Marker Gm20_38690822, primers and application for identifying β-conglycinin subunit deletion trait

Abstract

The present invention belongs to the technical field of SNP markers, specifically a marker Gm20_38690822, primers and applications for identifying the trait of β-conglycinin subunit deficiency, and a marker Gm20_38690822 for identifying the trait of β-conglycinin subunit deficiency, wherein the base of the marker Gm20_38690822 at position 38690822 of chromosome 20 of the soybean genome is C or T. The KASPSNP marker Gm20_38690822 of the present invention is detected using plant DNA as a sample, can be detected in various tissues and development stages of soybeans, and is not limited by the growth and development period of soybeans; there is no problem of damaging seeds and affecting seed vitality; at the same time, the technical method is simple, the detection equipment can be quantified, and the identification efficiency and identification cost are low.

Description

Technical Field

[0001] The invention relates to the technical field of SNP markers, and in particular to a marker Gm20_38690822 for identifying a beta-conglycinin subunit deletion trait, primers and applications thereof. Background Art

[0002] β-conglycinin is the main allergen protein of soybean. The absence of its three main subunits: α-, α′-, and β-subunits can alleviate the allergenic problem of soybean. At the same time, soybeans with low β-conglycinin have high contents of sulfur amino acids, isoflavones, daidzein, and genistein, and have more material processing value, nutritional value, and medical and health value.

[0003] For the identification of whether the β-conglycinin subunit is missing or not, the existing technology is to use SDS-PAGE protein gel electrophoresis for identification. This method can only use bean powder extracted from beans as samples, which limits the detection samples and damages seed vitality. The experimental steps of this method include eight steps: protein extraction, preparation of separation gel, preparation of concentrated gel, gel electrophoresis, gel unloading, staining, decolorization, and band reading. The technology is cumbersome, and the entire experiment takes eight hours. The identification process requires a lot of manpower, and there is a lack of commonly used instruments and equipment for throughput detection, making it difficult to achieve large-scale rapid detection. In summary, the existing technology has problems such as limited samples, damage to seeds, cumbersome identification technology, low identification efficiency and high identification cost, which seriously restricts the large-scale identification of the β-conglycinin subunit missing trait.

[0004] Single Nucleotide Polymorphism (SNP) refers to the variation of a single nucleotide in the genome, which is a variation form formed by the substitution, transversion, insertion or deletion of a single nucleotide pair. With the rapid development of genome sequencing technology and the throughput of SNP detection equipment, SNP molecular marker technology has accelerated the efficiency of target traits by identifying the genotype of target traits through flux, especially the KASP SNP technology with low technical requirements. Although some genes that regulate the deletion of β-conglycinin subunits have been found at home and abroad, the SNP identification markers for the complete deletion of these three subunits α-, α′-, and β-subunits have not been developed. Therefore, at this stage, the identification of the complete deletion of these three subunits of soybean β-conglycinin in China still remains at the SDS-PAGE protein gel electrophoresis phenotypic identification method, which seriously limits the development of research related to the large-scale identification of this trait. Summary of the invention

[0005] In order to provide an identification marker for complete deletion of three subunits, namely, α-, α′-, and β-subunits, the present invention provides a marker Gm20_38690822, primers and applications for identifying the β-conglycinin subunit deletion trait.

[0006] The marker Gm20_38690822 for identifying the β-conglycinin subunit deletion trait, wherein the base of the marker Gm20_38690822 at position 38690822 of chromosome 20 of the soybean genome is C or T.

[0007] Preferably, the soybean whose base at the KASPSNP marker Gm20_38690822 is C is a β-conglycinin-subunit complete deletion type.

[0008] Preferably, the whole genome sequence version of the soybean genome is Williams82v2.0.

[0009] The application of the marker Gm20_38690822 in the detection of β-conglycinin subunit deficiency trait.

[0010] The KASP primer set for detecting the KASPSNP marker Gm20_38690822 includes a FAM primer, a VIC primer, and a COM primer. The nucleotide sequence of the FAM primer is shown in SEQ NO.49, the nucleotide sequence of the VIC primer is shown in SEQ NO.50, and the nucleotide sequence of the COM primer is shown in SEQ NO.51.

[0011] The application of the KASP primer set in the detection of β-conglycinin subunit deletion trait.

[0012] A kit includes the KASP primer set.

[0013] The kit is used in detecting the β-conglycinin subunit deficiency trait.

