InDel molecular marker for identifying pepper purple fruit, primer thereof and application

Abstract

The application discloses an InDel molecular marker for identifying pepper purple fruits, primers and application thereof. The application locates a linkage candidate interval related to the purple fruits by constructing a genetic linkage map of different pepper fruit colors, screens candidate genes and InDel markers in the interval, and develops the InDel molecular marker for identifying pepper purple fruits and the primers, so that the pepper purple fruits can be accurately and quickly distinguished and identified. Meanwhile, the application also provides a method for identifying pepper purple fruits or typing of pepper purple fruit plants, which can more accurately type pepper parents and offspring, identify F1 generation and parental purple traits, solves the problems that only the purple traits can be identified in the prior art and the F1 generation traits of the pepper purple fruits are not accurately typed, shortens a screening period of pepper purple fruit germplasm resources, and provides technical support for identification of the pepper purple fruits and offspring.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular breeding technology. More specifically, it relates to an InDel molecular marker for identifying purple fruit of chili peppers, its primers, and its applications. Background Technology

[0002] Chili pepper (Capsicum annum L.) is a plant belonging to the genus Capsicum in the Solanaceae family and is an important vegetable and condiment. Besides its spiciness, fruit color is also a crucial commercial trait of chili peppers. Chili pepper fruits contain various pigments, including chlorophyll, anthocyanins, carotenoids, and flavonoids. Among these, anthocyanins are closely related to fruit color, and differences in the relative content of various anthocyanin components contribute to the diversity of chili pepper fruit colors. Chili pepper breeding requires not only red but also different fruit colors, such as purple. The formation and genetic mechanisms of chili pepper color are complex, posing a challenge to the genetic improvement of green-ripe purple varieties. Therefore, developing a molecular marker associated with green-ripe purple chili peppers will be of great significance for the genetic improvement of the green-ripe fruit color trait.

[0003] InDel markers (insertion-deletion) are a technique used in biological research and genetic engineering to mark and identify insertion and deletion events in specific DNA sequences. InDels are one of the most common forms of variation in DNA sequences. InDel molecular markers allow for the precise detection and analysis of insertion and deletion events in DNA sequences, enabling in-depth research into genome structure and function, and are important markers for molecular breeding applications. Existing research has used InDel markers to screen and identify different fruit colors in chili peppers. For example, existing technologies have disclosed molecular markers HS5 and HS6 linked to chili pepper fruit color genes, used to identify red or yellow chili pepper fruits. There are also molecular markers used to identify purple chili pepper fruits at the green-ripe stage, such as the CAMS-454 marker, which can be used to screen individual chili pepper plants with purple fruits, distinguishing between purple and green fruits. Another example is the CAPS-78-708 marker, which regulates the purple color of green chili pepper fruits, using three different genotypes to distinguish between purple and green chili pepper fruits.

[0004] Therefore, it is evident that there are currently few InDel molecular markers available for identifying purple pepper fruits and differentiating purple pepper plant types. Furthermore, existing molecular markers can only distinguish between green and purple, and cannot differentiate between parental and F1 generation plants, resulting in inaccurate phenotyping of the F1 generation of purple pepper fruit hybrids. Phenotyping of the F1 generation of purple pepper fruit hybrids would improve the accuracy and efficiency of marker-assisted breeding for purple green-ripening pepper varieties. Therefore, further research into marker-assisted selection is needed to develop more molecular markers, specifically InDel molecular markers that can more accurately identify purple pepper fruits and differentiate between parental and offspring plants, providing technical support for the identification of purple pepper fruit germplasm resources and molecular-assisted breeding. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of existing molecular markers for the differentiation of parental and offspring plants of purple pepper fruit, and to provide an InDel molecular marker for identifying purple pepper fruit, its primers and applications.

[0006] The first objective of this invention is to provide an InDel marker for the morphology of purple pepper fruits.

[0007] A second objective of this invention is to provide a primer for detecting InDel markers in purple fruit of chili peppers.

[0008] A third objective of this invention is to provide applications of the InDel marker or its detection primers.

[0009] The fourth objective of this invention is to provide a kit for identifying purple pepper fruits or for identifying / screening F1 generation hybrids of purple pepper fruits.

[0010] The fifth objective of this invention is to provide a method for identifying purple pepper fruits or purple pepper fruit plants and their progeny typing.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention provides an InDel marker for genotyping purple fruits of chili peppers. The InDel marker, InDel 67, is located at the 185664068bp site on chromosome 10 of chili peppers and has a 49bp deletion.

