A molecular marker primer combination for identifying rapeseed wax powder characteristics and its application

Abstract

This invention discloses a molecular marker primer combination and its application for identifying wax powder characteristics in rapeseed, belonging to the field of molecular marker technology. The molecular marker primer combination comprises primers for molecular marker A9-ID10 and primers for molecular marker C5-ID8; wherein the nucleotide sequences of the primers for molecular marker A9-ID10 are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the primers for molecular marker C5-ID8 are shown in SEQ ID NO.3 and SEQ ID NO.4. This invention solves the problem that no molecular markers have been developed for identifying wax powder characteristics in the whole rapeseed plant. The molecular markers of this invention can determine the absence of wax powder in the whole plant at an early stage.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to a molecular marker primer combination and its application for the identification of rapeseed wax powder characteristics. Background Technology

[0002] rape( Brassica napus Rapeseed (L.) is an important oilseed crop worldwide, with its seed oil being a high-quality source of edible oil and its oilseed meal an important source of protein feed. Improving rapeseed yield and stress resistance has always been a core objective of breeding efforts. Plant epidermal wax is a hydrophobic protective layer covering the surface of aerial organs, crucial for plants to cope with biotic and abiotic stresses. Plant epidermal wax is mainly composed of very long-chain fatty acids and their derivatives (such as alkanes, primary alcohols, and wax esters), playing a key role in reducing water loss, resisting ultraviolet radiation, and resisting diseases and pests. In rapeseed, this wax is often distributed as a white powder (called "wax powder") on the surface of leaves, stems, and siliques, and its content directly affects drought resistance, disease resistance (such as sclerotinia rot and downy mildew), and insect resistance (such as aphids). Therefore, wax powder content is an important indicator in rapeseed stress resistance breeding.

[0003] Currently, the identification of rapeseed wax powder characteristics mainly relies on traditional methods: field visual inspection is easily affected by environmental, growth stage and human factors, and is highly subjective and has low accuracy; solvent extraction is cumbersome to operate, damages samples and is inefficient, making it difficult to apply to the rapid and non-destructive screening of large-scale breeding materials.

[0004] Marker-assisted selection (MAS) technology provides a powerful tool for crop genetic improvement. This technology analyzes DNA molecular markers closely linked to target traits, enabling accurate genotyping at the seedling stage or even the cotyledon stage. It boasts significant advantages such as being unaffected by environmental factors, high selection accuracy, and the ability to perform early selection, thus significantly shortening the breeding cycle and improving selection efficiency.

[0005] Currently, despite the limited understanding of wax synthesis metabolic pathways and key genes (such as those in Arabidopsis thaliana and other model plants) in this field, CER1, CER2, KCS, LACS, WSD1 While there has been relatively in-depth research on wax powder traits (e.g., rapeseed, etc.), the formation mechanism of wax powder traits is more complex in rapeseed, an allotetraploid crop. Research and application of developing specific molecular markers closely linked to rapeseed wax powder traits for assisted breeding are still relatively lacking. Rapeseed breeding still relies on the aforementioned inefficient traditional methods for screening wax powder traits, hindering the breeding process of new rapeseed varieties with high stress resistance.

[0006] Although Chinese patent CN116622890A discloses molecular markers and their applications that are closely related to the waxy properties of rapeseed leaves, it can only determine the waxy properties of leaves and cannot determine the absence of wax powder in the whole plant, and it is not applicable to early screening.

[0007] Therefore, discovering molecular markers that are co-segregated or closely linked to rapeseed wax powder traits and establishing a rapid, accurate, and efficient molecular marker-assisted selection system is of great practical significance and application value for achieving technological innovation in rapeseed whole-plant stress resistance breeding, accelerating the selection and promotion of superior varieties.

