A molecular marker tightly linked to the light green trait of ripe pepper fruit and its application

Abstract

The present invention discloses a molecular marker tightly linked to the light green trait of green ripe pepper fruit and its application. The present invention provides the application of a substance for detecting whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the pepper genome is A, G, or both A and G for the following purposes: identifying the color trait of green ripe pepper fruit; identifying the depth of the peel color of green ripe pepper fruit. The dCAPS10-1 marker developed by the present invention had a phenotypic matching rate of 95.92% in the constructed F2 population. In addition, the marker dCAPS10-1 was used to perform genotyping on 126 pepper materials with known green ripe fruit colors stored in the laboratory. The results showed that the success rate of verification of the 126 inbred line materials using the dCAPS10-1 marker was 76.20%, of which the phenotypic matching rate was 79.37% for 63 materials with light green green ripe fruit and 73.00% for 63 materials with green green ripe fruit, indicating that this marker has strong applicability and can be used for rapid identification of green ripe fruit color in practical breeding applications.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a molecular marker tightly linked to the light green trait of ripe pepper fruit and an application thereof. Background Art

[0002] Green ripe fruit color (also called commercial ripe fruit color) as a symbol of fresh pepper ( Capsicum annuum One of the most important appearance qualities of pepper (L.) is a primary criterion for consumers when choosing fresh peppers. Five common color variations exist: yellow-white, creamy yellow, green, purple, and purple-black. Therefore, the identification of genes controlling fruit color in pepper green-ripe fruit and the development of relevant markers are crucial for the breeding of distinctive pepper varieties. Currently, although linked markers for fruit color of pepper green-ripe fruit (also known as commercial ripe fruit; see "Pepper Germplasm Resource Survey Standards and Description Specifications" edited by Li Xixiang) have been developed, such as InDelP146, CAMS-454, and CAPS-78-708 (Jiang Haikun et al., 2018; Shen Huolin and Liu Jinqiu, 2019; Le Jun, 2020), most of these markers are linked to the purple trait of pepper green-ripe fruit. Linked molecular markers for the green trait of pepper green-ripe fruit have yet to be reported.

[0003] According to the degree of color depth, the green color of pepper ripe fruit can be further divided into five distinct green categories: yellow-green, light green, green, dark green, and dark green. This indicates that the formation and genetic mechanism of pepper green ripe fruit is relatively complex, which has brought challenges to the genetic improvement of pepper green ripe fruit color. Until recent years, the gene related to the green color of pepper ripe fruit has been found. APPR2- Like (Pan et al., 2013), CaGLK2 (Brand et al., 2014) and CcLOL1 (Borovsky et al., 2019) have been reported, but molecular markers closely linked to them have not yet been developed. Therefore, the development of molecular markers linked to the gene controlling the light green color of pepper green-ripe fruit (also known as commercial ripe fruit) would be of great significance for the genetic improvement of pepper green-ripe fruit color. Summary of the Invention

[0004] The present invention aims to provide a molecular marker tightly linked to the light green trait of green ripe pepper fruit and its application.

[0005] In a first aspect, the present invention claims the use of a substance for detecting whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the pepper genome is A or G or both A and G in any of the following:

[0006] (A1) Identify or assist in identifying the color traits of green and ripe pepper fruits;

[0007] (A2) Preparation of products for identifying or assisting in identifying the color characteristics of green and ripe pepper fruits;

[0008] (A3) Identify or assist in identifying the color depth of the green peel of ripe pepper fruit;

[0009] (A4) Preparation of products for identifying or assisting in identifying the color depth of the peel of green, ripe pepper fruit.

[0010] In (A1) and (A2), the identification or assisted identification of the color trait of the green ripe pepper fruit can specifically be the identification or assisted identification of whether the peel color of the green ripe pepper fruit is green or light green. Here, the green ripe fruit peel color refers to the green ripe pepper fruit peel color being the color type coded as FAN3 141AB on the Royal Horticultural Society's Colour Chart; the light green ripe fruit peel color refers to the green ripe pepper fruit peel color being the color type coded as FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0011] In (A3) and (A4), the green ripe fruit specifically refers to ripe fruit with a green or light green peel. Here, the green ripe fruit peel color refers to the peel color of ripe peppers being the color code FAN3 141AB on the Royal Horticultural Society's Colour Chart; the light green ripe fruit peel color refers to the peel color of ripe peppers being the color code FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0012] Furthermore, the substance for detecting whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the pepper genome is A, G, or A and G can be a primer pair or a reagent or kit containing the primer pair.

