InDel molecular marker for identifying peanut shell hardness, primer pair and application thereof

Abstract

This invention discloses an InDel molecular marker, primer pair, and their applications for identifying peanut shell hardness, belonging to the field of molecular biology. The InDel molecular marker is located on peanut chromosome A16 and is characterized by a 2502 bp nucleotide sequence deletion, as shown in SEQ ID NO:5. This invention also provides specific primer pairs for detecting this marker, as shown in SEQ ID NO:1 and 2. PCR amplification using these primer pairs allows for rapid identification of peanut shell hardness by detecting amplified fragments of 877 bp (high hardness) or 3379 bp (low hardness). This molecular marker shows a significant correlation with shell hardness traits, and the detection method is simple, rapid, and low-cost, making it suitable for rapid identification, screening, and marker-assisted breeding of peanut germplasm resources with high shell hardness.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to an InDel molecular marker, primer pair, and their applications for identifying peanut shell hardness. 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] peanut( Arachis hypogaea L. Peanuts are an important grain and oilseed crop in China, but the increasing planting area and yield have led to rising production costs and other production problems. Improving the level of mechanized harvesting is an effective measure to solve this problem. Insufficient shell mechanical properties lead to pod damage during harvest, with shell strength showing a highly significant negative correlation with the breakage rate. The large number of peanut germplasm samples with significant differences in shell hardness collected in our laboratory can be used to discover the main genes controlling pod mechanical traits, which is an important foundation for optimization and improvement using molecular marker-assisted breeding technology.

[0004] As an allotetraploid plant (AABB, 2n=4x=40), peanut gene mapping is more complex and difficult than that of diploid plants, requiring overcoming polyploid redundancy and repetitive sequence interference. Currently, QTLs related to peanut shell hardness are rarely reported, and the phenotypic contribution of published QTLs is small, with poor reproducibility across different years and environments. Therefore, further research is needed on QTLs related to peanut shell hardness. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention identifies phenotypic data of shell hardness in 500 natural peanut populations under two environments and performs genome-wide association analysis using laboratory whole-genome resequencing data. The aim is to discover and locate SV signals and major genes significantly associated with target traits, and to develop functional molecular markers for germplasm improvement of peanut shell mechanical properties.

[0006] The primary objective of this invention is to provide an InDel molecular marker that is closely linked to the hardness trait of peanut shells.

[0007] Another object of the present invention is to provide specific primer pairs for detecting the above-mentioned InDel molecular markers.

[0008] Another objective of this invention is to provide the application of the above-mentioned InDel molecular markers and primer pairs in the identification of peanut germplasm resources and molecular marker-assisted breeding.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention, based on genome-wide association analysis (GWAS), identified a structural variant (SV) significantly associated with peanut shell hardness, namely the InDel molecular marker tightly linked to peanut shell hardness. This marker is essentially a structural variant located in a specific region of the peanut A16 chromosome, characterized by a 2502 bp (1998250~2000751 bp) nucleotide sequence deletion, as shown in SEQ ID NO:5. The complete reference sequence containing this deleted region (i.e., the amplified sequence) is shown in SEQ ID NO:3.

[0011] The InDel marker has two main alleles: the reference allele, which has a complete sequence with no deletions and a corresponding amplified fragment of 3379 bp; and the deletion allele, which has a 2502 bp deletion and a corresponding amplified fragment of 877 bp.

[0012] Genome-wide association analysis and haplotype analysis confirmed that the deleted allele was significantly positively correlated with the trait of high shell hardness.

[0013] Based on this, in a first aspect of the present invention, an InDel molecular marker for identifying peanut shell hardness is provided, the marker being located at position 1998250~2000751 bp on chromosome A16 of the peanut genome, and is represented by a deletion of a nucleotide sequence of length 2502 bp; the deleted nucleotide sequence is shown in SEQ ID NO:5.

