A molecular marker significantly related to soybean protein content, a kasp detection method and application thereof

Abstract

The application provides a molecular marker significantly related to soybean protein content, a KASP detection method and application. The nucleotide sequence of the molecular marker significantly related to soybean protein content is shown as SEQ ID NO. 1, wherein S at the 23th bp represents G or C. The KASP primer pair for detecting the molecular marker significantly related to soybean protein content contains sequences shown as SEQ ID NO. 2-4. The molecular marker and kit provided by the application can be directly used for early generation screening of soybean high-protein varieties in molecular breeding, shortens the breeding cycle, improves the selection efficiency, and accelerates the breeding process of excellent varieties.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and crop genetic breeding technology, and particularly relates to a molecular marker significantly related to soybean protein content, a KASP detection method and application thereof. BACKGROUND

[0002] Soybean is one of the most important crops for both food and oil in the world, and its yield and quality are directly related to agricultural production and economic value. Important agronomic traits of soybean, such as yield, disease resistance, quality (protein content, oil content), growth period (flowering period, maturation period), etc., are mostly complex traits controlled by multiple genes / QTLs (quantitative trait loci).

[0003] At present, the genotyping techniques of soybean include SSR (simple sequence repeat), chip, resequencing and PCR-based methods, etc. The KASP (Kompetitive Allele Specific PCR) technology is a new type of fluorescent PCR genotyping technology, which has the outstanding advantages of high throughput, high precision, low cost, high degree of automation, etc. It determines the genotype by fluorescence signal in the PCR process through two specific primers for different alleles and one common primer, and is very suitable for molecular marker-assisted selection (MAS) of large-scale breeding materials.

[0004] At present, there is no report on the identification of SNP markers related to protein content in soybean genes and the development of KASP markers for assisted breeding in the prior art. SUMMARY

[0005] One object of the present application is to provide a molecular marker significantly related to soybean protein content, another object of the present application is to provide primers and kits for detecting the molecular marker significantly related to soybean protein content, and a third object of the present application is to provide the application of the above-mentioned primer set or kit in soybean molecular marker-assisted breeding, germplasm identification, genetic diversity analysis and variety purity detection.

[0006] To achieve the above objects, the present application adopts the following technical solutions:

[0007] A molecular marker significantly related to soybean protein content, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and S at the 23rd bp represents G or C, wherein the soybean protein content of soybean carrying CC genotype is significantly higher than that of soybean carrying GG genotype.

[0008] A KASP detection primer pair for detecting a molecular marker significantly related to soybean protein content, comprising a FAM-labeled forward primer F1, a HEX-labeled forward primer F2 and a universal reverse primer R, the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the universal reverse primer R is shown as SEQ ID NO. 4.

[0009] A kit for detecting a molecular marker significantly related to soybean protein content, the kit comprising a KASP detection primer pair, the KASP detection primer pair comprising a FAM-labeled forward primer F1, a HEX-labeled forward primer F2 and a universal reverse primer R, the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the universal reverse primer R is shown as SEQ ID NO. 4.

[0010] The kit as described above, further comprising a KASP Master Mix, a PCR reaction buffer, a negative control, and a positive control, wherein the positive control is an allele Hap1, the sequence of which is shown as SEQ ID NO. 1, and the S at the 23rd bp is G; and an Hap2 sequence shown as SEQ ID NO. 1, and the S at the 23rd bp is C; and the negative control is nuclease-free water.

[0011] A method for detecting a molecular marker significantly related to soybean protein content, comprising the following steps:

[0012] (1) extracting genomic DNA of a soybean sample to be tested;

[0013] (2) using the extracted DNA as a template, performing PCR amplification using the KASP marker primer pair as described above or the kit described above;

[0014] (3) performing fluorescence detection on the PCR amplification product, determining the genotype and protein content of the amplification product of the sample to be tested by analyzing the fluorescence signal intensity of FAM and HEX; when the genotype of the nucleotide sequence at the 23rd bp of the amplification product is CC genotype, the soybean protein content is significantly higher than that of the soybean carrying GG genotype. Further, judging the genotyping and protein content of the soybean according to the fluorescence detection result after amplification includes: if the universal tag FAM fluorescence linker sequence is detected, the genotype is GG, and the corresponding soybean protein content is low; if the universal tag HEX fluorescence linker sequence is detected, the genotype is CC, and the corresponding soybean protein content is high.

