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CN-122012803-A - Pepper CMS fertility restoration trait related Indel marker, amplification primer and application thereof

CN122012803ACN 122012803 ACN122012803 ACN 122012803ACN-122012803-A

Abstract

The invention relates to a capsicum CMS fertility restoration trait related Indel marker, an amplification primer and application thereof. Specifically, the Indel marker provided by the invention is closely linked with the capsicum CMS restorer gene, has stable positioning effect, is simultaneously suitable for laboratory conditions and fertility detection of capsicum in any growth period, lays a foundation for cloning of the capsicum CMS restorer gene and research of capsicum pollen development molecular mechanism, and has higher application value in utilization of capsicum heterosis and cultivation of new varieties.

Inventors

  • SHI FENGYAN
  • ZOU CHUNLEI
  • LI MENG
  • ZHANG XI
  • WANG XIUXUE

Assignees

  • 辽宁省农业科学院

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. The Indel marker related to the fertility restoration trait of the capsicum CMS is characterized in that an insertion site of the Indel marker is at 213979230bp position of a genome 6 No. 6 chromosome of capsicum cv.Zun la-1v2.0, and the Indel marker is a specific insertion fragment with the length of 88bp and a nucleotide sequence shown as SEQ ID No. 323.
  2. 2. The pepper CMS fertility restoration trait related Indel marker of claim 1, wherein said Indel marker is used for molecular characterization of the pepper CMS fertility restoration trait.
  3. 3. A primer for amplifying an Indel marker associated with a fertility restoration trait of capsicum CMS according to claim 1 or 2, comprising: Indel 160-F:5'-CCTGAGACGCTAGAAGGTAAAA-3'(SEQ ID NO:160); Indel 160-R:5'-TTGAATACTCAGGCAGCACC-3'(SEQ ID NO:321)。
  4. 4. A method for molecular identification using the Indel marker of claim 1 or 2, characterized in that the identification step comprises: s1) designing a specific primer for the Indel label according to claim 1 or 2; S2) respectively carrying out PCR reactions by taking sterile line and restorer line peppers as materials; s3) identifying and analyzing the result, wherein the length of the amplified fragment is 790bp, the amplified fragment is 702bp, the amplified fragment is homozygous sterile, and the amplified fragment is 702bp and 790bp double-band, the amplified fragment is heterozygous fertile.
  5. 5. The method for molecular characterization according to claim 4, wherein the specific primer is as defined in claim 3.
  6. 6. The Indel label according to claim 1 or 2, the primer for amplification according to claim 3, or the use of the molecular characterization method according to claim 4 or 5, characterized in that the use comprises at least one of the following: 1) Identifying fertility of the pepper pollen; 2) Crossbreeding or molecular genetic breeding of capsicum; 3) Analyzing genetic diversity of the peppers; 4) Constructing a pepper fertility genetic linkage map; 5) Identifying a pepper cytoplasmic male sterility restoring gene; 6) Identification of cytoplasmic male sterility of capsicum, and/or, 7) And (5) breeding a pepper cytoplasmic male sterile restorer line.
  7. 7. A product for identifying the fertility restoration trait of capsicum CMS, comprising the Indel-labeled amplification primer of claim 1 or 2.
  8. 8. A product for identifying a pepper CMS fertility restoration trait as claimed in claim 7, wherein said product is a kit.
  9. 9. The product for identifying fertility restoration traits of pepper CMS as claimed in claim 8, wherein said product comprises a detection reagent, a kit, a genome chip or a liquid phase probe, said detection reagent being the amplification primer of claim 3.
  10. 10. A product for identifying pepper CMS fertility restoration traits as defined in any one of claims 7-9, wherein said product further comprises PCR reaction reagents.

Description

Pepper CMS fertility restoration trait related Indel marker, amplification primer and application thereof Technical Field The invention relates to the technical fields of molecular biology and plant molecular marker breeding, in particular to a capsicum CMS fertility restoration trait related Indel marker, and an amplification primer and application thereof. Background Capsicum annuum L is an important vegetable and flavoring crop of Solanaceae, and along with the increase of the demand for high-quality, high-yield and stress-resistant hybrid seeds, the utilization of heterosis has become an important way for breeding high-quality pepper varieties. Cytoplasmic male sterility (Cytoplasmic MALE STERILITY, CMS) is a high-efficiency breeding technology system widely applied to pepper hybrid seed production. The CMS can effectively avoid artificial emasculation, reduce seed production cost and improve seed production efficiency by the pollen abortion caused by the interaction of mitochondrial genes and nuclear genes. However, the application of the CMS system is limited by factors such as the great difficulty of selection and breeding of the restorer, the complex genetic mechanism of the restorer gene, the lack of molecular markers and the like. Capsicum is a typical "three-line" seed crop (CMS sterile line, maintainer line, restorer line). The restorer line is the only male parent capable of providing functional pollen to the hybrid (F1). Traditional breeding relies on phenotype measurement and allocation, and has long period and large environmental interference. After the restoring gene is cloned, co-dominant and high-flux functional markers can be developed, so that accurate screening in the seedling stage is realized, the breeding period is shortened by more than 50%, and the creation efficiency of an excellent restoring line is remarkably improved. By means of Rf gene molecular marker, the high yield, disease resistance, weak light resistance and other characters can be polymerized fast, and the novel restorer with high outcrossing rate and high grain weight can be cultivated, so that the F1 seed production cost is reduced by 30-40%. At least 5 different CMS sources have been reported from capsicum, whose fertility restoration is controlled by different Rf sites, cloning and verifying the interaction network of each Rf can provide greater genetic diversity for hybrids. Restoring CMS fertility is critical in hybrid production, and Rf genes typically affect protein expression of CMS-related genes, promoting fertility restoration. In recent 10 years, with the subsequent publication of plant reference genomes, the study of CMS restorer genes has become one of the hot spots. However, because the male sterile molecular mechanism of the capsicum is complex, the marking, positioning and cloning of the gene related to pollen fertility become the core link of molecular marker assisted selection of dominant varieties. Molecular marker assisted breeding is to screen the target region and even the whole genome of the individual by directly utilizing the molecular marker closely linked or co-separated with the target character gene, so as to improve the selection efficiency of the target character and shorten the breeding period. More than 10 Rf genes have been identified and reported from rice, maize, canola, wheat, soybean and other crops. The Rf gene generally affects transcription of the sterile gene in mitochondria, thereby restoring fertility to the sterile line. In the related studies of capsicum CMS, it was mostly reported that fertility restoration is a dominant trait controlled by 1 gene (Gulyas et al.2006), and researchers found that fertility restoration is a quantitative trait controlled by 1 major QTL and 4 minor QTL (Wang et al 2004), and that the sterile phenotype may also be affected by temperature (Kim et al 2013). With the development of bioinformatics and the completion of whole genome sequencing of capsicum, a plurality of capsicum restorer genes are cloned and reported sequentially, and corresponding molecular markers are developed simultaneously. However, the molecular markers available in these publications have certain limitations in practical application, especially have shortcomings in phenotype and genotype matching among various germplasm, which limits the application range of molecular marker-assisted breeding to a certain extent. Therefore, the development of a new molecular marker closely linked with the recovery of fertility of the capsicum has important significance for promoting molecular marker-assisted selective breeding of the capsicum. Disclosure of Invention In view of the above, the invention provides a capsicum CMS fertility restoration trait related Indel marker, and an amplification primer and application thereof, aiming at solving the technical problems currently faced. In one aspect, the invention provides a capsicum CMS fertility restoration trait related Indel marker. In so