An Indel molecular marker closely associated with the soluble sugar content of pepper fruit and its application

Abstract

This invention provides an Indel molecular marker closely related to the soluble sugar content of pepper fruit and its application, belonging to the technical field of nucleic acid-based assay methods. The nucleotide sequence of the Indel molecular marker is shown in SEQ ID NO.1 of the sequence listing; the Indel molecular marker is located at positions 5274716-5274727 of the full-length sequence of chromosome 3 of the pepper reference genome. Primers for amplifying the Indel molecular marker include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2 of the sequence listing, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3 of the sequence listing. This molecular marker can be directly applied in early generations of breeding, avoiding the destructive sampling of chemical detection, significantly reducing breeding costs, effectively shortening the breeding cycle, and greatly improving the selection efficiency of molecular marker-assisted breeding in peppers.

Description

Technical Field

[0001] This invention relates to an Indel molecular marker closely related to the soluble sugar content of chili pepper fruit and its application, belonging to the technical field of nucleic acid determination methods. Background Technology

[0002] Chili peppers, as an important vegetable and condiment, are rich in nutrients and have significant industrial value. The soluble sugar content of chili peppers directly affects their flavor quality and stress resistance. Through phenotypic selection or marker-assisted breeding, sugar content can be selectively improved, the sweet-spicy balance optimized, fruit storage resistance enhanced, and high-sugar, high-quality varieties cultivated to meet the diverse market demand for flavorful chili peppers and promote the precision development of breeding technology.

[0003] Traditional breeding methods for selecting high-soluble sugar pepper varieties suffer from drawbacks such as long cycles, reliance on phenotypic selection, and susceptibility to environmental interference. Molecular marker detection can precisely locate genes related to sugar metabolism, enabling early screening, shortening the breeding cycle, improving selection efficiency, overcoming the limitation of weak correlation between phenotype and genotype, and promoting the development of precision breeding.

[0004] Indel markers have the advantages of high polymorphism and whole genome coverage in the detection of soluble sugars in peppers. They can be rapidly genotyped by PCR-electrophoresis and accurately locate sugar metabolism-related genes. They are genetically stable and easy to detect, and can screen germplasm with high soluble sugar content at an early stage, shorten the breeding cycle, and improve selection efficiency. They are an efficient and economical tool in molecular marker-assisted breeding.

[0005] There are currently no reports on Indel molecular markers that are closely related to the soluble sugar content of pepper fruits. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an Indel molecular marker closely related to the soluble sugar content of chili pepper fruits and its application, enabling early, non-destructive screening of chili pepper germplasm with high soluble sugar content, achieving high accuracy and shortening the breeding cycle.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] An Indel molecular marker closely associated with the soluble sugar content of chili pepper fruit, the nucleotide sequence of which is shown in SEQ ID NO.1 in the sequence listing; the Indel molecular marker is located at positions 5274716-5274727 of the full-length sequence of chromosome 3 of the chili pepper reference genome.

[0009] The primers for amplifying the Indel molecular marker include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2 in the sequence listing, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3 in the sequence listing.

[0010] Application of primers amplifying the molecular marker in identifying the soluble sugar content of pepper fruits.

[0011] The application includes the following steps:

[0012] (1) Extract genomic DNA from the leaves of the chili peppers to be tested;

[0013] (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.

[0014] (3) If the molecular marker amplifies an amplification product with a length of 219 bp, the soluble sugar content of the fruit of the chili pepper germplasm is predicted to be ≥4.5%, which is a chili pepper germplasm material with high soluble sugar content. If the molecular marker does not amplify a band, the soluble sugar content of the fruit of the chili pepper germplasm is predicted to be <4.5%, which is a chili pepper germplasm material with low soluble sugar content.

[0015] The nucleotide sequence of the 219bp amplification product is shown in SEQ ID NO.6 of the sequence listing.

[0016] Compared with the prior art, the present invention achieves the following beneficial effects:

[0017] This invention develops an Indel molecular marker that is significantly associated with the soluble sugar content of pepper fruits, which can be used in marker-assisted selection (MAS) breeding of peppers to achieve early, non-destructive screening. In the early stages of pepper growth, PCR amplification can rapidly distinguish between genotypes with high and low soluble sugar content, thereby efficiently and directionally selecting pepper varieties with high soluble sugar content. This molecular marker can be directly applied in early generations of breeding. Compared to traditional screening methods that rely on mature fruit phenotypes, it avoids the destructive sampling required for chemical testing, significantly reduces breeding costs, effectively shortens the breeding cycle, and greatly improves the selection efficiency of marker-assisted breeding in peppers.