[0008] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide a molecular marker primer combination and its application for the identification of wax powder characteristics in rapeseed, which solves the problem that no molecular markers have been developed for the identification of wax powder characteristics in the whole rapeseed plant. The molecular markers of this invention can determine the absence of wax powder in the whole plant at an early stage.

[0010] To achieve the above objectives, the present invention provides a molecular marker primer combination for identifying the wax powder characteristics of rapeseed, the molecular marker comprising: a primer for molecular marker A9-ID10 and a primer for molecular marker C5-ID8; wherein the nucleotide sequence of the primer for molecular marker A9-ID10 is shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the primer for molecular marker C5-ID8 is shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] This invention clarifies that wax powder deficiency is controlled by two recessive genes, which have been finely mapped to the corresponding intervals of 49.265–49.788 Mb on ChrA09 and 17.047–17.289 Mb on ChrC05 in the ZS11 reference genome (https: / / yanglab.hzau.edu.cn / BnIR, ZS11_V0). Wax powder deficiency is determined by the combined use of two molecular markers, with both markers being homozygous for recessiveness. This method provides 100% accurate identification, and the molecular markers used in this application can detect wax powder deficiency in the entire plant at an early stage.

[0012] A second objective of this invention is to provide a kit for identifying the properties of rapeseed wax powder using molecular markers, wherein the primers in the kit employ the aforementioned molecular marker primer combination.

[0013] Preferably, the kit also includes a Taq enzyme premix.

[0014] A third objective of this invention is to provide the application of the aforementioned molecular marker primer combination, or the aforementioned kit, in identifying rapeseed wax powder traits or in molecular breeding.

[0015] A fourth objective of this invention is to provide a method for identifying rapeseed wax powder traits, the method comprising: using the aforementioned molecular marker primer combination or the aforementioned kit, and employing the DNA of the rapeseed plant to be tested as a template, performing PCR amplification, and analyzing the rapeseed wax powder traits based on band analysis.

[0016] If the rapeseed plant to be tested shows only one band in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 205bp and the band size of molecular marker C5-ID8 being 273bp, then it is a wax powder-deficient material.

[0017] If the rapeseed plant to be tested shows two bands in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 241bp and 205bp, and the band size of molecular marker C5-ID8 being 259bp and 273bp, then it is a homozygous genotype wax powder normal material.

[0018] If the rapeseed plant to be tested has a band size of 241bp and 205bp for molecular marker A9-ID10 and a band size of 259bp for molecular marker C5-ID8 during the amplification of molecular markers A9-ID10 and C5-ID8, or a band size of 241bp for molecular marker A9-ID10 and a band size of 259bp and 273bp for molecular marker C5-ID8, then it is a heterozygous genotype wax powder normal material.

[0019] Preferably, the PCR amplification system comprises: Taq enzyme premix, DNA template, forward and reverse primers, and ultrapure water.

[0020] More preferably, the PCR amplification system comprises: 5 μL of Taq enzyme premix, 1 μL of DNA template, 0.25 μL each of 10 μM forward and reverse primers, and 3.5 μL of ultrapure water.

[0021] Preferably, the PCR program is as follows: pre-denaturation at 94 °C for 5 min; followed by 9 cycles: denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s with a decrease of 0.5 °C per cycle, extension at 72 °C for 30 s; then 29 cycles: denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s; finally, final extension at 72 °C for 10 min, and incubation at 25 °C for 3 min.

[0022] Preferably, the materials required for DNA extraction from the rapeseed plant to be tested are collected from any part of the plant.

[0023] More preferably, the rapeseed plant to be tested can be collected when it is in the cotyledon stage.

[0024] The method of this invention can be applied to the detection of wax powder deficiency in the whole plant (leaves, stems, siliques, etc.), and is suitable for breeding of stress resistance in the whole plant, such as drought resistance, disease resistance, and insect resistance. Moreover, identification can be performed at the cotyledon stage, enabling early non-destructive high-throughput screening, avoiding redundant planting, greatly shortening the breeding cycle, and improving selection efficiency.

