A SNP molecular marker related to the regulation of anthocyanins in seed coat and its application

Abstract

The present invention relates to the field of plant genetic engineering technology, and in particular to a SNP site, primer and application of a gene related to regulation of anthocyanin in peanut seed coat color. The SNP site is located at the 03 chromosome of peanut. AhPSC1 Genetically, named AhPSC1‑203, specifically, located AhPSC1 The cDNA sequence of the gene is located at the 203rd base at the 5' end. AhPSC1 The SNP site AhPSC1‑203 on the gene was applied to the model organism Arabidopsis thaliana, which helps to reveal the molecular genetic basis of anthocyanin regulation in plant seed coats. It plays an important role in using genetic engineering technology to improve plants, especially peanuts, to quickly and efficiently cultivate varieties with dark seed coats rich in anthocyanins, and provides new technical support for the development of new plant germplasm resources.

Description

[0001] This application is a divisional application with application number 202311725963.3, application date December 15, 2023, and invention name “Peanut seed coat color regulation-related gene AhPSC1 and its related applications”. Technical Field

[0002] The invention relates to the technical field of plant genetic engineering, and in particular to a SNP molecular marker related to the regulation of anthocyanin in peanut seed coat and an application thereof. Background Art

[0003] Peanut, also known as groundnut, longevity fruit, is a treasure from head to toe. Peanut (Arachis hypogaea L.) is an economically important leguminous crop grown in more than 100 countries around the world, with a total output of 53.92 million tons in 2021 and an area of ​​32.72 million hectares (FAOSTAT 2021). Peanut seed coat is rich in various nutrients, such as vitamins B1, B3, B9 and E, biotin, resveratrol, isoflavones, phytic acid and proanthocyanidins. Seed coat color is an important quality feature, such as white, pink, red, purple, black and mixed colors affect the commodity value. In particular, the darker the color, the more antioxidants are contained in the seed coat. Anthocyanins are important plant pigments in plants, which not only play an important physiological function, but also have strong antioxidant capacity and important nutritional value. In particular, the darker the color, the more antioxidants are contained in the seed coat. Anthocyanins are important plant pigments in plants, which not only play an important physiological function, but also have strong antioxidant capacity and important nutritional value. Importantly, high-anthocyanin varieties have become one of the important directions of rice, corn and wheat breeding. Therefore, breeding new peanut varieties with different seed coat colors will have higher economic value and broader market prospects.

[0004] The seed coat color of peanuts is an important trait of peanuts, such as white, pink, red, dark purple and black. The content and composition of anthocyanins are important factors that determine the color of chestnuts. In higher plants, there are six types of anthocyanins, namely delphinidin, cyanidin, pelargonidin, peonyin, petuniain and malvidin. There are significant differences in the types and contents of anthocyanins in peanuts of different colors, among which the contents of delphinidin, cyanidin and teracanthus are considered to be closely related to the color.

[0005] The biosynthesis of anthocyanins is regulated by many factors, such as the influence of vacuolar pH, temperature, light and other environmental factors on the final color of anthocyanins, but the decisive factor is the structural genes and regulatory factors in the metabolic pathway and their interaction. In addition, the seed coat color of peanuts cannot be observed at the seedling stage, and it is difficult to make a preliminary selection of seed coat color by checking the appearance of buds or leaves at the seedling stage. Revealing the genetic mechanism that controls the red seed coat and developing molecular markers closely related to it will help us identify seed coat color in advance and accelerate the breeding process of red seed coat peanut cultivars. In the past 100 years, a lot of research has been done to improve the understanding of the genetics that determine the seed coat color of peanuts.