[0018] Large-sample validation (94 chili pepper samples) showed that the Indel-1 molecular marker developed in this invention can effectively distinguish between chili pepper germplasm with high and low soluble sugar content. Chili pepper fruits with high soluble sugar content obtained using this molecular marker showed an average 22.94% higher soluble sugar content compared to chili pepper fruits with low soluble sugar content, a difference that was statistically significant. Attached Figure Description

[0019] Figure 1 A bar chart showing the soluble sugar content of pepper germplasm materials P241098 and P241135;

[0020] Figure 2 Cluster heatmap of gene expression levels for pepper germplasm materials P241098 and P241135;

[0021] Figure 3 Principal component scatter plot of gene expression levels for pepper germplasm materials P241098 and P241135;

[0022] Figure 4 Volcano plot for DeSeq2 differential expression analysis;

[0023] Figure 5 Venn diagram of differentially expressed genes for DeSeq2;

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

[0025] Figure 7 Cluster diagram of the WGCNA analysis module for differentially expressed genes;

[0026] Figure 8 Heatmap of trait correlation coefficients for WGCNA analysis module of differentially expressed genes;

[0027] Figure 9 PCR amplification results of Indel-1 marker in 20 pepper germplasms;

[0028] Figure 10 PCR amplification results of Indel-1 marker in 94 pepper germplasms;

[0029] In the attached figure, CK1 is the band corresponding to germplasm P241098, and CK2 is the band corresponding to germplasm P241135. Detailed Implementation

[0030] Example 1: Detection of soluble sugars and transcriptome sequencing of pepper germplasm fruits with different contents

[0031] (1) Detection of soluble sugars in pepper germplasm fruits with different contents

[0032] This invention selected two chili pepper germplasms with significant differences in soluble sugar content as experimental materials, with germplasm numbers P241098 and P241135, respectively. These materials were provided by Shandong Yongsheng Agricultural Development Co., Ltd.

[0033] The soluble sugar content of pepper pulp was determined by anthrone colorimetric method at 12 days (DAA12), 27 days (DAA27), and 42 days (DAA42) after flowering. The experiment was conducted using a completely randomized design and was repeated 3 times.

[0034] The results of soluble sugar detection showed that the soluble sugar content of pepper fruits increased with the increase of development time, and there were significant differences in the soluble sugar content of fruits between the two pepper germplasms at all three developmental stages (P<0.05). Figure 1 ).

[0035] (2) Transcriptome sequencing

[0036] Transcriptome sequencing was performed on the pulp tissues of two pepper germplasms at 12 days (DAA12), 27 days (DAA27), and 42 days (DAA42) after flowering, with three biological replicates for each developmental stage. Sequencing was performed by Novogene Biotechnology Co., Ltd. using the Illumina NovaSeq 6000 platform; the raw data were re-filtered using the FASTQ program, yielding a total of 142.22 Gb of high-quality base data (results are shown in Table 1).

[0037] Table 1. Transcriptome sequencing results of fruits from two pepper germplasms.

[0038]

[0039] The pepper reference genome (ASM51225v2) was downloaded from the EnsemblPlants website (https: / / plants.ensembl.org / index.html). Transcriptome sequencing data from three developmental stages of the fruit of pepper lines P241098 and P241135 were aligned to this reference genome using the Hisat2 program. The results (Table 2) show that the number of paired consistent reads ranged from 33,758,378 to 78,806,164, the number of paired inconsistent reads ranged from 398,208 to 1,441,752, and the number of single-end aligned reads ranged from 2,090,075 to 4,456,196. The overall alignment rate for all samples was above 90%, indicating good alignment quality suitable for subsequent bioinformatics analysis.

[0040] Overall comparison rate = (Number of pairs of identical reads + Number of pairs of inconsistent reads + Number of single reads) / Total number of valid reads

[0041] Table 2. Hisat2 transcriptome alignment results

[0042]

[0043] Based on transcriptome alignment results, gene expression levels were counted using FeatureCounts software (v2.0.3), and expression clustering heatmaps for sequencing sample pairs were plotted using the R language Pheatmap package. Figure 2 ), and used the ggplot2 package to plot the principal component scatter plot of expression levels in the sequencing samples. Figure 3 The results showed that samples from P241098 and P241135 clustered together in the three periods of DAA12, DAA27, and DAA42, indicating that the sampling and experimental design of this study had good repeatability and rationality.

[0044] Example 2: Differential Gene Expression Analysis and Key Gene Mining

[0045] (1) Differential gene expression analysis

[0046] Differential gene expression analysis was performed on the raw gene expression count data using the DESeq2 package in R. A threshold of |log2FoldChange|≥1 (logarithmic change of expression greater than or equal to 2) and a Padj (p-value after multiple test correction) less than 0.05 were used to identify significantly differentially expressed genes. Volcano plots and Venn diagrams for differential gene expression analysis are shown below. Figure 4 and Figure 5 As shown, 4491, 3462, and 3891 significantly differentially expressed genes were identified at the DAA12, DAA27, and DAA42 stages, respectively. A total of 1626 genes showed significantly differential expression between samples P241098 and P241135 during these three developmental stages of the pepper fruit.