[0013] This invention utilizes BSA seq analysis to construct genetic linkage maps for different pepper fruit colors, locating candidate linkage intervals related to purple fruit. Genes associated with purple pepper fruit are preliminarily located on chromosomes 9 and 10. Candidate genes and SNP sites within these intervals are screened, preliminarily identifying the transcription factor MYB1 (Capann_59V1aChr10g016200), which influences anthocyanin synthesis, as a potential key candidate gene for poor anthocyanin trait in the exocarp of mature peppers. Further analysis of this key candidate gene yields an InDel marker, InDel 67. Polymorphism detection using InDel 67 primers in parents, F1, and F2 populations shows that InDel 67 is polymorphic in both parents and F1, and can be used for F2 genotyping. Comparison with field phenotypes shows a 92.4% concordance rate, indicating good accuracy. This method can better differentiate between parents and F1 generations, and can be used for genotyping of purple-fruited pepper plants and identification of germplasm resources. The method is simple, feasible, and easy to operate.

[0014] Therefore, the present invention provides a primer for detecting InDel markers for the genotyping of purple pepper fruits, comprising an upstream primer: 5'-TGGGAGGTGTGTGTATATAATGG-3'; and a downstream primer: 5'-GCCTCACCTTATCATATACCCT-3'.

[0015] This invention provides the application of the above-mentioned InDel marker or its detection primer in identifying plants with purple fruit in chili peppers, or in identifying / screening F1 generation hybrid plants with purple fruit in chili peppers.

[0016] This invention provides the application of the above-mentioned InDel marker or its detection primer in the typing of purple fruit plants of peppers and their progeny.

[0017] This invention provides the application of the above-mentioned InDel marker or its detection primer in the breeding of purple fruit of pepper.

[0018] This invention provides the application of the above-mentioned InDel marker or its detection primers in the preparation of kits for identifying purple pepper fruits, or for preparing F1 generation hybrid plants of purple pepper fruits, or for preparing kits for genotyping purple pepper fruit plants and their progeny.

[0019] This invention provides a kit for identifying purple pepper fruits or for identifying / screening F1 generation hybrid plants of purple pepper fruits. The kit contains primers for detecting InDel markers used to genotype purple pepper fruits, including an upstream primer: 5'-TGGGAGGTGTGTGTATATAATGG-3'; and a downstream primer: 5'-GCCTCACCTTATCATATACCCT-3'.

[0020] Preferably, the kit further includes reagents for extracting sample DNA.

[0021] This invention also provides a method for identifying purple pepper fruits or purple pepper fruit plants and their progeny typing, comprising the following steps:

[0022] S1. Extract DNA from the sample to be tested;

[0023] S2. Using the sample extracted in step S1 as a template, PCR detection was performed using primers with InDel markers for detecting purple fruit typing in chili peppers;

[0024] S3. Detect the PCR amplification products by agarose gel electrophoresis and analyze the results.

[0025] Furthermore, in step S3, the plants with a single amplified band and a short fragment are pepper plants with purple fruits, the plants with a single amplified band and a long fragment are pepper plants with green fruits, and the plants with two amplified bands are F1 generation hybrids of peppers with purple fruits.

[0026] Preferably, the PCR amplification reaction system consists of 5 μL Green Taq Mix (produced by Nanjing Novizan Biotechnology Co., Ltd.), 0.2 μL each of forward and reverse primers, 1 μL DNA template (DNA concentration of approximately 50 ng / μL), and finally ddH2O added to a final volume of 10 μL.

[0027] Preferably, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 30 s; repeat for 35 cycles; 72℃ final extension for 2 min.

[0028] The present invention has the following beneficial effects:

[0029] This invention, through the construction of a genetic map of purple pepper fruits and the development of InDel molecular marker primers, has yielded an InDel marker capable of identifying purple pepper fruits or genotyping purple pepper fruit plants and their offspring. Using this marker and its detection primers, purple pepper fruits and their F1 hybrid plants can be accurately and rapidly distinguished and identified, improving the accuracy and efficiency of marker-assisted breeding for purple green-ripening pepper varieties. Simultaneously, this invention provides a method for identifying purple pepper fruits or genotyping purple pepper fruit plants and their offspring, enabling more accurate genotyping of parent and offspring, and identifying the purple trait in F1, F2, and both parents. The method is simple, feasible, and easy to operate, solving the problems of existing technologies that can only identify the purple trait and are inaccurate in genotyping of the F1 generation of purple pepper fruits. This shortens the screening cycle for purple pepper germplasm resources and provides technical support for the identification of purple pepper fruits and their offspring. Attached Figure Description

[0030] Figure 1 Genetic diagram of pericarp color in the 59×Z81 hybrid combination.

