A SNP site associated with soybean high-density tolerance and high-yield plant type traits, CAPS molecular marker, primer set, and application thereof

Abstract

The present invention provides a SNP site associated with soybean plant-type traits of high-density tolerance and high yield, a CAPS molecular marker, and a primer set and application thereof, relating to the technical field of soybean molecular breeding. The present invention identified a high-density tolerance mutant, cp1, from an EMS-induced population. Through fine mapping, it was found that the mutant had a non-synonymous mutation from C to A at position 517 after the ATG of the Glyma.19g185600 gene. Based on this, a CAPS molecular marker was developed, the nucleotide sequence of which is shown in SEQ ID NO: 2. The analysis and utilization of cp1 in the present invention contributes to improving soybean plant type and high-density tolerance breeding, and has significant breeding application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of soybean molecular breeding, and specifically relates to a SNP site related to the high-density-tolerant and high-yield plant type trait of soybean, a CAPS molecular marker, and a primer set and application thereof. Background Art

[0002] As a key legume crop, soybeans are a significant source of high-quality protein and vegetable oil. Soybean plant architecture generally refers to the three-dimensional structure of the plant's aboveground tissues. It encompasses plant height, leaf shape, petiole length and angle, pod-setting habits, and the arrangement and spatial structure of individual plants within a population. Soybean plant architecture is crucial to growth and yield. The ideal high-yielding soybean plant is characterized by a well-developed main stem with numerous nodes, numerous pods, a strong stem, small leaves, and leaf differentiation between the upper and lower parts of the plant, with larger leaves at the lower part and smaller leaves at the upper part. The petiole, the organ connecting the stem to the leaves, plays a crucial role in determining leaf angle, plant canopy structure, and the transport and storage of photosynthetic products. Petiole length is a key factor in determining the ideal soybean plant architecture, influencing canopy structure, photosynthetic efficiency, and, consequently, yield. Short petioles significantly improve the efficiency of soybean leaves in intercepting light energy, thereby enhancing photosynthesis. Appropriate planting density increases the accumulation of organic matter per unit area, ultimately boosting yield.

[0003] The length of the petiole is related to the division and elongation of epidermal cells and vascular bundles. When differentiation is abnormal, the plant will show the characteristics of short petioles. Many studies have shown that the formation of short petioles is related to changes in the proximal-distal axis. Previous studies have found that the complex AS1-AS2 (asymmetric leaves) regulates the length of the proximal-distal leaves by directly inhibiting the expression of the Knotted1-like homebox (KNOX) gene in Arabidopsis. The transport and signal transduction of plant hormones also play an important role in the development of leaves and petioles by regulating the expression of key genes and transduction proteins. IAA can initiate and maintain the initiation of lateral organs by inhibiting the expression of KNOX genes and regulating the cytokinin signaling pathway.

[0004] Research on the petiole locus began in 1983, when Kilen discovered a short-petioled soybean line, D76-1609, and crossed it with the long-petioled line, Lee68. Genetic analysis revealed that short petiole is controlled by a single recessive nuclear gene, named lps. Previous researchers identified a short-petiole mutant, NJ90L-LSP, from the progeny of the round-leaved, long-inflorescence material NJ90L-1. Genetic analysis revealed that it is controlled by two pairs of recessive nuclear genes, named lps1 and lps2. The short-petiole gene, lps3, was mapped between markers Sat-234 and Sat-033 on chromosome 13. Given the limited discovery of genes associated with soybean petiole length, identifying new petiole-specific trait genes is of great theoretical and practical significance for soybean dense planting. Summary of the Invention

[0005] In view of this, the present invention aims to provide a SNP site associated with soybean high-density tolerance and high-yield plant type traits, a CAPS molecular marker, and a primer set and application thereof. The present invention identified a high-density tolerance mutant, cp1, from an EMS-induced population. This mutant exhibits a non-synonymous C-to-A mutation at position 517 after the ATG of the Glyma.19g185600 gene. Based on this, the present invention developed a CAPS molecular marker. The CAPS molecular marker of the present invention can identify wild-type and mutant genotypes, contributing to improved soybean plant type and high-density tolerance breeding.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a SNP site in identifying a dense-tolerant and high-yield soybean plant type. The SNP site is located at the 517th base after the ATG of the soybean gene Glyma.19g185600, and the polymorphism is C / A. The nucleotide sequence of the soybean gene Glyma.19g185600 is shown in SEQ ID NO: 1.

