SNP (Single Nucleotide Polymorphism) related to soybean plant height under shade-avoiding reaction and application thereof

Abstract

The invention discloses SNP (Single Nucleotide Polymorphism) related to soybean plant height under shade-avoiding reaction and application thereof. The SNP locus is positioned at the 248th basic group from the 5'tail end of a nucleotide sequence shown as SEQ ID NO: 1; the locus comprises two allele types: T type and A type; soybeans with different genotypes have different plant heights correspondingly, specifically, in a weak light environment, the plant height of the soybean with the genotype being a TT homozygous type is larger than or candidate larger than that of the soybean with the genotype being an AA homozygous type. Technical research results of the invention are expected to be used in molecular marker-assisted breeding of soybeans and related crops, and have important values in theory and application aspects for breeding soybean varieties suitable for close planting, enhancing plant lodging resistance, increasing soybean yield and other practical hot problems.

Description

Technical Field

[0001] This invention relates to molecular breeding technology, and in particular to a SNP related to soybean plant height under shade avoidance response and its application. Background Technology

[0002] Soybean (Glycine max (Linn.) Merr.) is my country's fourth largest food crop, and its production plays a vital role in ensuring food security. As an important oilseed crop, it also provides more than a quarter of the world's total protein for food and animal feed, holding a significant position in the international oilseed crop market and becoming one of the world's most important food and economic crops.

[0003] Increasing soybean yield is mainly achieved through two methods: increasing yield per individual plant or increasing planting density to increase overall yield. However, increasing planting density can trigger a shade avoidance response, leading to excessive vegetative growth, increased plant height, increased hypocotyl length, and thinner stems. Studies have shown that plant height directly affects final yield by influencing planting density, light capture efficiency, and photosynthetic efficiency. Generally, plants with a higher center of gravity are more prone to lodging, and the light absorption efficiency of the lower leaves decreases. Shorter plants, on the other hand, have a lower center of gravity and are less prone to lodging.

[0004] In summary, the discovery and development of molecular markers for soybean plant height under dense planting, especially SNPs closely related to crop phenotypes, and their application in molecular improvement and marker-assisted breeding of crops, is a current hot topic and a challenging issue in research and application. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a SNP related to soybean plant height under shade avoidance response and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0007] In a first aspect, the present invention includes a primer combination for detecting single nucleotide polymorphisms at SNP sites in the soybean genome, wherein the primer combination is the front primer F corresponding to SEQ ID NO: 2 and the back primer R corresponding to SEQ ID NO: 3 in the sequence listing;

[0008] The SNP site is located at the 248th base from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1; this site contains two allele types: T type and A type.

[0009] Different soybean genotypes correspond to different plant heights. Specifically, at least under low light conditions, soybeans with the TT homozygous genotype have a greater or candidate greater plant height than soybeans with the AA homozygous genotype.

[0010] Secondly, the present invention also includes a reagent or kit for identifying or assisting in the identification of soybean plant height traits, said reagent or kit being used to detect the SNP sites described in claim 1, and comprising at least the primer combination described in claim 1, template DNA, restriction endonuclease that recognizes and cuts template DNA, buffer required for PCR amplification, dNTPs, and other necessary components for gene detection.

[0011] As a preferred embodiment of the present invention, the restriction endonuclease is HindIII enzyme.

[0012] Thirdly, the present invention also includes a method for identifying or assisting in the identification of soybean plant height traits, comprising the following steps:

[0013] (1) Extract genomic DNA from the soybean sample to be identified;

[0014] (2) Detect the genotype of the SNP site in the soybean genomic DNA to be identified, wherein the polymorphism of the SNP site is W=T / A, corresponding to the 248th base from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1;

[0015] (3) Determine the plant height trait of the soybean to be identified based on the genotype: at least under low light conditions, when the genotype of the SNP locus is TT homozygous, the plant height of the soybean to be identified is greater than or candidate greater than that of the soybean with AA homozygous genotype.

