Characteristic sequence, primer group and application related to low-temperature germination trait of brassica campestris

Abstract

The application provides a characteristic sequence, a primer group and application related to a low-temperature germination resistance character of rapeseed, and belongs to the technical field of molecular marker assisted breeding. BnaC5.MFT The application develops a primer combination aiming at the SNP site, and the primer combination can distinguish the haplotype of the rapeseed variety gene, and can be simply, rapidly and high-throughput applied to screening and identification of the low-temperature germination and seedling growth ability phenotype of the rapeseed variety, and provides important technical support for molecular marker assisted breeding of the low-temperature germination and seedling growth rapeseed variety.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology, and in particular relates to a characteristic sequence, primer set and application related to the low-temperature germination trait of rapeseed. Background Technology

[0002] rape( Brassica napus Rapeseed (L.) is an important oilseed crop. Rapeseed germination and seedling emergence are highly sensitive to temperature, with an optimal germination temperature of 15-20℃. Excessively low temperatures significantly inhibit seed germination and seedling growth. Under multiple cropping systems, the rice-rice-rapeseed rotation is becoming increasingly strained, and the delayed harvest of late rice leads to postponed rapeseed sowing, often resulting in seeds encountering low temperatures after sowing. Low-temperature stress significantly reduces seed germination potential, germination rate, germination index, and vigor index, and prolongs the average germination time, leading to uneven emergence, missing seedlings, and gaps in rows, severely restricting rapeseed yield. Therefore, identifying superior gene resources related to low-temperature germination and seedling emergence in rapeseed, developing efficient and reliable molecular markers, and using molecular marker-assisted selection breeding to cultivate new rapeseed varieties resistant to low-temperature germination and seedling emergence are important ways to solve this industry problem.

[0003] Molecular marker-assisted selection is an important technique in crop breeding. Kompetitive allele-specific PCR (KASP) marker technology, with its advantages of high throughput, low cost, and ease of operation, has been widely used in rapeseed breeding. Currently, KASP markers have achieved good results in identifying early flowering traits, screening for oil content, breeding for resistance to clubroot disease, and constructing varietal fingerprints. For example, researchers have used early flowering genes... BnCRY2 The KASP marker BN900449 was developed based on functional site SNPs, and marker-assisted selection was used to create extra-early maturing rapeseed resources (Wang Churui, Li Kaixiang, Zhao Zhi, et al. Early flowering gene in Brassica napus). BnCRY2 Development and application of KASP markers for functional sites [J]. Acta Agronomica Sinica, 2026, 52(3):708-721.); In addition, some studies have developed KASP markers that co-segregate with major QTLs for secondary dormancy in rapeseed seeds, which are used to precisely improve the secondary dormancy characteristics of rapeseed seeds (Liu Fuxia, Liu Lei, Bai Shuwen, et al. Combining GWAS and WGCNA to mine candidate genes for secondary dormancy in rapeseed seeds [J]. Chinese Journal of Oil Crops, 2025, 47(3):601-615.). However, as of now, no studies have been found that use KASP markers based on rapeseed seeds for secondary dormancy. BnaC5.MFT Reports on the development of KASP markers for low-temperature germination seedlings.

[0004] Current methods for breeding rapeseed seedlings to withstand low temperatures during germination mainly rely on phenotypic identification and conventional breeding techniques, which suffer from problems such as long cycles, low efficiency, and significant susceptibility to environmental influences. Although some studies have focused on the molecular mechanisms of rapeseed cold tolerance, molecular markers for low-temperature tolerance traits during seed germination are still lacking. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a characteristic sequence, primer set, and application related to the low-temperature germination trait of rapeseed. This invention analyzes single-base nucleotide polymorphism variations in the genome of core germplasm resources of the Brassica napus type, uncovering new genetic loci affecting the low-temperature germination and seedling formation of rapeseed, and providing a new method for determining low-temperature germination and seedling formation varieties of rapeseed.

[0006] This invention provides a characteristic sequence related to the low-temperature germination trait of rapeseed, as shown in SEQ ID NO: 1.