[0014] A method for detecting β-conglycinin subunit-deficient soybean material, characterized by comprising the following steps:

[0015] (1) Extracting genomic DNA from soybean powder or leaves to be tested;

[0016] (2) Detection: Using the soybean genomic DNA to be detected as a template, a KASP primer set is used to perform a PCR amplification reaction; the KASP primer set includes a FAM primer, a VIC primer, and a COM primer, the nucleotide sequence of the FAM primer is shown in SEQ NO.49, the nucleotide sequence of the VIC primer is shown in SEQ NO.50, and the nucleotide sequence of the COM primer is shown in SEQ NO.51;

[0017] (3) Detect PCR amplification products and divide genotypes based on fluorescence signal performance.

[0018] Preferably, the genotype classification standard is: using the FAM data of the fluorescence signal as the X-axis and the HEX data as the Y-axis to make a scatter plot, the gene clusters close to the X-axis and the Y-axis are CC or TT genotypes, respectively, the CC genotype soybean sample is determined to be a homozygous type of β-conglycinin-subunit total deletion; the TT genotype soybean sample is determined to be a type without β-conglycinin subunit deletion, and the type located between the X-axis and the Y-axis is the CT heterozygous genotype, which is determined to be a heterozygous type of β-conglycinin-subunit total deletion.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention provides a SNP identification marker Gm20_38690822 for complete deletion of the three subunits α-, α′-, and β-subunits. The KASPSNP marker Gm20_38690822 is detected using plant DNA as a sample and can be detected in all tissues and development stages of soybeans without being restricted by the growth and development period of soybeans. There is no problem of damaging seeds or affecting seed vitality.

[0021] 2. The present invention is used to identify whether the β-conglycinin (α′+α+β)-subunit is missing, which only requires simple DNA extraction and 3.5 hours of PCR amplification. Compared with the SDS-PAGE protein gel electrophoresis detection method, the technology is simple, the time required for the entire detection process is reduced by half, and the instrument equipment can achieve throughput. It is a fast, efficient, and low-cost detection method that can perform throughput detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the Δ(SNP-index) fitting curve of SNPs on 20 chromosomes of soybean in genome-wide association analysis;

[0023] Figure 2 It is a preliminary positioning of the genetic linkage map;

[0024] Figure 3 It is a fine-mapping recombination analysis map;

[0025] Figure 4 Gm20_38690822 performed KASPPCR detection on 100 parental resources and DG2. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0027] Jiyu 86 is a new soybean variety bred by the soybean quality breeding team of Jilin Academy of Agricultural Sciences. It has passed the national variety approval with the approval number of Guoshendou2009007 and is currently stored in the National Soybean Resource Bank with the resource number of ZDD30908.

[0028] DG2 is a genetically stable mutant strain obtained by our team through cobalt mutagenesis of Jiyu 86. It is now stored in the resource library of the quality breeding team of the Soybean Research Institute of Jilin Academy of Agricultural Sciences.

[0029] Example 1

[0030] Genome-wide association analysis for the β-conglycinin (α′+α+β)-subunit deletion trait

[0031] (1) An F2 genetic population of Jiyu 86 and its cobalt-induced mutant DG2 was constructed. The F1 was obtained by hybridization with Jiyu 86 as the maternal parent and DG2 as the paternal parent. Only one F1 plant was selected for self-pollination to obtain an F2 genetic population. Jiyu 86 had no (α′+α+β)-subunit deletion, while DG2 had a complete (α′+α+β)-subunit deletion.

[0032] (2) SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis, abbreviated as SDS-PAGE, is the most commonly used protein expression analysis technique in polyacrylamide gel electrophoresis) was used to identify the protein subunit deletions of 302 individuals in the F2 population. Based on the protein bands, 30 individuals with complete (α′+α+β)-subunit deletion and those with no subunit deletion were screened out, and two BSA gene pools with and without subunit deletion were constructed.

[0033] (3) Whole genome resequencing was performed on Jiyu 86, DG2 and the above two BSA gene pools to identify SNP sites, and the Δ(SNP_index) method was used to perform association analysis between subunit deletion traits and SNP sites ( Figure 1 The (α′+α+β)-subunit deletion trait was associated with chromosome 20 between 38,069,324-39,309,951bp (Willianms82v2.0 version), with a physical distance of approximately 1.24Mb.

[0034] Example 2

[0035] Genetic Mapping of the (α′+α+β)-Subunit Deletion Trait in β-conglycinin

[0036] (1) Targeting the SNP loci in the subunit deletion-associated region of chromosome 20, 38,069,324-39,309,951bp (willianms82 v2.0 version), 13 KASPSNP markers were successfully synthesized, among which the typing of the amplified progeny of Gm20_38566824 was not obvious, and the polymorphism of Gm20_38670505 was poor in both parents. Finally, 11 KASPSNP markers were successfully designed, namely: Gm20_38099035, Gm20_38259585, Gm20_38425042, Gm20_38527876, Gm20_38606427, Gm20_38760645, Gm20_38832258, Gm20_38902888, Gm20_38979797, Gm20_39128168 and Gm20_39278663. Primer sets were designed for the above 11 KASPSNP markers, as shown in Table 1.

