Application of BnaCT3 gene and SNP molecular marker in regulating freezing tolerance of Brassica napus

Abstract

This application discloses the application of the BnaCT3 gene and its SNP molecular markers in regulating the cold resistance of rapeseed. The nucleotide sequence of the BnaCT3 gene is shown in SEQ ID NO.1. Overexpression of the BnaCT3 gene in rapeseed significantly improves the survival rate of plants under low-temperature stress, reduces electrolyte leakage rate and malondialdehyde and hydrogen peroxide content, increases proline content and antioxidant enzyme activity, and enhances the cold resistance of rapeseed. Furthermore, this application identifies two SNP sites located in the promoter region of the BnaCT3 gene, whose specific base combinations constitute H1 and H2 allele combinations. Rapeseed materials carrying the H2 allele exhibit significantly better cold resistance than those carrying the H1 allele, and all currently promoted varieties lack the H2 allele. This application provides effective gene resources and molecular markers for the genetic improvement of rapeseed cold resistance, which can be used for overexpression transgenic breeding and marker-assisted selection breeding.

Description

Technical Field

[0001] This application relates to the field of molecular biology breeding technology, specifically to the application of the BnaCT3 gene and its SNP molecular markers in regulating the frost resistance of rapeseed. Background Technology

[0002] Rapeseed (Brassica napus L.) belongs to the Brassicaceae family and is currently the most widely planted oilseed crop in my country, holding an important position in global trade and agricultural production. In recent years, frequent extreme cold waves and late spring frosts have often led to seedling death, frost damage during the flowering stage, and reduced seed setting rate, severely impacting yield.

[0003] However, current research on key cold-resistant genes in rapeseed is not systematic, cold-resistant germplasm resources are scarce, and existing varieties lack excellent cold-resistant alleles, making it difficult to meet the actual needs of cold-resistant molecular breeding. Therefore, identifying and characterizing key cold-resistant genes and related molecular markers in rapeseed is of great significance for cold-resistant molecular breeding of rapeseed. Summary of the Invention

[0004] Given the scarcity of existing rapeseed germplasm resources and the insufficient discovery of key cold-resistant genes, this application provides an application of the BnaCT3 gene and its SNP molecular markers in regulating rapeseed cold resistance. The BnaCT3 gene was cloned from Brassica napus and introduced into target rapeseed plants for overexpression. Overexpression of the BnaCT3 gene significantly improved the cold resistance of rapeseed seedlings, reduced electrolyte leakage rate and oxidative damage after frost damage, and increased plant survival rate, thus providing an effective gene resource for molecular breeding of rapeseed cold resistance.

[0005] To achieve the above objectives, this application provides the following technical solution:

[0006] In one aspect, this application provides an application of the BnaCT3 gene in improving the frost resistance of rapeseed, wherein the nucleotide sequence of the BnaCT3 gene is shown in SEQ ID NO.1.

[0007] Secondly, this application provides a method for improving the frost resistance of rapeseed, comprising overexpressing the BnaCT3 gene in rapeseed plants, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] In some preferred embodiments, the overexpression is achieved by transferring an expression vector containing the BnaCT3 gene into rapeseed plants; the expression vector is pCambia1302-BnaCT3, the nucleotide sequence of which is shown in SEQ ID NO.9.

[0009] Thirdly, this application provides a method for breeding cold-resistant rapeseed varieties, which includes introducing an overexpression vector containing the coding sequence of the BnaCT3 gene into recipient rapeseed plants and screening to obtain transgenic rapeseed plants overexpressing the BnaCT3 gene; wherein the nucleotide sequence of the BnaCT3 gene is shown in SEQ ID NO.1.

[0010] In some embodiments, the transgenic rapeseed plants exhibit improved frost resistance compared to the recipient rapeseed plants, specifically manifested in one or more of the following indicators: increased survival rate after low-temperature stress, decreased electrolyte leakage rate, decreased malondialdehyde content, decreased hydrogen peroxide content, increased proline content, increased catalase activity, and increased peroxidase activity.

