InDel marker co-1 for peanut bud opening character, detection primer pair and screening method

Abstract

The application discloses an InDel marker CO-1 of peanut bud open trait, a detection primer pair and a screening method, and belongs to the technical field of molecular biology. The application also discloses the detection primer pair of the peanut InDel marker CO-1, wherein the nucleotide sequence of an upstream primer is shown in SEQ ID NO:1, and the nucleotide sequence of a downstream primer is shown in SEQ ID NO:2. The peanut InDel marker CO-1 and primers thereof are disclosed for the first time, DNA of 200 natural population materials is subjected to PCR amplification, and electrophoresis is used for detection, and it is found that the amplification product of the CO-1 marker primer can be obviously distinguished from peanut bud open traits, which has important significance for the screening of peanut varieties in the production of buds.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an InDel marker CO-1 for the clove-opening trait of peanut sprouts, a detection primer pair, and a screening method. 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 should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Peanuts are an important oilseed crop in my country, and a significant source of high-quality protein and oil. Peanut sprouts are edible sprout vegetables that germinate from seeds in a short period of time. During this process, the large molecular storage substances in the seeds are broken down, facilitating the body's absorption of nutrients. In addition to being rich in protein and fat, peanut sprouts are also high in resveratrol, containing 4-5 times more than peanuts themselves, comparable to the resveratrol content in wine, making them a highly nutritious food. Therefore, sprout production has great potential in the later-stage processing of peanuts.

[0004] The evaluation of peanut sprouts mainly focuses on aspects such as sprout yield, nutrient composition, growth characteristics, and sensory evaluation. Among these, the splitting of the seed coat is a negative factor affecting both yield and appearance quality. After splitting, the hypocotyl growth of the sprout often slows down, while the terminal bud grows rapidly, frequently causing cotyledon shedding, leading to reduced yield and uneven appearance. Figure 1 Therefore, selecting peanut varieties that do not split open during the sprout cultivation process is the foundation of sprout production.

[0005] Molecular marker technology, as an important tool in modern genetic research, plays an irreplaceable role in germplasm resource identification. Its core advantage lies in its ability to directly reveal genetic diversity at the DNA level, overcoming the limitations of traditional morphological identification and providing a scientific basis for the accurate identification, classification, and utilization of germplasm resources. Molecular markers are diverse, mainly including PCR-based markers such as SSR (simple sequence repeat), SNP (single nucleotide polymorphism), and InDel (insertion-deletion). Among them, InDel molecular markers are a type of molecular marker technology developed based on insertion or deletion variations at specific locations in the genome. Due to their high polymorphism, ease of development, and low cost, they are widely used in fields such as genetic diversity analysis, variety identification, evolutionary research, and molecular breeding. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an InDel marker CO-1 closely linked to the peanut sprout clove phenotype and its application in peanut sprout variety screening. Using the InDel molecular marker of the present invention, the clove-opening trait of peanut sprouts can be detected and identified, enabling rapid and efficient screening of peanut germplasm resources suitable for sprout production.

[0007] The technical solution adopted in this invention is as follows:

[0008] In a first aspect of the invention, an InDel marker CO-1 for the splitting morphology of peanut sprouts is provided, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0009] In a second aspect of the invention, a primer pair is provided for detecting the clove-opening trait of peanut sprouts, for amplifying the InDel marker CO-1 of the peanut sprout clove-opening trait, the primer pair consisting of the following sequence:

[0010] Upstream primer: as shown in SEQ ID NO: 1;

[0011] Downstream primer: as shown in SEQ ID NO: 2.

[0012] In a third aspect of the invention, a kit for detecting the clove-opening trait of peanut sprouts is provided, comprising the primer pair described above.

[0013] In one or more embodiments of the present invention, the kit further comprises one or more of PCR reaction buffer, dNTPs, Taq DNA polymerase, and nucleic acid electrophoresis detection reagents.