[0016] As a preferred embodiment of the present invention, the method for detecting genotypic polymorphisms of SNP sites in the soybean genome is as follows:

[0017] (1) Using the genomic DNA of the soybean to be tested as a template, PCR amplification was performed using a primer combination to obtain PCR amplification products; the primer combination consisted of the front primer F corresponding to SEQ ID NO: 2 and the back primer R corresponding to SEQ ID NO: 3;

[0018] (2) After digesting the amplification product with enzymes, the enzyme digestion product is obtained; the enzyme digestion product is detected by electrophoresis, and the SNP site genotype is determined according to the band type of the enzyme digestion product.

[0019] As a preferred embodiment of the present invention, the method for determining the SNP genotype based on the band type of the enzyme digestion product is as follows: if the PCR product cannot be cleaved and the band length of the enzyme digestion product detected by electrophoresis is 273 bp, the genotype of the SNP site is TT; if the PCR product can be cleaved and the band length of the enzyme digestion product detected by electrophoresis is 247 bp and 26 bp, the genotype of the SNP site is AA.

[0020] Fourthly, the present invention also includes a soybean breeding method, comprising the following steps:

[0021] (1) Use the method described in any one of claims 4-6 to screen out dwarf soybean germplasm under low light conditions;

[0022] (2) The selected soybeans are used as parents for hybridization or self-pollination to obtain soybean offspring with stable plant height traits.

[0023] Finally, the present invention also includes the application of the primer combination, the reagent or kit, comprising any one or any combination thereof of (1)-(4):

[0024] (1) Application in screening or assisting in the screening of dwarf soybean varieties;

[0025] (2) Application in identifying or assisting in the identification of soybean plant height;

[0026] (3) Application in soybean breeding;

[0027] (4) Application in the preparation of soybean breeding products.

[0028] The beneficial effects of adopting the above technical solution are as follows: the technical research results of this invention are expected to be used in molecular marker-assisted breeding of soybeans and related crops. It has important theoretical and applied value for practical hot issues such as breeding suitable densely planted soybean varieties, enhancing plant lodging resistance, and increasing soybean yield. Attached Figure Description

[0029] Figure 1 This is a schematic diagram showing the frequency distribution of plant height in a RIL population under natural light.

[0030] Figure 2 This is a schematic diagram of the frequency distribution of plant height in a RIL population under low light conditions. In the diagram: A: Frequency distribution of plant height in the first replicate RIL population; B: Frequency distribution of plant height in the second replicate RIL population.

[0031] Figure 3This diagram illustrates the molecular marker detection method and results. In the diagram: A: Schematic diagram of parental plant height difference phenotypes; B: Schematic diagram of offspring difference phenotypes in the greenhouse RIL population; C: Schematic diagram of offspring plant height difference phenotypes in the field RIL population; D: Schematic diagram of parental difference gel analysis of marker dCAPS-1; E: Schematic diagram of difference gel analysis of marker dCAPS-1 in the offspring RIL population; F: Schematic diagram of statistical analysis of molecular markers in the offspring RIL population. Detailed Implementation

[0032] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase.

[0033] In the following description of the embodiments, specific details such as particular operating methods and process parameters are set forth for illustrative purposes and not for limitation, so as to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that this application can also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known technical methods are omitted so as not to obscure the description of this application with unnecessary details.

[0034] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0035] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0036] As used in this application specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]."

[0037] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0038] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0039] Example 1, Materials and Methods

[0040] 1.1 Test Materials

[0041] This experiment used Williams82 (W82) (genetic resource source disclosed) and GDL025 (ant egg) (genetic resource source disclosed) to construct a RIL population, which contains 151 families.

[0042] 1.2 Test Methods

[0043] This experiment used both low-light and natural light environments for cultivation. The low-light condition was set up in a greenhouse with a light path density of approximately 500 µmol / m². -2 s -1 The photoperiod was 13 hours of light / 11 hours of darkness. The control group plants were grown under natural light with a light flux density of approximately 1700 µmol / m². -2 s -1 Seeds were planted in 16 cm diameter pots (nutrient soil: vermiculite = 2:1) for seedling cultivation. Thirty days after planting, the plant height was measured with a ruler. Three plants were randomly selected from each pot for measurement. The plant height was measured in centimeters, accurate to 0.1 cm. Two biological replicates were set up.