[0011] Fourthly, this application provides a set of SNP molecular markers related to the frost resistance of rapeseed. The SNP molecular markers are located in the promoter region of the BnaCT3 gene BnaA07G0038800ZS of rapeseed, corresponding to positions 3480595 and 3480604 on chromosome A07 of the rapeseed reference genome ZS11.v0. The polymorphism at position 3480595 is C / T, and the polymorphism at position 3480604 is T / C.

[0012] In the above SNP molecular markers, two SNP sites constitute a haplotype, which includes haplotype H1 and haplotype H2; wherein haplotype H1 is C at position 3480595 and T at position 3480604, and haplotype H2 is T at position 3480595 and C at position 3480604.

[0013] Fifthly, this application provides the application of the SNP molecular markers described in the fourth aspect in identifying or assisting in the identification of rapeseed frost resistance. By detecting the genotype of the single nucleotide polymorphism sites at positions 3480595 and 3480604 on chromosome A07 of the BnaCT3 gene promoter region in rapeseed plants, corresponding to the single nucleotide polymorphism sites at positions 3480595 and 3480604 on chromosome A07 of the rapeseed reference genome ZS11.v0, the frost resistance of rapeseed plants can be determined.

[0014] Sixthly, this application provides a method for screening frost-resistant rapeseed, comprising detecting the genotype of the BnaCT3 gene BnaA07G0038800ZS promoter region in the rapeseed plant to be tested, corresponding to the single nucleotide polymorphism sites at positions 3480595 and 3480604 on chromosome A07 of the rapeseed reference genome ZS11.v0, and selecting plants carrying the H2 allele combination as frost-resistant breeding material; wherein the H2 type is T at position 3480595 and C at position 3480604.

[0015] Seventhly, this application provides a method for breeding cold-resistant rapeseed varieties, including introducing the H2 haplotype of the BnaCT3 gene BnaA07G0038800ZS into a target rapeseed variety through molecular marker-assisted selection. The H2 haplotype is a haplotype of the BnaCT3 gene BnaA07G0038800ZS promoter region that corresponds to the single nucleotide polymorphism sites at positions 3480595 and 3480604 on chromosome A07 of the Brassica napus reference genome ZS11.v0, which has a cold-resistant genotype advantage.

[0016] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0017] 1. This application is the first to discover that overexpression of the BnaCT3 gene derived from Brassica napus can significantly improve the frost resistance of rapeseed. After low-temperature stress, the survival rate of overexpressing lines was significantly increased, the electrolyte leakage rate was significantly reduced, malondialdehyde and hydrogen peroxide accumulation was decreased, proline content and antioxidant enzyme activity were increased, and overall frost resistance was significantly enhanced. This application provides an effective gene resource and overexpression technology solution for the genetic improvement of frost resistance in rapeseed.

[0018] 2. This application identified two key SNP sites in the promoter region of the BnaCT3 gene, corresponding to positions 3480595 and 3480604 on chromosome A07 of the reference genome ZS11.v0. Specific base combinations at these sites constitute two allele combinations: H1 and H2. Freezing tolerance testing showed that rapeseed materials carrying the H2 allele exhibited significantly better freezing tolerance than those carrying the H1 allele. This SNP molecular marker can be used for auxiliary identification of freezing tolerance and marker-assisted selection breeding, and is simple to operate and inexpensive.

[0019] 3. The SNP marker provided in this application can be rapidly introduced into existing varieties of H2 type to target and improve the frost resistance of rapeseed. Furthermore, the BnaCT3 gene used is an endogenous plant gene, which is environmentally friendly and poses no biosafety risks. Attached Figure Description

[0020] Figure 1 The expression levels of the rapeseed gene BnaCT3 in low-temperature sensitive and low-temperature resistant materials in the embodiments of this application are shown.

[0021] Figure 2 The expression levels of the rapeseed gene BnaCT3 in the embodiments of this application are shown at different times under normal and low temperature treatment.