[0031] Figure 2 The distribution of G-values ​​across chromosome sets is shown in Figure A (Δ(SNP-index) distribution); Figure B (Gprime distribution on chromosomes) is shown in Figure B.

[0032] Figure 3 Genetic linkage map of pepper pericarp color.

[0033] Figure 4 The expression levels of different candidate genes in the parents are shown in Figure 1 (A represents the expression level of Capann_59V1aChr10g016200, B represents the expression level of Capann_59V1aChr10g016210C, C represents the expression level of Capann_59V1aChr10g016220, and D represents the expression level of Capann_59V1aChr10g016230).

[0034] Figure 5 This is a sequence analysis diagram of key candidate genes.

[0035] Figure 6 The results show the banding patterns of InDel 67 in both parents and F1 (A) and the banding distribution in some plants in the F2 generation (B) (M in the figure is DNA Mark). Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] The chili peppers used in the following examples were all grown at South China Agricultural University.

[0039] Example 1: Construction of a genetic map

[0040] 1. Field trait statistics

[0041] The pepper materials used in this embodiment, No. 59 and No. Z81, are both high-generation inbred lines of more than 8 generations. No. 59 pepper has a green exocarp, and No. Z81 pepper has a purple exocarp. No. 59 pepper is used as the female parent (P1) and No. Z81 pepper is used as the male parent (P2). F1 generation plants were obtained by bagging and hybridization. F1 generation plants were then bagged and self-crossed to obtain F2 generation segregating population. When the F1 and F2 generation segregating population plants reached the green-mature stage, the field traits of all plants were counted.

[0042] The results are as follows Figure 1 As shown, the F1 generation of peppers obtained by crossing the green-skinned "59" (female parent) and the purple-skinned "Z81" (male parent) with bagged peppers all exhibited purple exocarps, indicating that purple exocarp color is dominant over green. In the F2 segregating population (466 plants) obtained by self-pollination of the bagged F1 plants, 363 plants had purple exocarps and 103 plants had green exocarps. Chi-square test revealed that the segregation ratio generally followed a purple:green ratio of 3:1, with a p-value of 0.28 > 0.05, consistent with the inheritance pattern controlled by a single gene.

[0043] 2. Genome resequencing

[0044] Then, genome resequencing was performed on two parental pools, namely pepper No. 59 (already sequenced) and pepper No. Z81, as well as on the two extreme F2 generation exocarps with "green" and "purple" exocarps. The sequencing depth of the parents was 20x, and the sequencing depth of the extreme F2 generation pools was 50x (approximately 10% of the F2 generation population). Based on the MGI-seq2000 high-throughput sequencing platform (manufactured by Shenzhen BGI Genomics Co., Ltd.), the results are shown in Table 1 below. A total of 371.85 Gb of data was generated, with an average Q20 of 96.9% and an average Q30 of 90.1%. The GC content was normal, indicating that the amount of data obtained through sequencing was sufficient and the data quality was high, which can be used for subsequent experimental analysis.

[0045] Table 1 Summary of Genome Resequencing Data

[0046]

[0047] 3. Initial localization of capsicum anthocyanins using BSA-seq

[0048] Using the “CA59” pepper genome from our research group as the reference genome, and employing genome retesting data from two extreme mixed pools of fruit color from the middle parents and F2 generation, we detected single nucleotide polymorphisms (SNPs) and short insertion / deletion (InDel) variations. We calculated SNP-index and Gprime values ​​using high-quality SNPs from the “purple” and “green” mixed pools of pepper exocarps. Then, we calculated the difference to obtain the Δ(SNP-index) value, and based on the results, we plotted the Δ(SNP-index) distribution map and the Gprime distribution map on the chromosome.

[0049] Based on Δ(SNP-index), using a 95% confidence level as the screening threshold, distinct peaks were found on chromosomes 9 and 10. The peak on chromosome 9 was located at 19.1 Mb-267.3 Mb, with a length of 248.2 Mb; the peak on chromosome 10 was located at 7585 bp-238330779 bp, with a length of 238.23 Mb. Figure 2 As shown in Figure A, these two peaks represent candidate regions for genes controlling anthocyanin synthesis in the pericarp of peppers in this population.

[0050] 4. Construct genetic linkage maps of the localization regions.