[0014] In a second aspect of the invention, a primer pair for detecting peanut shell hardness is provided, the primer pair being capable of specifically amplifying a genomic region containing the deletion variant site shown in SEQ ID NO: 5; the primer pair consisting of an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 2.

[0015] The primer pair is designed with specificity, with the upstream primer located at chr16:1997487~1997508bp (22bp in total) and the downstream primer located at chr16:2000845~2000865bp (21bp in total), which allows for direct identification of the genotype at this locus through the length polymorphism of the PCR amplification product.

[0016] Based on this primer pair, in a third aspect of the present invention, a kit for identifying the hardness of peanut shells is provided, comprising the primer pair described above.

[0017] In a fourth aspect of the invention, the application of the InDel molecular marker for identifying peanut shell hardness, the primer pair, or the kit described herein is provided in peanut breeding for identifying shell hardness.

[0018] In a fifth aspect of the present invention, a method for determining the hardness of peanut shells is provided, comprising the following steps:

[0019] (1) Extract genomic DNA from the peanut sample 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 fragment size of the PCR amplification product: If the main band size of the amplification product is 877 bp, the peanut sample to be tested is determined to be of the high shell hardness type, representing the deletion haplotype; if the main band size of the amplification product is about 3379 bp, the peanut sample to be tested is determined to be of the low shell hardness type, representing the reference haplotype.

[0022] In step (2), the PCR amplification reaction system includes: DNA template, upstream primer, downstream primer, ddH2O and 2×Taq Plus Master Mix.

[0023] In step (2), the PCR amplification program is as follows: pre-denaturation at 98 ℃ for 30 s; followed by 34 cycles, each cycle consisting of 98 ℃ for 10 s, 65 ℃ for 5 s, 72 ℃ for 4 min; and finally, final extension at 72 ℃ for 5 min.

[0024] In step (3), the PCR amplification products are detected by agarose gel electrophoresis. The concentration of the agarose gel is 1.2 w / v%, and the electrophoresis conditions are 120 V voltage and 140 mA current.

[0025] In a sixth aspect of the present invention, a method for screening peanut breeding materials with high shell hardness is provided, comprising the following steps: determining shell hardness by detecting the deletion of the sequence shown in SEQ ID NO: 5 in the peanut genome.

[0026] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0027] The InDel marker provided by this invention was identified through GWAS analysis of a large-scale natural population. It showed a highly significant association with the trait of pericarp hardness and exhibited good reproducibility in different environments. Using the primer pairs of this invention, genotyping can be completed rapidly, and pericarp hardness at maturity can be accurately predicted, overcoming the bottlenecks of traditional phenotypic identification methods, which are characterized by long cycles and significant environmental influences.

[0028] The InDel marker obtained by screening in this invention is located in a key region of a major gene (AhSHG1) that controls fruit shell hardness, and has a large contribution to the phenotype, resulting in high screening efficiency.

[0029] The molecular marker developed in this invention is of the InDel type. The detection method is based on conventional PCR and agarose gel electrophoresis. It does not require complicated instruments and expensive reagents. It is simple and quick to operate and easy to promote and apply in most molecular laboratories and breeding units. It is particularly suitable for large-scale germplasm resource screening and early generation selection in breeding.

[0030] The InDel markers obtained by screening in this invention provide a direct and effective tool for the genetic improvement of peanut shell mechanical strength, which plays an important role in breeding new peanut varieties suitable for mechanized harvesting, reducing production costs, and improving industrial efficiency. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, 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.

[0032] Figure 1 The distribution of shell hardness of different peanut varieties between two points over two years;

[0033] Figure 2 The GWAS correlation results for peanut shell hardness between two points over two years are shown. In the figure, a is the Manhattan plot of shell hardness under the GLM model in the 2022 environment, and b is the Manhattan plot of shell hardness under the MLM model in the 2023 environment.

[0034] Figure 3 The results of haplotype analysis of sites significantly associated with peanut shell hardness are shown, where a and b are box plots of two haplotypes of sites significantly associated with shell hardness in 2022 and 2023, respectively, using hardness typing.