[0015] The application of the molecular marker, the KASP detection primer pair or the detection kit or the method for detecting the molecular marker significantly related to the soybean protein content as described above in breeding.

[0016] Specifically, the breeding purpose is to screen soybean species with high soybean protein content.

[0017] The KASP detection primer pair or the detection kit as described above is applied in the detection and prediction of high soybean protein content.

[0018] The application has the following beneficial effects:

[0019] The molecular marker significantly related to the soybean protein content provided by the application has two alleles of G and C, and the protein content and the hundred-grain weight of the C allele are significantly higher than those of the G allele.

[0020] The primer pair for detecting the molecular marker significantly related to the soybean protein content provided by the application has high specificity and accuracy, and the consistency of the typing results and the sequencing results is 98% through a large number of experiments.

[0021] The method for detecting the molecular marker significantly related to the soybean protein content provided by the application is efficient and rapid, and is compatible with 384-well plates or even higher throughput by using the KASP technology, so that the detection of thousands of samples can be completed within 2-3 hours, and the efficiency of breeding selection is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a Manhattan plot of protein content and SNP;

[0023] Figure 2 It is a QQ plot according to the GWAS association result;

[0024] Figure 3 It is the typing detection result of the KASP marker on different genotypes of soybean samples in Example 2;

[0025] Figure 4 It is the typing detection result of the KASP marker on different genotypes of soybean samples in Example 3;

[0026] Figure 5 It is a comparative box plot of the phenotypic difference of soybean varieties carrying different alleles in protein content. DETAILED DESCRIPTION

[0027] The present application is based on whole genome association analysis (GWAS) to locate genetic loci significantly associated with soybean protein content and identify key SNP variations that can represent excellent alleles; then using allele-specific competitive PCR (KASP) technology, the SNP is designed as a specific primer system that can be distinguished by fluorescence, and the different genotypes are accurately determined by competitive amplification. Relying on the stable genetic association between the target site and protein content, the KASP marker developed by the present application can be used for rapid and accurate genotyping and auxiliary screening of high-protein breeding materials to achieve efficient genetic improvement of traits.

[0028] The following examples are used to further illustrate the present application, but should not be construed as limiting the present application. Modifications or substitutions made to the present application without departing from the spirit and essence of the present application shall fall within the scope of the present application.

[0029] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art, and the reagents used in the examples are analytical pure or above.

[0030] Example 1: Selection of soybean protein content significantly associated SNP and design and synthesis of KASP marker primer

[0031] Soybean population selection and phenotype identification: 1200 domestic and foreign natural population resources of Sanya Beinongchuangxin Gene Technology Co., Ltd. were used to collect soybean leaves in the 2024 growing season for whole genome resequencing, and the seeds were harvested for protein content determination using the Xingchuangzong G1200 protein determination instrument. Then, the genotype and protein content phenotype of the soybean materials were analyzed by whole genome association analysis (GWAS) using the PLINK software linear regression model, and the association results are shown in Table 1. Figure 1 As shown in Table 1, there is a region on the soybean Chr11 chromosome significantly associated with protein content, and 26 significant SNP loci are obtained on Chr11, and it is determined that there is a significant non-synonymous SNP locus (p=4.7355e-07) at position 4903434 on the soybean Chr11 chromosome.

[0032] Primer design: According to the KASP primer design principle, using snpgene software, the sequence of 100bp upstream and downstream of the non-synonymous SNP locus at position 4903434 on the soybean Chr11 chromosome as the target gene, allele-specific primers and universal reverse primers were designed. After verifying the specificity of the primers by Blast, they were synthesized by Shengong Biotechnology (Shanghai) Co., Ltd.

[0033] The designed specific primers and universal reverse primers are as follows:

[0034] Allele 1 specific forward primer F1 (FAM labeled) is recorded as FAM primer (SEQ ID NO. 2): 5'- GAAGGTGACCAAGTTCATGCTTTGGAAATGAACGCAAACTTGAG-3', and FAM labeled at the 5' end of the sequence.