[0051] Preliminary localization results based on the color of the pepper pericarp indicate that the candidate linkage regions are located at positions 19.1 Mb-267.3 Mb on chromosome 9 (248.2 Mb) and 7585 bp-238330779 bp on chromosome 10 (238.23 Mb). Figure 2 As shown in B. To further narrow down the candidate regions, DNA from the parents P1, P2, F1 generation, and 466 F2 generation plants was used as templates for subsequent PCR amplification. Based on the genotyping of polymorphic InDels in the F2 generation population, linkage genetic maps were constructed using QTL IicMapping software.

[0052] Eight polymorphic markers with roughly equal coverage of candidate regions on chromosome 9 were used. The results showed that no region on chromosome 9 was linked to pepper pericarp color, indicating that chromosome 9 is not linked to pepper pericarp color. Twenty-nine polymorphic markers were screened from candidate regions on chromosome 10 for constructing a linkage genetic map. The results showed that the linkage region was located between InDel 67 and InDel 75, with a physical location of 185664068 bp-186514350 bp, a genetic distance of 0.96 cM, a LOD value of 52.886, and a contribution rate of 50.61%. Figure 3 As shown, the subsequent experimental analysis was conducted using the linkage interval InDel 67-InDel 75.

[0053] Example 2: Analysis of candidate genes within the genetic candidate region for anthocyanins in pepper pericarps

[0054] 1. Sequence analysis and functional annotation of candidate genes

[0055] Comparing the candidate gene positions within the candidate regions screened in Example 1 with the variant detection results of genome resequencing in Example 1, it was found that Capann_59V1aChr10g016200 has 7 SNPs, Capann_59V1aChr10g016230 has 1 InDel, and the DNA sequences of Capann_59V1aChr10g016210 and Capann_59V1aChr10g016220 are completely identical in peppers No. 59 and No. Z81. Therefore, it is inferred that the difference in the color of the pepper pericarp is caused by the Capann_59V1aChr10g016200 or Capann_59V1aChr10g016230 gene.

[0056] Subsequently, the CDS sequences of the four candidate genes within the located candidate region were aligned to the NT, NR, and Swissprot databases using Diamond and BLAST software for functional annotation. The results are shown in Table 2, indicating that three of the four genes within the candidate region were annotated. A search for genes related to anthocyanin synthesis in pepper revealed a known transcription factor, MYB1 (Capann_59V1aChr10g016200), within the located region. This may be a key candidate gene for the anthocyanin trait in the exocarp of peppers in this population.

[0057] Table 2. Gene function annotations within candidate regions for capsicum anthocyanins.

[0058]

[0059] 2. Expression analysis of candidate genes

[0060] To identify the strongest candidate gene controlling anthocyanin synthesis in the pericarp of pepper, CDS sequences of all candidate genes within the candidate region were extracted, and specific primers were set for each gene. The ubiquitin extension protein gene CA12g20490 of pepper was used as an internal reference gene, and quantitative real-time PCR was performed using ChamQ Universal SYBR qPCR Master Mix from Nanjing Novizan Biotechnology Co., Ltd.

[0061] Quantitative real-time PCR experiments revealed that only MYB1 (Capann_59V1aChr10g016200) showed a significant difference in expression levels between the two parents, as shown in the results below. Figure 4As shown in A, the relative expression levels of the other three genes did not differ significantly between the parents. Figure 4 (BD), which further suggests that MYB1 is a key candidate gene controlling the anthocyanin trait in the pericarp of peppers in this population.

[0062] 3. Sequence analysis and cloning of the key gene MYB1

[0063] The MYB1 gene is considered a key gene controlling anthocyanin synthesis in this population. Located on chromosome 10 at positions 185765157bp-185766243bp, it contains three exons and has a coding length of 701bp. Parental genome resequencing revealed seven SNPs in the full-length MYB1 gene, four of which are located in the coding region and three in the non-coding region. Figure 5 As shown.

[0064] To further verify the authenticity of the SNPs in the MYB1 gene sequence, genomic DNA from peppers No. 59 and Z81 was used as templates for PCR amplification. The PCR products were then purified and recovered, and sequenced by Sangon Biotech (Shanghai) Co., Ltd., followed by sequence alignment using DNAMAN software. The comparison revealed that, compared to pepper No. 59, pepper No. Z81 indeed contained seven SNPs at positions 128bp, 256bp, 371bp, 374bp, 578bp, 991bp, and 1017bp, resulting in a two-amino acid change during subsequent translation. This indicates that the gene does indeed exist and differs between green and purple peppers.

