Search

CN-122012799-A - Molecular marker primer combination for rape wax powder character identification and application thereof

CN122012799ACN 122012799 ACN122012799 ACN 122012799ACN-122012799-A

Abstract

The invention discloses a molecular marker primer combination for rape wax powder sex-state identification and application thereof, and belongs to the technical field of molecular markers, wherein the molecular marker primer combination comprises a primer of a molecular marker A9-ID10 and a primer of a molecular marker C5-ID8, wherein the nucleotide sequence of the primer of the molecular marker A9-ID10 is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the primer of the molecular marker C5-ID8 is shown as SEQ ID NO.3 and SEQ ID NO. 4. The invention solves the problem that no molecular marker is developed for the wax powder character identification of the whole rape plant at present, and the molecular marker can judge the wax powder deficiency of the whole rape plant at early stage.

Inventors

  • GAO JINXIANG
  • ZHANG YUSONG
  • ZHANG LIFAN
  • YUAN XIAOYAN
  • FU MINGLIAN
  • LI JINFENG
  • DONG YUNSONG
  • CHEN WEI
  • LI GENZE
  • ZU FENG
  • TIAN ZHENGSHU
  • CHEN XIAOYAN
  • ZHU XUAN
  • LIU CUICUI
  • LUO YANQING
  • ZHAO KAIQIN
  • ZHANG YUNYUN

Assignees

  • 云南省农业科学院经济作物研究所
  • 大理白族自治州农业科学推广研究院

Dates

Publication Date
20260512
Application Date
20260410

Claims (10)

  1. 1. The molecular marker primer combination for the rape wax powder sex characteristic identification is characterized by comprising a primer of a molecular marker A9-ID10 and a primer of a molecular marker C5-ID8, wherein the nucleotide sequence of the primer of the molecular marker A9-ID10 is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the primer of the molecular marker C5-ID8 is shown as SEQ ID NO.3 and SEQ ID NO. 4.
  2. 2. A kit for molecular marker for wax powder sex identification of rape, which is characterized in that the primer in the kit adopts the molecular marker primer combination as defined in claim 1.
  3. 3. The kit of claim 2, further comprising a pre-mix of Taq enzyme.
  4. 4. Use of a molecular marker primer combination according to claim 1 or a kit according to claim 2 or 3 for identifying canola wax meal traits or molecular breeding.
  5. 5. A method for identifying the traits of canola wax meal, the method comprising: based on the molecular marker primer combination according to claim 1 or the kit according to claim 2 or 3, the DNA of the rape plant to be detected is taken as a template, and the rape wax powder character is analyzed according to the strip by PCR amplification: If the rape plant to be detected shows only one band in the amplification of the molecular markers A9-ID10 and C5-ID8, the band size of the molecular markers A9-ID10 is 205bp, and the band size of the molecular markers C5-ID8 is 273bp, the rape plant to be detected is a wax powder deletion material; If the rape plant to be detected shows two bands in the amplification of the molecular markers A9-ID10 and C5-ID8, the sizes of the bands of the molecular markers A9-ID10 are 241bp and 205bp, and the sizes of the bands of the molecular markers C5-ID8 are 259bp and 273bp, the rape plant to be detected is a homozygous genotype wax powder normal material; If the rape plant to be detected is amplified by the molecular markers A9-ID10 and C5-ID8, the heterozygous genotype wax powder normal material is obtained if the band size of the molecular markers A9-ID10 is 241bp and 205bp and the band size of the molecular markers C5-ID8 is 259bp, or the band size of the molecular markers A9-ID10 is 241bp and the band size of the molecular markers C5-ID8 is 259bp and 273 bp.
  6. 6. The method of claim 5, wherein the PCR amplification system comprises Taq enzyme premix, DNA template, forward and reverse primers, and ultrapure water.
  7. 7. The method according to claim 6, wherein the PCR amplification system comprises 1. Mu.L of Taq enzyme premix 5. Mu. L, DNA template, 0.25. Mu.L of 10. Mu.M forward and reverse primers, and 3.5. Mu.L of ultrapure water.
  8. 8. The method according to any one of claims 5 to 7, wherein the PCR procedure is 94℃pre-denaturation 5min, followed by 9 cycles of 94℃denaturation 30 s, 60℃initial annealing 30 s and 0.5℃decrease each cycle, 72℃extension 30 s, followed by 29 cycles of 94℃denaturation 30 s, 56℃annealing 30 s, 72℃extension 30 s, and final 72℃extension 10min, 25℃incubation 3 min.
  9. 9. The method of claim 5, wherein the material required for DNA extraction of the rape plant to be detected is collected from any part of the plant to be detected.
  10. 10. The method of claim 9, wherein the canola plant to be detected is harvested while in cotyledonary stage.