[0007] Preferably, the characteristic sequence contains three SNP sites related to the low-temperature germination trait, namely +252A / T, +151A / G, and -204A / G in SEQ ID NO: 1; when the three sites are T, G, and G respectively, the rapeseed exhibits the low-temperature germination seedling phenotype.

[0008] The present invention provides a primer combination for amplifying the feature sequence, comprising a first-round amplification primer and a second-round amplification primer; The first round of amplification primers include those shown in SEQ ID NO: 2. BnaC5.MFT KASP-F and as shown in SEQ ID NO: 3 BnaC5.MFT KASP-R; the first round of PCR amplification fragment was 859bp; The second round of amplification primers includes those for detection. BnaC5.MFT +252 primer set, used for detection BnaC5.MFT +151 primer set and for detection BnaC5.MFT -204 primer set; Used for detection BnaC5.MFT The primer set for +252 is BnaC5.MFT +252 F1-FAM-T, BnaC5.MFT +252F2-HEX-A and BnaC5.MFT +252 R, the sequences are shown in SEQ ID NO: 4~6 respectively, and the amplified fragment is 74bp; Used for detection BnaC5.MFT The primer set for +151 is BnaC5.MFT+ 151F1-FAM-C 、BnaC5.MFT+ 151F2-HEX-T 和BnaC5.MFT+151 R, the sequences are shown in SEQ ID NO: 7~9, and the amplified fragment is 117bp; Used for detection BnaC5.MFT The primer set for -204 is BnaC5.MFT -204 F1-FAM-T 、BnaC5.MFT -204F2-HEX-C 和BnaC5.MFT- 204 R ， The sequences are shown in SEQ ID NO: 10~12, and the amplified fragment is 64bp.

[0009] This invention provides the application of the aforementioned characteristic sequence and primer combination in molecular marker-assisted breeding of rapeseed with low-temperature germination and seedling formation traits.

[0010] This invention provides a method for detecting the ability of rapeseed to germinate and grow seedlings under low temperatures, comprising the following steps: (1) Extract genomic DNA from the rapeseed sample to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the first-round amplification primers in the primer combination to obtain PCR amplification products; (3) Using the PCR amplification product obtained in step (2) as a template, perform real-time PCR amplification with the second-round amplification primers to obtain the second-round amplification product; (4) Detect the fluorescence signal of the second-round amplification product, determine the genotype of the sample at the three loci based on the fluorescence signal, and then determine its haplotype: like BnaC5.MFT +252 position is A, BnaC5.MFT +151 is A, BnaC5.MFT If position -204 is A, then it is BnaC5.MFT 单倍型1 Genotype analysis determined that the rapeseed tested exhibited a phenotype that was intolerant to low-temperature germination and seedling formation. like BnaC5.MFT +252 position is T, BnaC5.MFT +151 site is G, BnaC5.MFT If position -204 is G, then it is BnaC5.MFT 单倍型2 The genotype was determined to be that the rapeseed tested exhibited a low-temperature germination and seedling phenotype.

[0011] This invention provides a method for screening rapeseed varieties that are resistant to low-temperature germination and seedling formation. The method uses the primer combination to detect the genotype of three SNP sites on the characteristic sequence in the rapeseed germplasm, and selects rapeseed varieties with T, G, and G at the three sites as target varieties that are resistant to low-temperature germination and seedling formation.

[0012] This invention provides a kit for detecting the ability of rapeseed to germinate and grow seedlings under low temperature, including the primer combination mentioned above.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a characteristic sequence related to the low-temperature germination trait of rapeseed, as shown in SEQ ID NO: 1; the characteristic sequence is located at BnaC5.MFT Genetically, this invention utilizes genome-wide association analysis (GWAS) based on single nucleotide polymorphisms (SNPs) to identify three SNP sites on the characteristic sequence that are significantly associated with low-temperature tolerance for germination and seedling formation in rapeseed. Primer combinations based on these three SNP sites were developed for detecting single nucleotide polymorphisms. These primer combinations can be used to detect rapeseed varieties at any time and in any tissue. BnaC5.MFT By differentiating and identifying haplotypes of genes, rapeseed varieties with good low-temperature germination and seedling emergence ability can be quickly screened.