[0065] Example 3: Development and validation of molecular markers related to anthocyanin inheritance in pepper pericarp

[0066] Screening experiments on InDel markers in the MYB1 gene sequence revealed that the molecular marker InDel 67 can effectively distinguish between purple and green pepper fruits. InDel 67 is located at 185664068bp on chromosome 10 of pepper, with a 49bp deletion. It is speculated that this molecular marker is linked to the MYB1 gene that controls anthocyanin synthesis in pepper.

[0067] To further validate the InDel 67 molecular marker, primers for this marker were designed and their sequences synthesized, as shown in Table 3 (primer synthesis was commissioned to Beijing Qingke Biotechnology Co., Ltd.). PCR detection and analysis were performed on P1, P2, F1 generation, and 466 F2 generation plants. The PCR reaction system consisted of 5 μL Green Taq Mix (produced by Nanjing Novizan Biotechnology Co., Ltd.), 0.2 μL each of forward and reverse primers, 1 μL DNA template (DNA concentration approximately 50 ng / μL), and finally ddH2O added to a final volume of 10 μL. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 30 s; repeated for 35 cycles; final extension at 72℃ for 2 min. The amplified products were detected by gel electrophoresis on a 3% agarose gel in 180V electrophoresis buffer. The electrophoretic bands were observed using a gel imaging system, and the detection results were analyzed.

[0068] Table 3 Primer information for InDel 67 markers

[0069]

[0070] Testing revealed that InDel 67 exhibits polymorphism in both parents and F1 cells, such as... Figure 6 As shown in Figure A, plants with a single amplified band and a short fragment are pepper plants with purple fruits, plants with a single amplified band and a long fragment are pepper plants with green fruits, and plants with two amplified bands are F1 generation hybrids of peppers with purple fruits. These can be used for genotyping detection of F1 generation hybrids and subsequent F2 generation populations.

[0071] Further genotyping analysis of the 462 F2 plants revealed that 105 F2 plants were genotyped the same as P1 (59), 126 plants were genotyped the same as P2 (Z81), and 227 plants were genotyped the same as F1. Some results are shown below. Figure 6 As shown in B. Combined with field phenotypic findings, among the 466 F2 generation plants, 462 had a known phenotype, and 427 plants were phenotypically identical, resulting in a concordance rate of 92.4%. Therefore, the InDel 67 molecular marker can be used to identify purple fruits in peppers and for genotyping between different pepper parents and F1 and F2 generations.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of an InDel-labeled primer for detecting purple fruit morphology in peppers for identifying plants with purple fruit or in identifying F1 generation hybrids of purple fruit peppers, characterized in that, The primers consist of an upstream primer: 5'-TGGGAGGTGTGTGTATATAATGG-3'; and a downstream primer: 5'-GCCTCACCTTATCATATACCCT-3'.

2. A kit for identifying purple-fruited peppers, used in identifying plants with purple-fruited peppers or in identifying F1 generation hybrids of purple-fruited peppers, characterized in that... The kit contains primers for detecting InDel markers used in the genotyping of purple pepper fruits, including an upstream primer: 5'-TGGGAGGTGTGTGTATATAATGG-3'; and a downstream primer: 5'-GCCTCACCTTATCATATACCCT-3'.

3. The application of an InDel-marked primer for detecting purple fruit morphology in pepper breeding, characterized in that... The primers consist of an upstream primer: 5'-TGGGAGGTGTGTGTATATAATGG-3'; and a downstream primer: 5'-GCCTCACCTTATCATATACCCT-3'.

4. The application of an InDel-labeled primer for detecting the genotype of purple pepper fruit in the preparation of a kit for identifying purple pepper fruit, or for preparing F1 generation hybrids of purple pepper fruit, or for preparing genotypes of purple pepper fruit plants and their progeny, characterized in that, The primers consist of an upstream primer: 5'-TGGGAGGTGTGTGTATATAATGG-3'; and a downstream primer: 5'-GCCTCACCTTATCATATACCCT-3'.

5. The application according to claim 4, characterized in that, The kit also includes reagents for extracting DNA from samples.

6. A method for identifying plants with purple-fruited chili peppers, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA from the sample in step S1 as a template, PCR detection was performed using InDel-labeled primers for detecting purple fruit morphology in chili peppers; The primers comprise an upstream primer: 5'-TGGGAGGTGTGTGTATATAATGG-3'; and a downstream primer: 5'-GCCTCACCTTATCATATACCCT-3'. S3. Perform agarose gel electrophoresis on the PCR amplification products and analyze the results. In the results, the plants with purple fruit peppers are those with single amplification bands and short fragments, the plants with green fruit peppers are those with single amplification bands and long fragments, and the plants with double amplification bands are F1 generation hybrid plants with purple fruit peppers.

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