SNP sites, CAPS molecular markers and applications associated with the chili pepper CMS recovery gene

Abstract

This invention relates to the field of chili pepper molecular markers, disclosing SNP sites, CAPS molecular markers, and applications related to chili pepper CMS restorer genes. The applicant constructed an F2 segregating population using the chili pepper CMS restorer line 'CA1403' and the sterile line '1310A'. Addressing the issue that some chili pepper CMS restorer genes could not be mapped to second-generation reference genomes, the applicant utilized a newly released high-quality third-generation reference genome combined with BSA-seq and chromosome walking to map the restorer genes... Rfa The gene was precisely mapped to a 377 kb physical region on chromosome 6 of the CA59 reference genome. Simultaneously, a co-segregating molecular marker was developed based on sequence differences between the candidate region and the restorer line. Using this marker, the presence or absence of the CMS restorer gene can be accurately identified at the seedling stage, clarifying the restorer-protection relationship between different materials and the sterile line, significantly improving breeding efficiency and reducing seed production costs.

Description

Technical Field

[0001] This invention relates to the field of chili molecular markers, specifically to SNP sites, CAPS molecular markers, and applications related to the chili CMS recovery gene. Background Technology

[0002] Hybrid pepper varieties are widely used in pepper production, significantly improving yield, resistance, and quality. However, the production of hybrid peppers still relies heavily on manual emasculation, a technically demanding and labor-intensive process with high labor costs, and the purity of seeds is difficult to guarantee. Utilizing male-sterile lines can effectively solve the problem of manual emasculation, simplifying the seed production process, reducing production costs, and facilitating the commercial and efficient production of hybrid peppers. Cytoplasmic male sterility (CMS) is one of the main pathways for utilizing heterosis in higher plants, possessing unique advantages in hybrid seed production due to its maternal inheritance characteristics. Therefore, conducting research on CMS fertility restoration genes in peppers, clarifying the distribution of restoration genes in natural populations, discovering CMS fertility restoration regulatory genes, and developing practical markers are of great significance for the three-line system of pepper seed production.

[0003] The CMS sterile line '1310A' involved in this invention is a naturally occurring mutant material discovered in pepper breeding materials. It exhibits complete and stable sterility. Through restorer-protector relationship testing, it was identified as the matching maintainer line '1310B' and restorer line 'CA1403', successfully achieving three-line seed production for pepper. Due to limitations in second-generation reference genome assembly, currently finely mapped candidate CMS restorer genes in pepper, such as CaPPR6 (BAC-Contig 85), CaRf032 (CA00g82510), and Rfu (CA00g30080), cannot be mapped to the second-generation reference genome Zunla-1 or CM334 chromosomes. Therefore, to better identify the restorer-protector relationship between different breeding materials and sterile lines, this invention utilizes newly released high-quality third-generation genome fine mapping of CMS restorer genes and develops efficient and convenient molecular markers. This allows for accurate identification of the presence or absence of CMS restorer genes at the seedling stage, saving significant planting space, manpower, and material resources, significantly improving breeding efficiency, and reducing seed production costs. Summary of the Invention

[0004] The purpose of this invention is to provide a reagent for detecting SNP sites associated with the CMS restorer gene in the breeding of CMS restorer lines of pepper. The SNP site is located at position 241,817,582 on the pepper P06 chromosome. The pepper reference is the pepper CA59 reference genome, Ver 2024-02-21, https: / / db.cngb.org / search / project / CNP0001129 / .

[0005] Another objective of this invention is to provide CAPS molecular marker primers for detecting SNP sites associated with the CMS recovery gene in chili peppers, wherein the primers are F: 5'-CGAAGTTGATTGAAAATGAA-3', R: 5'-CTTAGTTGCTTGGACTCTACG-3'.