[0013] The primer pair consists of an upstream primer and a downstream primer; the upstream primer is designed based on the upstream sequence of the 174795925th deoxyribonucleotide on chromosome 10 in the pepper genome (such as positions 174795744 to 174795768); the downstream primer is designed based on the downstream sequence of the 174795925th deoxyribonucleotide on chromosome 10 in the pepper genome (such as positions 174795924 to 174795951).

[0014] In a specific embodiment of the present invention, the primer pair is a dCAPs primer. The upstream primer is the DNA molecule shown in SEQ ID No. 1; the downstream primer is the DNA molecule shown in SEQ ID No. 2. In addition, the kit also contains the restriction endonuclease HinfI.

[0015] In a second aspect, the present invention claims a method for identifying or assisting in identifying the color traits of green and ripe pepper fruits to be tested.

[0016] The method for identifying or assisting in identifying the color trait of green and ripe pepper fruits to be tested, as claimed in the present invention, may comprise the following steps:

[0017] (B1) Detect whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is A, G, or both A and G, and then determine the genotype as follows:

[0018] If the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is homozygous for A, then the genotype of the pepper to be tested is A:A type;

[0019] If the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is homozygous for G, then the genotype of the pepper to be tested is G:G type;

[0020] If the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is a hybrid of A and G, then the genotype of the pepper to be tested is A:G type;

[0021] (B2) Determining the green and ripe fruit color trait of the pepper to be tested according to the genotype of the pepper to be tested.

[0022] Furthermore, step (B2) can specifically be determining whether the peel color of the green and ripe fruit of the pepper to be tested is green or light green as follows: if the genotype of the pepper to be tested is G:G type, then the peel color of the green and ripe fruit of the pepper to be tested is or can be green; if the genotype of the pepper to be tested is A:A type or A:G type, then the peel color of the green and ripe fruit of the pepper to be tested is or can be light green.

[0023] Here, the green color of the peel of the green ripe fruit means that the color of the peel of the green ripe fruit of pepper is the color type coded as FAN3 141AB on the standard color card of The Royal Horticultural Society's Colour Chart; the light green color of the peel of the green ripe fruit means that the color of the peel of the green ripe fruit of pepper is the color type coded as FAN3 140A on the standard color card of The Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0024] In a third aspect, the present invention claims protection for a method for identifying or assisting in identifying the color depth of the peel of green, ripe pepper fruit. The green, ripe fruit specifically refers to a ripe pepper fruit having a peel color of green or light green. Here, the green peel color refers to the peel color of the ripe pepper fruit being the color coded FAN3 141AB on the Royal Horticultural Society's Colour Chart; the light green peel color refers to the peel color of the ripe pepper fruit being the color coded FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0025] The method for identifying or assisting in identifying the color depth of the peel of green ripe pepper fruit claimed in the present invention may include the following steps:

[0026] (C1) Detect whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the pepper genome to be tested is A, G, or both A and G, and then determine the genotype as follows:

[0027] If the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is homozygous for A, then the genotype of the pepper to be tested is A:A type;

[0028] If the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is homozygous for G, then the genotype of the pepper to be tested is G:G type;

[0029] If the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is a hybrid of A and G, then the genotype of the pepper to be tested is A:G type;

[0030] (C2) The color depth of the peel of the green ripe fruit of the pepper to be tested is determined as follows: the peel color of the green ripe fruit of the pepper to be tested with the genotype of A:A or A:G is lighter than or has the potential to be lighter than the peel color of the green ripe fruit of the pepper to be tested with the genotype of G:G (the peel color of the green ripe fruit of the pepper to be tested with the genotype of G:G is darker than or has the potential to be darker than the peel color of the green ripe fruit of the pepper to be tested with the genotype of A:A or A:G).

[0031] Furthermore, in steps (B1) and (C1), whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is A, G, or both A and G can be detected by a method comprising any of the following steps:

[0032] P1, using the genomic DNA of the pepper to be tested as a template, using the primer pair described above (SEQ ID No.1 and SEQ ID No.2) PCR amplification is performed to obtain an amplified product; the amplified product is then digested with a restriction endonuclease HinfI, and based on the digestion result, whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the tested pepper is A, G, or A and G is determined as follows: if the digestion product contains a 208 bp target band and does not contain a 183 bp target band, the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the tested pepper is G; if the digestion product contains a 208 bp target band and a 183 bp target band, the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the tested pepper is A and G; if the digestion product does not contain a 208 bp target band but contains a 183 bp target band, the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the tested pepper is A;

[0033] P2. Using the genomic DNA of the pepper to be tested as a template, PCR amplification is performed using the primer pair described above (such as SEQ ID No. 1 and SEQ ID No. 2) to obtain an amplified product; then, the amplified product is sequenced, and based on the sequencing results, it is determined whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the genome of the pepper to be tested is A, G, or both A and G.