[0006] Zhao used QTL-seq to identify genes that control seed coat color Ah The gene was located in the 4.7 Mb region of chromosome A10. Molecular markers were developed through BSR, and finally a marker pTesta1089 related to seed coat color was screened out; Wan et al. showed that hormone synthesis, signal transduction, and competition between flavonoid synthase and dihydroflavonol-4-reductase regulated by the MBW complex (MYB, bHLH, and WDR) are key molecular control nodes for regulating the white seed coat phenotype; Zhang et al. used BSA sequencing analysis to preliminarily identify genes related to peanut red seed coat on chromosome 12, and further located them to a 530 kb interval through linkage mapping using recombinant inbred lines (RILs). AhRt2 To identify the gene controlling the red seed coat color in peanuts, Chen et al. constructed a hybrid F2 population of pink and red seed coat varieties, and accurately mapped it to the 580 kb genomic region of chromosome A03 through BSA sequencing analysis and segregation population mapping, and finally obtained a gene encoding a bHLH transcription factor. AhRt1 Gene. However, so far, few new genes related to the control of peanut seed coat color have been cloned. Therefore, it is of great significance to isolate new genes related to the control of seed coat color and study their functions and analyze their molecular mechanisms. Summary of the invention

[0007] The present invention provides a gene related to the regulation of anthocyanin in seed coat AhPSC1 , encoding proteins, primers and their application in breeding, in order to solve the problems of lack of genes related to the regulation of anthocyanin content in plant seed coat, lack of new genes related to the purple-red seed coat color of peanuts and lack of molecular markers, and provide more options for breeding of crop varieties with high anthocyanin content.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] In the first aspect, a gene related to the regulation of anthocyanin in seed coat is provided. AhPSC1 , characterized in that its nucleotide sequence is one of the following sequences:

[0010] (1) the genomic nucleotide sequence shown in SEQ ID NO.1;

[0011] (2) the CDS nucleotide sequence as shown in SEQ ID NO.2;

[0012] (3) A nucleic acid sequence having equivalent function derived from the nucleic acid sequence shown in SEQ ID NO.2.

[0013] In a second aspect, the present invention provides a seed coat anthocyanin regulation-related gene AhPSC1 The promoter is characterized in that the nucleotide sequence is one of the following sequences:

[0014] (1) the nucleotide sequence shown in SEQ ID NO.4;

[0015] (2) Adding, deleting or replacing one or more nucleic acids on the basis of the nucleic acid sequence shown in SEQ ID NO.4 to obtain the sequence of an active fragment or conservative variant.

[0016] In a third aspect, the present invention provides an AhPSC1 protein encoded by a gene related to the regulation of anthocyanins in seed coat, characterized in that the amino acid sequence is one of a series of sequences:

[0017] (1) the amino acid sequence shown in SEQ ID NO.3;

[0018] (2) The sequence of an active fragment or conservative variant is obtained by adding, deleting or replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO.3.

[0019] In a fourth aspect, the present invention provides a pair of primers for detecting the seed coat anthocyanin regulation-related gene AhPSC1, characterized in that the primer nucleotide sequence of the primer pair is:

[0020] AhPSC1-1F: 5'-ATGGCTGACGATGAGGAT-3' (SEQ ID NO. 5);

[0021] AhPSC1-1R: 5'-TCTTAGAAGTGCTCCTCCGTAG-3' (SEQ ID NO. 6).

[0022] In a fifth aspect, the present invention provides a method for producing a gene containing anthocyanin regulation-related genes in seed coat. AhPSC1 and / or genes related to seed coat anthocyanin regulation AhPSC1 An expression vector, recombinant bacteria or expression cassette constructed with a promoter.

[0023] Furthermore, the expression vector or recombinant bacteria or expression box is characterized in that it is obtained by inserting the nucleotide sequence into the pCAMBIA1300-GFP overexpression vector.

[0024] In a sixth aspect, the present invention provides a method for producing a gene related to the regulation of anthocyanin in seed coat. AhPSC1 and / or genes related to seed coat anthocyanin regulation AhPSC1 Application of promoter in preparing transgenic plant cells.

[0025] In a seventh aspect, the present invention also provides genes related to the regulation of anthocyanins in seed coat AhPSC1 and / or genes related to seed coat anthocyanin regulation AhPSC1 promoter and / or AhPSC1 protein and / or AhPSC1 Application of primers in plant breeding.