[0014] In a fourth aspect of the invention, the use of the InDel-labeled CO-1, the primer pair, or the kit is provided in any of the following:

[0015] (a) Screening peanut germplasm resources suitable for peanut sprout production;

[0016] (b) To identify or assist in the identification of the clove-like characteristics of peanut sprouts;

[0017] (c) Peanut molecular marker-assisted breeding.

[0018] In a fifth aspect of the present invention, a method for screening peanut germplasm resources suitable for peanut sprout production is provided, comprising the following steps:

[0019] (1) Extract genomic DNA from the peanut plants to be tested;

[0020] (2) Using the genomic DNA obtained in step (1) as a template, perform PCR amplification using the primer pair described above;

[0021] (3) Detect the band size of the PCR amplification product obtained in step (2) and determine the clove-opening trait of peanut sprouts based on the band size; if the electrophoresis pattern shows a 172bp band, it indicates that the peanut plant to be tested contains a clove-opening gene, corresponding to the clove-opening trait.

[0022] If the electrophoresis pattern shows only a 172bp band, it indicates that the peanut plant being tested has a homozygous open petal gene; if it contains both 172bp and 192bp bands, it indicates that the peanut plant being tested has a heterozygous open petal gene; if it shows only a 192bp band, it indicates that the peanut plant being tested does not contain the open petal gene.

[0023] According to a further preferred embodiment of the present invention, in step (1), the genomic DNA of the peanut plant to be tested is extracted using a modified CTAB method.

[0024] According to a further preferred embodiment of the present invention, in step (2), the PCR reaction system comprises the following components: template DNA, upstream primer, downstream primer, dNTPs, Taq DNA polymerase, 1×PCR buffer containing 1.5-2.5 mM MgCl2 and ddH2O.

[0025] Specifically, the PCR reaction system is as follows, with a total volume of 10 μL:

[0026] 2 μL of 10 ng / μL template DNA, 1 μL of 10×PCR buffer containing 20 mM MgCl2, 0.25 μL of 2.5 mM dNTPs, 0.2 μL of 10 μM upstream primer, 0.2 μL of 10 μM downstream primer, 0.1 μL of 1 U Taq DNA polymerase, and ddH2O to a final volume of 10 μL.

[0027] According to a further preferred embodiment of the present invention, in step (2), the PCR amplification procedure is as follows:

[0028] Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 15 s, for a total of 35 cycles; extension at 72℃ for 5 min.

[0029] According to a further preferred embodiment of the present invention, in step (3), the fragment size of the PCR amplification product is detected by polyacrylamide gel electrophoresis, agarose gel electrophoresis or capillary electrophoresis.

[0030] The polyacrylamide gel electrophoresis was performed using 8% denaturing polyacrylamide gel electrophoresis.

[0031] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0032] This invention discloses for the first time the InDel marker CO-1 associated with peanut clove-opening genes and its detection primers. By performing PCR amplification on the DNA of inbred lines of peanuts with and without cloves, as well as the F2 segregating population materials produced by hybridization of the two, and detecting it using 8% denaturing polyacrylamide gel electrophoresis, it was found that the InDel marker CO-1 associated with peanut clove opening can clearly distinguish between peanut materials with and without cloves, and can be used for screening non-clove varieties in sprout production. Attached Figure Description

[0033] 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.

[0034] Figure 1 Phenotypes of open and closed peanut sprouts.

[0035] Figure 2 The nucleotide sequence of the InDel marker CO-1 of the peanut valve-opening gene;

[0036] Caption: The underlined area in the figure is the InDel marker CO-1; compared with open peanut (DL057), unopened peanut (SA890) has a 20 bp DNA insertion at this site.

[0037] Figure 3 Application of the peanut valve-opening gene InDel marker CO-1 in molecular-assisted breeding;

[0038] Caption: The image shows the results of molecular marker detection of the primers in unopened peanut (SA890) and open peanut (DL057) and the F1 generation population produced by their hybridization; Lane 1: Parent P1 (SA890, unopened), Lane 2: Parent P2 (DL057, open), Lanes 3-12: F1 generation single plants from the hybridization of SA890 and DL057.

