InDel molecular marker sl-12 for identifying peanut leaf size, primer pair and application thereof
By developing the InDel molecular marker SL-12 and its primer pair, the precise localization and early selection of peanut leaf size genes were achieved, solving the problems of high difficulty in developing molecular markers and long breeding cycles in peanut breeding, and realizing an efficient and low-cost breeding method.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANDONG ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2025-11-25
- Publication Date
- 2026-05-15
AI Technical Summary
Developing molecular markers for peanut leaf size-related genes is challenging. Traditional breeding methods rely on long-term field phenotypic identification, which is also affected by the environment, making it difficult to achieve early selection and efficient breeding.
Develop an InDel molecular marker SL-12 and its primer pair for PCR amplification and gel electrophoresis detection to accurately locate the peanut leaf size gene. Provide kits and identification methods to enable genotyping and early selection in the seedling stage.
By using molecular marker-assisted breeding, the breeding cycle can be shortened, the accuracy and efficiency of selection can be improved, and the cost can be reduced, thus enabling the rapid breeding of new small-leaf peanut varieties and improving photosynthetic efficiency.
Smart Images
Figure CN121183030B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology and relates to an InDel molecular marker SL-12 for identifying peanut leaf size, primer pairs, and their applications. Background Technology
[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.
[0003] Molecular markers are specific DNA fragments that reflect certain differences in the genomes of individuals or populations. They are commonly used for gene mapping and are widely applied in genetic breeding. InDel markers are a commonly used type of molecular marker. They are insertion and deletion variant markers based on genomic sequences and are characterized by high stability, rich polymorphism, and strong universality.
[0004] Peanuts are an important oilseed and economic crop in my country, rich in nutrients such as oil and protein. Cultivated peanuts are allotetraploids with complex genomes and high homology, making molecular marker development challenging and hindering genetic research progress. Leaf size is one of the important traits of peanuts. Traditional large-leaf peanuts significantly shade the middle and lower leaves, reducing the overall photosynthetic efficiency of the plant, while smaller leaves allow for better light penetration and improve photosynthetic balance within the plant population. Currently, almost no genes related to peanut leaf size have been reported. Using molecular markers for selective breeding of peanut small-leaf genes can effectively improve the efficiency of peanut plant architecture genetic improvement, thereby exploring its potential application value for yield enhancement in densely planted environments. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention precisely locates and develops an InDel marker SL-12 for a peanut leaf size-related gene from the complex tetraploid peanut genome and its application in molecular-assisted breeding.
[0006] The technical solution adopted in this invention is as follows:
[0007] In a first aspect of the invention, an InDel molecular marker SL-12 for identifying the size of peanut leaves is provided, the nucleotide sequence of which is shown in SEQ ID NO: 1.
[0008] The InDel molecular marker SL-12 for peanut leaf size-related genes provided by this invention is located in the 2.8 kb interval of chr15:156,936,257~156,939,086. The corresponding sequence of small-leaved peanut SA288 is shown in SEQ ID NO: 1, and the corresponding sequence of normal-leaved peanut Weihua 25 (WH25) is shown in SEQ ID NO: 2.
[0009] In a second aspect of the invention, a primer pair for identifying the size of peanut leaves is provided, the primer pair being designed based on the InDel molecular marker SL-12, the forward primer sequence being shown in SEQ ID NO: 3, and the reverse primer sequence being shown in SEQ ID NO: 4.
[0010] In a third aspect of the invention, a kit for identifying the size of peanut leaves is provided, comprising the primer pair described above.
[0011] In one or more embodiments of the present invention, the kit further comprises PCR reaction reagents and / or electrophoresis detection reagents.
[0012] In a fourth aspect of the invention, the application of the InDel molecular marker SL-12, the primer pair, or the kit is provided in marker-assisted breeding of peanuts.
[0013] Specifically, the application is in the breeding of peanut varieties with a small-leaf phenotype.
[0014] In a fifth aspect of the present invention, a method for identifying peanut leaf size-related genotypes is provided, comprising the following steps:
[0015] (1) Extract genomic DNA from the peanut sample to be tested;
[0016] (2) Perform PCR amplification using the primer pairs described above;
[0017] (3) Detect the fragment size of the PCR amplification product;
[0018] (4) Determine the genotype based on the size of the fragment: if a 459 bp fragment is amplified, the genotype is small-leaf homozygous; if a 2499 bp fragment is amplified, the genotype is large-leaf homozygous; if both 459 bp and 2499 bp fragments are amplified, the genotype is small-leaf heterozygous.