[0026] Furthermore, the plant breeding is to cultivate transgenic plants with increased anthocyanin content in the seed coat.

[0027] Furthermore, the plant is peanut, and the increased anthocyanin content in the seed coat appears as purple-red on the peanut seed coat.

[0028] In the eighth aspect, the present invention provides an application of a SNP molecular marker AhPSC1-203 related to the regulation of anthocyanins in seed coat in plant molecular marker-assisted selection breeding. The nucleotide sequence containing the SNP molecular marker is shown in SEQ ID NO.2. The 203rd base from the 5' end of the sequence shown in SEQ ID NO.2 is a SNP site, and the SNP site is G or A.

[0029] Furthermore, the base of the SNP site is A, which is a phenotype with a high anthocyanin content in the seed coat, and the base of the SNP site is G, which is a phenotype with a low anthocyanin content in the seed coat.

[0030] Furthermore, the seed coat anthocyanin high content phenotype appears purple-red on the peanut seed coat, and the seed coat low anthocyanin content phenotype appears pink on the peanut seed coat.

[0031] In a ninth aspect, the present invention provides an application of a SNP molecular marker AhPSC1-203 related to the regulation of anthocyanins in seed coat for identifying the high and low anthocyanin content in plant seed coats.

[0032] In a tenth aspect, the present invention provides a primer pair, characterized in that the primers are used to amplify and / or detect the SNP molecular marker AhPSC1-203 related to the regulation of anthocyanins in seed coat according to claim 1, and the primer nucleotide sequence of the primer pair is:

[0033] AhPSC1-SNPF: 5'-CTTAAGTTGGGTTTTAGACTTTA-3' (SEQ ID NO. 7);

[0034] AhPSC1-SNPR: 5'-ATCGTCCAGAGCATATTTGATGCAG-3' (SEQ ID NO. 8).

[0035] In an eleventh aspect, the present invention provides a kit for identifying SNP molecular markers related to the regulation of anthocyanins in seed coat, comprising the above-mentioned primer pair.

[0036] In a twelfth aspect, the present invention provides an application of the above-mentioned primer pair and / or a kit containing the primer pair in plant molecular marker-assisted selection breeding.

[0037] Furthermore, the application is the application of identifying anthocyanin content in plant seed coats.

[0038] Furthermore, the plant is peanut.

[0039] Compared with the prior art, the beneficial technical effects of the present invention are:

[0040] 1. The present invention discloses for the first time and confirms a new gene related to the regulation of anthocyanins in seed coat that can be used in the cultivation of new plant varieties, named AhPSC1 The gene is located on peanut chromosome 03, with a CDS sequence length of 1401bp, containing 7 exons, and the protein it expresses is 466 amino acids long.

[0041] 2. The present invention clarifies the genes related to the regulation of anthocyanins in seed coat AhPSC1 The DNA sequence, CDS sequence and coding protein sequence lay the foundation for the application practice of peanut breeding.

[0042] 3 The present invention discloses and confirms for the first time a gene SNP site related to the regulation of anthocyanins in seed coat, named AhPSC1-203. It is also clarified that the AhPSC1-203 site is at the 203rd base from the 5' end of the cDNA sequence of the AhPSC1 gene.

[0043] 4. The present invention helps to reveal the molecular genetic basis of anthocyanin regulation in plant seed coat by applying AhPSC1 gene and its related SNP sites to the model organism Arabidopsis thaliana. It plays an important role in improving the use of genetic engineering technology to quickly and efficiently cultivate dark seed coat varieties rich in anthocyanins, especially peanuts, and provides new technical support for the development of new germplasm resources of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 are the characteristics of ND_F and ND_Z; among them, Figure 1 a is the seed coat color representation of ND_F and ND_Z, Figure 1 b is the anthocyanin content of ND_F and ND_Z; the error bars represent the standard deviation of three biological replicates. ** P < 0.01 (two-tailed t test);

[0045] Figure 2 It is the characteristics of AhPSC1 gene and G / A population at AhPSC1-203 locus;