[0014] The method for detecting the ability of rapeseed to germinate and grow seedlings under low temperature provided by this invention is convenient and fast, and is not limited by weather and environmental conditions. The primer combination has the characteristics of convenient detection, stable amplification products, and high specificity. It can be applied to the screening and identification of phenotypic traits of rapeseed varieties to germinate and grow seedlings under low temperature in a simple, rapid and high-throughput manner, providing important technical support for molecular marker-assisted breeding of rapeseed varieties that are resistant to low temperature germination and seedling growth. Attached Figure Description

[0015] Figure 1 The analysis results of low-temperature germination tolerance of 273 rapeseed germplasms are shown, where A is the frequency histogram of the low-temperature germination index, B is the Manhattan plot based on genome-wide association analysis (GWIA) of single nucleotide polymorphisms (SNPs), C is the QQ plot based on GWIA of SNPs, and D is... qLTGI-4 Local Manhattan plot of 5Mb upstream and downstream of the site, with E as the candidate gene. BnaC5.MFT Local Manhattan plots of internal and upstream / downstream 2kb variant sites, F is a linkage disequilibrium heatmap, and G is... BnaC5.MFT Haplotype analysis in core germplasm resources, and H represents the difference in low-temperature germination index among different haplotypes.

[0016] Figure 2 The results of the analysis of different haplotype germplasms are shown, where A represents the selected haplotypes. BnaC5.MFTA shows a schematic diagram of the three SNP sites; B shows the difference analysis of low-temperature germination rate and low-temperature germination index among different haplotypes of the 71 selected rapeseed germplasm resources; C shows the results of the first round of routine PCR amplification of the 71 germplasm resources; D shows the difference analysis of low-temperature germination rate and low-temperature germination index among different haplotypes of another 48 validation germplasm resources; E shows the results of the first round of routine PCR amplification; and F shows the typing results of FAM and HEX fluorescence quantitative PCR obtained from three sets of KASP primers.

[0017] Figure 3 The results of validation for 48 germplasm resources are presented, where A represents the phenotypic validation results of some germplasm resources among the 48 validated germplasm resources, and B represents the analysis of low-temperature germination rate and low-temperature germination index among germplasm resources. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1

[0020] Development of molecular markers related to low-temperature germination and seedling formation in rapeseed

[0021] This invention analyzed 273 natural populations of Brassica napus (see Table 1 for the germplasm list). The germination index (LTGI) of the rapeseed was determined using an artificial climate incubator with low-temperature conditions of 10℃ / 5℃ (12 hours light / 12 hours darkness, ℃ being degrees Celsius), a light intensity of 20,000 lux (lux being illuminance), and a relative humidity of 60%. (gi represents the number of seeds that germinate on day i, and ti represents the number of seeds germinated on day i after sowing), to measure the germination and seedling emergence ability of rapeseed at low temperatures. The germination index of rapeseed under low-temperature conditions was measured, and it was found that the variation range of the germination index under low-temperature conditions was 10.93-30.09, with an average value of 19.73. Figure 1 GWAS mapping analysis was performed on the low-temperature germination index trait based on single nucleotide polymorphisms (SNPs) in the genome, with the threshold P for screening significantly associated sites set at 3.32 × 10⁻⁶. -7 (1 / 3010041), and defined a QTL as a variant site within a 75kb interval. Ultimately, six sites were identified in rapeseed, such as... Figure 1 As shown, where qLTGI-4 It contains 622 variant sites, forming a continuous accumulation peak. This site is located on chromosome C5 of the rapeseed reference genome ZS11.v0, covering a genomic region of approximately 500kb. Figure 1 ). Specific examination of candidate genes BnaC5.MFTAfter identifying variant sites within 2kb upstream and downstream of the gene, a dense distribution and strong linkage of these variant sites were found. Using the most significant SNP (C5_9747315) as the core site, a total of 16 strongly linked (…) r 2 The variant sites were >0.6, including 13 SNPs and 3 InDels ( Figure 1 These sites are mostly distributed in promoter and intron regions, with only the core site (C5_9747315) located in... BnaC5.MFT On the first exon, and replace the 51st amino acid from serine with proline ( Figure 1 (S51P). Haplotype analysis revealed that... BnaC5.MFT In the natural population, it was mainly divided into two haplotypes, with haplotype 1 being the majority haplotype and haplotype 2 being the minority haplotype, both exhibiting low-temperature germination phenotypes. The genotypes of 162 materials in the population showed the following expression: BnaC5.MFT 单倍型1 39 materials showed the following genotypic expression: BnaC5.MFT 单倍型2 Further analysis revealed differences in low-temperature germination indices between materials possessing two different haplotypes within the population, and found that... BnaC5.MFT 单倍型1 Compared to materials, it has BnaC5.MFT 单倍型2 The materials have a significantly higher low-temperature germination index ( Figure 1 ).illustrate BnaC5.MFT Natural variations in the genome sequence are closely related to the low-temperature tolerance and germination rate of Brassica napus seedlings.