[0025] Preferably, the PCR amplification system comprises: Taq enzyme premix, DNA template, forward and reverse primers, and ultrapure water.

[0026] More preferably, the PCR amplification system comprises: 5 μL of Taq enzyme premix, 1 μL of DNA template, 0.25 μL each of 10 μM forward and reverse primers, and 3.5 μL of ultrapure water.

[0027] Preferably, the PCR program is as follows: pre-denaturation at 94 °C for 5 min; followed by 9 cycles: denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s with a decrease of 0.5 °C per cycle, extension at 72 °C for 30 s; then 29 cycles: denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s; finally, final extension at 72 °C for 10 min, and incubation at 25 °C for 3 min.

[0028] The molecular marker primer combination and its application for identifying wax powder characteristics of rapeseed in this invention solves the problem that no molecular markers have been developed for identifying wax powder characteristics of the whole rapeseed plant, and has the following advantages:

[0029] (1) The method of the present invention overcomes the shortcomings of traditional field visual inspection, which is highly subjective and has low accuracy, as well as the cumbersome operation, sample damage, and low efficiency of solvent extraction. At the same time, compared with the molecular markers disclosed in Chinese Patent No. CN116622890A, which can only determine the wax content of leaves, the method of the present invention can more comprehensively determine the wax content of the whole plant, and has higher application value for the whole plant stress resistance breeding of rapeseed;

[0030] (2) In practical applications, this invention can quickly and accurately screen rapeseed breeding materials with excellent wax powder traits by simply collecting DNA samples of plants in the cotyledon stage and using the primers of the molecular marker developed in this invention for PCR amplification and band analysis, thus providing strong technical support for the breeding of new rapeseed varieties with high stress resistance.

[0031] (3) The molecular markers A9-ID10 and C5-ID8 of the present invention are closely linked to the wax powder trait in rapeseed. By detecting the amplification bands of these two molecular markers, it is possible to accurately determine whether a rapeseed plant is a wax powder-deficient material. This method of judgment is not affected by environmental factors and can be carried out in the early stage of the plant's cotyledon stage, avoiding redundant planting due to unsuitable wax powder traits, further shortening the breeding cycle, and greatly improving the efficiency of breeding material screening. It has a significant promoting effect on accelerating the breeding and promotion of superior rapeseed varieties. Attached Figure Description

[0032] Figure 1 The phenotypes of M9 (a wax-deficient material from Brassica napus) and Westar (a wax-normal material from Brassica napus) and their hybrid F1 are shown in this invention.

[0033] Figure 2 These are cryo-scanning electron micrographs of the leaf epidermis of the M9 leaf (lacking wax powder) and the Westar leaf (with normal wax powder) of the present invention; wherein, A is a frontal scan of the Westar leaf (with normal wax powder); B is a frontal scan of the M9 leaf; C is a back scan of the Westar leaf; and D is a back scan of the M9 leaf.

[0034] Figure 3 The results of BSA resequencing analysis of rapeseed leaf wax powder traits are presented in this invention.

[0035] Figure 4 This is a banding diagram of the molecular marker primer set amplification of the present invention; wherein lanes 1 to 96 are 96 individual plants randomly selected from the BC2 population. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] It should be noted that: Unless otherwise specified in the examples, standard conditions or conditions recommended by the manufacturer should be followed. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0038] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0039] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0040] In the following embodiments, the plants grown from rapeseed germplasm M9 are referred to as rapeseed wax powder-deficient material M9; the normal materials referred to below are all rapeseed wax powder normal materials. In this experiment, the rapeseed wax powder normal material was selected from the inbred line Westar.