[0034] In a fourth aspect, the present invention claims protection for a substance having the following functions (A) or (B):

[0035] (A) Identify or assist in identifying the color characteristics of green and ripe pepper fruits;

[0036] (B) Identify or assist in identifying the color depth of the peel of green, ripe pepper fruit

[0037] In (A), the identification or assisted identification of the color trait of the green ripe pepper fruit can specifically be the identification or assisted identification of whether the peel color of the green ripe pepper fruit is green or light green. Here, the green ripe fruit peel color refers to the green ripe pepper fruit peel color being the color type coded as FAN3141AB on the Royal Horticultural Society's Colour Chart; the light green ripe fruit peel color refers to the green ripe pepper fruit peel color being the color type coded as FAN3140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0038] In (B), the green ripe fruit specifically refers to ripe fruit with a green or light green peel. Here, the green ripe fruit peel color refers to the peel color of ripe peppers being the color code FAN3 141AB on the Royal Horticultural Society's Colour Chart; the light green ripe fruit peel color refers to the peel color of ripe peppers being the color code FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0039] The substance claimed for protection in the present invention may specifically be the substance described above for detecting whether the deoxyribonucleotide at position 174795925 on chromosome 10 in the pepper genome is A, G, or both A and G.

[0040] In a fifth aspect, the present invention claims protection for any of the following applications:

[0041] (D1) Use of the substance described in the fourth aspect above in identifying or assisting in identifying the color characteristics of green and ripe pepper fruits, or in preparing a product for identifying or assisting in identifying the color characteristics of green and ripe pepper fruits.

[0042] The identification or auxiliary identification of the color trait of the green ripe pepper fruit can specifically be the identification or auxiliary identification of whether the peel color of the green ripe pepper fruit is green or light green. Here, the green ripe fruit peel color refers to the green ripe pepper fruit peel color being the color type coded as FAN3141AB on the Royal Horticultural Society's Colour Chart; the light green ripe fruit peel color refers to the green ripe pepper fruit peel color being the color type coded as FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0043] (D2) Use of the substance described in the fourth aspect above in identifying or assisting in identifying the color depth of the peel of green, unripe pepper fruit, or in preparing a product for identifying or assisting in identifying the color depth of the peel of green, unripe pepper fruit.

[0044] The term "green ripe fruit" specifically refers to ripe fruit with a green or light green peel. Here, the term "green ripe fruit peel color" refers to ripe pepper peel color that is the color code FAN3 141AB on the Royal Horticultural Society's Colour Chart; the term "light green ripe fruit peel color" refers to ripe pepper peel color that is the color code FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0045] (D3) Use of the method described in the second or third aspect or the substance described in the fourth aspect in pepper breeding.

[0046] In the above aspects, the chili peppers are green or light green in color. Here, the green color of the green peel refers to the color of the green peel of the chili peppers being the color code FAN3 141AB on the Royal Horticultural Society's Colour Chart; and the light green color of the green peel refers to the color of the green peel of the chili peppers being the color code FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001).

[0047] In a specific embodiment of the present invention, the pepper is specifically selected from: the hybrid offspring (such as F2 generation) of pepper materials 17C827 (green-ripe fruit light green) and 17C658 (green-ripe fruit green), or the 63 pepper materials in Table 1.

[0048] In the present invention, the physical position in the pepper genome is based on the genome of Zunla No. 1 (version V2.0) as the reference sequence, the website is: http: / / peppersequence.genomics.cn / page / species / download.jsp#5.