[0044] 1.3 Statistics and Analysis

[0045] SPSS 27.0 was used to perform maximum, minimum, coefficient of variation, skewness, kurtosis, and normality analyses on the obtained phenotypic data. The heritability of related traits was analyzed using ICIM-ADD. Analysis of variance was performed on haplotype phenotypic differences.

[0046] Example 2: QTL localization and molecular marker development

[0047] Based on the constructed high-density genetic linkage map, QTLs were detected using the interval mapping (IM) method in the Mapping QTL6 software, combined with population phenotypic data. A LOD greater than 2.5 was used as the presence threshold. The additive effect and phenotypic contribution rate of each QTL were calculated based on the software results. A positive additive effect indicates that the enhancing allele originates from the paternal parent, while a negative additive effect indicates that it originates from the maternal parent. The QTL naming principle is 'q' + the abbreviation of the target trait + chromosome or linkage group band.

[0048] Based on the initial localization, a SNP locus was found in soybean genome sequencing data of this RIL population, located at position 12336540 on chromosome 17 (reference genome version number: Glycine max Wm82.a2.v1), with polymorphism A / T; experimental verification showed that it has major effect.

[0049] Using online enzyme identification software (website: http: / / indcaps.kieber.cloudapps.unc.edu / results Select the restriction endonuclease HindIII and design primer sequences. The underlined bases in primer R are artificially introduced mismatched bases. When the base at the mutation site is T, the amplification product of primer pair F / R does not contain the HindIII recognition sequence and cannot be digested by HindIII. When the base at the mutation site is A, the amplification product of primer pair F / R contains the HindIII recognition sequence and can be digested by HindIII. See the table below for more details.

[0050]

[0051] 2.1 Analysis of genetic variation in plant height phenotype in RIL population

[0052] 2.1.1 Analysis of phenotypic genetic variation in plant height of RIL population under natural light

[0053] To clarify the genetic characteristics of soybean plant height under natural light and low light conditions, this study conducted a genetic variation analysis on the plant height of the RIL population of soybeans under natural light. The results showed that the plant height ranged from 12.3 cm to 43.2 cm, with a coefficient of variation of 23.39%. The kurtosis of the plant height length phenotype in the RIL population was 0.49, and the skewness was -0.165, exhibiting an approximately normal distribution, indicating that the plant height of soybeans under natural light conditions follows a typical quantitative pattern (Table 1). Figure 1 ).

[0054] Under single conditions of low light or natural light, the heritability of plant height in the RIL population was 0.94 and 0.96, respectively; the heritability of plant height in the RIL population was 0.80 under both conditions, indicating that the plant height of soybeans in the RIL population is regulated by both environment and genetics.

[0055] Table 1. Phenotypic variation of plant height in RIL populations under natural light conditions.

[0056]

[0057] 2.1.2 Phenotypic Genetic Variation Analysis of Plant Height in RIL Population under Low Light Conditions

[0058] Phenotypic identification of plant height in RIL populations grown in greenhouses in 2024 was performed using SSPS.27 and ICIM-AOD. The results showed that the kurtosis values ​​of the plant height phenotype in the RIL population were -0.166 and -0.257, and the skewness values ​​were 0.685 and 0.510, respectively, exhibiting an approximately normal distribution. Figure 2 (Table 2).

[0059] Table 2. Phenotypic variation of plant height in RIL population under low light conditions.

[0060]

[0061] 2.2 QTL Locating and Analysis of RIL Population

[0062] 2.2.1 QTL localization of RIL population under low light conditions

[0063] Based on the plant height phenotype and genotype data of the RIL population, QTL analysis was performed on the plant height of the RIL population planted in greenhouse using the IM method of Mapping QTL6.0 software. The data from both greenhouse replicates were located on chromosome 17, and qPH17-1 was reproducibly located in both replicates, indicating that the additive effect originated from the maternal parent (Table 3).