[0022] Figure 3 This is a map of the binary expression vector pCambia1302 used in this application.

[0023] Figure 4This is a map of the overexpression vector pCambia1302-BnaCT3 in the embodiments of this application.

[0024] Figure 5 The expression level of BnaCT3 in transgenic rapeseed in the embodiments of this application is shown, and ZS11 is wild type.

[0025] Figure 6 The following are the frozen phenotype (A), survival rate statistics (B), and electrolyte leakage rate statistics (C) of the BnaCT3-OE transgenic material seedlings in the embodiments of this application.

[0026] Figure 7 The values ​​for malondialdehyde (A), hydrogen peroxide (B), proline (C), antioxidant reductase CAT (D), POD (E), and SOD (F) of the BnaCT3-OE transgenic material seedlings after freezing during the seedling stage in this application are shown in the examples.

[0027] Figure 8 This example shows the haplotype analysis of BnaCT3 in a Brassica napus population; where A is the p-value matrix of survival differences between haplotypes, B is the cold stress survival distribution of haplotypes H1 and H2, and C is the location of key SNP sites in the BnaCT3 gene region. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0029] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0030] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0031] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. To enable those skilled in the art to better understand this application, the following explanation of terminology is provided.

[0033] 1. Overexpression: This refers to the use of genetic engineering techniques to significantly increase the expression level of a target gene in transgenic plants compared to its endogenous expression level in recipient plants. In the embodiments of this application, the expression level of the BnaCT3 gene in the overexpression lines was upregulated by 2-4 times compared to the recipient control.

[0034] 2. Haplotype / Allele Combination: Brassica napus is an allotetraploid (AACC) containing two subgenomes, A and C. For accuracy, the term "haplotype" or "allele combination" used in this application specifically refers to the base combination at two specific SNP sites (positions 3480595 and 3480604) in the promoter region of the BnaCT3 gene BnaA07G0038800ZS on chromosome A07 of the A subgenome, wherein:

[0035] H1 type: The 3480595th bit is C, and the 3480604th bit is T;

[0036] H2 type: The 3480595th bit is T, and the 3480604th bit is C.

[0037] The genomic coordinates of Brassica napus may differ depending on the database version. For clarity, this application uses the physical location disclosed in version ZS11.v0 of the BnIR database (see https: / / yanglab.hzau.edu.cn / BnIR / germplasm_info?id=ZS11.v0) to determine the location. Specifically, the BnaCT3 gene BnaA07G0038800ZS described in this application is located on chromosome A07, and the definitions of SNP sites 3480595 and 3480604 in its promoter region are based on the ZS11.v0 reference genome. However, those skilled in the art will understand that when using other reference genome versions, the location may be mapped to different physical coordinates, but the corresponding SNP sites are equivalent variations of this application and still fall within the scope of protection of this application.

[0038] The following are specific examples:

[0039] Example 1: Obtaining and Sequence Analysis of the BnaCT3 Gene

[0040] 1.1 Plant materials

[0041] The rapeseed germplasm used in this study includes 2AF006, 2AF207, and Zhongshuang 11 (ZS11), all of which were provided by the National Oil Crop Germplasm Resource Mid-term Bank (Wuhan).

[0042] The germplasm information of the tested rapeseed is shown in Table 1.

[0043] Table 1. Names of rapeseed varieties tested

[0044]

[0045] 1.2 Identification and Nomenclature of the BnaCT3 Gene

[0046] Using previously screened cryogenically resistant materials (2AF006, 2AF207) and cryogenically sensitive materials (ZS11), transcriptome analysis under low-temperature stress was performed, identifying the core candidate gene BnaA07G0038800ZS (BnIR database: https: / / yanglab.hzau.edu.cn / BnIR). The expression level of this gene differed significantly between the cryogenically resistant and cryogenically sensitive materials (see...). Figure 1 Based on the annotation information of homologous genes in the Arabidopsis thaliana genome, this gene was found to be involved in abiotic stress response in Arabidopsis. Therefore, this application names this candidate gene Cold Tolerant gene 3 (CT3), which is abbreviated as BnaCT3 gene in this application. The nucleotide sequence of the CDS of BnaCT3 gene is shown below:

[0047] BnaCT3 gene CDS (SEQ ID NO.1):

[0048] .