[0041] Example 1: Obtaining Molecular Markers for Identifying Rapeseed Wax Powder Properties

[0042] 1. Obtaining and phenotypic identification of rapeseed wax powder-deficient materials

[0043] After planting, rapeseed germplasm M9 exhibited a waxy powder-deficient phenotype in its leaves, petioles, stems, siliques, and other organs. Figure 1 The material with a glossy appearance was named M9, a rapeseed germplasm lacking wax powder. To clarify the differences in wax structure between rapeseed germplasm M9 and the wax-powder-normal material Westar, cryo-scanning electron microscopy was performed on leaves at the five-leaf stage. The results showed that both sides of the leaves of the wax-powder-normal material Westar were covered with a large amount of wax crystals, while almost no wax crystals were observed on either side of the leaves of M9. Figure 2 This confirms that the wax powder is missing phenotype.

[0044] 2. Precise localization of the presence or absence of sites in the control of wax powder properties.

[0045] Reciprocal crosses were performed between the wax-deficient material M9 and the wax-normal material Westar. All F1 plants exhibited normal wax production, indicating that this trait is controlled by a recessive nuclear gene. An investigation of the four F2 segregating populations showed that the segregation ratio of wax-normal plants to wax-deficient plants was approximately 15:1, indicating that the wax-promoting trait is controlled by two recessive nuclear genes. To further locate the target gene, 15 plants each with the wax-normal and wax-deficient traits were selected from the F2 populations, a DNA pool was constructed, and 35X deep resequencing was performed. Analysis results showed that significant association signals were identified on chromosomes A09 and C05. Figure 3 ).

[0046] Subsequently, polymorphic markers were developed within candidate regions on these two chromosomes, and initial mapping was performed using 956 recessive individuals from the F2 population. This narrowed the target regions to a 2.56 Mb region on ChrA09 and a 1.98 Mb region on ChrC05 in the corresponding ZS11 reference genome (https: / / yanglab.hzau.edu.cn / BnIR, ZS11_V0). Further fine mapping was performed using a total of 3016 recessive individuals from the F2, BC1, and BC2 populations. The region on ChrA09 was ultimately narrowed to 49.265–49.788 Mb (approximately 523 kb), yielding the co-segregating marker A9-ID10; simultaneously, the region on ChrC05 was narrowed to 17.047–17.289 Mb (approximately 242 kb), yielding the co-segregating marker C5-ID8.

[0047] The primer sequences for the molecular marker A9-ID10 are as follows:

[0048] SEQ ID NO.1: ATTCAATCCACACCAAAAACCG;

[0049] SEQ ID NO. 2: CAACGGAATTTAAATAGGTTATTCG.

[0050] The primer sequences for the molecular marker C5-ID8 are as follows:

[0051] SEQ ID NO.3: AGCTAACTCATGAGCTTGGTCG;

[0052] SEQ ID NO. 4: GCTCGGATGCCGCATGAG.

[0053] By combining the two pairs of markers mentioned above, the genotype of a single plant can be accurately determined: a single plant showing only one band in both A9-ID10 and C5-ID8 amplification is a recessive homozygous genotype with a wax powder deletion phenotype; a single plant showing two amplification bands in both is a dominant homozygous genotype with a normal wax powder phenotype; and the remaining band combinations indicate a heterozygous genotype with a normal wax powder phenotype.

[0054] See electrophoresis results Figure 4 In the A9-ID10 band pattern, the large fragment is 241 bp (SEQ ID NO.5), and the small fragment is 205 bp (SEQ ID NO.6); in the C5-ID8 band pattern, the large fragment is 273 bp (SEQ ID NO.7), and the small fragment is 259 bp (SEQ ID NO.8); lanes 1-96 represent 96 randomly selected individuals from the BC2 population. The electrophoresis results and corresponding phenotypes of these 96 individuals are consistent with the aforementioned discrimination criteria. Figure 4In some cases, A9-ID10 may not amplify, while C5-ID8 may amplify only one band (lane 67), or A9-ID10 may amplify two bands while C5-ID8 may not amplify (lane 76). The absence of a band is due to an operational error, such as improper DNA template addition. This type of error is common in large-scale population genotyping and is understandable to industry professionals. If A9-ID10 does not amplify, but C5-ID8 amplifies two bands, the phenotype is normal. If C5-ID8 amplifies only one band, A9-ID10 needs to be amplified again to determine the phenotype of this individual. If A9-ID10 also shows only one band, then the phenotype is the wax-deficient phenotype. In large-scale population identification, this error leading to an inaccurate identification of an individual can result in the individual being discarded, as there are still many individuals available for selection. Conversely, the same logic applies if A9-ID10 amplifies but C5-ID8 does not.

