An Indel molecular marker for identifying the gene controlling light-induced purple color in pepper pericarps and its application.

Abstract

This invention relates to the field of molecular markers for peppers, and discloses an Indel molecular marker for identifying genes controlling light-induced purple color in pepper pericarps and its application. The applicant used pooled transcriptome sequencing (BSR-seq) combined with linkage mapping to identify the relevant sites controlling light-induced purple color in pepper pericarps, and designed the marker A_836 and detection primers based on the inserted Indel. By detecting the deletion or insertion of the provided specific fragment, the fruit color of peppers can be accurately and rapidly detected at the seedling stage, eliminating the need to wait until the fruit setting stage. This reduces labor, significantly improves breeding efficiency, and lowers breeding costs.

Description

Technical Field

[0001] This invention relates to the field of molecular markers for chili peppers, specifically to an Indel molecular marker for identifying the gene controlling the light-induced purple color of chili pepper pericarps and its application. Background Technology

[0002] Chili peppers (Capsicum spp.) are the most widely cultivated vegetable crop in my country and an important spicy condiment. The fruits of chili peppers come in a variety of colors, from green, yellow, orange, milky white to purple, mainly due to variations in the relative content of pigments such as chlorophyll, anthocyanins, carotenoids, and flavonoids in the fruit peel. These natural pigments are beneficial to human health. Furthermore, the rich diversity of fruit colors also makes chili peppers a suitable ornamental crop.

[0003] Purple peppers are primarily due to the biosynthesis and accumulation of anthocyanins in the exocarp. CaAn2 and CaAn3 are two key genes controlling anthocyanin synthesis in peppers, located on chromosome P10, both encoding the R2R3-MYB transcription factor. CaAn2, also known as CaMYBA or the A gene, exhibits purple coloration in purple peppers when a retrospinus is inserted into its promoter region. CaAn2 expression is induced by low temperature and light; plants with the CaAn2 genotype exhibit purple leaves, stems, flowers, and fruits. The CaAn3 gene, adjacent to CaAn2, allows for alternative splicing in purple peppers, enabling the formation of a complete R2R3 domain protein, which in turn forms the MBW transcription factor complex, activating the expression of genes related to the anthocyanin synthesis pathway. CaAn3 expression is not significantly affected by environmental factors; the CaAn3 genotype pepper accumulates purple color only specifically in the fruit.

[0004] The purple pepper involved in this invention accumulates anthocyanins only in the pericarp, exhibiting photoinduced purple coloration, which differs from the phenotypes of CaAn2 and CaAn3 genotype peppers. Furthermore, the purple coloration of the pericarp is strongly induced by light. The application of this purple pepper gene resource is of great significance for pepper quality breeding and ornamental pepper cultivation, and also provides a new case study for the research of pepper anthocyanin synthesis mechanisms. To better conduct the breeding of purple-fruited pepper varieties, developing an efficient and convenient molecular marker is crucial, especially one that does not require identification during the fruiting period and is unaffected by external environmental factors. This marker can accelerate breeding efficiency and improve the accuracy of progeny selection, and is of significant value for screening pepper breeding populations. Summary of the Invention

[0005] The purpose of this invention is to provide an Indel molecular marker for identifying the gene controlling the light-induced purple color of pepper pericarp. The molecular marker is a DNA fragment, as shown in SEQ ID NO.4.

[0006] The purpose of this invention is to provide the application of a reagent for detecting the DNA fragment shown in SEQ ID NO.4 in the light-induced purple breeding of pepper pericarps.

[0007] A final objective of this invention is to provide primers for detecting the DNA fragment shown in SEQ ID NO.4, namely F: 5'-ATGTCCGATCCACAAGACACC-3', R: 5'-AAACCATACTTCGCCCCGAC-3'. To achieve the above objectives, this invention employs the following technical measures:

[0008] This invention utilizes F2 segregating populations constructed from a photosensitive pepper material '20170909-16-M' (with a purple pericarp specifically induced by light) and a pepper material '20190325-001w-F3' (with a milky-white pericarp). Using BSR-seq combined with population mapping, candidate regions of the gene controlling the purple pericarp in photosensitive peppers were located within the interval of 187.48 to 191.11 Mb on the pepper P10 chromosome (the reference genome of pepper CM334, Ver2.0, http: / / peppergenome.snu.ac.kr). Based on gene annotation information from the reference genome CM334 (Ver1.55 and Ver2.0), markers were further developed near and within the located interval to identify exchanger plants. Combined with analysis of differential gene expression within the candidate interval in mixed pools of purple and white peppers, candidate genes controlling the purple pericarp in photosensitive peppers were identified. Ultimately, an 8bp Indel insertion was found to be associated with the purple pericarp of peppers. The Indel insertion is located at position 182,957,332 on the pepper P10 chromosome (CM334 reference genome, Ver1.55). When this Indel insertion is present, the pepper pericarp is light-induced purple; when it is absent, the pepper pericarp is not light-induced purple.

[0009] Furthermore, the applicant designed a molecular marker for targeted sequencing of this Indel insertion. The detection primers for this molecular marker are: F: 5'-ATGTCCGATCCACAAGACACC-3', R: 5'-AAACCATACTTCGCCC CGAC-3'.

[0010] The parent ginkgo '20190325-001w-F3' amplified an 836bp fragment (sequence shown in SEQ ID NO.3), and the photoinduced purple parent '20170909-16-M' amplified an 851bp fragment (sequence shown in SEQ ID NO.4).

[0011] Therefore, the presence of the DNA fragment shown in SEQ ID NO.4 in chili peppers can be used to determine whether the pepper peel is photoinduced purple.

[0012] The scope of protection of this invention includes:

[0013] An Indel molecular marker for identifying the gene controlling light-induced purple color in pepper pericarps, wherein the molecular marker is a DNA fragment as shown in SEQ ID NO.4.

[0014] Application of reagents for detecting DNA fragments containing the DNA fragment shown in SEQ ID NO.4 in the breeding of light-induced purple pericarp in peppers;

[0015] In the above-described applications, preferably, the reagent is a primer;

[0016] In the above-described applications, the preferred primers are: F: 5'-ATGTCCGATCCACAAGACACC-3', R: 5'-AAACCATACTTCGCCCCGAC-3';

[0017] In the above applications, the preferred method for determining whether the pepper pericarp is photoinduced purple is: amplifying the genomic DNA of the pepper to be tested using the above primers, and then performing Sanger sequencing on the PCR amplification primers.

[0018] If the PCR product is an 851bp fragment (as shown in SEQ ID NO.4), then the pericarp of the pepper material to be tested will be photoinduced purple;

[0019] If the PCR product is only an 836bp fragment (as shown in SEQ ID NO.3), the pericarp of the tested pepper material will not have a purple hue. Compared with the prior art, the present invention has the following advantages:

[0020] This invention utilizes an F2 segregating population of light-induced purple-skinned peppers and normal non-purple-skinned peppers. Using BSR-seq combined with population mapping, candidate genes regulating the light-induced purple-skinned pepper appearance are precisely located and identified through pooled transcriptome analysis. Based on the sequence differences of these candidate genes between parents, a novel Indel-targeted sequencing gene marker, A_836, is developed that completely co-segregates with the light-induced purple-skinned pepper appearance. This Indel marker achieves 100% detection efficiency in the F2 segregating population of light-induced purple-skinned peppers and can rapidly detect the presence or absence of purple skin in light-induced peppers.

[0021] The molecular markers developed in this invention can be used as an auxiliary selection breeding technique to detect whether the pericarp of peppers is purple under light induction. There is no need to wait for the peppers to set fruit; screening can be carried out at the seedling stage using molecular markers. It is not affected by the environment and can greatly reduce the workload of field identification, thus accelerating the breeding process of new purple pepper varieties with good quality and ornamental qualities. Attached Figure Description

[0022] Figure 1 A schematic diagram of the phenotypic characteristics of the two parental purple peppers '20170909-16-M' and milky white peppers '20190325-001w-F3';

[0023] Among them: a is a plant with purple fruit '20170909-16-M', b is a plant with milky white fruit '20190325-001w-F3', and c is an F1 generation pepper plant of '20170909-16-M×20190325-001w-F3' with purple fruit;

[0024] Figure 2 The BSR-seq localization results are for the candidate intervals. Detailed Implementation

[0025] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in the present invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not as limiting the scope of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the instruments, materials and reagents used are commercially available unless otherwise specified.