[0037] Table 1 11 KASP SNP primers designed in the association region

[0038]

[0039]

[0040] (2) KASP SNP PCR amplification was performed on 302 F2 individuals to identify their genotypes. PCR amplification reaction system (5uL): DNA 1.5uL (5-30ng / uL), 2×KASP Master MIX buffer 1.5uL, KASP primer set 0.05uL, ddH2O 2uL; PCR reaction conditions: 94℃ pre-denaturation for 15 minutes; 94℃ denaturation for 20 seconds, 61℃ annealing and extension for 60 seconds (0.6 degree reduction per cycle), 10 cycles; 94℃ denaturation for 20 seconds, 55℃ annealing and extension for 60 seconds, 36 cycles; 35℃ for 2min. Finally, the temperature was lowered to below 40℃. The signal was excited at 35℃ for 1 minute during reading, and the RT of QIAGEN Rotor-Gene Q Series Software V2.0 was used. 2 Profiler TM The PCR Arrays analysis module detects PCR amplification products, divides genotypes according to the fluorescence signal performance, and distinguishes individuals with and without subunit deletion.

[0041] (3) JoinMap4.1 software was used to conduct linkage analysis on the genotypes and phenotypes of 302 F2 individuals. Figure 2), the (α′+α+β)-subunit deletion gene was preliminarily genetically located between Gm20_38606427 and Gm20_38760645 on chromosome 20 (willianms82 v2.0 version), with a positioning distance of 1.1 cM and a physical distance of approximately 154 kb.

[0042] (4) Based on the preliminary genetic positioning between Gm20_38606427 and Gm20_38760645, four primer sets were successfully designed, including four primer sets for KASPSNP markers: Gm20_38639043, Gm20_38662447, Gm20_38675773, and Gm20_38697396. The primer set sequences are shown in Table 2.

[0043] Table 2 Four KASP SNP primers designed in the localization region

[0044]

[0045]

[0046] (5) The F3 was obtained by self-fertilization of individuals in the F2 genetic population, and 12 F3 were screened for heterozygous KASPSNP markers in the preliminary genetic positioning interval. 2：3 The 380 F3 individuals of the line were identified by genotype and phenotype. The phenotype was identified by SDS-PAGE protein gel electrophoresis, and the genotype was identified by KASP SNP PCR method. The reaction system, reaction conditions and genotype analysis method were carried out according to the conditions (2) in Example 2.

[0047] (6) Recombination analysis of phenotypes and genotypes of 380 F3 individuals ( Figure 3 ), the (α′+α+β)-subunit complete deletion trait was finely located to the 34.95kb range between 38662447-38697396bp on soybean chromosome 20 (willianms82 v2.0 version).

[0048] Example 3

[0049] Development of markers for the (α′+α+β)-subunit deletion trait of β-conglycinin

[0050] (1) In the NJAU355kb soybean SNP chip, 17 SNP sites within 38662447-38697396bp were screened, of which 7 were tested and KASPSNP markers were successfully designed and synthesized, namely Gm20_38666133, Gm20_38667159, Gm20_38669170, Gm20_38670424, Gm20_38670505, Gm20_38690822 and Gm20_38692748. The primer set information designed for the above 7 markers is shown in Table 3.

[0051] Table 3 Seven SNP primers designed based on soybean SNP chip loci

[0052]

[0053]

[0054] (2) The KASPSNPPCR genotyping of 100 soybean breeding resources and DG2 was performed for the 7 successfully synthesized KASPSNP primer sets, and the reaction system, reaction conditions and genotyping analysis method were performed according to the conditions of (2) in Example 2. Among the 7 markers, except for the typing failure and the marker identification efficiency lower than 90%, the Gm20_38690822 marker could clearly type the 100 parental resources and DG2 with strong amplification signals, and the identification efficiency was 100%.

[0055] Effect verification

[0056] The application methods include:

[0057] (1) Material preparation: The genomic DNA of soybean powder or leaves to be tested was extracted using a DNA extraction kit produced by Dingguo Company. It should be noted that the DNA can be extracted using conventional DNA extraction methods or commercially available DNA extraction kits that can be used to extract soybean powder and leaves, such as the DNA extraction kits produced by Quanshijin, Tiangen, Dingguo Company, etc.

