SEQ ID NO. 17 in the sequence listing;

[0076] Indel-4 amplified a 313bp band in high vitamin C line P241098, the nucleotide sequence of which is shown as SEQ ID NO. 18 in the sequence listing, and a 289bp band in low vitamin C line P241135, the nucleotide sequence of which is shown as SEQ ID NO. 19 in the sequence listing.

[0077] Example 2 Preliminary verification

[0078] The preliminary verification population of molecular markers consisted of 20 pepper advanced germplasm (F6) materials, and the material numbers are shown in Table 7. The germplasm materials were provided by Shandong Yongsheng Agricultural Development Co., Ltd.

[0079] Table 7 Numbering information of 20 pepper germplasms

[0080]

[0081] Using the genomic DNA of the primary verification population consisting of 20 pepper advanced germplasms (F6) as a template, the primers of the above-mentioned four Indel sites were used for PCR amplification and product detection, and the results showed that the electrophoresis results of Indel-2 and Indel-4 in combination could significantly distinguish the vitamin C content of pepper fruits; the pepper germplasm with Indel-2 amplifying a 264bp band and Indel-4 amplifying a 313bp band had a higher vitamin C content than the pepper germplasm with Indel-2 amplifying a 249bp band and Indel-4 amplifying a 289bp band, and the vitamin C content of the former was increased by 22.88% (P<0.05) (Table 8, Table 9). Figure 10 Figure 11

[0082] Table 8 Amplification results and vitamin C content

[0083]

[0084] Table 9 Preliminary verification results of molecular markers of 20 pepper germplasms

[0085]

[0086] Finally, the combination of Indel-2 and Indel-4 sites was selected for distinguishing the vitamin C content of pepper fruits.

[0087] Example 3 Detection of Indel-2 and Indel-4 molecular markers in multiple pepper germplasm materials

[0088] Ninety-four pepper materials were identified using Indel-2 and Indel-4 molecular markers.

[0089] ​​Molecular marker verification of large population consisted of 94 randomly selected pepper advanced homozygous lines, the material number was shown in Table 10, Table 11. The above materials were provided by Shandong Yongsheng Agricultural Development Co., Ltd.

[0090] Table 10 Numbering information of the first 47 pepper germplasm

[0091]

[0092] Table 11 Numbering information of the 48th to 94th pepper germplasm

[0093]

[0094] The identification method was:

[0095] A, extraction of pepper leaf genomic DNA

[0096] The genomic DNA of pepper leaves was extracted by using plant genomic DNA extraction kit (Solarbio).

[0097] B, PCR amplification

[0098] The primers of Indel-2 and Indel-4 molecular markers were used for PCR amplification of 94 materials.

[0099] The PCR amplification reaction system was 20 μL, specifically: 2 × Rapid Tap Master Mix 10 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, genomic DNA 1 μL, ddH2O 7 μL.

[0100] The PCR amplification program was as follows:

[0101] (1) 95℃, 3min;

[0102] (2) 95℃, 30s;

[0103] (3) 55℃, 30s;

[0104] (4) 72℃, 30s;

[0105] (5) Steps (2)-(4), repeat 34 times;

[0106] (6) 72℃, 5min;

[0107] (7) 12℃ preservation.

[0108] C, PCR product detection

[0109] The PCR amplification products were detected by agarose gel electrophoresis, and after electrophoresis, the gel imaging system was photographed to observe the specific conditions of the amplification products.

[0110] The electrophoretic detection results are shown in Figures 12-15

[0111] The above molecular marker identification results were compared with the vitamin C content of the ripe stage (60 days after flowering) of the pepper fruits. The results showed that among the 94 materials, 15 pepper germplasms simultaneously amplified the 264 bp band of Indel-2 and the 313 bp band of Indel-4, and the average content of vitamin C was 116.48 mg / 100g, 22 pepper germplasms simultaneously amplified the 249 bp band of Indel 2 and the 289 bp band of Indel 4, and the average content of vitamin C was 97.53 mg / 100g (Table 12). Using R language for two independent sample t test, the results showed that |t|=8.785, P=2.84x10 -9 , indicating that the result has significant statistical significance.

[0112] The combination of Indel-2 and Indel-4 molecular markers can effectively screen high-vitamin C pepper planting materials, and significantly improve the efficiency of molecular marker assisted selection in pepper breeding.

[0113] Table 12 Verification results of molecular markers of pepper germplasm materials

[0114]

[0115] The amplification results and statistical results of vitamin C content of the above 15 pepper germplasm materials are shown in Table 13, and the amplification results and statistical results of vitamin C content of the above 22 pepper germplasm materials are shown in Table 14.

[0116] Table 13 Amplification results and statistical results of vitamin C content of the above 15 pepper germplasm materials

[0117]

[0118] Table 14 Amplification results and statistical results of vitamin C content of the above 22 pepper germplasm materials

[0119] .​

Claims

1. A molecular marker for controlling the vitamin C content of chili pepper fruits, characterized in that: The molecular marker is a combination of two Indel molecular markers, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3 in the sequence listing, respectively; the molecular marker shown in SEQ ID NO.2 is located at positions 260909281-260909295 of the full-length sequence of chromosome 3 of the pepper reference genome, and the molecular marker shown in SEQ ID NO.3 is located at positions 210356054-210356077 of the full-length sequence of chromosome 11 of the pepper reference genome; the version of the pepper reference genome is ASM51225 v2.

2. The application of primers amplifying the molecular marker described in claim 1 in identifying the vitamin C content of pepper fruits, characterized in that: The primers for amplifying the molecular marker shown in SEQ ID NO.2 include a forward primer a and a reverse primer a, the nucleotide sequence of which is shown in SEQ ID NO.6 of the sequence listing, and the nucleotide sequence of which is shown in SEQ ID NO.7 of the sequence listing; the primers for amplifying the molecular marker shown in SEQ ID NO.3 include a forward primer b and a reverse primer b, the nucleotide sequence of which is shown in SEQ ID NO.10 of the sequence listing, and the nucleotide sequence of which is shown in SEQ ID NO.11 of the sequence listing.

3. The application according to claim 2, characterized in that: Includes the following steps: (1) Extract genomic DNA from the leaves of the chili peppers to be tested; (2) Using the genomic DNA of pepper leaves as a template, PCR amplification was performed using primers for amplifying molecular markers, and the PCR amplification products were detected. (3) If the molecular marker shown in SEQ ID NO.2 amplifies an amplification product with a length of 264 bp and the molecular marker shown in SEQ ID NO.3 amplifies an amplification product with a length of 313 bp, then the vitamin C content of the fruit of the chili pepper germplasm is predicted to be ≥110 mg / 100 g. If the molecular marker shown in SEQ ID NO.2 amplifies an amplification product with a length of 249 bp and the molecular marker shown in SEQ ID NO.3 amplifies an amplification product with a length of 289 bp, then the vitamin C content of the fruit of the chili pepper germplasm is predicted to be <110 mg / 100 g.

4. The application according to claim 3, characterized in that: The nucleotide sequence of the 264bp amplification product is shown in SEQ ID NO.14 of the sequence listing; the nucleotide sequence of the 249bp amplification product is shown in SEQ ID NO.15 of the sequence listing; the nucleotide sequence of the 313bp amplification product is shown in SEQ ID NO.18 of the sequence listing; and the nucleotide sequence of the 289bp amplification product is shown in SEQ ID NO.19 of the sequence listing.

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