[0022] Finally, the core locus (C5_9747315, hereinafter referred to as...) was selected. BnaC5.MFT +151) and two sites highly linked to the core site (C5_9747669, hereinafter referred to as... BnaC5.MFT -204; C5_9747214, hereinafter referred to as BnaC5.MFT +252), a total of 3 SNP sites were used for molecular marker development ( Figure 1 The nucleotide sequence of the SNP site is shown in SEQ ID NO: 1 below. BnaC5.MFT +252 is A and BnaC5.MFT +151 is A and ​ When -204 is A, that is, as ​ 单倍型1 Rapeseed varieties. When ​ +252 is T and ​ +151 is G and ​ -204 is G, that is, as ​ 单倍型2 rapeseed germplasm ( ​The violin plot of the low-temperature germination index corresponding to the population genotype is shown below. ​ Among them, the genotype is ​ 单倍型1 The rapeseed variety exhibits a phenotype of intolerant to low-temperature germination and seedling formation, with the genotype being: ​ 单倍型2 The rapeseed varieties exhibited a low-temperature resistant germination and seedling emergence phenotype, and their genotype was... ​ 单倍型2 The low-temperature germination index of rapeseed varieties was significantly higher than that of genotype [missing information]. ​ 单倍型1 The rapeseed varieties, that is, the two varieties, showed significant differences in their ability to germinate and grow seedlings in low temperatures.

[0023] Specifically, the nucleotide polymorphism is contained in the following 859 bp sequence amplified by the first round of PCR, as shown in SEQ ID NO: 1. In SEQ ID NO: 1... ​ The start codon T is +1, where ​ A single nucleotide polymorphism (SNP) of A / T exists at position +252, A / G exists at position +151, and A / G exists at position -204. Therefore, ​ 单倍型1 It is a combination of single nucleotides: +151A, +252A, and -204A. ​ 单倍型2 The sequences are single nucleotide combinations of +151T, +252G, and -204G. The underlined sequences at the beginning and end are SEQ ID NO: 2 and SEQ ID NO: 3 sequences used in the first round of PCR amplification. The underlined sequences are the sequences coupled to the three sets of KASP fluorescent tags in the second round (SEQ ID NO: 4 and 5, 7 and 8, 10 and 11).

[0024] ​ AACTCCAAAAAAACCATGAACTGAAAAGTCAGTGAAAATACCAGCATATATTAAAGAATGAATGCATGTTCCTGTGGGCTGGTATAATCATGATCATTGAACAAGAGGAAGAGGAAACATGTAACGCATGCGTGTGTATATTCAAAGATATGTTTGC[+252A / T] ​ TATAAGTGATAAGATGAATAAATAAATATACGAGGGTGTAAAGCTCATCAGAGTTGCCGGAGATGTTGACTTTGGGAG[+151A / G] ​GGGTTTGATCTCGCAGCCGTTAGTGATGTGTTTGGGGCCAACGTAGACTGACATGTTGGCAGTGGGGATGTACATGTCCAACACATCTCCAATCACTCTTCCAACCACCAAAGGATCAACCGAAGCTGCTGC ​ CGATCCACGGAAAGAGAAAGAGAAGTAGAGATGGAGAAAGAGAGAACTAGAGATTCTTTTATTTATAGAAGAAGAATCGTTGGGCGCATGTCCTAATTCGGATGGACATCACTGTAATGATTTATATATAGAGATAAATTTTCTTACAACAAAAAATAATATTAAATTGCATCGACAAATACAATGAATTTGAACAATGAT[-204A / G] ​ ​ GTTAAACTATAATACACTTCGATGTTTTCTTTTGTCTCTGCGTGTTTGCGTTTTATATAGGGCCGGGCAAAATATCTAGAACCGAAGAACCGATCTCAGGAGGATCCTAATTGATGTATCCGAAGAACCGGTACCTAATCCGATCTGGACTGAAAACCA ​ (SEQ ID NO: 1)

