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KR-20260065728-A - NOVEL ONION FERTILITY RESTORING GENE AND MOLECULAR MARKERS FOR DISCRIMINATING GENOTYPING THEREOF

KR20260065728AKR 20260065728 AKR20260065728 AKR 20260065728AKR-20260065728-A

Abstract

The present invention relates to a novel gene for restoring onion fertility and a molecular marker for determining the genotype thereof, and more specifically, to a composition and method capable of determining male sterility or male fertility of onions. By using the composition or kit of the present invention, onion individuals capable of restoring male fertility can be selected and removed early to prevent them from being introduced into onion breeding lines. Furthermore, the primer set of the present invention can rapidly determine male sterility or male fertility of onions with excellent accuracy.

Inventors

  • 김성길
  • 김건중

Assignees

  • 전남대학교산학협력단

Dates

Publication Date
20260511
Application Date
20251023
Priority Date
20241101

Claims (12)

  1. An SNP marker composition for determining male sterility or male fertility in onions, comprising a polynucleotide consisting of 8 to 100 consecutive nucleotides or a polynucleotide complementary thereof, comprising a single nucleotide polymorphism (SNP) site located 5' upstream from the start codon in the AcPPR876 gene or its allele.
  2. A composition for determining male sterility or male fertility of onions, comprising a preparation that detects or amplifies a single nucleotide polymorphism (SNP) site located in the 5' upstream region from the start codon in the AcPPR876 gene or its allele.
  3. A composition for determining male sterility or male fertility of an onion, wherein the AcPPR876 gene or its allele comprises any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO. 72 to SEQ ID NO. 77.
  4. A composition for determining male sterility or male fertility of an onion, wherein the single nucleotide polymorphism site is any one selected from the group consisting of the 2876th nucleotide of the nucleotide sequence of SEQ ID NO. 72, the 2392nd nucleotide of the nucleotide sequence of SEQ ID NO. 73, the 65th nucleotide of the nucleotide sequence of SEQ ID NO. 74, the 30th nucleotide of the nucleotide sequence of SEQ ID NO. 75, the 30th nucleotide of the nucleotide sequence of SEQ ID NO. 76, and the 30th nucleotide of the nucleotide sequence of SEQ ID NO. 77.
  5. A composition for determining male sterility or male fertility of an onion, wherein the formulation is any one selected from the group consisting of a primer set, a probe, and a combination thereof.
  6. A kit for determining male sterility or male fertility of an onion, comprising a composition of any one of claims 2 to 5.
  7. A kit for determining male sterility or male fertility of an onion according to claim 6, further comprising at least one selected from the group consisting of DNA polymerase, dNTPs, and a buffer.
  8. A method for determining male sterility or male fertility of an onion, comprising the step of amplifying a polynucleotide containing a single nucleotide polymorphism (SNP) site located 5' upstream from the start codon in the AcPPR876 gene or its allele using the gDNA (genomic DNA) of the onion as a template.
  9. A method for determining male sterility or male fertility of an onion, wherein the single nucleotide polymorphism site is any one selected from the group consisting of the 2876th nucleotide of the nucleotide sequence of SEQ ID NO. 72, the 2392nd nucleotide of the nucleotide sequence of SEQ ID NO. 73, the 65th nucleotide of the nucleotide sequence of SEQ ID NO. 74, the 30th nucleotide of the nucleotide sequence of SEQ ID NO. 75, the 30th nucleotide of the nucleotide sequence of SEQ ID NO. 76, and the 30th nucleotide of the nucleotide sequence of SEQ ID NO. 77.
  10. A method for determining male sterility or male fertility of an onion according to claim 8, wherein the amplification is performed using a primer set consisting of the nucleotide sequence of SEQ ID NO. 1 and the nucleotide sequence of SEQ ID NO. 2.
  11. A method for determining male sterility or male fertility of an onion, further comprising: a step of determining the base of the single nucleotide polymorphism site by analyzing the product obtained by the amplification in claim 8; and a step of determining the individual as male fertile if the base of the single nucleotide polymorphism site is T.
  12. A primer set for determining male sterility or male fertility of an onion, consisting of the nucleotide sequence of SEQ ID NO. 1 and the nucleotide sequence of SEQ ID NO. 2.