[0006] To achieve the above objectives, the present invention adopts the following technical measures:

[0007] This invention constructs an F2 segregating population from the chili pepper CMS restorer line 'CA1403' (Li Ning, Wang Fei, Yin Yanxu, Yao Minghua, Jiao Chunhai, Zou Xiong, Zhao Rongqiu, Gao Shenghua. Analysis of genetic diversity of chili pepper germplasm [J]. Chili Journal, 2017, 15(04): 1-8+13) and the sterile line '1310A' (Li Ning, Wang Fei, Yin Yanxu, Yao Minghua, Jiao Chunhai, Zou Xiong, Zhao Rongqiu, Gao Shenghua. Analysis of genetic diversity of chili pepper germplasm [J]. Chili Journal, 2017, 15(04): 1-8+13). Addressing the issue that some chili pepper CMS restorer genes cannot be mapped to the second-generation reference genome, this invention utilizes the newly released high-quality third-generation reference genome combined with BSA-seq and chromosome walking to locate candidate regions of chili pepper CMS restorer genes on chili pepper chromosome P06 (chili pepper CA59 reference genome, Ver...). On February 21, 2024, the CNP0001129 gene was located within the interval of 241.57 to 241.94 Mb (https: / / db.cngb.org / search / project / CNP0001129 / ). Based on the gene annotation information of the reference genome CA59, markers were further developed and exchanged individual plants were searched within the located interval. Combined with comparative sequencing and expression pattern analysis, candidate genes for the CMS restorer gene in pepper were identified. Finally, a co-segregating SNP site with the restorer gene was identified at position 241,817,582 on the pepper P06 chromosome. When the base is A, the tested pepper material contains the restorer gene; when the base is G, the material does not contain the restorer gene.

[0008] Furthermore, the applicant designed a CAPS molecular marker for the aforementioned SNP site. The detection primers for this molecular marker are: F: 5'-CGAAGTTGATTGAAAATGAA-3', R: 5'-CTTAGTTGCTTGGACTCTACG-3'.

[0009] The scope of protection of this invention includes: the application of reagents for detecting SNP sites associated with the CMS recovery gene in pepper in pepper breeding, wherein the SNP sites are located at positions 241,817,582 on the pepper P06 chromosome, referencing the pepper CA59 reference genome, Ver 2024-2-21, https: / / db.cngb.org / search / project / CNP0001129 / .

[0010] The breeding described above refers to the breeding of the CMS restorer line for chili peppers.

[0011] In the above-described applications, preferably, the reagent is a primer. Primers commonly used in existing SNP detection methods in the art can all be used to complete this invention. The methods described include, but are not limited to, TaqMan probe method, ARMS-PCR method, high-resolution melting curve method, CAPS method, SNaPshot method, or KASP method.

[0012] In the above-described applications, the preferred primers are: F: 5'-CGAAGTTGATTGAAAATGAA-3', R: 5'-CTTAGTTGCTTGGACTCTACG-3';

[0013] In the above-described applications, the preferred method for determining whether the chili pepper CMS recovery gene is present using the aforementioned molecular markers is as follows: The genomic DNA of the chili pepper to be tested is amplified using the aforementioned primers, and the PCR amplification primers are digested with TaqI enzyme.

[0014] If the enzyme digestion products are 228bp and 234bp fragments, then the pepper material to be tested does not contain the restorer gene;

[0015] If the enzyme digestion product is only a 462bp fragment, then the pepper material to be tested contains the restorer gene;

[0016] If the enzyme digestion products are 228bp, 234bp, and 462bp fragments, then the pepper material being tested contains the restorer gene.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This invention addresses the current situation where some pepper CMS restorer genes cannot be mapped to second-generation reference genomes by constructing an F2 segregating population. Utilizing a newly released high-quality third-generation reference genome combined with BSA-seq and chromosome walking, candidate genes regulating the restoration of fertility in pepper cytoplasmic male sterility were precisely located and identified. Based on the sequence SNP differences between sterile and restorer lines within candidate regions, the molecular marker CaCMS-CAPS170, which completely co-segregates with the pepper CMS restorer gene, was developed. This CAPS molecular marker is a co-dominant marker, accurately distinguishing between homozygous and heterozygous genotypes of the restorer gene. Furthermore, it completely co-segregated with the fertility phenotype in a segregating population of 3120 F2 plants with and without the pepper CMS restorer gene, achieving a detection accuracy of 100%.