[0019] In one or more embodiments of the present invention, in step (2), the PCR amplification reaction system is as follows: 2 × Phanta Flash Master Mix (Dye Plus), DNA template, primer SL-12-F, primer SL-12-R and ddH2O.
[0020] The PCR amplification reaction program is as follows: pre-denaturation at 98℃ for 30 seconds; followed by 30 cycles at 98℃ for 10 seconds, 56℃ for 5 seconds, and 72℃ for 10 seconds; and finally extension at 72℃ for 1 minute.
[0021] In one or more embodiments of the present invention, in step (3), the fragment size of the PCR amplification product is detected by agarose gel electrophoresis; the agarose gel electrophoresis is performed using a gel with a concentration of 1 w / v% (meaning the mass of solute (g) contained in 100 ml of solution) at a voltage of 160 V for 25-35 minutes.
[0022] In a sixth aspect of the invention, a method for breeding small-leaf peanut varieties is provided, wherein the method is used to identify the genotype of individuals in a breeding population and to select offspring carrying the sequence shown in SEQ ID NO: 1 or those amplified with a 459 bp fragment for breeding.
[0023] In one or more embodiments of the present invention, the breeding population is an F2 generation segregating population, a backcross population, or a mutation population.
[0024] In summary, this invention provides a mutant small-leaf material SA288 and the DNA of its hybrid offspring population with the normal-leaf material WH25. The DNA template was amplified by PCR and gel electrophoresis using InDel marker SL-12. The genotype was determined based on the PCR amplification and gel electrophoresis results. If the marker amplification size of the offspring is the same as that of WH25, the genotype of the leaf size-related gene in that individual is the large-leaf parent type, and its phenotype is large-leaf. If the marker amplification size of the offspring is the same as that of SA288, the genotype of the leaf size-related gene in that individual is the small-leaf parent type, and its phenotype is small-leaf. Since small leaves are controlled by a dominant gene, when the marker amplification size of the offspring shows both WH25 and SA288, the individual's genotype is heterozygous, and its phenotype is small-leaf.
[0025] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:
[0026] (1) This invention is the first to develop the InDel molecular marker SL-12, which is closely linked to the major gene for peanut leaf size. By PCR amplification and gel electrophoresis detection of the DNA of the F2 segregating population generated after crossing small-leaf and normal-leaf plants, it can effectively distinguish between small-leaf and large-leaf materials and can be used for the breeding of new small-leaf peanut varieties. This marker is located in a specific 2.8Kb interval on chromosome chr15, which can accurately locate and control the genetic locus of leaf size, filling the gap in the development of molecular markers for this trait.
[0027] (2) Traditional field phenotypic identification relies on morphological observation during the middle and late stages of plant growth, which is time-consuming and highly susceptible to environmental influences. This invention can perform genotypic identification during the seedling stage or even using seed DNA, shortening the breeding selection cycle from several months to several days, enabling early selection, significantly accelerating the breeding process, and saving a lot of field labor and management costs.
[0028] (3) The marker is based on insertion / deletion variations in the genomic DNA sequence, and has high stability and good reproducibility. The PCR amplification results are clear and distinct, and can accurately distinguish between the three genotypes of homozygous large-leaf, homozygous small-leaf, and heterozygous small-leaf. The results are easy to interpret and the accuracy rate can reach 100%, effectively avoiding the interference of environmental factors and human experience misjudgment on the selection results.
[0029] (4) The detection method provided by this invention is based on conventional PCR and other technologies, and does not require expensive experimental equipment (such as sequencers and fluorescence PCR instruments) or complex operating skills. The reagents used are inexpensive and can be widely used, with low technical and economic barriers.
[0030] (5) This marker can be used to achieve targeted tracking and efficient aggregation of the dominant trait of "small leaves". Through molecular marker-assisted selection, new peanut germplasm or varieties with ideal plant type (small leaves, compact) can be bred quickly. This plant type is conducive to ventilation and light penetration in the population, improves the photosynthetic efficiency of the population under dense planting conditions, and provides key germplasm resources and technical support for breaking through the bottleneck of peanut yield.