[0025] Because rapeseed (Brassica napus) is an allotetraploid species, its genome is highly complex, containing numerous repetitive sequences and multicopy gene families. This invention targets... ​ The gene has highly homologous copies in the rapeseed genome (e.g. ​ If rapeseed genomic DNA is used directly as a template for KASP genotyping amplification, the following technical problems will be encountered: 1) Cross-amplification between homologous sequences is prone to producing non-specific products, leading to errors in fluorescence signal interpretation; 2) KASP reaction requires high purity and concentration of template DNA, but residual polysaccharides and polyphenols in rapeseed leaves may inhibit polymerase activity, often resulting in weak signals or amplification failure during direct amplification; 3) Even with optimized reaction system, direct KASP is difficult to efficiently distinguish three SNP sites simultaneously. To solve the above problems, this invention, after extensive experimentation, innovatively adopts a method of first using conventional PCR primer pairs (… ​The first round of amplification of KASP-F / R (SEQ ID NO: 2~3) yielded an 859bp target DNA fragment containing three SNP sites. Using the first-round PCR product as a template, qualitative and quantitative typing of KASP was performed, achieving for the first time the identification of a specific copy of this gene. ​ The efficient and accurate detection of SNP sites significantly improves the accuracy and stability of genotyping, enabling high-throughput and low-cost detection of low-temperature germination and seedling traits.

[0026] The primer sequences are as follows.

[0027] ​ KASP-F: TTAATTTCAGATATCTTCGGATACTCATT (SEQ ID NO: 2)

[0028] ​ KASP-R: GTAGTCCAATATTTAGCTAAACCGTGTCT (SEQ ID NO: 3)

[0029] The primers were used to perform the first round of routine PCR amplification on the test samples.

[0030] For each SNP locus, a KASP detection primer set was designed, consisting of three primers: two allele-specific forward primers and one universal reverse primer. The 5' ends of the two forward primers are each linked to a different fluorescent adapter sequence to distinguish between the two different allele loci. The specific primer design and assembly are as follows.

[0031] Used for detection ​ The primer set at +252 position is ​ +252 F1-FAM-T, ​ +252 F2-HEX-A and ​ +252 R.

[0032] ​ +252 F1-FAM-T: ​ TCCATTTATGTGCAATCTCCCTT

[0033] (SEQ ID NO: 4)

[0034] ​ +252 F2-HEX-A: ​ TCATTTATGTGCAATCTCCCTA (SEQ ID NO: 5)

[0035] ​+252 R:GGAAGAGGAAACATGTAACGACA (SEQ ID NO: 6)

[0036] Used for detection ​ The primer set at +151 site is ​ +151 F1-FAM-C ​ +151 F2-HEX-T and ​ +151 R.

[0037] ​ +151 F1-FAM-C: ​ TCCGTCGCAGTCAACC (SEQ IDNO: 7)

[0038] ​ +151 F2-HEX-T: ​ TCCGTCGCAGTCAACT (SEQ IDNO: 8)

[0039] ​ +151 R: TGAATAAATAAATATACGAGGGTGTAAAGC (SEQ ID NO: 9)

[0040] Used for detection ​ The primer set at position -204 is ​ -204 F1-FAM-T, BnaC5.MFT -204 F2-HEX-C and BnaC5.MFT -204 R.