[0035] Figure 4 For amplified sequences, SV and AhSHG1 The relative positions of the three on peanut chromosome 16;

[0036] Figure 5 Agarose gel electrophoresis images of primer pairs TH-1 and TH-2 used to identify high-hardness and low-hardness peanut varieties. Detailed Implementation

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 1. Plant material and phenotypic identification

[0041] Population Construction: 500 peanut germplasm resources collected in our laboratory were used as the association analysis population. This population exhibits rich genetic diversity, encompassing all five peanut subspecies: fastigiata, hypogaea, vulgaris, peruviana, and hirsuta. The germplasm materials originated from multiple countries and regions worldwide, including China, Argentina, Brazil, India, Bolivia, Zambia, Israel, Peru, Sudan, Japan, Mexico, the United States, Vietnam, Hungary, Ethiopia, Myanmar, Chile, and Morocco.

[0042] This population of germplasm resources covers major cultivation types and geographical ecotypes, representing a wide distribution of the global genetic background of peanut, and providing a reliable material basis for subsequent molecular marker association analysis.

[0043] Phenotypic data collection: Field cultivation experiments were conducted in two experimental fields in Wangbian Community, Heshan Township, Ningyang County, Tai'an City, Shandong Province in 2022 and 2023, respectively. The positive pressure shell-breaking force (unit: Newton, N) of peanut pods was measured using a texture analyzer as a quantitative indicator of shell hardness. Twelve pods were measured repeatedly for each material, and the average value was taken as the phenotypic value of that material.

[0044] Phenotypic data analysis: The phenotypic data distribution at two points over two years is as follows Figure 1As shown, the shell hardness phenotypic values ​​ranged from 29.45 to 141.28 N, exhibiting a continuous distribution consistent with quantitative traits. Significant differences were observed across different environments and years, but the relative hardness ranking among materials within the population remained stable, indicating that this trait is strongly controlled by genetic factors and is suitable for genome-wide association analysis.

[0045] 2. Genome sequencing and variant identification

[0046] DNA sequencing: Genomic DNA was extracted from the leaves of 500 peanut materials and whole-genome resequencing was performed using the Illumina HiSeq platform, with an average sequencing depth of 10X.

[0047] Data alignment and variant detection: The raw sequences obtained from sequencing were aligned with the peanut reference genome (Arachishypogaea L. Tifrunner.gnm1). Genotyping of single nucleotide polymorphisms (SNPs), insertions / deletions (InDels), and structural variations (SVs) was performed using bioinformatics software (such as GATK and SVcaller).

[0048] 3. Genome-wide association analysis (GWAS)

[0049] Analysis methods: Association analysis was performed between fruit shell hardness phenotypic data and genome-wide genetic variation data. Generalized linear model (GLM) and mixed linear model (MLM) were used to control for the influence of population structure.

[0050] Correlation results: GWAS analysis results are as follows Figure 2 As shown, in both models and under both years, a highly significant association signal was detected in the region approximately 2.0 Mb below chromosome A16. This signal was dominated by a structural variant (SV), whose -log10 (P-value) far exceeded the significance threshold (>7), indicating a highly significant association between this site and the pericarp hardness trait.

[0051] 4. Fine localization and haplotype analysis of associated loci

[0052] Site confirmation: Further analysis of the significantly associated region identified a nucleotide deletion of 2502 bp (the sequence of which is shown in SEQ ID NO:5), which was defined as a candidate InDel molecular marker.

[0053] Haplotype analysis: Based on the genotype of the SV locus, the 500 samples were divided into reference haplotypes (without deletion) and deletion haplotypes (with deletion). For example... Figure 3As shown, statistical analysis revealed that, in both years, the average shell hardness of the missing haplotype was significantly higher than that of the reference haplotype (P < 0.001). This further confirms the close linkage between the InDel marker and the high shell hardness trait.