[0055] The sequence of SEQ ID NO.5 is as follows:

[0056] ATTCAATCCACACCAAAAACCGAATAATTTTTTTGAAAAAATGTTTTAAAAAATAATATTAATATATTCTGTTAAATATATATATATATATATATATATATATATATATATATATATATATGGGTTAATATGATCTTCAGTAACTTTAAGTAAAATCATATTTAATAAATATTTTTAATCGAATAACCTATTTAAATTCCGTTG.

[0057] The sequence of SEQ ID NO.6 is as follows:

[0058] ATTCAATCCACACCAAAAACCGAATAATTTTTTTGAAAAAATGTTTTAAAAAAATAATATTAATATATTCTGTTAAATATATATATATATATATGGGTTAATATGATCTTCAGTAACTTTAAGTAAAATCATATTTAATAAATATTTTTAATCGAATAACCTATTTAAATTCCGTTG.

[0059] The sequence of SEQ ID NO. 7 is as follows:

[0060] AGCTAACTCATGAGCTTGGTCGTTTTTATACTTATATATATATATATATATATATATATATATATATTGATGACATCTACTTTTCTTTTGTAAAACAAAAATAGTTTCTATATTAATCATATTATCTTCTAAAGT TAAAATGTAAAACCAATTTTTTTTAAATATACATAAAAATGTAAAAGATTATTGATAGCAATGCTCGTATCATATATATTTTTAGAATTAAAATGAAAAATAAAATATTCCTAAGATTCTCATGCGGCATCCGAGC.

[0061] The sequence of SEQ ID NO. 8 is as follows:

[0062] AGCTAACTCATGAGCTTGGTCGTTTTTATACTTATATATATATATATATATATATTGATGACATCTACTTTTCTTTTGTAAAACAAAAATAGTTTCTATATTAATCATATTATCTTCTAAAGTTAAAATGTAAAACCAATTTTTTTTAAATATACATAAAAATGTAAAAGATTATTGATAGCAATGCTCGTATCATATATATTTTTAGAATTAAAATGAAAAATAAAATATTCCTAAGATTCTCATGCGGCATCCGAGC.

[0063] Therefore, by utilizing the two pairs of markers and discrimination criteria of the present invention, 100% accurate early identification of rapeseed wax powder characteristics can be achieved.

[0064] Example 2: Application of molecular marker primer set closely linked to rapeseed wax powder trait in rapeseed molecular breeding.

[0065] Using 524 individuals from the F2 population and 466 individuals from the BC1 population in Example 1 as materials, including 169 individuals with missing wax powder and 821 individuals with normal wax powder, genotyping was performed using the primer sets A9-ID10 and C5-ID8 developed in Example 1. The steps are as follows:

[0066] (1) DNA extraction

[0067] DNA was extracted from any part of the plant tissue using the CTAB method.

[0068] (2) PCR amplification

[0069] Using the DNA extracted in step (1) as a template, PCR amplification was performed using primer sets A9-ID10 and C5-ID8, respectively.

[0070] The total PCR reaction volume was 10 μL, including: 5 μL Taq enzyme mix, 1 μL DNA template, 0.25 μL each of 10 μM forward and reverse primers, and 3.5 μL ultrapure water.