[0019] The molecular markers developed in this invention can provide effective technical support for assisted selection breeding based on the presence or absence of the CMS restorer gene in chili peppers and for subsequent three-line seed production. Molecular markers can be used to screen chili peppers during the seedling stage, greatly reducing the workload of field identification and accelerating the breeding process. Attached Figure Description

[0020] Figure 1 The results of observations on the fertility phenotypes of restorer lines, sterile lines, and their F1 generations;

[0021] Wherein: P1 is a plant of the male-sterile line material '1310A' of pepper, P2 is a plant of the restorer line material 'CA1403' of pepper, and F1 is an F1 generation plant constructed with '1310A' as the female parent and 'CA1403' as the male parent. The upper figure shows the observation of the open flowers and flower buds of the corresponding plants, and the lower figure shows the acetocarmine staining results of the pollen of the corresponding plants.

[0022] Figure 2 To locate the Rfa gene for CMS recovery in chili peppers;

[0023] (A) Results of whole-genome resequencing analysis of the recovery gene Rfa; (B) Fine mapping of the recovery gene Rfa.

[0024] Figure 3 To mark the genotyping results of CaCMS-CAPS170 on some individual plants in the parental, F1 and F2 populations;

[0025] Among them: lane M is the marker, and the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp respectively; lanes 1-4 and 6-7 are fertile single plants of the F2 population; lanes 5, 8 and 9 are sterile single plants of the F2 population; lanes 10-12 are single plants of the F1 generation, 'CA1403' and '1310A' respectively. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] Example 1:

[0029] The development and application of CAPS molecular markers linked to the CMS recovery gene in pepper include the following steps:

[0030] 1. Obtaining CMS sterile and restorer lines for chili peppers

[0031] The CMS sterile line '1310A' is a naturally occurring mutant material discovered in pepper breeding materials. Through restorer-conservator relationship testing, the matching maintainer line '1310B' and restorer line 'CA1403' were identified, successfully achieving three-line breeding for peppers. The anthers of '1301A' are degenerate, significantly shorter than the stigma. In contrast, the anthers and stigmas of 'CA1403' and the F1 generation are more closely related. Pollen staining results showed that the mature anthers of 'CA1403' and F1 contained a large number of pollen grains, which were round, large, and deeply stained. The mature anthers of '1301A' contained no stained pollen grains. Figure 1 To improve the efficiency of identifying restorer relationships in field breeding materials, linkage molecular markers were developed for this CMS restorer gene.

[0032] 2. Construction, localization, and candidate gene analysis of chili pepper CMS recovery gene segregating populations.

[0033] This invention constructs an F2 segregating population from the chili pepper CMS restorer line 'CA1403' (Li Ning, Wang Fei, Yin Yanxu, Yao Minghua, Jiao Chunhai, Zou Xiong, Zhao Rongqiu, Gao Shenghua. Analysis of genetic diversity of chili pepper germplasm [J]. Chili Journal, 2017, 15(04): 1-8+13) and the sterile line '1310A' (Li Ning, Wang Fei, Yin Yanxu, Yao Minghua, Jiao Chunhai, Zou Xiong, Zhao Rongqiu, Gao Shenghua. Analysis of genetic diversity of chili pepper germplasm [J]. Chili Journal, 2017, 15(04): 1-8+13). Addressing the current situation where some chili pepper CMS restorer genes cannot be mapped to the second-generation reference genome, this invention utilizes the newly released high-quality third-generation reference genome combined with BSA-seq and chromosome walking to locate candidate regions of chili pepper CMS restorer genes on chili pepper chromosome P06 (chili pepper CA59 reference genome, Ver). February 21, 2024, https: / / db.cngb.org / search / project / CNP0001129 / ) within the physical location range of 241.57 to 241.94 Mb ( Figure 2 Based on the gene annotation information of the reference genome CA59, markers were further developed and exchanged single plants were searched within the localization region. Combined with comparative sequencing and expression pattern analysis, candidate genes for the chili pepper CMS recovery gene were identified.