[0046] Figure 3 is the expression level of AhPSC1 in different plant parts of peanut materials;

[0047] Figure 4 The results are a comparison of seed coat color between wild type (WT) and Arabidopsis thaliana expressing AhPSC1 gene, as well as anthocyanin quality detection; Figure 4 a is the comparison of seed coat color characterization, Figure 4 b Anthocyanin quality test; significant differences were assessed using one-way analysis of variance (ANOVA) followed by post hoc Tukey's multiple comparison test and are indicated by lowercase letters above each bar graph. DETAILED DESCRIPTION

[0048] The specific implementation modes of the present invention are described below in conjunction with the accompanying drawings and examples. However, the following examples are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way.

[0049] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; unless otherwise specified, the reagents involved are all commercially available conventional reagents; unless otherwise specified, the test methods involved are all conventional methods. The quantitative tests in the following examples were repeated three times, and the results were averaged. Primer synthesis and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0050] Example 1: Acquisition of seed coat color and anthocyanin regulation-related gene AhPSC1.

[0051] Forward genetics combined with genetic engineering was used to mine genes related to the regulation of anthocyanins in peanut seed coat, as follows:

[0052] (1) In order to identify the genes that control peanut seed coat color and anthocyanin regulation, we selected ND_F (pink seed coat) as the female parent and ND_Z (purple seed coat) as the male parent for hybridization. Figure 1As shown, the ND_Z seed coat contains 0.6 mg / g anthocyanin, while the ND_F seed coat contains only 0.2 mg / g anthocyanin. Compared with the genes that control the purple-red seed coat color that have been discovered, as shown in Table 1, these two materials are a new mutation site. The seed coat of peanut is developed from the integuments of the ovule, so 80 F1 seeds show the same color as the mother seed coat, and 701 F2 individuals show light purple-red seed coat. In the F3 population, the separation ratio of these two populations conforms to the Mendel model of 1:3 (162 pink seeds: 458 purple-red seeds; χ2 = 0.421 < 20.05, 1 = 3.84). These results indicated that the purple-red seed coat phenotype of ND_Z was controlled by a dominant gene. Then, 20 purple and pink individuals and leaves of the parents were selected for resequencing and aligned to the reference genome of cultivated peanut (Arachis hypogaea cv. Tifrunner, gnm1.ann1, Tif1, https: / / www.peanutbase. org / peanut_genome and Arachis. hypogaea var. Shitouqi, http: / / peanutgr.fafu.edu.cn) for BSA analysis. Finally, we took the intersection and located the candidate interval within a 3.61 Mb interval on chromosome 3.

[0053] Table 1 Specific identification of ND_F and ND_Z materials

[0054]

[0055] (2) In order to further precisely locate the target gene, we continued to develop more precise molecular markers InDel 52, InDel 32, InDel 16, InDel 60, InDel 70, InDel 48, SNP_7J, InDel 49 and InDel 51 in the candidate interval, and then used these molecular markers to perform association analysis on the F3 population. Finally, we narrowed the candidate interval to the 84.9 kb interval between InDel 48 and SNP_7J. According to the Tif1 reference genome assembly results, there are 4 annotated genes in this interval: This interval contains four predicted genes: a serine / threonine protein phosphatase gene ( Arahy.ZVWI94 ), a gene encoding a bHLH transcription factor ( Arahy.Y5CARX ), a quinone reductase family protein gene ( Arahy.QHE7IT ) and a receptor-like serine / threonine kinase gene ( Arahy.7J7FTE In addition, we studied the association between SNPs and traits in a recombinant inbred line (RIL) population. The results showed that Figure 2 As shown in a, the SNP at -1306 bp of the Arahy.Y5CARX promoter ("G" genotype in all pink individuals and "A" genotype in all purple individuals) co-segregated with seed coat color. AhPSC1 Encoding a bHLH transcription factor, containing a myc bHLH conserved domain, we finally identified it as the target gene AhPSC1 ,like Figure 2 As shown in b.