[0054] Gene annotation: Gene annotation of the SV region revealed that the deleted region is located in a key regulatory region or intron of a gene (named AhSHG1, the coding sequence of which is shown in SEQ ID NO:4) presumably related to secondary cell wall synthesis. This gene was identified as a major candidate gene controlling pericarp hardness. The relative positions of the amplified sequence, SV, and AhSHG1 gene are shown below. Figure 4 As shown.

[0055] Example 2: Development of detection primers

[0056] 1. Specific primer design

[0057] To convert the identified 2502 bp InDel into a usable molecular marker, specific PCR primers were designed on conserved sequences flanking the SV deletion region.

[0058] Upstream primer TH-1: 5'-GTGCCCAAACAAGCACTAAATC-3' (SEQ ID NO:1)

[0059] Downstream primer TH-2: 5'-GTTGCCTTCTTCACGTGACTG-3' (SEQ ID NO:2).

[0060] Primer design principle: This primer pair is designed to be specific, with the upstream primer located at chr16: 1997487~1997508bp (22bp in total) and the downstream primer located at chr16: 2000845~2000865bp (21bp in total), thus enabling direct identification of the genotype at this locus through the length polymorphism of the PCR amplification product. This design ensures:

[0061] For the reference allele (without deletion), the amplified fragment size is 3379 bp (sequence shown in SEQ ID NO:3).

[0062] For deletion alleles (with deletion), the amplified fragment size is 877 bp (3379 bp - 2502 bp).

[0063] 2. Establishment and optimization of PCR detection system

[0064] DNA extraction: Take young peanut leaves and extract high-quality genomic DNA using a commercial plant genomic DNA extraction kit (such as TIANGEND P305).

[0065] PCR reaction system (20 μL): DNA template (~50 ng / μL) 1.0 μL, TH-1 primer (10 μM) 0.5 μL, TH-2 primer (10 μM) 0.5 μL, 2×Taq Plus Master Mix 10.0 μL, ddH2O to make up to 20.0 μL.

[0066] PCR amplification program: 98 ℃ pre-denaturation for 30 s; 34 cycles: 98 ℃ for 10 s, 65 ℃ for 5 s, 72 ℃ for 4 min; final extension at 72 ℃ for 5 min, followed by incubation at 4 ℃.

[0067] 3. Genotyping methods

[0068] PCR products were separated by 1.2 w / v agarose gel electrophoresis. Electrophoresis conditions: 120 V, 35-40 min.

[0069] Genotype determination:

[0070] If a single bright band appears at ~3379 bp in the amplified product, it is identified as a low shell hardness type (reference homozygote).

[0071] If a single bright band appears at ~877bp in the amplified product, it is identified as a high-shell-hardness type (deletion homozygote).

[0072] If two bands, ~3379 bp and ~877 bp, appear simultaneously, the individual is heterozygous.

[0073] Example 3: Validation of the application of molecular markers

[0074] To verify the applicability of the molecular markers described in this invention to peanut materials with different hardness levels, seven varieties with high shell hardness and seven varieties with low shell hardness were selected as experimental materials, as detailed below:

[0075] High-hardness grades include DL307, DL015, SA429, SA435, SA481, SA543, and SA626;

[0076] Low hardness grades include SA282, SA523, SA431, SA398, SA729, SA529, and SA070.

[0077] (1) Experimental materials and DNA extraction

[0078] Young leaves of various plant varieties were used as experimental materials, and genomic DNA was extracted using the TIANGEN Plant Genomic DNA Extraction Kit (model: DP305) according to the manufacturer's instructions. The extracted DNA solution was diluted 10-fold with nuclease-free water and used as a template for PCR amplification.

[0079] (2) PCR amplification system and procedure

[0080] The PCR reaction system (20 μL) includes:

[0081] 1 μL DNA template; 0.5 μL upstream primer TH-1 (10 μM); 0.5 μL downstream primer TH-2 (10 μM); 10 μL 2×Taq Plus Master Mix (containing reaction buffer and Taq enzyme); ddH2O to bring the total volume to 20 μL.