[0071] The PCR program was as follows: pre-denaturation at 94 ℃ for 5 min; followed by 9 cycles: denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s (decreasing by 0.5 ℃ per cycle), extension at 72 ℃ for 30 s; then 29 cycles: denaturation at 94 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 30 s; finally, final extension at 72 ℃ for 10 min, and incubation at 25 ℃ for 3 min.

[0072] (3) Electrophoretic detection and banding analysis

[0073] Add 10 μL of loading buffer to the PCR products and separate them by electrophoresis using a 6% polyacrylamide gel.

[0074] The results showed that among the 169 paraffin-deleted monoclonal strains, each monoclonal strain showed only one band in both A9-ID10 and C5-ID8 amplification; the 821 paraffin-normal monoclonal strains all showed two bands in the electrophoresis results, or a combination of "two bands + one band" (i.e., two bands of A9-ID10 + one band of C5-ID8, or one band of A9-ID10 + two bands of C5-ID8).

[0075] (4) Self-crossing validation genotype-phenotype association analysis

[0076] The plants with different banding patterns were self-pollinated, and their phenotypes were observed. The results showed that: plants showing one band in both A9-ID10 and C5-ID8 did not segregate in their self-pollinated offspring, and their phenotypes all lacked wax powder, thus they were identified as recessive homozygous; plants showing two bands did not segregate in their self-pollinated offspring, and their phenotypes all had normal wax powder, thus they were identified as dominant homozygous; plants with a banding pattern of "two bands + one band" or "one band + two bands" showed phenotypic segregation in their self-pollinated offspring, and their phenotypes had normal wax powder, thus they were identified as heterozygous.

[0077] The above results indicate that the primer set of the present invention can accurately identify different genotypes at an early stage: A9-ID10 and C5-ID8 both have single bands, indicating recessive homozygosity and absence of wax powder; both have double bands, indicating dominant homozygosity and normal wax powder; "2+1" or "1+2" ​​band combinations indicate heterozygous genotypes and normal wax powder.

[0078] The experimental results above demonstrate that this invention clearly identifies wax powder deficiency as being controlled by two recessive genes, and these genes have been precisely mapped, resulting in a clearer understanding of the genetic mechanism. Wax powder deficiency is determined only when both molecular markers are homozygous recessive, ensuring 100% accurate identification.

[0079] By applying the primer set of this invention, combined with seed tray sowing and DNA extraction from cotyledon stage sampling for genotyping, genotyping can be completed at the seedling stage, allowing for early screening of target individual plants for transplanting to the field. This avoids redundant planting of non-target genotype materials, significantly improves selection efficiency, reduces field sowing and phenotypic identification costs, and effectively accelerates the breeding process of rapeseed varieties with wax powder specific traits.

[0080] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Use of a molecular marker primer combination in identifying the rapeseed waxy trait or in the molecular breeding of the rapeseed waxy trait, characterized in that, The molecular marker primer combination comprises: a primer for molecular marker A9-ID10 and a primer for molecular marker C5-ID8; wherein the nucleotide sequence of the primer for molecular marker A9-ID10 is shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the primer for molecular marker C5-ID8 is shown in SEQ ID NO.3 and SEQ ID NO.4; Using the DNA of the rapeseed plant to be tested as a template, the above-mentioned molecular marker primer combination was used for PCR amplification, and the wax powder trait of rapeseed was analyzed based on the bands: If the rapeseed plant to be tested shows only one band in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 205bp and the band size of molecular marker C5-ID8 being 273bp, then it is a wax powder-deficient material. If the rapeseed plant to be tested shows two bands in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 241bp and 205bp, and the band size of molecular marker C5-ID8 being 259bp and 273bp, then it is a homozygous genotype wax powder normal material. If the rapeseed plant to be tested has a band size of 241bp and 205bp for molecular marker A9-ID10 and a band size of 259bp for molecular marker C5-ID8 during the amplification of molecular markers A9-ID10 and C5-ID8, or a band size of 241bp for molecular marker A9-ID10 and a band size of 259bp and 273bp for molecular marker C5-ID8, then it is a heterozygous genotype wax powder normal material.