[0056] (3) To verify whether the above-mentioned difference in seed coat color between ND_F and ND_Z is caused by genes AhPSC1 The promoter (see SEQ ID NO.4) and CDS sequence (see SEQ ID NO.2) of the gene were cloned and sequenced in ND_F and ND_Z, respectively.

[0057] The specific steps are as follows:

[0058] 1.1 DNA extraction

[0059] The DNA of ND_F and ND_Z were extracted using the CTAB method.

[0060] 1.2 Total RNA extraction and first-strand cDNA synthesis

[0061] RNA extraction from different peanut tissues was performed using the RNA room temperature extraction kit (RC411, Novozyme, Nanjing, China) of Nanjing Novozyme Biotechnology Co., Ltd., and total RNA from ND_F and ND_Z was extracted respectively. RNA that met the quality standards was reverse transcribed and synthesized into cDNA using a reverse transcription kit, and then the RNA was synthesized using the TransScript® One-Step gDNA Removal and cDNASynthesis SuperMix Kit (AT311-02, Quanshijin, Beijing, China).

[0062] 1.3 AhPSC1 Gene cloning and sequence analysis

[0063] Design PCR primers. The primers are designed at both ends of the start codon ATG and the stop codon TAA as follows:

[0064] AhPSC1-1F: 5'-ATGGCTGACGATGAGGAT-3'; (SEQ ID NO.5)

[0065] AhPSC1-1R: 5'-TCTTAGAAGTGCTCCTCCGTAG-3'; (SEQ ID NO. 6)

[0066] The DNA of ND_F and ND_Z was used as template and AhPSC1-1F / R primer pair was used for PCR amplification. The PCR products were subjected to 1% agarose gel electrophoresis and connected to 5 x TA / Blunt-Zero Cloning Kit (C601-01, Novozymes, Nanjing, China) after recovery. After bacterial inspection, the correct monoclonal clone was selected and sent to Biotechnology for sequencing. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min in a PCR instrument; then 30 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 30 sec, extension at 72°C for 5 min, and then storage at 4°C.

[0067] The cDNA of ND_F and ND_Z was used as template and the high-fidelity enzyme Phanta® Uc Super-Fidelity DNA Polymerase for Library Amplification (P507-01, Novozymes, Nanjing, China) was used to perform PCR products with AhPSC1-1F / R primer pair. The products were then electrophoresed on 1% agarose gel and connected to 5 x TA / Blunt-Zero Cloning Kit (C601-01, Novozymes, Nanjing, China) after recovery. After bacterial testing, the correct single clones were selected and sent to Biotechnology for sequencing. The PCR reaction system and parameters were the same as the above method.

[0068] The sequencing results showed that the DNA fragment obtained by PCR amplification using peanut ND_Z DNA as a template was AhPSC1 The genome level sequence of the gene is shown in the sequence listing SEQ ID NO.1, and its length is 2792 bp.

[0069] PCR amplification using peanut ND_F and ND_Z cDNA as template AhPSC1 The cDNA sequence of the gene, the cloned CDS length is 1401 bp SEQ ID NO. 2. A G / A synonymous SNP mutation occurred at position 203 in its coding region ( Figure 2 b) Its amino acid sequence is as follows: SEQ ID NO.3 AhPSC1 Predicting the structure of genes, AhPSC1 It contains a myc bHLH conserved domain and is a bHLH transcription factor.

[0070] Example 2: Development and population validation of SNP molecular marker primers.

[0071] According to the two materials AhPSC1 Develop SNP markers based on SNP differences in genes:

[0072] AhPSC1-SNPF: 5'-CTTAAGTTGGGTTTTAGACTTTA-3'; (SEQ ID NO.7)

[0073] AhPSC1-SNPR: 5'-ATCGTCCAGAGCATATTTGATGCAG-3' (SEQ ID NO.8)

[0074] We then selected 30 extreme individuals from the RIL population and 60 natural varieties for verification, and the results showed that the SNP variation in the AhPSC1 promoter was strongly associated with the color of the peanut seed coat, such as Figure 2 As shown in c.