[0064] Table 3. QTL mapping results of soybean plant height under low light conditions

[0065]

[0066] 2.2.2 QTL localization of RIL populations under natural light

[0067] Based on the plant height phenotype and genotype data of the RIL population, QTL analysis was performed on the plant height of the RIL population planted under natural light using the IM method of Mapping QTL 6.0 software. No reliable QTL intervals with LOD values ​​greater than 2.5 were detected. Therefore, the shade avoidance response QTL loci located under low light conditions regulate soybean plant height in low light environments.

[0068] Example 3: Genotyping

[0069] The test materials were derived from 51 accessions (F6 generation) of the RIL population. The RIL population was constructed by crossing Williams82 (W82, T genotype), which has a taller plant height, and GDL025 (ant egg, A genotype), which has a shorter plant height. The RIL population was then self-crossed for 5 generations to achieve homozygous genotypes for each individual.

[0070] 3.1 DNA Extraction

[0071] The method for extracting soybean genomic DNA is as follows:

[0072] (1) Take about 100-500 mg of fresh soybean leaves into a 2 ml centrifuge tube, add three 3 mm steel balls, cool with liquid nitrogen and then grind quickly with a grinder to ensure that the sample is fully pulverized.

[0073] (2) Add 800 µL of crude extraction buffer to the sample centrifuge tube and vortex the sample for 1 minute to mix it thoroughly. Let it stand at room temperature for 10 min to allow the soybean leaf tissue to fully lyse.

[0074] (3) Centrifuge at 12000 rpm for 10 min, take 450 µL and put it into a new centrifuge tube, add 450 µL of pre-cooled isopropanol, shake vigorously, place in a -20℃ refrigerator to precipitate for 30 min, and then centrifuge at 12000 rpm for 10 min.

[0075] (5) After centrifugation, discard the liquid, add 1 ml of 75% ethanol, shake vigorously, then centrifuge at 12000 rpm for 5 min and discard the supernatant.

[0076] (6) Repeat step (5)

[0077] (7) After drying at 65℃ for 20 min, add 50 µL of ultrapure water.

[0078] (8) Quantitative detection of DNA was performed using an ultraviolet spectrophotometer. Qualified DNA was stored at -20 ℃ for later use.

[0079] 3.2 PCR amplification and enzyme digestion

[0080] Configure the PCR reaction system and set up the PCR program for amplification:

[0081]

[0082]

[0083] After the PCR reaction was completed, 10 μL of the PCR product was subjected to 1% agarose gel electrophoresis, and the results were then scanned using a gel imaging system.

[0084] The enzyme digestion system is shown in the table below (enzyme digestion reaction conditions: 37℃ for 2 h, 65℃ for 20 min inactivation):

[0085]

[0086] The enzyme digestion products were subjected to agarose gel electrophoresis, and the specific steps are as follows:

[0087] (1) Electrophoresis buffer preparation: 1×TAE buffer (powder, Solarbio) added to 1 L of distilled water

[0088] (2) Preparation of 6% agarose gel: Weigh 6 g agarose and add 100 mL 1×TAE buffer. Heat in a microwave oven until completely dissolved. After the solution cools down, add 4 μL nucleic acid dye, pour into the gel tank, and insert two rows of 10 μL combs.

[0089] (3) Electrophoresis: Use a pipette to add 4 μL of 2000 DNA Marker to the first well, followed by 8 μL of the enzyme digestion product to the subsequent wells. Turn on the electrophoresis apparatus and set the electrophoresis parameters: constant voltage 120 volts, electrophoresis time approximately 2 hours. After electrophoresis, scan the results using a gel imaging system.

[0090] Because the amplification products of the TT genotype soybean material do not contain the HindIII restriction enzyme recognition sequence, the digestion products cannot be digested by HindIII, and the band length of the digested products detected by electrophoresis is 273 bp. In contrast, the amplification products of the AA genotype soybean material contain the HindIII restriction enzyme recognition sequence, so the digestion products can be digested by HindIII, and the band lengths of the digested products detected by electrophoresis are 247 bp and 26 bp, respectively. The TT genotype refers to the homozygous type with deoxyribonucleotide T at position 12336540 of chromosome 17 in the soybean genome, and the AA genotype refers to the homozygous type with deoxyribonucleotide A at position 12336540 of chromosome 17 in the soybean genome.