[0049] Example 2: Low-Temperature Response Expression Analysis of the BnaCT3 Gene

[0050] 2.1 Material handling and sampling

[0051] Select plump and uniform ZS11 seeds (provided by the National Oil Crop Germplasm Resource Mid-term Bank) and allow them to germinate and grow to the 4-5 leaf stage. Select 8 plants with consistent growth and place them in an artificial climate incubator for low-temperature treatment. Treatment conditions: temperature -2 ℃, relative humidity 55%, light intensity 10000 lx, photoperiod 16 h / 8 h (day / night). Samples were taken before treatment (0 h) and at 2 h, 4 h, 8 h, 12 h, and 24 h after treatment. Functional leaves from the same part of the plant were taken and immediately flash-frozen in liquid nitrogen and stored at -80 ℃ for later use.

[0052] 2.2 RNA extraction and cDNA synthesis

[0053] Total RNA was extracted from the frozen samples using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit (AT311) according to the manufacturer's instructions.

[0054] 2.3 Quantitative Real-Time PCR (qPCR)

[0055] Using cDNA obtained through reverse transcription as a template, qPCR was performed on a real-time PCR instrument using the Novizan Real-Time PCR Kit (catalog number Q711). The reaction system and procedure were performed according to the kit instructions. The Actin gene was used as an internal control, with primer pairs q-Actin-F and q-Actin-R. The primer pairs q-CT3-F and q-CT3-R were used to detect BnaCT3 gene expression. The primer sequences used are shown in Table 2.

[0056] Table 2 Primer sequences for quantitative real-time PCR

[0057]

[0058] Use 2 -△△Ct The relative expression level of the BnaCT3 gene was calculated. Three technical replicates were set up for each sample.

[0059] 2.4 Results

[0060] The results are as follows Figure 2 As shown in the figure, the expression level of the BnaCT3 gene was low before low temperature treatment (0 h); after low temperature stress treatment, the expression level of the BnaCT3 gene increased significantly, and the relative expression level gradually increased with the extension of treatment time (2 h to 24 h). This result indicates that the expression of the BnaCT3 gene is induced by low temperature stress and participates in the low temperature response process of rapeseed.

[0061] Example 3 Construction of BnaCT3 gene overexpression vector

[0062] 3.1 Vector backbone and enzyme digestion

[0063] The plant binary expression vector pCambia1302 (carrying a 35S promoter, a GFP tag, a kanamycin prokaryotic resistance gene, and a hygromycin eukaryotic resistance gene) was constructed and preserved by the applicant's laboratory. The vector map is shown below. Figure 3As shown in SEQ ID NO.6, the vector sequence is as follows. The pCambia1302 plasmid was double-digested with restriction endonucleases SpeI and PstI. The digestion products were purified by agarose gel electrophoresis to obtain the linearized vector.

[0064] 3.2 Amplification of the CDS fragment of the BnaCT3 gene

[0065] Using ZS11 cDNA as a template (obtained from Example 2), the coding sequences (CDS) of CT3-F and CT3-R were amplified using novizan high-fidelity enzyme (Vazyme #P526) and primer pairs. Primer sequences are shown in Table 3.

[0066] Table 3. Primer sequences for BnaCT3 gene CDS amplification

[0067]

[0068] The PCR product was recovered and purified by agarose gel electrophoresis to obtain the BnaCT3-CDS fragment, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0069] 3.3 Homologous recombination linkage

[0070] Using the Novizan ClonExpress II One Step Cloning Kit (catalog number C112), the purified BnaCT3-CDS fragment was homologously ligated into the linearized vector pCambia1302. The ligation product was transformed into competent E. coli cells, plated on LB agar plates containing kanamycin, and positive clones were screened. Plasmids of positive clones were extracted and sequenced for verification. The correctly sequenced recombinant plasmid was named pCambia1302-BnaCT3, and the vector map is shown below. Figure 4 As shown, the vector sequence is shown in SEQ ID NO. 9.