Description

Novel Onion Fertility Restoring Gene and Molecular Markers for Discriminating Genotyping Thereof The present invention relates to a novel onion fertility recovery gene and a molecular marker for determining the genotype thereof, and more specifically, to a composition and method capable of determining male sterility or male fertility of onions. As of 2023, domestic seed sales for onions amounted to 36 billion won, making them one of the most marketable vegetable crops after chili peppers. However, net seed imports also stood at 5.2 billion won, ranking second among all vegetable crops, indicating a high dependence on imports. Furthermore, since almost all onion seeds are imported from Japan, dependence on Japan is the highest. Therefore, investment in research and development for the creation of domestic varieties is urgently required. Currently, most onion varieties distributed are F1 varieties that utilize hybrid vigor. F1 refers to the first generation of offspring produced from different parents. Therefore, euthanasia to prevent maternal self-pollination is essential for the stable production of F1 varieties. Since onion flowers lack self-incompatibility and possess an umbel inflorescence structure, male sterility is utilized as the only method of euthanasia. Male sterility, in which fertilizable pollen is not produced normally, is caused by male sterility-inducing genes located in the cytoplasm and is suppressed by fertility restoration genes located in the nucleus. The Ms locus is known as the fertility restoration locus in onions. The Ms locus stably restores fertility regardless of the onion's cytoplasmic type. Meanwhile, it was recently reported that a new Rf locus, the Ms2 locus, was discovered in onions (Yu and Kim (2021), Identification of Ms2, a novel locus controlling male-fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.), and development of tightly linked molecular markers. Euphytica 217:191). The Ms2 locus has been observed to exhibit unstable fertility restoration (MF) in some onion genetic resources. However, since it is impossible to confirm the presence of a dominant Ms2 locus within onion genetic resources solely through phenotypic analysis, there is a risk that the dominant Ms2 locus, which can cause unstable fertility restoration, may be introduced into breeding lines through crosses during the variety development process. Therefore, it is necessary to select and remove individuals possessing the dominant Ms2 locus at an early molecular level. However, the gene determining the Ms2 locus has not yet been accurately identified. Therefore, it is necessary to identify the verified gene of the Ms2 locus and develop reliable molecular markers for determining the Ms2 genotype. Figure 1 is a diagram illustrating the boundary setting of a genomic region containing the Ms2 locus. (A) A physical map of the Ms2 adjacent region in the NWPU genomic sequence. Filled horizontal bars represent additionally boundaryd genomic regions containing the Ms2 locus determined by recombination analysis. The numbers under the marker names indicate the number of recombinants between the Ms2 locus and the corresponding marker. (B) The genotypes of the molecular markers of 12 recombinants. Green boxes indicate the genotypes of the molecular markers that match the phenotype of the Ms2 locus. 'a': Homozygous dominant; 'h': Heterozygous; 'b': Homozygous recessive; 'd': Homozygous dominant or heterozygous. The 'a' genotype of the recombinants selected from the TUMS4-C9 population may appear due to heterozygosity of the tagged region in a male-sterile maternal line. Figure 2 is a diagram showing the genomic structure of a 3.19 Mb bounded region containing the Ms2 locus. The arrow-shaped box indicates the 5' to 3' direction of the annotated gene. The candidate gene for the Ms2 locus is shown in light blue. Figure 3 shows the inferred amino acid sequence alignment of MF, MS, and NWPU AcPPR876 proteins from candidate genes of the Ms2 locus. The square box below the alignment represents the PPR motif. The red triangle indicates the location of polymorphic amino acid residues between the MF and MS AcPPR876 proteins. Figure 4 is a diagram analyzing the phylogenetic relationships between PPR proteins from onions and known Rf and Rf-like PPR proteins isolated from other plant species. The red and blue curves represent the P and PLS subfamilies, respectively. For non-Rf-like PPR proteins, only one representative protein randomly selected from each clade was included for a highlighted visual representation. Figure 5 is a diagram analyzing the phylogenetic relationships between the PPR protein of green onion and known Rf and Rf-like PPR proteins isolated from other plant species. The red and blue curves represent the P and PLS subfamilies, respectively. For non-Rf-like PPR proteins, only one representative protein randomly selected from each clade was included for a highlighted visual representation. Fi