2. Use of a kit of molecular markers for identifying the traits of Brassica napus wax powdery in the identification of the traits of Brassica napus wax powdery or in the molecular breeding of the traits of Brassica napus wax powdery, characterized in that, This kit contains a molecular marker primer set, which includes a primer for molecular marker A9-ID10 and a primer for molecular marker C5-ID8; wherein the nucleotide sequences of the primers for molecular marker A9-ID10 are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the primers for molecular marker C5-ID8 are shown in SEQ ID NO.3 and SEQ ID NO.4; Using the DNA of the rapeseed plant to be tested as a template, the above-mentioned molecular marker primer combination was used for PCR amplification, and the wax powder trait of rapeseed was analyzed based on the bands: If the rapeseed plant to be tested shows only one band in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 205bp and the band size of molecular marker C5-ID8 being 273bp, then it is a wax powder-deficient material. If the rapeseed plant to be tested shows two bands in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 241bp and 205bp, and the band size of molecular marker C5-ID8 being 259bp and 273bp, then it is a homozygous genotype wax powder normal material. If the rapeseed plant to be tested has a band size of 241bp and 205bp for molecular marker A9-ID10 and a band size of 259bp for molecular marker C5-ID8 during the amplification of molecular markers A9-ID10 and C5-ID8, or a band size of 241bp for molecular marker A9-ID10 and a band size of 259bp and 273bp for molecular marker C5-ID8, then it is a heterozygous genotype wax powder normal material.

3. A method for identifying the waxy trait in Brassica napus, characterized in that, The method includes: Based on the molecular marker primer combination as described in claim 1, or the kit as described in claim 2, using the DNA of the rapeseed plant to be tested as a template, the molecular marker primer combination is used for PCR amplification, and the wax powder morphology of rapeseed is analyzed based on the bands: If the rapeseed plant to be tested shows only one band in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 205bp and the band size of molecular marker C5-ID8 being 273bp, then it is a wax powder-deficient material. If the rapeseed plant to be tested shows two bands in both molecular marker A9-ID10 and C5-ID8 amplification, with the band size of molecular marker A9-ID10 being 241bp and 205bp, and the band size of molecular marker C5-ID8 being 259bp and 273bp, then it is a homozygous genotype wax powder normal material. If the rapeseed plant to be tested has a band size of 241bp and 205bp for molecular marker A9-ID10 and a band size of 259bp for molecular marker C5-ID8 during the amplification of molecular markers A9-ID10 and C5-ID8, or a band size of 241bp for molecular marker A9-ID10 and a band size of 259bp and 273bp for molecular marker C5-ID8, then it is a heterozygous genotype wax powder normal material.

4. The method of claim 3, wherein, The PCR amplification system includes: Taq enzyme premix, DNA template, forward and reverse primers, and ultrapure water.

5. The method of claim 4, wherein, The PCR amplification system comprises: 5 μL of Taq enzyme premix, 1 μL of DNA template, 0.25 μL each of 10 μM forward and reverse primers, and 3.5 μL of ultrapure water.

6. The method according to any one of claims 3 to 5, characterized in that, The PCR program was as follows: pre-denaturation at 94 ℃ for 5 min; followed by 9 cycles: denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s with a 0.5 ℃ decrease per cycle, extension at 72 ℃ for 30 s; then 29 cycles: denaturation at 94 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 30 s; and finally, final extension at 72 ℃ for 10 min, followed by incubation at 25 ℃ for 3 min.

7. The method of claim 3, wherein, The materials required for DNA extraction from the rapeseed plant to be tested were collected from any part of the plant.

8. The method of claim 7, wherein, The rapeseed plants to be tested can be collected when they are in the cotyledon stage.

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