[0041] BnaC5.MFT -204 F1-FAM-T: GAAGGTGACCAAGTTCATGCT CGCGAAAAATATCAAATAAAATGGACTGT (SEQ ID NO: 10)

[0042] BnaC5.MFT -204 F2-HEX-C: GAAGGTCGGAGTCAACGGATT CGCGAAAAATATCAAATAAAATGGACTGC (SEQ ID NO: 11)

[0043] BnaC5.MFT -204 R: TGCATCGACAAATACAATGAATTTGA (SEQ ID NO: 12)

[0044] The underlined sections indicate fluorescent linker sequences, while the bolded sections represent SNP sites.

[0045] Example 2

[0046] The application of a KASP molecular marker associated with the low-temperature germination index in rapeseed in identifying the low-temperature tolerance for germination and seedling formation includes the following steps:

[0047] (1) DNA extraction

[0048] We selected 71 rapeseed varieties out of 273 (the varieties in bold in Table 1) and extracted their genomic DNA using conventional methods.

[0049] (2) Sequence amplification

[0050] Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO: 2 and 3 according to the following PCR reaction system: The PCR reaction system was 10 μl, including 5 μl of 2×Taq PCR Mix, 0.2 μl of 10 μM forward primer, 0.2 μl of 10 μM reverse primer, 0.5 μl of the extracted genomic DNA template, and sterile water was added to bring the total to 10 μl. The PCR reaction was performed on a conventional PCR amplification instrument, with the following reaction program: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, for a total of 35 cycles; and a final extension at 72℃ for 2 min. After PCR amplification, 1 μl of the amplification product was analyzed by 1% agarose gel electrophoresis. By comparing with the DNA molecular weight standard, the amplified target fragment was 859 bp, thus determining whether the fragment was correctly amplified. The PCR product was used for subsequent experimental operations. Figure 2 ).

[0051] (3) Genotyping

[0052] The first-round PCR products were used for the second round of KASP PCR amplification. Three sets of KASP primers (SEQ ID NO: 4-6, 7-9, 10-12) were used for quantitative real-time PCR amplification. Each test was performed in at least four technical replicates. The reaction system included 5 μl of 2×KASP Master Mix, 0.25 μl each of 10 μM forward primers F1 and F2, 0.75 μl of 10 μM reverse primer R, 0.2 μl of the PCR product from step (2), and sterile water to a final volume of 10 μl. The reaction program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, annealing / extension at 61℃-55℃ (decreasing by 0.6℃ per cycle) for 40 s, for a total of 10 cycles; 95℃ denaturation for 20 s, annealing / extension at 55℃ for 40 s, for a total of 28 cycles; and finally, fluorescence scanning at 30℃ for 30 s. FAM uses 485 nm excitation for fluorescence, while HEX uses 535 nm. KASP genotyping using other unrelated DNA templates or without a DNA template is considered a negative control. RFU stands for Relative Fluorescence Unit in qPCR (real-time quantitative PCR), used to detect fluorescence signal intensity in real time, reflecting the accumulation of target products during DNA amplification. Its value is directly proportional to the signal released by the fluorescent probe or dye and is a key indicator for determining whether amplification has occurred.

[0053] (4) Result determination

[0054] To avoid primer misuse, specifically BnaC5.MFT The three haplotype 1 SNP sequences were labeled with two different fluorescent markers, instead of using the same fluorescent marker. When using... BnaC5.MFT +252 F1-FAM-T, BnaC5.MFT +252F2-HEX-A and BnaC5.MFT The second round of KASP typing was performed using the +252 R (SEQ ID NO: 4-6) primer set. The fragment size should be 74 bp (excluding fluorescent sequences and not displayed after gel electrophoresis). If only FAM fluorescence signal is present, it indicates that... BnaC5.MFT The +252 site indicates homozygous haplotype 1; the presence of only HEX fluorescence signal indicates... BnaC5.MFT The +252 site indicates a homozygous haplotype 2; if both FAM and HEX fluorescence signals are present simultaneously, it means... BnaC5.MFT The +252 site indicates a heterozygous genotype; the absence of fluorescence indicates genotyping failure; the signal of the negative control is close to the 0 position on both the x and y axes.