[0075] Example 3: Fluorescence quantitative analysis of AhPSC1.

[0076] Fluorescence quantitative analysis was performed on the tissues of materials of different tissues of ND_F and ND_Z; the extraction of total RNA and the reverse transcription of cDNA were the same as in Example 1.

[0077] against AhPSC1 The specific primers for fluorescence quantitative analysis were designed based on the gene sequence as follows:

[0078] AhPSC1-qF: 5'-TATGATCCACAGCAGCAGCC-3';

[0079] AhPSC1-qR: 5'-GTGCTTTGCCATTGGGGGAAG-3';

[0080] The internal reference gene was Ahactin 7 (XM_025826875), and the primer sequences were as follows:

[0081] Ahactin7-F: 5'-GATTGGAATGGAAGCTGCTG;

[0082] Ahactin7-R: 5'-CGGTCAGCAATACCAGGGAA;

[0083] The PerfectStart® Green qPCR SuperMix Kit (AQ101-01, TransGenBiotech, Beijing, China) and the Bio-Rad CFX96 Touch Real-Time PCR System (Bio-Rad, Hercules, CA, USA) were used for the analysis.

[0084] 20 μl reaction system, cDNA 1 μl, upstream and downstream primers 0.5 μl each, 2 x PerfectStart®Green qPCR SuperMix 10 μl, nuclease-free water 8 μl.

[0085] PCR program: 95°C for 3 min, 95°C for 5 s, 55°C for 30 s, 72°C for 30 s, 40 cycles.

[0086] Take 5 μl of PCR reaction product and run it on 2.0% agarose gel electrophoresis, and send it to Bio-Tech to sequence to determine whether the target fragment is consistent. △△CT The relative expression of AhPSC1 gene was calculated by Figure 3 The gene expression pattern reflects the gene function to a certain extent. We used RT-qPCR to detect the expression of genes in different tissues of ND_F and ND_Z. AhPSC1 The relative expression of genes was shown in AhPSC1 The expression pattern of is constitutive expression. It is expressed in the roots, stems, leaves and seed coat of peanut, but the expression level only reaches a significant difference level in the seed coat. The expression level in ND_Z is higher than that in ND_F and reaches a difference level.

[0087] Embodiment 4: AhPSC1 Obtaining and identifying transgenic Arabidopsis thaliana.

[0088] (1) Construction of plant overexpression vector

[0089] Overexpression vector construction: Sequencing the correct junction AhPSC1 The T-vector plasmid of the gene was used as a template and PCR amplification was performed using pCAMBIA1300-PSC1-GFP F / R plus homology arm primers.

[0090] pCAMBIA1300-PSC1-GFP_F: 5'-AGAGAACACGGGGGACGAGCTCATGGCTGACGATGAGGAT-3';

[0091] pCAMBIA1300-PSC1-GFP_R: 5'-AGCTCCTCGCCCTTGCTCACCATTCTTAGAAGTGCTCCTCCG-3';

[0092] The overexpression vector pCAMBIA1300-GFP was digested with restriction endonucleases KpnI-HF (Star Select) and SacI-HF (Star Select), digested at 37℃ for 30 min, and heat-inactivated at 65℃ for 20 min for double digestion. The PCR product and digested product were then run on 1% agarose and the product was recovered. According to the homologous recombination kit (C112, Novozymes, Nanjing, China), 4 μl of the insert fragment, 3 μl of the linear vector, 2 μl of 5 × CE II Buffe, and 1 μl of Exnase I were reacted in a metal bath at 37℃ for 30 min for ligation. The ligated vector was transferred into DH5α according to 2.2.6 and coated on a culture dish containing 50 mg / L kanamycin (Kanamycin, Kan). Then, the 1300_GFP-F / R primer was used for bacterial detection, and the correct monoclonal bacterial solution was sent to Shanghai Shenggong Biological Company for sequencing.