Description

Molecular marker primer combination for rape wax powder character identification and application thereof Technical Field The invention belongs to the technical field of molecular markers, and particularly relates to a molecular marker primer combination for rape wax powder sex identification and application thereof. Background Rape (Brassica napus l.) is an important worldwide oil crop, its rapeseed oil is a good quality source of edible oil, and the cakes are important protein feeds. Improving the yield and stress resistance of rape has been the core goal of breeding efforts. The plant epidermis wax is a hydrophobic protective layer covering the surface of aerial organs, and is important for the plant to cope with biotic and abiotic stress. The plant epidermis wax is mainly composed of super long chain fatty acid and derivatives thereof (such as alkane, primary alcohol, wax ester, etc.), and plays a key role in reducing water loss, resisting ultraviolet radiation, resisting diseases and insects, etc. In oilseed rape, the wax is often distributed on the surfaces of leaves, stems and fruits in the form of white powder (called "wax powder"), and the content of the wax directly influences drought resistance, disease resistance (such as sclerotinia sclerotiorum and downy mildew) and insect resistance (such as aphid). Therefore, the wax powder content is an important index in rape stress-tolerant breeding. At present, the rape wax powder characteristic identification mainly depends on the traditional method that the field visual inspection method is easily influenced by environment, growth period and artificial factors, has strong subjectivity and low accuracy, and the solvent extraction method has complicated operation, damages samples and has low efficiency, so that the method is difficult to be suitable for rapid and nondestructive screening of large-scale breeding materials. Molecular Marker-assisted selection (Marker-Assisted Selection, MAS) technology provides a powerful tool for crop genetic improvement. The technology can accurately select genotypes in seedling stage and even cotyledon stage by analyzing DNA molecular markers closely linked with target traits, has the outstanding advantages of no environmental influence, high selection accuracy, capability of early selection and the like, can obviously shorten breeding period and improve selection efficiency. At present, although wax anabolic pathways and key genes (such as CER1, CER2, KCS, LACS, WSD1 and the like) of plants in modes such as Arabidopsis thaliana and the like are intensively studied, the formation mechanism of wax powder characters is more complex in the heterotetraploid crop of rape. The development of specific molecular markers closely linked to the wax powder character of rape is still relatively lacking for the research and application of auxiliary breeding. The rape breeding still depends on the low-efficiency traditional method when screening wax powder characters, and restricts the breeding process of new varieties of high stress resistance rape. Although a molecular marker closely related to the waxy character of rape leaves and application thereof are disclosed in Chinese patent publication No. CN116622890A, the marker can only judge the waxy of the leaves, cannot judge the loss of wax powder of the whole plant and is not applicable to early screening. Therefore, the molecular marker which is separated from the rape wax powder in a sex-state or is closely linked is discovered, and a set of rapid, accurate and efficient molecular marker auxiliary selection system is established, so that the method has important practical significance and application value for realizing the technical innovation of the whole-plant stress-resistant breeding of rape and accelerating the breeding and popularization of good varieties. It should be noted that the information disclosed in the above background section is only for enhancing understanding of the background of the present disclosure and thus may include information that does not constitute prior art known to those of ordinary skill in the art. Disclosure of Invention The invention aims to provide a molecular marker primer combination for rape wax powder character identification and application thereof, which solve the problem that a molecular marker is not developed for rape wax powder character identification at present. In order to achieve the aim, the invention provides a molecular marker primer combination for rape wax powder sex characteristic identification, which comprises a primer of a molecular marker A9-ID10 and a primer of a molecular marker C5-ID8, wherein the nucleotide sequences of the primer of the molecular marker A9-ID10 are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the primer of the molecular marker C5-ID8 are shown as SEQ ID NO.3 and SEQ ID NO. 4. The application makes clear that the wax powder deficiency is controlled by two recessive genes and