[0035] Allele 2 specific forward primer F2 (HEX labeled) is recorded as HEX primer (SEQ ID NO. 3): 5'- GAAGGTCGGAGTCAACGGATTTTGGAAATGAACGCAAACTTGAC-3', and HEX labeled at the 5' end of the sequence. Universal reverse primer R is recorded as reverse primer (SEQ ID NO. 4): 5'- CATTGCCAGCCTATCCTTTCTT-3'. The sequence of the primer amplification product is shown in SEQ ID NO. 1, and S at the 23rd bp represents G or C. SEQ ID NO. 1: TTGGAAATGAACGCAAACTTGASTCCGAAAGAAAGGATAGGCTGGCAATG. Among them, as Figure 1 and Figure 2 Manhattan plot and QQ plot of association results were drawn using R language according to GWAS association results, Figure 1 The X axis of the Manhattan plot represents the chromosome region, and the Y axis represents -log10(p). The red dotted line represents the significance threshold. There are multiple SNPs in the Chr11 region that exceed the threshold. Figure 2 The X axis (expected -log 10 (P value)) of the QQ plot represents the expected P value negative logarithm under the condition of no association. It represents the level of "random noise". The Y axis (observed -log 10 (P value)): represents the actually observed P value negative logarithm. It represents the level of "real signal + noise". The QQ plot represents the deviation level of observed p value and expected p value. It is shown that the overall level of the association results is good. The QQ plot shows that the reliability of the association results is high.

[0036] Example 2 Verification of effectiveness of KASP marker

[0037] Selection of KASP verification population: 16 soybean germplasm resources with known genotypes (known by resequencing) (10 of which are Hap I GG homozygous, and 6 are Hap II CC homozygous) were randomly selected as verification materials and protein content was measured.

[0038] DNA extraction: The genomic DNA of the above soybean young leaf was extracted by CTAB method, and the concentration and purity were determined by NanoDrop, mainly measuring the absorbance values A260 and A280 at 260 nm and 280 nm, when A260 / A280 was between 1.7-2.0, and it was diluted to the working concentration (50 ng / µL) as KASP detection DNA template.

[0039] and the primer F1, F2, R synthesized in Example 1 were diluted to the working concentration 100 µmol / mL, and the primer configuration was mixed according to the volume ratio: F1:F2:R:water=12:12:30:46 to obtain the KASP primer mixture.

[0040] KASP-PCR reaction: reaction system (5 μL): DNA template 1.5 µL, KASP mix 2.5 µL, KASP primer mixture (F1+F2+reverse primer) 0.07 µL, ddH2O to 5 µL; the reaction program is shown in Table 1.

[0041] Table 1 Reaction program

[0042]

[0043] Result detection and analysis: after the reaction was completed, the fluorescence signal was collected using the software of QuantStudio 5 real-time fluorescent PCR instrument, and cluster analysis was carried out. The results are shown in Figure 3 , the KASP typing results of all 16 samples are completely consistent with the known WGS sequencing genotype results, the accuracy reaches 100%, which proves that the KASP marker primer designed in the application has high reliability and accuracy.

[0044] The protein content of soybean was determined by Star Creative Spectrum G1200 protein determination instrument; the average protein content of all GG genotypes was 40.01%; the average protein content of all CC genotypes was 47%; the Welch's t-test statistical test reached the significant difference level (p=0.00020); the protein content of the CC genotype soybean population was significantly higher than that of the GG genotype soybean population.

[0045] Example 3

[0046] The KASP kit for detecting the protein content related molecular marker of soybean comprises a KASP marker primer pair, a KASP Master Mix, a PCR reaction buffer, a positive control DNA and a negative control (nuclease-free water). The KASP marker primer pair comprises an allele F1 specific forward primer (FAM labeled) FAM primer (SEQ ID NO. 2):

[0047] 5'- GAAGGTGACCAAGTTCATGCTTTGGAAATGAACGCAAACTTGAG-3', and FAM labeled at the 5' end of the sequence.

[0048] Allele 2 specific forward primer F2 (HEX labeled) is noted as HEX primer (SEQ ID NO. 3): 5'- GAAGGTCGGAGTCAACGGATTTTGGAAATGAACGCAAACTTGAC-3', and HEX labeled at the 5' end of the sequence.

[0049] Universal reverse primer R is noted as Reverse primer (SEQ ID NO. 4): 5'- CATTGCCAGCCTATCCTTTCTT-3'.

[0050] KASP Master Mix (containing premixed Taq enzyme, dNTPs, Mg 2+ and the like, LGC: KASP TF V4.0 can be used), positive control DNA: allele Hap1, the sequence of which is shown as SEQ ID NO. 1, and the S at the 23rd bp is G; and Hap2 sequence is shown as SEQ ID NO. 1, and the S at the 23rd bp is C.