[0082] The amplification procedure is as follows:

[0083] Pre-denaturation at 98 ℃ for 30 s; 34 cycles were performed: 98 ℃ for 10 s, 65 ℃ for 5 s, and 72 ℃ for 4 min;

[0084] Finally, extend at 72 °C for 5 minutes, then hold at 4 °C.

[0085] (3) Detection of amplification products

[0086] The PCR products were detected by 1.2% (w / v, representing the mass of solute (g) per 100 mL of solution) agarose gel electrophoresis. The electrophoresis conditions were 120 V voltage and 140 mA current. The dye was added at a working concentration of 1×Ultra GelRed when preparing the agarose gel. After electrophoresis, the bands were observed directly under a UV imaging system.

[0087] (4) Results and Analysis

[0088] like Figure 5 As shown, the PCR amplification products of seven high-hardness peanut materials showed a single bright band at 877 bp; while the PCR amplification products of seven low-hardness peanut materials showed a single bright band at 3379 bp. The results indicate that the molecular marker described in this invention can effectively distinguish the differences in peanut shell hardness, and has good stability and applicability.

[0089] (5) Effect description

[0090] The experimental verification in this embodiment proves that the molecular marker method provided by the present invention can accurately identify different shell hardness types in peanut germplasm resources, and the detection results are highly consistent with the phenotype, providing a feasible means for early molecular screening of peanut shell hardness traits and molecular marker-assisted breeding.

[0091] 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 for identifying peanut shell hardness, characterized in that, The marker is located at position 1998250~2000751 bp on chromosome A16 of the peanut genome Arachis hypogaea L. Tifrunner.gnm1, and is represented by a deletion of a nucleotide sequence of length 2502 bp; the deleted nucleotide sequence is shown in SEQ ID NO:

5.

2. The application of the InDel molecular marker for identifying peanut shell hardness, the primer pair for detecting peanut shell hardness, or the kit for identifying peanut shell hardness as described in claim 1 in peanut breeding for identifying shell hardness, characterized in that, The primer pair is capable of specifically amplifying genomic regions containing the deletion variant sites shown in SEQ ID NO:

5. The primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

2. The kit contains the primer pair.

3. A method for determining the hardness of peanut shells, characterized in that, Includes the following steps: (1) Extract genomic DNA from the peanut sample to be tested; (2) Using the genomic DNA obtained in step (1) as a template, perform PCR amplification using the primer pair described in claim 2; (3) Detect the fragment size of the PCR amplification product: If the main band size of the amplification product is 877 bp, the peanut sample to be tested is determined to be of the high shell hardness type; if the main band size of the amplification product is 3379 bp, the peanut sample to be tested is determined to be of the low shell hardness type.

4. The method for determining the hardness of peanut shells as described in claim 3, characterized in that, In step (2), the PCR amplification reaction system includes: DNA template, upstream primer, downstream primer, ddH2O and 2×Taq Plus Master Mix.

5. The method for determining the hardness of peanut shells as described in claim 3, characterized in that, In step (2), the PCR amplification program is as follows: pre-denaturation at 98 ℃ for 30 s; followed by 34 cycles, each cycle consisting of 98 ℃ for 10 s, 65 ℃ for 5 s, 72 ℃ for 4 min; and finally, final extension at 72 ℃ for 5 min.

6. The method for determining the hardness of peanut shells as described in claim 3, characterized in that, In step (3), the PCR amplification products are detected by agarose gel electrophoresis.

7. A method for screening flowering plants with high pericarp hardness for breeding, characterized in that, Includes the following steps: Peanut shell hardness is determined by detecting the deletion of the sequence shown in SEQ ID NO: 5 in the peanut genome; when the sequence shown in SEQ ID NO: 5 is deleted, the peanut has high shell hardness.

Images