[0047] (2) WGCNA analysis

[0048] For the 1626 genes that were significantly differentially expressed simultaneously at three developmental stages of pepper fruit, weighted gene co-expression network analysis (WGCNA) was performed using the corresponding soluble sugar content data. The results showed that setting the soft threshold to 18 in the R language WGCNA package was effective. Figure 6 ), 1626 genes were divided into 15 co-expression modules ( Figure 7 , Figure 8 ).

[0049] Among the 15 co-expression modules, ME1, ME6, and ME8 showed relatively high correlation coefficients with soluble sugar content, at 0.70, 0.90, and 0.76, respectively, with corresponding P-values ​​of 1×10⁻⁶. 3 4×10 7 and 2×10 4 All three parameters reached a highly significant level, indicating that the genes in these three modules may be significantly associated with the soluble sugar content in pepper fruits.

[0050] (3) Discovery of potential key genes controlling the soluble sugar content of pepper fruits

[0051] Genes in modules ME1, ME6, and ME8 were further screened based on MM (Module Membership) and GS (Gene Significance) values ​​generated by the WGCNA program. The MM threshold was set at 0.9, and the GS threshold at 0.8. The screening yielded 40 genes that met the criteria, which were identified as potential key genes significantly associated with the soluble sugar content of pepper fruits (Table 3).

[0052] Table 3. MM and GS values ​​of 40 potential key genes

[0053]

[0054] Example 3 Indel variant detection, primer design and screening

[0055] Indel variants based on transcriptome sequencing data were detected using GATK 4.0 (Genome Analysis Toolkit). Location information of the 40 potential key genes controlling soluble sugar content in pepper fruits was identified using the annotation file of the pepper reference genome (ASM51225v2). Indel variants located within the genes and within a 1500bp upstream and downstream region were extracted using the VCFtools program, ultimately yielding 423 Indel variants.

[0056] Each of the 423 Indel loci was analyzed individually, and Indel loci meeting the following criteria were selected: ① Indel fragment length ≥ 6 bp; ② 100% identity of the Indel genotype at P241098 and P241135 across the three time points of DAA12, DAA27, and DAA42; ③ 100% concordance between the Indel sequence and GATK4 results across the three time points when the sequencing data was visualized in IGV. Two Indel loci were ultimately selected (Table 4).

[0057] Table 4 shows information on two Indel sites associated with soluble sugar content in pepper fruits.

[0058]

[0059] Using the Fasta Extract tool in Tbtools software, DNA sequences of 1000 bp upstream and downstream of the two Indel sites were extracted from the reference genome. Primers were designed using the Primer3 online program (https: / / ginkgo.zju.edu.cn / genome / tools / primer3 / ) (Table 5). Electronic PCR was performed using the Simple e-pcr program in Tbtools software.

[0060] Table 5 Primer sequences designed for the Indel site

[0061]

[0062] The Indel-1 site is located on chromosome 3 of the chili reference genome at positions 5274716bp-5274727bp; the nucleotide sequence of the Indel-1 molecular marker is shown in SEQ ID NO.1 of the sequence listing.

[0063] 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.2 in the sequence listing, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3 in the sequence listing.

[0064] The Indel-2 site is located on chromosome 11 of the pepper reference genome, at positions 13010447bp-13010455bp; the nucleotide sequence of the Indel-2 molecular marker is ACTAGGCTA.

[0065] 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 in SEQ ID NO.4 of the sequence listing; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.5 of the sequence listing.

[0066] Electronic PCR and bioinformatics analysis showed that:

[0067] Indel-1 amplified a 219bp band in the high soluble sugar content strain P241098, and its nucleotide sequence is shown in SEQ ID NO.6 in the sequence listing. No amplified band was observed in the low soluble sugar content strain P241135 (indicated by "-").

[0068] Indel-2 amplified a 358bp band in the high-soluble sugar strain P241098, the nucleotide sequence of which is shown in SEQ ID NO.7 in the sequence listing, and a 349bp band in the low-soluble sugar strain P241135, the nucleotide sequence of which is shown in SEQ ID NO.8 in the sequence listing;

[0069] Example 4 Preliminary Verification

[0070] Twenty F6 generation pepper germplasm materials were selected to form a preliminary validation population to verify the effectiveness of the Indel molecular marker. The material numbers are shown in Table 6. These pepper germplasm materials were provided by Shandong Yongsheng Agricultural Development Co., Ltd.