[0055] When using BnaC5.MFT +151 F1-FAM-C BnaC5.MFT +151 F2-HEX-T andBnaC5.MFT The +151 R (SEQ ID NO: 7-9) primer set was used for the second round of KASP typing. The fragment size should be 117bp (excluding fluorescent sequences, not displayed after gel electrophoresis). If only HEX fluorescence signal appears, it indicates that... BnaC5.MFT The +151 site indicates homozygous haplotype 1; if only FAM fluorescence signal is present, it indicates that... BnaC5.MFT +151 site indicates homozygous haplotype 2; if both HEX and FAM fluorescence signals are present simultaneously... BnaC5.MFT The +151 site indicates a heterozygous genotype; the absence of fluorescence indicates genotyping failure; the signal of the negative control is close to the 0 position on both the x and y axes.

[0056] When using BnaC5.MFT -204 F1-FAM-T, BnaC5.MFT -204 F2-HEX-C and BnaC5.MFT The second round of KASP typing was performed using the -204 R (SEQ ID NO: 10-12) primer set. The fragment size should be 64 bp (excluding fluorescent sequences and not displayed after gel electrophoresis). If only FAM fluorescence signal appears, it indicates that... BnaC5.MFT -204 is a homozygous haplotype 1; if only HEX fluorescence signal is present, it indicates... BnaC5.MFT -204 is a homozygous haplotype 2; if both FAM and HEX fluorescence signals are present simultaneously, it indicates... BnaC5.MFT -204 locus indicates heterozygosity; if no fluorescence is observed, the genotyping has failed; the signal of the negative control is close to the 0 position on both the x and y axes.

[0057] The PCR fragments amplified in the second round of KASP are relatively short, making it difficult to distinguish their size during gel electrophoresis. In KASP, a single FAM or HEX fluorescence signal is located near the x or y axis; a mixed fluorescence signal of two FAM and HEX signals is located near the y=x axis (i.e., at a 45-degree angle); no signal or amplification failure signal is located near the 0 position of the x and y axis coordinates. Therefore, the above method can be used to... BnaC5.MFT The KASP signals of the top three SNPs are used to determine their haplotypes, which are then used to determine the phenotype of low-temperature germination tolerance. KASP is used to determine... BnaC5.MFT 单倍型1 Homozygous genotypes are identified as germplasm with poor cold-hardy germination and seedling emergence ability. This is determined by KASP. BnaC5.MFT 单倍型2 The homozygous genotype was determined to be a germplasm with good low-temperature germination and seedling formation ability.

[0058] (5) Verification results

[0059] This study developed markers in 71 rapeseed varieties, and ultimately selected 48 varieties for result validation (as shown in Table 1, the bolded marker varieties are the 71 rapeseed germplasms used for marker development, and the underlined marker varieties are the 48 rapeseed germplasms used for result validation). Among these, 24 rapeseed varieties were identified as homozygous using KASP combinatorial markers. BnaC5.MFT 单倍型1 The genotypes of the other 24 rapeseed varieties were determined to be homozygous using KASP combinatorial markers. BnaC5.MFT 单倍型2 (See Table 1 for details) (rapeseed germplasm). The T-test showed that (ns indicates no significance). It is 0.001< P ≤0.01, yes P ≤0.0001), BnaC5.MFT 单倍型1 and BnaC5.MFT 单倍型2 Significant differences were found in the germination phenotypes of the low-temperature tolerant varieties. The detection results were consistent with the genotypes and actual low-temperature germination and seedling formation traits. Figure 2 A selection of materials was used for standard germination testing to verify the results, and the phenotypic observations were conducted. Figure 3 Consistent with the genotype prediction results, this confirms that R5049 and R5039 are... BnaC5.MFT 单倍型1 R4845 and R5044 are BnaC5.MFT 单倍型2 Furthermore, the low-temperature germination ability of R5049 and R5039 is inferior to that of R4845 and R5044. The above experiments confirm the accuracy and reliability of the molecular markers of the present invention.

[0060] Table 1. Germplasm names of 273 accessions of Brassica napus

[0061] The above-mentioned germplasm is core rapeseed germplasm from different regions around the world. See the literature “The intronic structure variation of rapeseed BnaC3.LEAFY regulates the timing of inflorescence formation and flowering” (Gong et al., Plant communications 2025) and “Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals the Genetic Basis of Ecotype Divergence” (Wu et al., 2019 Molecular plant).