[0039] Figure 4 Detection of molecular markers in the F2 segregating population of peanut pod-opening gene InDel marker after crossing non-pod-opening peanut (SA890) and pod-opening peanut (DL057);

[0040] Caption: The image shows the molecular marker detection results of the F2 segregating population generated after crossing non-opening peanut (SA890) and open-opening peanut (DL057) using the InDel gene marker for promoting opening; Lane 1: Parent P1 (SA890, non-opening), Lane 2: Parent P2 (DL057, open), Lanes 3-14: F2 non-opening population of SA890 and DL057, Lanes 15-26: F2 open-opening population of SA890 and DL057.

[0041] Figure 5 Photograph of PCR amplification electrophoresis results of peanut germplasm resources using the peanut valve-opening gene InDel marker CO-1;

[0042] Caption: The image shows the results of detecting 26 peanut germplasm resources using the InDel gene marker to promote petal opening; parent P1 (SA890, non-penetrating), lane 2: parent P2 (DL057, petal opening); lanes 3-14: non-penetrating germplasm resources; lanes 15-26: petal opening germplasm resources. Detailed Implementation

[0043] 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.

[0044] 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.

[0045] 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.

[0046] biomaterial sources

[0047] The 433 natural populations, including SA890 and DL057, are preserved by the Institute of Crop Germplasm Resources, Shandong Academy of Agricultural Sciences. The F1 and F2 generation materials are offspring obtained by our laboratory through hybridization of the two germplasms, SA890 and DL057.

[0048] Example 1: Development of InDel-labeled primers

[0049] Using 433 natural peanut populations, 50 plump peanut seeds were selected from each germplasm. These seeds were soaked in water for 4 hours to allow them to swell, washed twice with sterile water, and placed in petri dishes lined with moistened filter paper. Germination was carried out in the dark at 28°C. After 24-36 hours, most seeds showed signs of sprouting. These sprouted seeds were then transplanted, radicle-side down, into seedling trays with 1.5cm spacing between holes. Water was added just above the radicle, and the trays were placed in an artificial climate chamber at 26°C in the dark. Six days after transplanting, the peanut sprout phenotype was examined. Genome-wide association analysis (GWAS) was performed on the cotyledon opening phenotype of peanut sprouts based on our laboratory's peanut resequencing data. One significant locus was identified, falling within the gene... AhCO1 Within the genome, this gene was listed as a candidate gene for promoting petal opening. Based on the resequencing results, the genome sequences of the non-peeling germplasm SA890 and the peteling germplasm DL057 were compared, and InDel primers were designed in the flanking region of the petal opening gene. The designed InDel primers were tested using 20 peteling germplasms and 20 non-peeling germplasms, revealing a close linkage between the molecular marker CO-1 and the petal opening phenotype. The sequence alignment diagram of the peanut InDel marker CO-1 is shown below. Figure 2 As shown in the figure, the underlined area is the InDel region. Compared to open-lobed peanuts (DL057), unopened peanuts (SA890) have a 20 bp DNA insertion. Primers for detecting peanut InDel-labeled CO-1 were designed, and the primer sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0050] Example 2: Application of InDel-labeled primers

[0051] 2.1 Extraction of plant material and template DNA

[0052] Leaves from peanut parents SA890 and DL057, as well as F1 hybrid plants, were selected as experimental materials.

[0053] Total DNA was extracted using a modified CTAB method, with the following specific steps:

[0054] 1) Take 1.0 g of fresh peanut leaves and grind them into powder quickly in liquid nitrogen;

[0055] 2) Transfer the frozen powder into a pre-chilled centrifuge tube, add an equal volume (w / v, g / ml) of 2×CTAB extraction buffer, and incubate at 65℃ for 20 minutes;

[0056] 3) Add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1), invert the centrifuge tube to mix, and centrifuge at 12000 r / min at room temperature for 10 minutes;

[0057] 4) Transfer the supernatant to another centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1), invert the centrifuge tube to mix, and centrifuge at 12000 r / min at room temperature for 10 minutes.