[0071] Example 4: Genetic transformation and positive line screening of Brassica napus

[0072] 4.1 Genetic transformation

[0073] The constructed overexpression vector of the BnaCT3 gene was transformed into rapeseed ZS11. The genetic transformation method for Brassica napus followed the method described by the State Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University (Cheng Dai et al., An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus, Mol Breeding, 2020, 40:96). Hypocotyls of sterile Brassica napus seedlings were used as explants, and the exogenous fragment was introduced into Brassica napus using Agrobacterium infection.

[0074] 4.2 Molecular detection of positive strains

[0075] After the transgenic plants were regenerated, genomic DNA and total RNA were extracted from the leaves, and the expression level of the BnaCT3 gene was detected by qPCR. The qPCR primers were q-CT3-F and q-CT3-R as shown in Table 2, and the internal reference gene was Actin.

[0076] Three overexpression lines were obtained through screening and named OE-2, OE-9, and OE-12, respectively. The qPCR results are shown below. Figure 5 As shown, compared with wild-type ZS11, the expression level of BnaCT3 gene in OE-2, OE-9 and OE-12 lines was significantly upregulated, with an upregulation fold of 2-4 times (p<0.05).

[0077] Example 5: Verification of the cold resistance of transgenic rapeseed seedlings

[0078] 5.1 Low-temperature treatment and phenotypic observation

[0079] Seeds of wild-type ZS11 and overexpression lines OE-2, OE-9, and OE-12 were collected and allowed to germinate until they reached the 4-5 leaf stage. Healthy plants with uniform growth were selected. The above materials were placed in an artificial climate incubator for low-temperature treatment: temperature -2 ℃, relative humidity 55%, light intensity 10000 lx, photoperiod 16 h / 8 h (day / night), treatment time 24 h.

[0080] After the low-temperature treatment, the plants were transferred to a normal temperature environment (22 °C) for recovery culture, while other culture conditions remained unchanged. After 3 days of recovery culture, the plant phenotype was observed, and the survival rate was calculated. Phenotypes of the plants before and after treatment were photographed. The survival rate was calculated using the following formula:

[0081] Survival rate (%) = Number of surviving plants / Total number of plants in each treatment × 100%.

[0082] 5.2 Electrolyte Leakage Rate Measurement

[0083] The electrolyte leakage rate of leaves was determined using a Leici DDS-307A conductivity meter (Shanghai Yidian Scientific Instruments Co., Ltd.). Functional leaves from the same part of 3-5 plants from each line were taken and punched into uniform leaf discs using a 1 cm diameter punch as the test samples.

[0084] After immersing leaf discs in deionized water for a thorough soaking, the initial conductivity was measured. Conductivity was then measured at 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h after soaking. Following the measurements, the samples were boiled in a water bath for 15 min to fully disrupt the cell membrane. After cooling, the total conductivity was measured. The ratio of conductivity at each time point to total conductivity was used to represent electrolyte extravasation, reflecting the stability and degree of cell membrane damage.

[0085] Electrolyte leakage rate is calculated using the following formula:

[0086] Electrolyte leakage rate (%) = (L t / L total )×100%;

[0087] In the formula, L t L represents the conductivity values ​​measured at different soaking times (1 h, 2 h, 3 h, 6 h, 12 h, 24 h). total The total conductivity value is measured after boiling water bath.

[0088] 5.3 Results

[0089] The results are as follows Figure 6 As shown. Among them Figure 6 Figure A shows the phenotypic results. As can be seen from the figure, before the low-temperature treatment, there was no significant difference in phenotype among all materials. After low-temperature treatment and recovery culture for 3 days, the wild-type ZS11 plant had completely died, and all leaves of the plant had withered. In contrast, the overexpression lines OE-2, OE-9, and OE-12 were still alive, with only some leaves withered, and some plants were able to produce new leaves.