[0049] Experiments have demonstrated that the dCAPS10-1 marker developed in this invention has a phenotypic matching rate of 95.92% in the constructed F2 population. Furthermore, the dCAPS10-1 marker was used to perform genotyping on 126 pepper lines with known green-ripe fruit color stored in the laboratory. The results showed a 76.20% success rate in verifying the 126 inbred lines using the dCAPS10-1 marker, demonstrating the marker's strong applicability and its potential for rapid identification of green-ripe fruit color in practical breeding applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Figure 3 HinfI digestion results of the amplified fragments of dCAPS10-1 in the two parents and different mixed pools (A, 7% native polyacrylamide gel electrophoresis, silver nitrate staining) and the linkage map in F2 (B). DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0052] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0053] Example 1. Acquisition of molecular marker dCAPS10-1

[0054] The physical positions in the pepper genome involved in this embodiment are all based on the genome of Zunla No. 1 (version V2.0) as the reference sequence, the website is: http: / / peppersequence.genomics.cn / page / species / download.jsp#5.

[0055] 1. Construction of positioning groups

[0056] In this experiment, pepper material 17C827 (green-ripe fruit light green) was used as the female parent, and 17C658 (green-ripe fruit green) was used as the male parent (both parents are recorded in Appendix 2 of the article “Wu Lang, Wang Peng, Wang Yihao, Cheng Qing, Lu Qiaohua, LiuJinqiu, Li Ting, Ai Yixin, Yang Wencai, Sun Liang and Shen Huolin, 2019. Genome-wide correlation of 36 agronomic traits in the 287 pepper (capsicum)accessions obtained from the slaf-seq-based gwas. International Journal of Molecular Sciences, 20(22). ”, which is available to the public from the applicant and can only be used to repeat the experiments of the present invention and cannot be used for other purposes). F1 was obtained by hybridization, and 941 F2 segregating populations were obtained by self-pollination of F1. The color of the green-ripe pepper fruit was investigated by visual inspection. A total of 726 strains had light green fruit (identified as FAN3 140A on the Royal Horticultural Society's Colour Chart according to the "Quality Control Standards for Pepper Germplasm Resources" and the principle of maximum similarity, respectively (Royal Horticultural Society, 2001)). 215 strains had green fruit (identified as FAN3 141AB on the Royal Horticultural Society's Colour Chart according to the "Quality Control Standards for Pepper Germplasm Resources" and the principle of maximum similarity, respectively (Royal Horticultural Society, 2001)). The chi-square test showed that the segregation ratio (χ2) was 3:1. 2 =2.324<6.64 (df=1,p=0.01)). Genomic DNA of pepper was extracted using the modified CTAB method.

[0057] 2. Molecular Marker Development

[0058] The green-ripe fruit light green and green-ripe fruit green individuals in the F2 population obtained above were mixed and simplified resequencing was performed, wherein the number of individuals in the green-ripe fruit light green pool was 30, and the number of individuals in the green-ripe fruit green pool was 30. Relevant markers were developed based on the simplified resequencing results.

[0059] 3. Obtaining the molecular marker dCAPS10-1

[0060] Using the candidate genes located by simplified resequencing results of the green-ripe fruit light green pool and the green-ripe fruit green pool in the F2 population, the cloning of candidate genes showed that a GA substitution occurred at deoxyribonucleotide position 174795925 on chromosome 10 in the pepper genome in the green-ripe fruit light green material (17C827) compared with the green-ripe fruit green (17C658) material.

[0061] Zunla-1:

[0062] 5'-CTTTGTTTGCATATTTGGCAGGGGTTGATTTCCATGTGTTTCT-3'

[0063] 17C827:

[0064] 5'-CTTTTGTTTGCATATTTGGCAGGGATTGATTTCCATGTGTTTCT-3'

[0065] 17C658:

[0066] 5'-CTTTGTTTGCATATTTGGCAGGGGTTGATTTCCATGTGTTTCT-3'

[0067] Based on the replacement base, a dCAPS marker dCAPS10-1 was designed. The primer sequence for the marker dCAPS10-1 is shown below:

[0068] dCAPS10-1F: 5'-TTCTTCATGTCTAAATTCCCAGAGG-3' (SEQ ID No. 1);

[0069] dCAPS10-1R: 5′-GTATGTGAAGAAACACATGGAAATG (mismatched base / enzyme cleavage site) AA-3′ (SEQ ID No. 2).

[0070] When the SNP at position 174795925 on chromosome 10 in the pepper genome is A, the amplified product will form a HinfI recognition sequence, and after HinfI digestion, two target fragments of 183 and 25 bp will be obtained. When the SNP at position 174795925 on chromosome 10 in the pepper genome is G, the amplified product will not form a HinfI recognition sequence, and the product after HinfI digestion will be a 208 bp fragment.