[0027] Example 1:

[0028] Obtaining the InDel marker, which is closely related to the gene controlling purple color in pepper fruit, involves the following steps:

[0029] 1. Preliminary mapping of the gene controlling purple color in pepper fruit

[0030] This invention constructs an F2 segregating population using a light-induced purple-fruited pepper material '20170909-16-M' as the female parent and a milky-white-fruited pepper material '20190325-001w-F3' as the male parent (these two parents are inbred lines segregated from germplasm resources). In the autumn of 2021, the two parents, F1 generation plants, and F2 generation segregating populations were planted in a greenhouse. The fruit color of the two parents and their offspring segregating populations was observed, and only the presence or absence of purple fruit was counted and recorded. The results show that the trait controlling the presence or absence of purple fruit in light-induced peppers is controlled by a single gene dominant inheritance. Mixed pools of purple and white fruit were constructed separately. Using BSR-seq, the candidate segment of the gene controlling the light-induced purple fruit color in peppers was located on chromosome 10 of pepper (the pepper CM334 reference genome, Ver2.0, http: / / peppergenome.snu.ac.kr) within the interval from 187.48 to 191.11 Mb (specifically as shown in the image). Figure 2 (As shown).

[0031] 2. Development of markers closely related to genes controlling light-induced purple color in pepper fruit and analysis of candidate genes.

[0032] Based on the SNP differences between purple and white mixed pools in BSR-seq sequencing, CAPS markers were designed on both sides and within candidate intervals. Exchanged individual plants were screened using the F2 population. Combined with the analysis of differential gene expression within candidate intervals in purple and white mixed pools, candidate genes controlling photoinduced purple coloration of pepper fruits were identified.

[0033] Based on the reference genome sequence information of pepper CM334 (Ver1.55, Ver2.0, http: / / peppergenome.snu.ac.kr and VerUCD 10×, https: / / solgenomics.net / ftp / genomes / Capsicum_annuum / C.annuum_UCD10X / ), specific primers A_836 were designed to amplify the candidate gene. The A_836 primer pair consists of two single-stranded DNA molecules.

[0034] Forward primer A_836_F:5'-ATGTCCGATCCACAAGACACC-3' (SEQ ID No. 1);

[0035] Reverse primer A_836_R:5'-AAACCATACTTCGCCCCGAC-3' (SEQ ID No. 2);

[0036] The gDNA of both parents was amplified, and the PCR products were sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for Sanger sequencing. The sequencing results showed that the ginkgo parent '20190325-001w-F3' amplified an 836bp fragment (containing the sequence shown in SEQ ID NO. 3), and the photoinduced purple phenotype parent '20170909-16-M' amplified an 851bp fragment (containing the sequence shown in SEQ ID NO. 4). The photoinduced purple phenotype parent fragment contained an 8bp insertion at position 142 and a 7bp insertion at position 350. Further amplification and targeted sequencing of individual plants in the F2 population using the A_836 marker showed that the 8bp Indel insertion at position 142 was closely related to the loss of the photoinduced purple phenotype in pepper pericarps.

[0037] The PCR amplification system consisted of: a total of 20.0 μL, 1.0 μL of 50 ng / μL DNA, 10.0 μL of 2×Taq Master Mix, 1.5 μL of 10 μmol mixed primers (i.e., upstream primer and two downstream primers), and the remainder of ddH2O.

[0038] The PCR amplification program was as follows: 94℃ pre-denaturation for 1 min 30 s, 94℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 30 s, 30 cycles, 72℃ extension for 5 min, and storage at 16℃ for 5 min.

[0039] PCR amplification product detection: The above PCR products were sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for Sanger sequencing detection.

[0040] Example 2:

[0041] Application of markers closely related to genes controlling purple color in pepper fruits:

[0042] The test materials consisted of 13 purple-fruited pepper germplasm resources (see Table 1 for details): Each test material in Table 1 was provided by the Institute of Economic Crops, Hubei Academy of Agricultural Sciences, or was published in the literature, or was a commercially available variety.