[0090] Figure 6 B represents the survival rate statistics, which show that the survival rate of the overexpression line was significantly higher than that of the wild-type ZS11 (p < 0.05).

[0091] Figure 6 C represents the electrolyte leakage rate. The results show that the electrolyte leakage rate of the overexpression line was significantly lower than that of the wild-type ZS11 at all time points (p < 0.05).

[0092] The above results indicate that overexpression of the BnaCT3 gene significantly improves the frost resistance of rapeseed seedlings.

[0093] Example 6: Analysis of Oxidative Damage and Antioxidant System in Transgenic Rapeseed under Low Temperature Stress

[0094] 6.1 Sample preparation

[0095] Wild-type ZS11 and overexpression lines OE-2, OE-9, and OE-12 were grown to the 4-5 leaf stage according to the method in Example 5, and then subjected to -2 ℃ low-temperature stress treatment for 12 h. Functional leaves from the same part of the plant were taken, immediately flash-frozen with liquid nitrogen, and stored in an ultra-low temperature freezer at -80 ℃ for the determination of various physiological indicators.

[0096] 6.2 Methods for Measuring Physiological Indicators

[0097] All physiological parameters were measured using the corresponding kits from BOXBIO, following the instructions for each kit. The measured parameters and corresponding kit numbers are as follows:

[0098] Hydrogen peroxide (H2O2) content: Item No. AKAO009M, Malondialdehyde (MDA) content: Item No. AKFA013M, Proline (Pro) content: Item No. AKAM003M, Catalase (CAT) activity: Item No. AKAO003-2M, Peroxidase (POD) activity: Item No. AKAO005M, Superoxide dismutase (SOD) activity: Item No. AKAO001M-50S.

[0099] 6.3 Results

[0100] Results of various physiological indicators as follows Figure 7 As shown, from Figure 7 It can be seen that:

[0101] Malondialdehyde (MDA) content: The MDA content of the overexpression lines OE-2, OE-9, and OE-12 was significantly lower than that of the wild-type ZS11 (p < 0.05). Figure 7 A);

[0102] Hydrogen peroxide (H2O2) content: The H2O2 content of the overexpression lines was significantly lower than that of the wild-type ZS11 (p < 0.05). Figure 7 B);

[0103] Proline (Pro) content: The Pro content of the overexpression lines was significantly higher than that of the wild-type ZS11 (p < 0.05). Figure 7 C);

[0104] Catalase (CAT) activity: The CAT activity of the overexpression lines was significantly higher than that of the wild-type ZS11 (p < 0.05). Figure 7 D);

[0105] Peroxidase (POD) activity: The POD activity of the overexpression lines was significantly higher than that of the wild-type ZS11 (p < 0.05). Figure 7 E);

[0106] Superoxide dismutase (SOD) activity: There was no significant difference between the overexpression lines and wild-type ZS11. Figure 7 F).

[0107] The above results indicate that under low temperature stress, rapeseed plants overexpressing the BnaCT3 gene exhibit lower levels of membrane lipid peroxidation (lower MDA content), less reactive oxygen species accumulation (lower H2O2 content), stronger osmotic regulation (higher Pro content), and higher reactive oxygen species scavenging enzyme activity (higher CAT and POD activities), thus demonstrating stronger frost resistance than the wild type.

[0108] Example 7: Identification and Breeding Application of SNP Markers in the BnaCT3 Gene Promoter

[0109] 7.1 Identification of SNP sites and definition of haplotype

[0110] Sequence analysis of the promoter region of the BnaCT3 gene in a natural population of Brassica napus revealed two key single nucleotide polymorphism (SNP) sites, such as... Figure 8 As shown. The positions of the two SNP sites correspond to the promoter region of Brassica napus BnaA07G0038800ZS, and to positions 3480595 and 3480604 on chromosome A07 of the Brassica napus reference genome ZS11.v0. The polymorphism at position 3480595 is C / T, and the polymorphism at position 3480604 is T / C.