[0071] In practical applications, the pepper genome to be tested is used as a template and the dCAPS10-1 primer sequence is used for PCR amplification. The amplified product is then digested with HinfI to obtain the digested product, which is then tested on agarose gel (the 25bp target band is too small to be easily lost during agarose gel running, so it can be ignored and does not affect the result determination).

[0072] If the enzyme digestion product contains the 208 bp target band and does not contain the 183 bp target band, then the 174795925th deoxyribonucleotide on chromosome 10 in the genome of the pepper to be tested is G, that is, the G:G type.

[0073] If the enzyme digestion product contains a 208 bp target band and a 183 bp target band, the deoxyribonucleotides at position 174795925 on chromosome 10 in the genome of the pepper to be tested are A and G, that is, A:G type.

[0074] If the enzyme digestion product does not contain the 208 bp target band but contains the 183 bp target band, then the 174795925th deoxyribonucleotide on chromosome 10 in the genome of the pepper to be tested is A, that is, A:A type.

[0075] The dCAPS10-1 marker was used to detect different mixed pools (for specific methods, see step 2 of Example 2). The results showed that the dCAPS10-1 marker had stable polymorphism between the two parents and the two extreme pools, and the bands amplified by the marker were clear and significantly different between them ( Figure 1 The amplification product of the molecular marker dCAPS10-1 in the genomic DNA of the green extreme pool of unripe fruit contained a 208 bp fragment (excluding the 183 bp fragment). The amplification product of the DNA of the light green extreme pool of unripe fruit contained both 208 bp and 183 bp fragments (the 25 bp target band was lost during gel running because it was too small, and there was heterozygosity in the light green extreme pool!).

[0076] Statistical analysis showed that the phenotypic matching rate of the dCAPS10-1 marker in the constructed F2 population was 95.92%.

[0077] 4. Genetic linkage analysis

[0078] The polymorphic molecular marker dCAPS10-1 obtained by screening was used to analyze the genotype of the F2 population (for specific methods, see step 2 of Example 2), and linkage analysis was performed in combination with the field color phenotype observation results of the population. The results showed that the molecular marker dCAPS10-1 was closely linked to the light green trait of green ripe pepper fruit ( Figure 1 (B) can be used as a molecular marker.

[0079] The pepper genome to be tested was used as a template and dCAPS10-1 primer sequence was used for PCR amplification. The amplified product was then digested with HinfI to obtain the digested product.

[0080] If the enzyme digestion product contains the 208 bp target band and does not contain the 183 bp target band, that is, the G:G type, the corresponding pepper fruit is green.

[0081] If the enzyme digestion product contains a 208 bp target band and a 183 bp target band, that is, the A:G type, the corresponding pepper fruit is light green.

[0082] If the enzyme digestion product does not contain the 208 bp target band but contains the 183 bp target band, that is, type A:A, the corresponding green and ripe pepper fruit is light green.

[0083] Example 2: Application of molecular marker dCAPS10-1

[0084] 1. Experimental Materials

[0085] The present invention uses 126 pepper materials (Table 1) whose green and ripe fruits are known to be light green or green (after years of investigation, with reference to the "Pepper Germplasm Resources Quality Control Standards", according to the maximum similarity principle, 63 samples were identified as belonging to the color category coded as FAN3 140A on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001), i.e., light green; and 63 samples were identified as belonging to the color category coded as FAN3 141AB on the Royal Horticultural Society's Colour Chart (Royal Horticultural Society, 2001), i.e., green). The molecular marker dCAPS10-1 was verified. The 126 materials used were obtained from the College of Horticulture, China Agricultural University (all recorded in Table 2 of “Wu Lang, Wang Peng, Wang Yihao, Cheng Qing, Lu Qiaohua, Liu Jinqiu, Li Ting, Ai Yixin, Yang Wencai, Sun Liang and Shen Huolin, 2019. Genome-wide correlation of 36 agronomic traits in the 287 pepper (capsicum) accessions obtained from the slaf-seq-based gwas. International Journal of Molecular Sciences, 20(22). ”, which are available to the public from the applicant and may only be used to repeat the experiments of the present invention and may not be used for any other purpose).

[0086] Table 1. Identification results of 126 pepper inbred lines and varieties

[0087]

[0088] Note: Band A: light green; Band B: green; Band H: light green. Band A contains only the 183 bp band (excluding the 208 bp band), Band B contains only the 208 bp band (excluding the 183 bp band), and Band H contains both the 208 bp and 183 bp bands.