[0031] (6) This marker can be used as a powerful tool to quickly screen and identify whether existing peanut germplasm resources carry the small leaf gene, deeply explore and utilize potential superior alleles, and provide rich parental selection for peanut plant type improvement breeding. Attached Figure Description
[0032] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0033] Figure 1 The image shows the leaf size phenotypic diagrams of the peanut parents WH25, SA288, F1 and F2 involved in this invention.
[0034] Figure 2 This is a diagram showing the initial localization BSA-seq analysis results of the invention.
[0035] Figure 3 This is an electrophoretic image of the InDel marker SL-12 used in this invention on the parents and the F2 population. Detailed Implementation
[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.
[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0039] Example 1: Group Construction
[0040] The small-leaved mutant SA288 was found in natural populations, with leaf length, leaf width, and leaf area all significantly smaller than the normal leaf WH25. Figure 1 Using SA288 as the male parent and WH25 as the female parent, the F1 generation showed a phenotype identical to SA288, indicating that leaflet size is controlled by a dominant gene. Figure 1 ), self-crossing produces the F2 segregating population.
[0041] Example 2: Preliminary mapping of leaflet genes
[0042] Fifty plants with extremely large leaves were selected from the F2 segregating population, and their DNA was extracted and mixed in equal amounts to form a pool of plants with extremely large leaves. Similarly, 50 plants with extremely small leaves were selected, and their DNA was extracted and mixed in equal amounts to form a pool of plants with extremely small leaves. Both pools were then sequenced at a depth of 40X. BSA-seq results showed that the major gene was located within a 6 Mb range of 153.00-159.00 Mb at chr15. Figure 2 ).
[0043] The DNA extraction method using the kit is as follows:
[0044] (1) Peanut leaves are ground into fine powder in liquid nitrogen and quickly transferred to a centrifuge tube containing 600 μL Buffer ATS. Add 4 μL RNase A (10 mg / ml), shake to mix, and homogenize thoroughly (avoiding any clumps of tissue). Let stand at room temperature for 10 min, inverting and mixing 2-3 times during this period.
[0045] (2) Centrifuge at 12,000 rpm for 3 min, and transfer the supernatant into a 1.5 ml centrifuge tube.
[0046] (3) Add 0.5 times the volume of the supernatant of isopropanol and mix thoroughly (precipitation may occur at this time). Transfer the resulting solution and any precipitate to the adsorption column (Spin Columns AC) that has been loaded into the collection tube. Centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.
[0047] (4) Add 600 μL of anhydrous ethanol buffer WB2 to the adsorption column AC, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.
[0048] (5) Repeat step (4).
[0049] (6) Put the adsorption column AC back into the empty collection tube, centrifuge at 12,000 rpm for 2 min, and discard the collection tube.
[0050] (7) Place the adsorption column AC into a new centrifuge tube, add 100 μL of Buffer EB to the middle of the adsorption membrane, incubate at room temperature for 3-5 min, centrifuge at 12,000 rpm for 1 min, and collect the DNA solution. Store the DNA at -20℃.
[0051] Example 3: Development and Application of Molecular Markers
[0052] 3.1 Molecular marker design
[0053] Based on the parental resequencing and BSA-seq results, the InDel marker SL-12 was designed in the 153.00-159.00 Mb region of chr15, with primer sequences as shown in SEQ ID NO:3 and SEQ ID NO:4.
[0054] 3.2 DNA Extraction
[0055] (1) Two 4mm steel balls and leaves of SA288, WH25 and F2 groups were placed in 2ml centrifuge tubes respectively, frozen with liquid nitrogen, and ground on a machine.
[0056] (2) Quickly transfer to a centrifuge tube containing 600 μL Buffer ATS and mix well. Add 4 μL RNase A, shake to mix, and homogenize thoroughly (avoiding any clumps of tissue). Let stand at room temperature for 10 min, inverting and mixing 2-3 times during this period.
[0057] (3) Centrifuge at 12,000 rpm for 3 min, and transfer the supernatant into a 1.5 ml centrifuge tube;
[0058] (4) Add 300 μL of isopropanol, mix thoroughly, and transfer the resulting solution and any precipitate that may form into the adsorption column (Spin Columns AC) already loaded into the collection tube. Centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and return the adsorption column to the collection tube;
[0059] (5) Add 600 μL of Buffer WB2 containing anhydrous ethanol to the adsorption column AC, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.