[0058] 5) Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, gently invert to mix, and let stand at room temperature for 30 minutes.

[0059] 6) Centrifuge at 12000 rpm for 5-10 minutes at room temperature, then discard the supernatant;

[0060] 7) Rinse with 70% ethanol, centrifuge at 12000 r / min at room temperature for 5-10 minutes, and discard the supernatant;

[0061] 8) Repeat step 7) once;

[0062] 9) After the precipitate has dried, add 100 μL of ultrapure water to dissolve the DNA and store at -20℃ for later use.

[0063] 2.2 PCR Amplification

[0064] Based on the peanut InDel-labeled CO-1 detection primers (SEQ ID NO: 1 and SEQ ID NO: 2) designed above, peanut InDel-labeled CO-1 primers were synthesized at Sangon Biotech Co., Ltd., dissolved, and diluted to 10 μM for later use. Using the extracted DNA as a template, PCR amplification was performed using the peanut InDel-labeled primers to obtain PCR products.

[0065] The PCR reaction system is as follows, with a total volume of 10 μL: 2 μL of 10 ng / μL template DNA, 1 μL of 10×PCR buffer containing 20 mM MgCl2, 0.25 μL of 2.5 mM dNTPs, 0.2 μL of 10 μM upstream primer, 0.2 μL of 10 μM downstream primer, 0.1 μL of 1 U Taq DNA polymerase, and ddH2O added to a final volume of 10 μL.

[0066] The amplification procedure is as follows:

[0067] Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 15 s, for a total of 35 cycles; extension at 72℃ for 5 min.

[0068] 2.3 Polyacrylamide gel electrophoresis separation and polymorphism detection

[0069] The PCR products were subjected to denaturing polyacrylamide gel electrophoresis at a mass concentration of 8%, and then detected by EB staining.

[0070] After mixing the PCR product with the loading buffer, pre-denatured it at 95°C for 10 min, then took 2 μL of the sample and loaded it onto a Bio-Rad (USA) nucleic acid electrophoresis system. The electrode buffer was 1×TBE, and the electrophoresis was performed at 100 volts at room temperature for 2 h. The results were then stained with EB, photographed, and recorded.

[0071] Figure 3 The image shows the results of 8% denaturing polyacrylamide gel electrophoresis of PCR amplification products from SA890, DL057, and two F1 populations of germplasm hybrids using primers with peanut InDel-labeled CO-1.

[0072] Figure 4 The image shows the results of 8% denaturing polyacrylamide gel electrophoresis of PCR amplification products from SA890, DL057, and their hybrid F2 populations using primers with peanut InDel-labeled CO-1.

[0073] Depend on Figures 3-4 It can be seen that the primers for peanut InDel-labeled CO-1 can effectively amplify the DNA of both open and closed peanuts, with clear banding patterns and good polymorphism in open and closed peanut germplasm resources. The amplified band of closed germplasm is 192 bp, and the amplified band of open germplasm is 172 bp, which can effectively distinguish between open and closed peanuts. It can be used to screen for closed peanut germplasm resources suitable for sprout production.

[0074] Example 3: Validation of InDel-labeled CO-1 in multiple varieties of resources

[0075] To verify the broad applicability of the InDel marker CO-1, this embodiment selected 200 natural population germplasm resources of known peanut sprout open-lobed phenotypes (including 100 non-open-lobed materials and 100 open-lobed materials) and used CO-1 marker for detection.

[0076] The detection method was the same as in Example 2. Statistical analysis of the detection results for all 200 materials showed that: in 100 non-lobed germplasm samples, 100 amplified a 192 bp band, with a concordance rate of 100%; in 100 lobe-opened germplasm samples, 100 amplified a 172 bp band, with a concordance rate of 100%. The overall validation accuracy of this marker reached 100% in 200 materials with different genetic backgrounds, indicating that the CO-1 marker has high reliability and broad applicability, and can be used for rapid screening of large-scale germplasm resources.