[0089] 2. Experimental Methods

[0090] The molecular marker dCAPS10-1 was used to perform genotyping on the above test materials, and the template was the genomic DNA of the above test materials.

[0091] The PCR reaction system (10 μl) was as follows: 1 μL DNA working solution, 5 μL 10× Buffer, 0.4 μL dNTPs, 0.2 μL DNA polymerase, 0.5 μL each of the forward primer (SEQ ID No. 1) and the reverse primer (SEQ ID No. 2) (10 μM), and the total volume was made up to 10 μL with 2.9 μL ddH2O.

[0092] PCR amplification reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 30 s, 35 cycles; and extension at 72°C for 5 min.

[0093] Enzyme digestion system: Add 0.2 μL of HinfI enzyme, 1.5 μL of CutSmart, and 3.3 μL of water to each reaction system, and then incubate the reaction at 37°C for 3 h.

[0094] The enzyme digestion products were electrophoresed on 7% non-denaturing polyacrylamide gel, stained with silver nitrate, and photographed. The enzyme digestion products were then sequenced.

[0095] 3. Experimental Results

[0096] The marker dCAPS10-1 was used to genotype 126 pepper accessions with known green-ripe fruit color stored in the laboratory. The results showed a 76.20% success rate for dCAPS10-1 verification of the 126 inbred lines. Among these, the phenotypic match rate was 79.37% for 63 accessions with light green green fruit and 73.00% for 63 accessions with green green fruit. This demonstrates the strong applicability of this marker and its potential for rapid identification of green-ripe fruit color in practical breeding applications.

[0097] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. <110> China Agricultural University <120> A molecular marker tightly linked to the light green trait of ripe pepper fruit and its application <130> GNCLN211127 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial sequence <400> 1 ttcttcatgt ctaaattccc agagg 25 <210> 2 <211> 27 <212> DNA <213> Artificial sequence <400> 2 gtatgtgaag aaacacatgg aaatgaa 27

Claims

1. Use of a primer pair or a reagent or kit containing the primer pair in any of the following: (A1) Identify or assist in identifying the color traits of green and ripe pepper fruits; (A2) preparing a product for identifying or assisting in identifying the color characteristics of green and ripe pepper fruits; (A3) Identify or assist in identifying the color depth of the peel of green, ripe pepper fruit; (A4) preparing a product for identifying or assisting in identifying the color depth of the peel of green ripe pepper fruit; The primer pair consists of an upstream primer and a downstream primer; the upstream primer is the DNA molecule shown in SEQ ID No. 1; the downstream primer is the DNA molecule shown in SEQ ID No. 2; The pepper is a green ripe pepper.

2. The use according to claim 1, characterized in that: The kit also contains restriction endonuclease HinfI.

3. A method for identifying or assisting in identifying the color traits of green and ripe pepper fruits to be tested, comprising the following steps: P1. Using the genomic DNA of the pepper to be tested as a template, PCR amplification is performed using the primer pair of claim 1 to obtain an amplified product; then, the amplified product is digested with the restriction endonuclease HinfI, and the color trait of the green-ripe fruit of the pepper to be tested is determined according to the digestion result as follows: If the enzyme digestion product contains the 208 bp target band and does not contain the 183 bp target band, the peel color of the green-ripe pepper to be tested is or may be green; If the enzyme digestion product contains a 208 bp target band and a 183 bp target band, the peel color of the green-ripe pepper to be tested is or may be light green; If the enzyme digestion product does not contain the 208 bp target band but contains the 183 bp target band, the peel color of the green-ripe fruit of the pepper to be tested is or may be light green; The green color of the peel of the green ripe fruit refers to the color of the peel of the green ripe pepper according to the color code FAN3 141AB on the standard color card of The Royal Horticultural Society's Colour Chart; the light green color of the peel of the green ripe fruit refers to the color of the peel of the green ripe pepper according to the color code FAN3 140A on the standard color card of The Royal Horticultural Society's Colour Chart; The pepper is a green ripe pepper.

4. A primer pair, characterized in that: The primer pair consists of an upstream primer and a downstream primer; the upstream primer is a DNA molecule shown by SEQ ID No.1; the downstream primer is a DNA molecule shown by SEQ ID No.

2.

5. Use of the method according to claim 3 or the primer pair according to claim 4 in pepper breeding; the pepper is green, ripe fruit pepper.

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