[0008] The present invention provides a CAPS molecular marker related to the soybean high-yield plant type trait, wherein the CAPS molecular marker comprises the above-mentioned SNP site and the nucleotide sequence of its upstream and downstream components; the nucleotide sequence of the CAPS molecular marker is shown in SEQ ID NO: 2.

[0009] The present invention provides a primer set for detecting the CAPS molecular marker, which comprises a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4.

[0010] The present invention provides a kit for detecting the CAPS molecular marker, which comprises the primer set.

[0011] The present invention provides a method for identifying a high-yield and high-density soybean plant type, comprising the following steps: (1) using genomic DNA of a soybean plant to be identified as a template and performing PCR amplification using a primer set to obtain an amplified product; (2) performing enzyme digestion on the amplified product and detecting the polymorphism of the enzyme digestion product; (3) when the enzyme digestion product contains only one main band, the soybean to be identified is a homozygous high-yield and high-density soybean plant type; when the enzyme digestion product contains three bands, the soybean to be identified is a heterozygous high-yield and high-density soybean plant type; and when the enzyme digestion product contains two bands, the soybean to be identified is a wild type.

[0012] Preferably, the soybean plant type that is tolerant to high density and high yield is a plant type in which the upper petiole length is shorter and the lower petiole length is longer.

[0013] Preferably, the enzyme digestion is performed using the restriction endonuclease AluI.

[0014] The present invention also provides an application of the CAPS molecular marker, the primer set or the kit in identifying a high-yield soybean plant type that is resistant to high density.

[0015] Preferably, the cultivation row spacing of the dense-tolerant and high-yield soybean plant type is less than 40 cm.

[0016] The present invention also provides an application of the CAPS molecular marker, the primer set or the kit in breeding of dense-density-tolerant soybean plants.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention identified a soybean mutant library with a significant change in the spatial configuration of petiole length, resulting in plants with short upper petioles and long lower petioles. Due to its high pod conspicuousness, it was designated cp1 (Conspicuous pods 1). Through fine mapping, it was found that this mutant was caused by a non-synonymous C-to-A mutation in the Glyma.19g185600 gene, leading to the development of a CAPS molecular marker. The CAPS molecular marker of the present invention can identify wild-type and mutant genotypes, contributing to improved soybean plant architecture and dense tolerance breeding.

[0019] (2) The present invention uses the cp1 mutant and the conventional variety WH506 in a high-density yield with a row spacing of 30 cm and a plant spacing of 10 cm. cp1 has a significant single-plant yield advantage. cp1 is further sexually hybridized with the nationally approved soybean variety Zhongdou 63 (abbreviated as ZD63) to obtain a near-isogenic line. Compared with NIL ZD63 Its NIL cp1 The yield performance of high density with a row spacing of 30 cm and a plant spacing of 10 cm is better. The utilization of cp1 in the present invention is helpful to improve soybean plant type and density-tolerant breeding, and has important breeding application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Phenotypic analysis of the cp1 mutant. In the figure, a is the plant phenotype of the mutant cp1; b is the plant phenotype of the wild-type WH506; cd are the corresponding pattern diagrams; e f are the petiole length analysis of cp1 and WH506, L1-L10 are the trifoliate compound leaves from the top to the bottom, scale bar: 10 cm;

[0021] Figure 2 Sequencing peak analysis of the mutation sites of wild-type WH506 and cp1 mutant;

[0022] Figure 3 This is a molecular marker developed for the cp1 mutant CAPS. In the figure, M is a StarMarker D2000 marker. The pod-invisible material can be digested by AluI endonuclease into two bands of 271 bp and 51 bp. The homozygous pod-visible material is not digested by the endonuclease and shows a single 322 bp band. The heterozygous pod-visible material shows three band patterns.