[0043] Table 1. Identification results of 13 purple-fruited pepper germplasm resources and their fruit color molecular markers.

[0044]

[0045] The Indel marker primer A_836 from Example 1 was used to amplify the parental '20170909-16-M', '20190325-001w-F3', and 11 other different genotypes of pepper germplasm to obtain PCR amplification products. The PCR amplification system was as follows: 20 μL total, 1.0 μL 50 ng / μL DNA, 10.0 μL 2×Taq Master Mix, 1.5 μL 10 μmol mixed primers (i.e., upstream primer and two downstream primers), and the remainder ddH2O.

[0046] The PCR amplification program was as follows: 94℃ pre-denaturation for 1 min 30 s, 94℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 30 s, 30 cycles, 72℃ extension for 5 min, and storage at 16℃ for 5 min.

[0047] PCR amplification product detection: The above PCR products were sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for Sanger sequencing detection.

[0048] Table 1 shows the selection of pepper germplasm with different genotypes. 1, 2, and 13 are pepper germplasm with non-purple pericarps, with 2 being a milky-white parental control. 4, 11, and 12 are pepper germplasm with uniquely purple pericarps, identified by molecular markers as carrying the CaAn3 gene, which specifically accumulates anthocyanins in the pericarp. 5 is pepper germplasm with purple stripes on the pericarp, identified by molecular markers as carrying the CaPs gene, which forms the pattern of purple stripes on the pericarp. 6 is pepper germplasm with purple stems, leaves, flowers, and pericarps, whose anthocyanin accumulation is light-induced, and identified by molecular markers as carrying the CaAn2 gene. 3, 7, 8, 9, and 10 are pepper germplasm with light-induced uniquely purple pericarps, with 10 being a light-induced purple pepper parental control. The genotypic data for the above materials can be found in "Li N, Liu Y, Yin Y, Gao S, Wu F, Yu C, Wang F, Kang BC, Xu K, Jiao C and Yao M (2023) Identification of CaPs locus involving in purple stripe formation on unripe fruit, reveal allelic variation and alternative splicing of R2R3-MYB transcription factor in pepper (Capsicum annuum L.). Front. Plant Sci. 14:1140851. doi:10.3389 / fpls.2023.1140851".

[0049] As shown in Table 1, primer A_836 can effectively distinguish the germplasm of light-induced purple peppers. The PCR amplification products were sequenced by Sanger sequencing to obtain a fragment of SEQ ID NO.4 with a size of 851bp (which can be amplified in commercial varieties, Guangxi Qicai Paojiao, potted Wucaijiao, Qicaijiao, and Wucai Xiaomijiao), while the other germplasm samples yielded a fragment of 836bp.

[0050] This marker can be used as an auxiliary selection breeding technique to detect whether the pericarp of peppers is purple under light induction. It is not affected by the environment and does not require waiting for results. Molecular markers can be used for screening at the seedling stage, shortening the breeding process for quality and colored pepper varieties.

[0051] It should be noted that the above embodiments are merely explanations of the present invention and are not intended to limit the invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of reagent for Indel molecular marker of Capsicum annuum L. fruit skin light-induced purple control gene in Capsicum annuum L. light-induced purple fruit breeding, wherein the molecular marker is shown as SEQ ID NO.

4.

2. The application of claim 1, wherein the reagent is a primer.

3. The application of claim 2, wherein the primer is F: 5'-ATGTCCGATCCACAAGACACC-3' and R: 5'-AAACCATACTTCGCCCCGAC-3'.

4. The application of claim 1, wherein the application process is: amplifying the genomic DNA of the Capsicum annuum L. to be tested by using the primer of claim 3, and performing Sanger sequencing on the PCR amplification primer: if the PCR product is a 851 bp fragment, i.e. SEQ ID NO. 4, then the fruit skin of the Capsicum annuum L. to be tested is light-induced purple; if the PCR product is only a 836 bp fragment, i.e. SEQ ID NO. 3, then the fruit skin of the Capsicum annuum L. to be tested is non-light-induced purple. ​ ​

Images