[0093] 1300_GFP-F: 5'-ATGGTGAGCAAGGGCGAGGAGCT-3';

[0094] 1300_GFP-R: 5'-ACCGATGATACGAACGAAAG-3';

[0095] Finally, the pCAMBIA1300-AhPSC1-GFP vector was constructed and transformed into Shanghai Weidi GV3101 Agrobacterium tumefaciens, spread on YEP solid medium containing 50 mg / L Kan and 25 mg / L rifampin (Rif) resistance, cultured at 28°C for 2 to 3 days, and single clones were picked for inspection.

[0096] (2) AhPSC1 Obtaining transgenic Arabidopsis

[0097] pCAMBIA1300-AhPSC1-GFP was transformed into Arabidopsis thaliana by Agrobacterium infection.

[0098] (3) PCR identification of genetically modified peanuts

[0099] The hygromycin tag (Hyg) was used for screening on 30 mg / L hygrovetine (hyg) screening medium, and the T1 generation transgenic plants were positively identified using the Hyg-1F / R primers. The primer sequences are as follows:

[0100] Hyg-F: 5'-CGAGTACTTCTACACAGCCATC-3';

[0101] Hyg-R: 5'-TCCACTATCGGCGAGTACTTCT -3';

[0102] A total of 9 positive transgenic plants were obtained. After additional generations, the T3 generation homozygous transgenic lines were obtained: OE-52-4-4, OE-52-4-6 and OE-52-10-6. Figure 4 As shown, the seed coat color of the overexpression plants OE-52-4-4, OE-52-4-6 and OE-52-10-6 was darker than that of the wild type (WT).

[0103] In summary, the seed coat anthocyanin regulation-related genes of the present invention AhPSC1 It can not only regulate the anthocyanin content in peanut seed coat, and further regulate the purple-red seed coat color, but also regulate the anthocyanin content and seed coat color in the model organism Arabidopsis seed coat. AhPSC1 In the future, this gene can be used to study the molecular mechanism of seed coat anthocyanin regulation, which has important application value in peanut breeding. It can also be extended to the development and research of increasing the seed coat anthocyanin content and seed coat color typing of other plants.

[0104] The present invention has been described in detail above with reference to the accompanying drawings and embodiments. However, those skilled in the art will appreciate that, without departing from the spirit of the present invention, the specific parameters in the above embodiments may be changed to form a plurality of specific embodiments, which are all within the common variation range of the present invention and will not be described in detail herein.

Claims

1. Application of a SNP molecular marker related to seed coat anthocyanin regulation in peanut molecular marker-assisted selection breeding, characterized in that: The SNP molecular marker site is located at the 696th position of the nucleotide sequence of SEQ ID NO.4, and the polymorphism is G / A.

2. The use according to claim 1, characterized in that: The base of the SNP molecular marker is A, which indicates a high anthocyanin content phenotype in the seed coat, and the base of the SNP molecular marker is G, which indicates a low anthocyanin content phenotype in the seed coat.

3. The use according to claim 2, characterized in that: The seed coat anthocyanin high content phenotype appears purple-red on the peanut seed coat, and the seed coat low anthocyanin content phenotype appears pink on the peanut seed coat.

4. Use of the SNP molecular markers related to the regulation of anthocyanins in seed coat as described in claim 1 in identifying the anthocyanin content in peanut seed coat.

5. A primer pair, characterized in that: The primers are used to amplify and / or detect the SNP molecular markers related to the regulation of anthocyanins in seed coat described in claim 1, and the primer nucleotide sequences of the primer pairs are: 5'-CTTAAGTTGGGTTTTAGACTTTA-3' and 5'-ATCGTCCAAGCATATTTGATGCAG-3'.

6. A kit for identifying SNP molecular markers related to anthocyanin content in peanut seed coat, characterized in that The kit comprises the primer pair according to claim 5.

7. Use of the primer pair according to claim 5 and / or the kit according to claim 6 in peanut molecular marker-assisted selection breeding.

8. The use according to claim 7, characterized in that: The application is the application of identifying the anthocyanin content in peanut seed coat.

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