[0023] Figure 4 This is the single plant yield performance of the cp1 mutant under high density conditions.

[0024] Figure 5 This is the yield performance of the hybrid offspring of the cp1 mutant under high-density conditions. The scale bar in the figure is 10 cm. DETAILED DESCRIPTION

[0025] The present invention provides an application of a SNP site in identifying a dense-density-tolerant and high-yield soybean plant type. The SNP site is located at position 585 of the soybean gene Glyma.19g185600 and has a C / A polymorphism. The nucleotide sequence of the soybean gene Glyma.19g185600 is shown in SEQ ID NO: 1. The present invention identified a soybean mutant library in which the spatial configuration of petiole length was significantly altered, with the plant exhibiting short petioles at the top and long petioles at the bottom. Due to its high pod visibility, it was codenamed cp1 (Conspicuous pods 1). Through fine mapping, it was found that the mutant was caused by a non-synonymous mutation from C to A at the 517th base after the ATG of the Glyma.19g185600 gene. This mutation caused the 174th amino acid to change from leucine to isoleucine.

[0026] Based on the above-mentioned cp1 mutant, the present invention provides a CAPS molecular marker related to the soybean high-density tolerance and high-yield plant type trait, the nucleotide sequence of the CAPS molecular marker is shown in SEQ ID NO: 1; or the CAPS molecular marker includes the above-mentioned SNP site and the nucleotide sequence of its upstream and downstream components; the nucleotide sequence of the CAPS molecular marker is shown in SEQ ID NO: 2, the SNP site exists at the 272nd position of the sequence, and the diversity is C / A.

[0027] The present invention provides a primer set for detecting the CAPS molecular marker, comprising a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4. The present invention designs specific primers based on the location of the cp1 mutant gene. Because the AluI endonuclease cleavage site is too close to the 3' end, which can result in unclear band separation, to address this issue, the present invention randomly adds 17 bp of the base sequence ATGCTTGATGAACTGAA to the 5' end of the reverse primer, as shown in SEQ ID NO. 5. Consequently, the amplified sequence is 322 bp in length, as shown in SEQ ID NO. 2.

[0028] The present invention provides a kit for detecting the CAPS molecular marker, which comprises the primer set.

[0029] The present invention provides a method for identifying a high-yield and high-density soybean plant type, comprising the following steps: (1) using genomic DNA of a soybean plant to be identified as a template and performing PCR amplification using a primer set to obtain an amplified product; (2) performing enzyme digestion on the amplified product and detecting the polymorphism of the enzyme digestion product; (3) when the enzyme digestion product contains only one main band, the soybean to be identified is a homozygous high-yield and high-density soybean plant type; when the enzyme digestion product contains three bands, the soybean to be identified is a heterozygous high-yield and high-density soybean plant type; and when the enzyme digestion product contains two bands, the soybean to be identified is a wild type.

[0030] The identification method of the present invention can identify wild-type and mutant genotypes. Based on the locus of the cp1 mutant gene, the present invention designed specific primers. Amplification results and electrophoresis analysis revealed that the wild-type can be cleaved by the AluI endonuclease into two bands of 271 bp and 51 bp; homozygous soybean high-density-tolerant and high-yield plants (homozygous pod-forming materials) are not cleaved by the endonuclease and display a single 322 bp band; and heterozygous soybean high-density-tolerant and high-yield plants (heterozygous pod-forming materials) display three band patterns. This indicates that the CAPS molecular marker of the present invention can identify wild-type and mutant genotypes and can be used for molecular breeding to assist molecular identification.