[0051] Application of Example 4 in screening of new population materials

[0052] 1. Materials: 180 soybean materials from natural population were selected.

[0053] 2. Allele detection: according to the method of Example 2, DNA of 180 natural population was extracted and genotyped by KASP detection kit of Example 3.

[0054] 3. Phenotype detection: the protein content of 180 soybeans was determined by Xingchuang Zhongpu G1200 protein determination instrument.

[0055] 4. KASP detection results: 180 materials were successfully divided into two groups: 98 carrying excellent allele Hap1 (homozygous GG type), 82 carrying non-excellent allele Hap2 (homozygous CC type), and the results are shown as Figure 4 , in which the X axis is the FAM fluorescence signal intensity; the Y axis is the HEX fluorescence signal intensity; clusters: two clusters are clearly shown in the figure, representing homozygous genotype GG (cluster 1) and homozygous genotype CC (cluster 2), and invalid data points (lower left corner), indicating that the primer genotyping effect is good and the resolution is high.

[0056] Results analysis: According to the protein content determined in step 3 and the molecular marker genotyping results determined in step 4, the protein content of different soybean allele populations was compared, and it was found that the average protein content of Hap2 population was 44.64 ± 2.87 (%) which was significantly higher than the average protein content of Hap1 population which was 38.83 ± 1.43. The non-parametric test U test results showed that the difference of protein content between the two groups reached a significant level, as shown in Figure 5 Figure 3, the protein content box plot of different genotypes. X axis is group, Hap1 represents GG genotype population, Hap2 represents CC genotype population; Y axis represents protein content. Statistical test significance: p<0.0001 (****), p<0.001 (***), p<0.01 (**), p<0.05 (*)).

[0057] According to the genotyping results, resources carrying homozygous excellent allele Hap2 were preferentially selected as high protein content variety breeding hybridization parent reserves. And further screening of strains carrying the excellent CC genotype can be carried out in the generations of their hybrid offspring. This greatly improves the target nature and efficiency of selection, and saves the cost of human, material and land in field test.

Claims

1. Use of a KASP detection primer pair or a kit containing the primer pair for detecting and predicting high protein content in soybean, characterized in that, The KASP detection primer pair comprises a FAM-labeled forward primer F1, a HEX-labeled forward primer F2, and a universal reverse primer R, the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the universal reverse primer R is shown as SEQ ID NO.

4.

2. A method for detecting a molecular marker significantly related to soybean protein content, comprising the following steps: (1) extracting genomic DNA of a soybean sample to be tested; (2) using the extracted DNA as a template, performing PCR amplification by using the KASP marker primer pair shown as SEQ ID NO. 2-4 or a kit containing the KASP marker primer pair; (3) performing fluorescence detection on the PCR amplification product, determining the genotype and protein content of the amplification product of the sample to be tested by analyzing the fluorescence signal intensity of FAM and HEX; the soybean protein content in the soybean with the genotype CC at the 23rd bp in the nucleotide sequence shown as SEQ ID NO. 1 is significantly higher than that in the soybean with the genotype GG.

3. The method of claim 2, wherein, The PCR amplification program adopts pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing + extension at 61℃ for 60 s, 10 cycles with a decrease of 0.6℃ for each cycle; denaturation at 94℃ for 20 s, annealing + extension at 55℃ for 60 s, 35 cycles; and final extension at 72℃ for 5 min.

4. The method of claim 2, wherein, According to the fluorescence detection results after amplification, the genotyping and protein content of the soybean are determined, including: if the universal tag FAM fluorescence linker sequence is detected, the genotype is GG, and the corresponding soybean protein content is low; if the universal tag HEX fluorescence linker sequence is detected, the genotype is CC, and the corresponding soybean protein content is high.

5. Use of a KASP detection primer pair or a kit containing the primer pair in soybean breeding, characterized in that, The KASP detection primer pair comprises a FAM-labeled forward primer F1, a HEX-labeled forward primer F2, and a universal reverse primer R, the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the universal reverse primer R is shown as SEQ ID NO. 4, The breeding purpose is to screen soybean species with high soybean protein content, and the soybean protein content in the soybean with the genotype CC at the 23rd bp in the amplification product SEQ ID NO. 1 is significantly higher than that in the soybean with the genotype GG.

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