[0077] To clearly present the results, Figure 5 The PCR amplification and electrophoresis results of 26 germplasm resources (including 12 non-valved and 14 valved materials) are listed as representative. (See figure below.)

[0078] Lane 1: Parent P1 (SA890, unopened, 192bp);

[0079] Lane 2: Parent P2 (DL057, open petal, 172bp);

[0080] Lanes 3-14: All 12 non-open germplasm resources amplified 192 bp bands;

[0081] Lanes 15-26: 14 open-petal germplasm resources, all amplified to 172 bp bands.

[0082] The test results of these 26 representative materials are consistent with the overall statistical conclusions mentioned above.

[0083] 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 marker CO-1 for the clove-opening trait of peanut sprouts, characterized in that, The molecular marker is a genomic fragment containing the nucleotide sequence shown in SEQ ID NO:3, which has an insertion / deletion polymorphism of 20 bp in length, and the polymorphic fragment is TTTTGTTTATTCAATTTCAA.

2. The use of the InDel marker CO-1 as described in claim 1 in any of the following: (a) Screening peanut germplasm resources suitable for peanut sprout production; (b) To identify or assist in the identification of the clove-like characteristics of peanut sprouts; (c) Marker-assisted breeding of peanut sprouts with clove-opening trait; The application is achieved by detecting the size of the InDel-tagged CO-1 fragment in the genomic DNA of the peanut plant to be tested. The detection method includes the following steps: (1) Extract genomic DNA from the peanut plants to be tested; (2) Using the genomic DNA obtained in step (1) as a template, perform PCR amplification using the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2; (3) Detect the fragment size of the PCR amplification product obtained in step (2) and determine the petal opening trait of peanut sprouts based on the fragment size; if the electrophoresis pattern only has a 172bp band, it indicates that the petal opening gene in the peanut plant to be tested is homozygous; if it contains both 172bp and 192bp bands, it indicates that the petal opening gene in the peanut plant to be tested is heterozygous; if it only has a 192bp band, it indicates that the peanut plant to be tested does not contain the petal opening gene.

3. A method for screening peanut germplasm resources suitable for peanut sprout production, characterized in that, Includes the following steps: (1) Extract genomic DNA from the peanut plants to be tested; (2) Using the genomic DNA obtained in step (1) as a template, perform PCR amplification using the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2; (3) Detect the fragment size of the PCR amplification product obtained in step (2) and determine the petal opening trait of peanut sprouts based on the fragment size; if the electrophoresis pattern only has a 172bp band, it indicates that the petal opening gene in the peanut plant to be tested is homozygous; if it contains both 172bp and 192bp bands, it indicates that the petal opening gene in the peanut plant to be tested is heterozygous; if it only has a 192bp band, it indicates that the peanut plant to be tested does not contain the petal opening gene.

4. The method for screening peanut germplasm resources suitable for peanut sprout production as described in claim 3, characterized in that, In step (2), the PCR reaction system contains the following components: template DNA, upstream primer, downstream primer, dNTPs, Taq DNA polymerase, 1×PCR buffer containing 1.5-2.5 mM MgCl2 and ddH2O.

5. The method for screening peanut germplasm resources suitable for peanut sprout production as described in claim 3, characterized in that, In step (2), the PCR amplification procedure is as follows: Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 15 s, for a total of 35 cycles; extension at 72℃ for 5 min.

6. The method for screening peanut germplasm resources suitable for peanut sprout production as described in claim 3, characterized in that, In step (3), the fragment size of the PCR amplification product is detected by polyacrylamide gel electrophoresis, agarose gel electrophoresis or capillary electrophoresis; the polyacrylamide gel electrophoresis is denaturing polyacrylamide gel electrophoresis with a mass percentage of 8%.

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