[0031] In the present invention, the soybean plant type that is tolerant to high density and high yield is a plant type in which the upper petiole length of the plant is shorter and the lower petiole length is longer.

[0032] In the present invention, the enzyme digestion is performed using the restriction endonuclease AluI. The present invention does not specifically limit the source of the restriction endonuclease AluI, and a conventional commercially available product can be used.

[0033] The present invention also provides an application of the CAPS molecular marker, the primer set or the kit in identifying a high-yield soybean plant type that is resistant to high density.

[0034] In the present invention, the cultivation row spacing of the soybean high-yield plant type is less than 40 cm. In the present invention, the wild type WH506 and the cp1 mutant were planted at a density of 30 cm in row and 10 cm in plant. The results showed that the mutant cp1 had a significant single-plant yield advantage under dense planting conditions, further proving that cp1 is a mutant with potential application in soybean high-yield breeding for high-density tolerance.

[0035] The present invention also provides an application of the CAPS molecular marker, the primer set or the kit in breeding of dense-density-tolerant soybean plants.

[0036] The technical solutions provided by the present invention are 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.

[0037] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without manufacturer specified were conventional products that can be purchased through regular channels.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0039] Example 1

[0040] 1. Identification and phenotypic analysis of the cp1 pod-forming mutant

[0041] A soybean EMS mutant library was screened to identify a mutant with a significant change in petiole spatial configuration and significantly improved light transmittance under dense planting conditions. This mutant, codenamed cp1, has a characteristic of observable top pods ( Figure 1 a, c), while WH506 is the opposite, the top pod is not obvious ( Figure 1 b, d). Further observation revealed that the main reason for this difference was the difference in the length of the upper petiole. The length of the petiole of the first seven trifoliate leaves from the top to the bottom of the pod-forming mutant cp1 was significantly longer than that of the non-pod-forming mutant WH506 ( Figure 1 e, f).

[0042] 2. Gene mapping of the cp1 pod-forming mutant

[0043] Fine mapping confirmed that the cp1 mutant was caused by a non-synonymous mutation from C to A in the first exon of the Glyma.19g185600 gene, which resulted in a change from leucine to isoleucine at position 174. Figure 2 ).

[0044] Example 2 Design of CAPS molecular markers for wild type and cp1 mutant

[0045] The genomic DNA of wild-type WH506 and cp1 mutants were extracted using the CTAB method, and after the quality was qualified by electrophoresis, they were used for subsequent PCR analysis. According to the site of the cp1 mutant gene, specific primers were designed. Since the restriction site is too close to the 3' end, the band separation may not be obvious. To solve this problem, the present invention randomly added 17bp bases (ATGCTTGATGAACTGAA) to the 5' end of the reverse primer, as shown in SEQ ID NO.5. Therefore, the amplified sequence length is 322bp, as shown in SEQ ID NO.2. The primer sequence is as follows:

[0046] SEQ ID NO.3: cp1-F:5'-CAACGAGGTAACACCACCCTTTG-3';

[0047] SEQ ID NO.4: cp1-R:

[0048] 5'-ATGCTTGATGAACTGAAGAGAGTTATGATTACCATAGAAAGCAT GG-3'

[0049] PCR amplification technology was used to detect wild-type WH506 and cp1 mutants using specific primers. The PCR amplification system included 12.5 μL of TaqMasterMix (2×), 1.0 μL each of forward and reverse primers, 2.0 μL of genomic DNA and 8.5 μL of ddH2O, with a total system of 25 μL. The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, extension at 72°C for 10 seconds, 36 cycles (0.2°C drop per round); final extension at 72°C for 5 minutes, and storage at 16°C for 2 hours. After the PCR reaction was completed, 1.2% agarose gel electrophoresis was used for detection. The product size was consistent with the theoretical fragment size, the product was highly specific, and there were no mixed bands. The PCR product was sent to a biological sequencing company for sequencing, and the sequencing results were analyzed for the mutation site using SeqMan software. The results showed that the mutation site of the Cp1 mutant was base A, and the wild-type WH506 had a base C at the corresponding site ( Figure 3 ).