[0034] 3. Development of CAPS markers co-segregating with the pepper CMS recovery gene

[0035] Based on BSA-seq results, CAPS markers were designed for differentially expressed SNP sites in candidate regions. A SNP site closely related to the recovery gene was finally identified at positions 241,817,582 on the pepper P06 chromosome. When the base is A, the tested pepper material contains the recovery gene (the sequence is shown in SEQ ID NO.1); when the base is G, the material does not contain the recovery gene (the sequence is shown in SEQ ID NO.2). Therefore, based on the sequence difference of this SNP site, the molecular marker CaCMS-CAPS170, which completely co-segregates with the pepper CMS recovery gene, was developed. The sequence of the SNP site closely related to the pepper CMS recovery gene can be recognized by the restriction enzyme TaqI. This CAPS molecular marker is a co-dominant marker, which can accurately distinguish between homozygous and heterozygous genotypes of the recovery gene. Furthermore, it completely co-segregated with the phenotype in a segregating population of 3120 F2 plants with and without the pepper CMS recovery gene, achieving 100% accuracy in marker identification.

[0036]

[0037] The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s (35 cycles in total), 72℃ final extension for 5 min, and storage at 16℃ for 5 min.

[0038] The CAPS digestion system consisted of 10 μL of total volume, 5.0 μL of PCR product, 1.0 μL of 2×TaqI buffer, 0.2 μL of TaqI, and 3.8 μL of ddH2O; digestion was performed at 65℃ for 2 h.

[0039] The enzyme digestion products were detected by 2% agarose gel electrophoresis. It was observed that the CMS male-sterile pepper material '1310A' showed detectable fragments of 228 bp and 234 bp, while the CMS restorer pepper material 'CA1403' could not be digested by the restriction enzyme, and only a 462 bp fragment was detected. The F1 generation plants showed all three bands simultaneously: 228 bp, 234 bp, and 462 bp. (Details are shown below.) Figure 3 As shown (228bp and 234bp overlap in the figure). The CAPS marker CaCMS-CAPS170 can accurately identify the presence of the restorer gene and the homozygous status of the gene.

[0040] Example 2:

[0041] Application of CAPS markers associated with the CMS restorer gene in pepper breeding:

[0042] The CAPS molecular marker CaCMS-CAPS170 from Example 1 was used to amplify the sterile line '1310A' and the restorer line 'CA1403', their F1 generation, and 3120 F2 plants to obtain PCR amplification products. The PCR amplification system consisted of 11 μL of total volume, 1.0 μL of DNA at a concentration of 20-100 ng / μL, 5.0 μL of 2×Taq Master Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer, and 4.0 μL of ddH2O.

[0043] The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s (35 cycles in total), 72℃ final extension for 5 min, and storage at 16℃ for 5 min.

[0044] The CAPS digestion system consisted of 10 μL of total volume, 5.0 μL of PCR product, 1.0 μL of 2×TaqI buffer, 0.2 μL of TaqI, and 3.8 μL of ddH2O; digestion was performed at 65℃ for 2 h.

[0045] The enzyme digestion products were subjected to gel electrophoresis. Due to space limitations, the detection results for some materials are as follows: Figure 3 As shown: Lane M is the marker; lanes 1-4 and 6-7 are fertile F2 plants; lanes 5, 8, and 9 are sterile F2 plants; lanes 10-12 are F1 plants, 'CA1403', and '1310A' plants, respectively. Figure 3 It can be seen that CaCMS-CAPS170 can effectively distinguish whether the CMS restorer gene is present in chili pepper materials. Specifically, chili pepper materials containing the CMS restorer gene can be detected with a 462bp fragment, or 228bp, 234bp and 462bp fragments, while chili pepper materials without the CMS restorer gene can be detected with 228bp and 234bp fragments.

[0046] The presence or absence of the CMS fertility restoration gene in the F2 segregating population of 3120 pepper plants was verified using the CaCMS-CAPS170 marker. The marker completely co-segregated with the CMS fertility restoration trait in pepper, with a detection accuracy of 100%.

[0047] 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 primers for detecting SNP sites associated with the CMS restorer gene in pepper in the breeding of pepper CMS restorer lines, wherein the SNP site is located at position 501 of the sequence shown in SEQ ID NO.1, and the primers are: F: 5'-CGAAGTTGATTGAAAATGAA-3', R: 5'-CTTAGTTGCTTGGACTCTACG-3'; the application process includes: Genomic DNA of the pepper to be tested was amplified, and the PCR amplification primers were digested with TaqI enzyme: If the enzyme digestion products are 228bp and 234bp fragments, then the pepper material to be tested does not contain the restorer gene; If the enzyme digestion product contains a 462bp fragment, then the pepper material being tested contains the restorer gene.

Images