[0058] (2) PCR amplification: Using the soybean genomic DNA to be detected as a template, the KASP primer set was used for PCR amplification reaction. The KASP primer set includes FAM primer, VIC primer, and COM primer. The nucleotide sequence of the FAM primer is shown in SEQ NO.49, the nucleotide sequence of the VIC primer is shown in SEQ NO.50, and the nucleotide sequence of the COM primer is shown in SEQ NO.51. PCR amplification reaction system (5uL): DNA 1.5uL (5-30ng / uL), 2×KASP Master MIX buffer 1.5uL, KASP primer set 0.05uL, ddH2O 2uL. PCR reaction conditions are: 94℃ pre-denaturation for 15 minutes; 94℃ denaturation for 20 seconds, 61℃ annealing and extension for 60 seconds (0.6 degree reduction per cycle), 10 cycles; 94℃ denaturation for 20 seconds, 55℃ annealing and extension for 60 seconds, 36 cycles; 35℃ 2min. Finally, cool to 4℃. (3) PCR product detection: The signal was stimulated at 35°C for 1 minute. The RT was performed using QIAGEN Rotor-Gene Q Series Software V2.0. 2 Profiler TM The PCR Arrays analysis module detects the fluorescence signal value of the PCR amplification product.

[0059] (4) Genotyping and result determination: The fluorescence signal FAM and HEX data can be plotted on the X-axis and Y-axis respectively using Kluster Caller TM (LGC) data analysis software. Alternatively, the FAM and HEX data can be imported into an EXCEL table, with the FAM data as the X-axis and the HEX data as the Y-axis, and the X-axis and Y-axis data can be expressed using a scatter plot function. A typical sample typing diagram has three clear genotyping clusters. The two side gene clusters (close to the X-axis and Y-axis, respectively) are CC or TT genotypes. The β-conglycinin-subunit complete deletion type control belongs to the CC genotype, and this type of soybean sample is determined to be a homozygous type for the β-conglycinin-subunit complete deletion; the β-conglycinin-subunit non-deletion type control belongs to the TT genotype, and this type of soybean sample is determined to be a non-deletion type for the β-conglycinin subunit. The middle type (located between the X-axis and the Y-axis) is the CT heterozygous genotype, which is a heterozygous type for the β-conglycinin-subunit complete deletion.

[0060] The results of KASPPCR test on 100 parental resources and DG2 in Gm20_38690822 are as follows Figure 4 As shown, there are two genotyping clusters, close to the X-axis and Y-axis respectively, that is, 100 parental resources were judged to be β-conglycinin subunit non-deficient type, and DG2 was judged to be β-conglycinin subunit completely deleted homozygous type.

[0061] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0062] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of the marker Gm20_38690822 for identifying the β-conglycinin subunit deficiency trait in detecting the β-conglycinin subunit deficiency trait, characterized in that: The base of the marker Gm20_38690822 at position 38690822 of chromosome 20 of the soybean genome is C or T; The whole genome sequence version of the soybean genome is Williams82 v2.

0.

2. Use of the marker Gm20_38690822 for identifying the β-conglycinin subunit deficiency trait according to claim 1 in detecting the β-conglycinin subunit deficiency trait, characterized in that: The soybeans whose base at KASP SNP marker Gm20_38690822 is C are of β-conglycinin subunit complete deletion type.

3. A KASP primer set for detecting the marker Gm20_38690822 according to any one of claims 1 to 2, characterized in that: It comprises a FAM primer, a VIC primer and a COM primer. The nucleotide sequence of the FAM primer is shown in SEQ NO.49, the nucleotide sequence of the VIC primer is shown in SEQ NO.50, and the nucleotide sequence of the COM primer is shown in SEQ NO.

51.

4. Use of the KASP primer set according to claim 3 in detecting the β-conglycinin subunit deficiency trait.

5. A kit, characterized in that: The kit comprises the KASP primer set described in claim 3.

6. Use of the kit according to claim 5 in detecting the β-conglycinin subunit deficiency trait.

7. A method for detecting β-conglycinin subunit-deficient soybean material, characterized in that: The following steps are involved: (1) Extracting genomic DNA from soybean powder or leaves to be tested; (2) Detection: using the soybean genomic DNA to be detected as a template, and performing a PCR amplification reaction using the KASP primer set described in claim 3; (3) Detect PCR amplification products and divide genotypes based on fluorescence signal performance.

8. The method for detecting β-conglycinin subunit-deficient soybean material according to claim 7, characterized in that: The genotype division standard is: with the FAM data of the fluorescence signal as the X-axis and the HEX data as the Y-axis, a scatter plot is made, and the gene clusters close to the X-axis and Y-axis are CC or TT genotypes, respectively. The CC genotype soybean sample is judged to be the homozygous type of β-conglycinin-subunit complete deletion; The soybean samples with TT genotype were judged as the type with no β-conglycinin subunit deletion, and the type between the X-axis and the Y-axis was the CT heterozygous genotype, which was judged as the heterozygous type with complete β-conglycinin subunit deletion.

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