[0050] The present invention further successfully developed CAPS markers using mutation sites. Using the analysis of individual plants and phenotypes of the isolated population, the normal plant type can be cut by AluI endonuclease into two bands of 271bp and 51bp; the homozygous high-yield pod-forming material is not cut by the endonuclease and shows a 322bp band; the heterozygous high-yield pod-forming material shows three band types ( Figure 3 The results indicate that the CAPS marker of the present invention can effectively identify the genotype of dense-density-tolerant, high-yielding, and pod-showing material, and can be used for molecular breeding to assist molecular identification.

[0051] Example 3 Breeding application of cp1 mutant

[0052] To evaluate the application of cp1 mutant in dense planting breeding, wild type WH506 and cp1 mutant were planted at a density of 30 cm row spacing and 10 cm plant spacing. Figure 4 As shown, Figure 4 The results showed that the mutant cp1 had a significant single-plant yield advantage under dense planting conditions. CP1 was further sexually hybridized with the nationally approved soybean variety Zhongdou 63 (abbreviated as ZD63) to obtain a near-isogenic line. Compared with NIL ZD63 Its NIL cp1 The yield performance of high density with row spacing of 30cm and plant spacing of 10cm is better ( Figure 5 This further proves that cp1 is a mutant with potential for application in soybean breeding for high-density tolerance and high yield, and has prospects for industrial promotion.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of SNP sites in identifying soybean high-yield and high-density tolerant plant types, characterized in that: The SNP site is located at position 585 of the soybean gene Glyma.19g185600, and the polymorphism is C / A; the nucleotide sequence of the soybean gene Glyma.19g185600 is shown in SEQ ID NO:

1.

2. A CAPS molecular marker related to soybean high-yield plant type traits, characterized by: The CAPS molecular marker comprises the SNP site according to claim 1 and the nucleotide sequence of its upstream and downstream components; the nucleotide sequence of the CAPS molecular marker is shown in SEQ ID NO:

2.

3. A primer set for detecting the CAPS molecular marker according to claim 2, characterized in that: The primer set includes a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

4.

4. A kit for detecting the CAPS molecular marker according to claim 2, characterized in that: The kit comprises the primer set according to claim 3.

5. A method for identifying soybean plant types that are tolerant to high density and high yield, characterized in that: The method comprises the following steps: (1) using the genomic DNA of the soybean plant to be identified as a template and performing PCR amplification using the primer set according to claim 3 to obtain an amplified product; (2) after enzyme digestion of the amplified product, detecting the polymorphism of the enzyme digestion product; (3) when the enzyme digestion product contains only one main band, the soybean to be identified is a homozygous high-density-resistant and high-yield plant type; when the enzyme digestion product contains three bands, the soybean to be identified is a heterozygous high-density-resistant and high-yield plant type; when the enzyme digestion product contains two bands, the soybean to be identified is a wild type; The enzyme digestion is performed using restriction endonuclease AluI.

6. The method according to claim 5, characterized in that The soybean plant type with high density tolerance and high yield is a plant type in which the upper petiole of the plant is shorter and the lower petiole is longer.

7. Use of the CAPS molecular marker according to claim 2, the primer set according to claim 3, or the kit according to claim 4 in identifying high-density-tolerant and high-yielding soybean plant types.

8. The use according to claim 7, characterized in that The planting row spacing of the dense-density-tolerant and high-yield soybean plant type is less than 40 cm.

9. Use of the CAPS molecular marker according to claim 2, the primer set according to claim 3, or the kit according to claim 4 in breeding of dense-density-tolerant soybean plants.

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