[0091] Based on the initial localization, dCAPS-1 markers linked to QTLs were developed using variations in SNPs between parents (Table S1). First, detection was performed between parents: dCAPS-1 markers showed differences between parents, with W82 corresponding to the A band type (TT genotype) and GDL025 corresponding to the B band type (AA genotype). Figure 3 (A and D).

[0092] Table S1. Related information about dCAPS-1 sites

[0093]

[0094] The results were analyzed in the pedigrees of the RIL population in the field. Among them, 25 materials corresponded to band A and 26 materials corresponded to band B (Table s2). Figure 3 (C and E), the plant height of the two strip-shaped materials differed significantly (P<0.001). Figure 3 F).

[0095] Table S2. Results of molecular markers and plant height detection in the progeny RIL population

[0096]

[0097] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0098] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A primer combination, characterized by: The primer combination is a forward primer F corresponding to SEQ ID NO: 2 and a reverse primer R corresponding to SEQ ID NO: 3 in the sequence listing for detecting the single nucleotide polymorphism of the T248A SNP site in the soybean genome; The T248A SNP site is located at the 248th base from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1; the site contains two allelic types: T type and A type; Soybeans of different genotypes correspond to different plant heights, specifically: at least in a weak light environment, soybeans of genotype TT homozygote have a plant height greater than or candidate greater than soybeans of genotype AA homozygote.

2. An agent or a kit for identifying or aiding in the identification of a soybean plant height trait, characterized by: The reagent or kit is used for detecting the T248A SNP site described in claim 1, and at least contains the primer combination described in claim 1, template DNA, restriction endonuclease for recognizing and cutting the template DNA, buffer, dNTPs and other necessary components for gene detection.

3. The reagent or kit of claim 2, wherein: The restriction endonuclease is HindIII enzyme.

4. A method of identifying or aiding in the identification of soybean plant height traits, characterized by: The method comprises the following steps: (1) extracting the genomic DNA of the soybean sample to be identified; (2) detecting the genotype of the T248A SNP site in the soybean genomic DNA to be identified, wherein the polymorphism of the T248A SNP site is W=T / A, corresponding to the 248th base from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1; (3) determining the plant height trait of the soybean to be identified according to the genotype: at least in a weak light environment, when the genotype of the T248A SNP site is TT homozygote, the plant height of the soybean to be identified is greater than or candidate greater than that of the soybean of genotype AA homozygote.

5. The method of claim 4, wherein: The method for detecting the genotype polymorphism of the T248A SNP site in the soybean genome comprises the following steps: (1) using the genomic DNA of the soybean to be tested as a template, performing PCR amplification with a primer combination to obtain a PCR amplification product; the primer combination consists of a forward primer F corresponding to SEQ ID NO: 2 and a reverse primer R corresponding to SEQ ID NO: 3; (2) cutting the amplification product to obtain a cut product; performing electrophoresis detection on the cut product to determine the genotype of the T248A SNP site according to the band type of the cut product.

6. The method of claim 5, wherein: The method for determining the genotype of the T248A SNP site according to the band type of the cut product is specifically: the PCR product cannot be cut, and the band length of the cut product detected by electrophoresis is 273 bp, at which time the genotype of the T248A SNP site is TT; the PCR product can be cut, and the band length of the cut product detected by electrophoresis is 247 bp and 26 bp, at which time the genotype of the T248A SNP site is AA.

7. A method of breeding soybeans, characterized by: The method comprises the following steps: (1) screening out dwarf soybean germplasm in a weak light environment by the method of any one of claims 4-6; (2) crossing or self-crossing the screened soybean as a parent to obtain soybean offspring with stable plant height traits.

8. Use of the primer combination according to claim 1, the reagent or kit according to any one of claims 2-3, comprising any one or any combination of the following (1)-(4): (1) use in screening or assisting screening of soybean varieties with short plant height; (2) use in identifying or assisting identifying plant height of soybean; (3) use in soybean breeding; (4) use in preparing products of soybean breeding.

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