[0111] Based on the base combinations at these two SNP sites, two main allele combinations (haplotypes) were identified, named H1 type and H2 type, respectively.

[0112] H1 type: The 3480595th bit is C, and the 3480604th bit is T;

[0113] H2 type: the 3480595th bit is T, and the 3480604th bit is C.

[0114] 7.2 Correlation analysis between haplotype and frost resistance

[0115] The frost resistance of different haplotype materials in natural populations was evaluated, with overwintering survival rate as the indicator of frost resistance. The results showed that the average survival rate of rapeseed materials carrying the H2 allele combination was significantly higher than that of materials carrying the H1 allele combination (p=0.0066), indicating that the H2 allele combination is the dominant allele combination associated with frost resistance.

[0116] 7.3 Evaluation of the breeding value of BnaCT3

[0117] The haplotypes of the BnaCT3 gene in some currently cultivated and promoted varieties of Brassica napus were analyzed, and the results are shown in Table 4. All the tested varieties carried the H1 type (freezing-resistant but not dominant) allele combination, while the freezing-resistant dominant H2 type was completely absent in the existing varieties.

[0118] Table 4. Allelic typing of different Brassica napus varieties

[0119]

[0120] The above results indicate that the H2 type (T at position 3480595 and C at position 3480604) is a naturally occurring combination of frost-resistant alleles, but it is completely absent in currently cultivated varieties. Therefore, the SNP molecular markers provided in this application (i.e., positions 3480595 and 3480604 on chromosome A07) can be directly used for:

[0121] Screening of freeze-resistant germplasm: Detect the genotypes of the two SNP loci mentioned above in the test materials, and select materials carrying the H2 type as parents for freeze-resistant breeding.

[0122] Molecular marker-assisted selection breeding: By introducing H2 type allele combinations into existing main cultivated varieties through backcrossing or marker-assisted selection of hybrid offspring, their frost resistance can be improved in a targeted manner.

[0123] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. Application of SNP molecular markers in identifying or assisting in the identification of frost resistance in rapeseed, wherein the SNP molecular markers are located in the promoter region of the BnaCT3 gene BnaA07G0038800ZS in Brassica napus, corresponding to positions 3480595 and 3480604 on chromosome A07 of the Brassica napus reference genome ZS11.v0, where the polymorphism at position 3480595 is C / T and the polymorphism at position 3480604 is T / C; the two SNP sites constitute a haplotype, which includes haplotype H1 and haplotype H2; wherein haplotype H1 has C at position 3480595 and T at position 3480604, and haplotype H2 has T at position 3480595 and C at position 3480604; The application comprises: By detecting the BnaCT3 gene BnaA07G0038800ZS promoter region in rapeseed plants, corresponding to the single nucleotide polymorphism sites at positions 3480595 and 3480604 on chromosome A07 of the Brassica napus reference genome ZS11.v0, if the genotype is H2 haplotype, it is determined to be frost-resistant rapeseed; if the genotype is H1 haplotype, it is determined to be non-frost-resistant rapeseed.

2. A method for screening frost-resistant rapeseed, comprising detecting the genotype of the BnaCT3 gene BnaA07G0038800ZS promoter region in the rapeseed plant to be tested, corresponding to the single nucleotide polymorphism sites at positions 3480595 and 3480604 on chromosome A07 of the rapeseed reference genome ZS11.v0, and selecting plants carrying the H2 allele combination as frost-resistant breeding material; wherein the H2 allele is T at position 3480595 and C at position 3480604.

3. A method for breeding cold-resistant rapeseed varieties, comprising introducing the H2 haplotype of the BnaCT3 gene BnaA07G0038800ZS into a target rapeseed variety through molecular marker-assisted selection, wherein the H2 haplotype is: in the promoter region of the BnaCT3 gene BnaA07G0038800ZS, the haplotype corresponding to position 3480595 of chromosome A07 of the rapeseed reference genome ZS11.v0 is T and position 3480604 is C.

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