[0062] The above results fully demonstrate that the three sets of molecular marker primers provided by this invention can accurately, stably, and efficiently identify the dominant haplotypes of rapeseed traits related to low-temperature germination and seedling formation.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A characteristic sequence related to the low-temperature germination trait of rapeseed, characterized in that, The characteristic sequence is shown in SEQ IDNO:

1.

2. The feature sequence according to claim 1, characterized in that, The characteristic sequence contains three SNP sites associated with low-temperature germination traits, namely +252A / T, +151A / G, and -204A / G in SEQ ID NO: 1; when the three sites are T, G, and G respectively, the rapeseed exhibits the low-temperature germination seedling phenotype.

3. A primer combination for amplifying a sequence containing the characteristic sequence of claim 1, characterized in that, Including primers for the first round of amplification and primers for the second round of amplification; The first round of amplification primers include those shown in SEQ ID NO:

2. BnaC5.MFT KASP-F and as shown in SEQ ID NO:3 BnaC5.MFT KASP-R; The fragment amplified by the first round of PCR was 859bp; The second round of amplification primers includes those for detection. BnaC5.MFT +252 primer set, used for detection BnaC5.MFT +151 primer set and for detection BnaC5.MFT -204 primer set; Used for detection BnaC5.MFT The primer set for +252 is BnaC5.MFT +252 F1-FAM-T, BnaC5.MFT +252 F2-HEX-A and BnaC5.MFT +252 R, the sequences are shown in SEQ ID NO: 4~6 respectively, and the amplified fragment is 74bp; Used for detection BnaC5.MFT The primer set for +151 is BnaC5.MFT+ 151F1-FAM-C BnaC5.MFT+ 151 F2-HEX-T and BnaC5.MFT+ 151 R, the sequences are shown in SEQ ID NO: 7~9, and the amplified fragment is 117bp; Used for detection BnaC5.MFT The primer set for -204 is BnaC5.MFT -204 F1-FAM-T BnaC5.MFT -204 F2-HEX-C and BnaC5.MFT- 204 R ， The sequences are shown in SEQ ID NO: 10~12, and the amplified fragment is 64bp.

4. The application of the characteristic sequence as described in claim 1 or 2 and the primer combination as described in claim 3 in molecular marker-assisted breeding of rapeseed with low-temperature germination and seedling formation traits.

5. A method for detecting the ability of rapeseed to germinate and grow seedlings under low temperatures, characterized in that, Includes the following steps: (1) Extract genomic DNA from the rapeseed sample to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the first-round amplification primers in the primer combination described in claim 3 to obtain PCR amplification products; (3) Using the PCR amplification product obtained in step (2) as a template, the second round of amplification products are obtained by performing real-time PCR amplification using the second round amplification primers described in claim 3. (4) Detect the fluorescence signal of the second-round amplification product, determine the genotype of the sample at the three loci based on the fluorescence signal, and then determine its haplotype: like BnaC5.MFT +252 position is A, BnaC5.MFT +151 is A, BnaC5.MFT If position -204 is A, then it is BnaC5.MFT 单倍型1 Genotype was used to determine the phenotype of rapeseed that was intolerant to low temperatures during germination and seedling formation. like BnaC5.MFT +252 position is T, BnaC5.MFT +151 site is G, BnaC5.MFT If position -204 is G, then it is BnaC5.MFT 单倍型2 Genotype was used to determine the low-temperature germination and seedling phenotype of the rapeseed to be tested.

6. A method for screening rapeseed varieties that can germinate and grow seedlings under low temperatures, characterized in that, The genotypes of three SNP sites on the characteristic sequences described in claim 1 or 2 in rapeseed germplasm were detected using the primer combination described in claim 3. Rapeseed varieties with T, G, and G sites were selected as target varieties for low-temperature germination and seedling formation.

7. A reagent kit for detecting the ability of rapeseed to germinate and grow seedlings under low temperatures, characterized in that, Includes the primer combination described in claim 3.

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