[0060] (6) Repeat step (4);
[0061] (7) Replace the adsorption column AC back into the empty collection tube, centrifuge at 12,000 rpm for 2 min, and discard the collection tube;
[0062] (8) Place the adsorption column AC into a new centrifuge tube, add 100 μL of Buffer EB to the middle of the adsorption membrane, place at room temperature for 3-5 min, centrifuge at 12,000 rpm for 1 min, and collect the DNA solution.
[0063] 3.3 PCR amplification
[0064] Peanut InDel-labeled SL-12 primers were synthesized at Sangon Biotech Co., Ltd., dissolved, and diluted to 10 μM for later use.
[0065] PCR amplification was performed using SA288, WH25, and F2 population DNA as templates and SL-12 primers. The PCR reaction volume was 20 μL, as shown in Table 1.
[0066] Table 1
[0067]
[0068] The PCR amplification reaction procedure is shown in Table 2:
[0069] Table 2
[0070]
[0071] 3.4 Agarose gel electrophoresis analysis
[0072] Prepare an agarose gel with a concentration of 1 w / v. Load 3 μL of PCR products from the SA288, WH25, and F2 populations onto the agarose gel. Perform electrophoresis at 160 V and 220 mA for 30 minutes, then irradiate the gel and record the results.
[0073] Electrophoretic results of InDel-labeled SL-12 on SA288, WH25, and F2 populations are shown in [the table below]. Figure 3As shown in the figure, the SA288 product amplified by SL-12 primers shows a 459bp band, and the WH25 product amplified by SL-12 primers shows a 2499bp band. If the F2 individual amplification product is a 459bp band, it indicates a small-leaf phenotype; if the F2 individual amplification product is a 2499bp band, it indicates a large-leaf phenotype; if the F2 individual amplification product shows both 459bp and 2499bp bands, it indicates a heterozygous small-leaf phenotype.
[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. An InDel molecular marker SL-12 for identifying peanut leaf size, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1 or SEQ ID NO:
2. The alleles corresponding to the small leaf phenotype contain the sequence shown in SEQ ID NO: 1, and the alleles corresponding to the normal leaf phenotype contain the sequence shown in SEQ ID NO:
2. The peanut varieties are SA288, WH25 and their progeny populations.
2. The application of the InDel molecular marker SL-12 as described in claim 1 in marker-assisted breeding of peanut leaf size, characterized in that, The peanut varieties mentioned are SA288, WH25 and their progeny populations.
3. The application as described in claim 2, characterized in that, The application is in the selection and breeding of peanut varieties with a small leaf phenotype.
4. A method for identifying genotypes related to peanut leaf size, characterized in that, The peanut varieties mentioned are SA288, WH25 and their progeny populations, and the method includes the following steps: (1) Extract genomic DNA from the peanut sample to be tested; (2) PCR amplification is performed using the primer pair for amplifying the InDel molecular marker SL-12 of claim 1, the primer pair comprising a forward primer and a reverse primer, the forward primer sequence being shown in SEQ ID NO: 3 and the reverse primer sequence being shown in SEQ ID NO: 4; (3) Detect the fragment size of the PCR amplification product; (4) Determine the genotype based on the size of the fragment: if a 459 bp fragment is amplified, the genotype is small-leaf homozygous; if a 2499 bp fragment is amplified, the genotype is large-leaf homozygous; if both 459 bp and 2499 bp fragments are amplified, the genotype is small-leaf heterozygous.
5. The method for identifying peanut leaf size-related genotypes as described in claim 4, characterized in that, In step (2), the PCR amplification reaction system is as follows: 2 × Phanta Flash Master Mix, DNA template, forward primer as shown in SEQ ID NO: 3, reverse primer as shown in SEQ ID NO: 4, and ddH2O.
6. The method for identifying peanut leaf size-related genotypes as described in claim 4, characterized in that, In step (3), the fragment size of the PCR amplification product is detected by agarose gel electrophoresis; the agarose gel electrophoresis uses a gel with a concentration of 1 w / v% and is performed at a voltage of 160 V for 25-35 minutes.
7. A method for breeding small-leaf peanut varieties, characterized in that, Genotyping of individuals in the breeding population is performed using the method described in any one of claims 4 to 6, and offspring carrying the sequence shown in SEQ ID NO: 1 or amplified with a 459 bp fragment are selected for breeding; the peanut varieties are SA288, WH25 and their offspring populations.