[0071] Table 6. Numbering information of 20 chili pepper germplasms

[0072]

[0073] Using genomic DNA from the leaves of 20 F6 generation pepper seedlings at the three-leaf-one-heart stage as templates, PCR amplification and product detection were performed using primers for the two Indel loci mentioned above. The results showed that Indel-1 could significantly distinguish the soluble sugar content of pepper fruits at the color-changing stage (DAA42); pepper germplasm amplified by Indel-1 with a 219bp band had a 22.66% higher soluble sugar content than pepper germplasm without amplified bands. Figure 9 (Tables 7 and 8).

[0074] Table 7 Amplification results and soluble sugar content

[0075]

[0076] Table 8. Preliminary validation results of molecular markers in 20 pepper germplasms.

[0077]

[0078] In a primary validation population of 20 F6 generation pepper germplasm materials, Indel-2 showed little amplification variation and poor performance.

[0079] Ultimately, the Indel-1 site was selected as the Indel site to distinguish the soluble sugar content of pepper fruits.

[0080] Example 5: Detection of Indel-1 molecular marker in multiple pepper germplasm materials

[0081] Ninety-four chili pepper samples were identified using the Indel-1 molecular marker.

[0082] The molecular marker validation population consisted of 94 randomly selected F6 generation high-generation homozygous pepper lines, with material numbers shown in Tables 9 and 10. These materials were provided by Shandong Yongsheng Agricultural Development Co., Ltd.

[0083] Table 9. Numbering information for pepper germplasm accessions 1-47

[0084]

[0085] Table 10: Numbering information for pepper germplasm accessions 48-94

[0086]

[0087] The identification method is as follows:

[0088] A. Extraction of genomic DNA from pepper leaves

[0089] Genomic DNA was extracted from leaves of pepper seedlings at the three-leaf-one-heart stage using a plant genomic DNA extraction kit (Solarbio).

[0090] B. PCR amplification

[0091] PCR amplification was performed on 94 samples using primers labeled with Indel-1.

[0092] PCR amplification products were detected using agarose gel electrophoresis. After electrophoresis, the amplification products were photographed and observed using a gel imaging system to determine their specific characteristics.

[0093] Electrophoresis results as follows Figure 10 As shown.

[0094] The results of the molecular marker identification were compared with the soluble sugar content of pepper fruits at the color-changing stage (DAA42). The results showed that among the 94 tested materials, 65 pepper germplasms amplified a 219 bp band at the Indel-1 locus, with an average soluble sugar content of 4.93%; the remaining 29 pepper germplasms did not amplify the band, with an average soluble sugar content of 4.01% (Table 11). A two-independent-samples t-test was performed using R language, and the results showed |t|=15.52, P=5.28×10⁻⁶. -19 This indicates that the result is statistically significant.

[0095] The Indel-1 molecular marker can effectively screen high-soluble sugar germplasm materials of chili peppers, significantly improving the efficiency of molecular marker-assisted selection in chili pepper breeding.

[0096] Table 11 Results of molecular marker validation in pepper germplasm materials

[0097]

[0098] The amplification results and statistical results of soluble sugar content of the above 65 chili germplasm materials are shown in Tables 12 and 13, and the amplification results and statistical results of soluble sugar content of the above 29 chili germplasm materials are shown in Table 14.

[0099] Table 12. Amplification results and statistical results of soluble sugar content of 65 pepper germplasm materials.

[0100]

[0101] Table 13. Amplification results and statistical results of soluble sugar content of 65 pepper germplasm materials.

[0102]

[0103] Table 14. Amplification results and statistical results of soluble sugar content of 29 pepper germplasm materials.

[0104] .

Claims

1. An Indel molecular marker closely associated with the soluble sugar content of chili pepper fruit, characterized in that: The nucleotide sequence of the Indel molecular marker is shown in SEQ ID NO.1 in the sequence listing; the Indel molecular marker is located at positions 5274716-5274727 of the full-length sequence of chromosome 3 of the pepper reference genome; the version of the pepper reference genome is ASM51225v2.

2. The Indel molecular marker closely related to the soluble sugar content of chili pepper fruit according to claim 1, characterized in that: The primers for amplifying the Indel molecular marker include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2 in the sequence listing, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3 in the sequence listing.

3. The application of the primers amplifying the molecular marker described in claim 2 in identifying the soluble sugar content of pepper fruits, characterized in that: Includes the following steps: (1) Extract genomic DNA from the leaves of the pepper germplasm to be tested; (2) Using the genomic DNA of pepper germplasm 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 amplifies an amplification product with a length of 219 bp, the soluble sugar content of the fruit of the chili pepper germplasm is predicted to be ≥4.5%; if the molecular marker does not amplify a band, the soluble sugar content of the fruit of the chili pepper germplasm is predicted to be <4.5%.

4. The application according to claim 3, characterized in that: The nucleotide sequence of the 219bp amplification product is shown in SEQ ID NO.6 of the sequence listing.

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