KASP molecular marker related to prematurity of peanut pods and application of KASP molecular marker

Abstract

The invention relates to the technical field of molecular genetic breeding, and discloses a KASP molecular marker related to prematurity of peanut pods and application of the KASP molecular marker related to prematurity of peanut pods. The KASP molecular marker comprises a forward primer F1 as shown in SEQ ID NO.1: 5 '-GGTATTTAGAATTTCACAAATGCTGAGGTA-3'; a forward primer F2 as shown in SEQ ID NO. 2; a reverse primer R as shown in SEQ ID NO.3; the KASP molecular marker is designed on the basis of an SNP (Single Nucleotide Polymorphism) site at 12750868bp on a peanut A01 chromosome. Through whole genome association analysis, a functional SNP site of a peanut A01 chromosome AhEBF1 promoter region is positioned, a target SNP is amplified by using a primer combination for marking a fluorophore, a genotype is judged by combining fluorescence analysis, and direct association between the genotype and a phenotype is established, so that the linkage tightness of a molecular marker and a target character is remarkably improved, and high-throughput typing is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetic breeding, and specifically relates to a KASP molecular marker related to early maturity of peanut pods and its application. Background Art

[0002] Peanut (Arachis hypogaea L.) is an important oil and cash crop in China. The total annual output and planting area in China rank among the top in the world. Its high and stable yield is of great significance for ensuring the safe supply of plant-derived edible oil in China. Early maturity is one of the important directions for peanut variety improvement, and it is an important way to alleviate the contradiction between grain and oil for land, reduce the threat of climate, and meet market demand. Early-maturing peanut varieties have low requirements for light and temperature, and are crucial for areas with shorter frost-free periods (such as high-latitude regions in the north and sub-optimal cotton-growing areas in Xinjiang) or regions that require multiple cropping and rotation. In addition, early-maturing peanut varieties have a short growth period and a wider sowing window, providing flexibility for farmers to avoid extreme weather (such as spring cold or autumn drought). Therefore, cultivating early-maturing peanut varieties is an important way to tap the potential for expanding peanut cultivation, and it has important significance and application prospects for enhancing peanut production capacity and promoting the development of the peanut industry. Exploring peanut early-maturity trait loci and developing related molecular markers are effective means to accelerate the cultivation of new peanut germplasms with early maturity.

[0003] With the completion of peanut genome sequencing (Chen et al., 2016; Bertioli et al., 2016; Lu et al., 2018; Chen et al., 2019; Bertioli et al., 2019; Zhuang et al., 2019), selection breeding based on the genome and known loci related to excellent traits has become a new direction for peanut breeding, making genotype selection breeding based on molecular markers / chips possible. However, there are few reports on peanut early-maturity related loci, and most are designed based on non-functional regions, which cannot directly associate with key phenotypic regulatory loci, resulting in low screening efficiency and being easily interfered by the genetic background, restricting the progress of molecular marker-assisted peanut early-maturity breeding. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a KASP molecular marker related to early maturity of peanut pods and its application, solving the problems of low screening efficiency and being easily interfered by the genetic background due to the inability to directly associate with key phenotypic regulatory loci.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A KASP molecular marker related to early maturity of peanut pods and its application, including:

[0006] Forward primer F1 shown in SEQ ID NO.1: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTA-3';

[0007] Forward primer F2 shown in SEQ ID NO.2: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTG-3';

[0008] Reverse primer R shown in SEQ ID NO.3: 5'-TTCCCCACCCTCCCTCTCTC-3';

[0009] The KASP molecular marker is designed based on the SNP locus at 12750868 bp on chromosome A01 of peanut. The nucleotide polymorphism of the SNP locus is A / G, and the nucleotide polymorphism of the corresponding antisense strand is T / C. The SNP locus is located in the promoter region of the candidate gene AhEBF1.

[0010] Preferably, the 5' ends of the forward primers F1 and F2 are respectively labeled with FAM and HEX fluorescent groups.

[0011] An application of a KASP molecular marker related to early maturity of peanut pods, comprising the following steps:

[0012] S1. Extract the genomic DNA of the peanut sample to be tested;

[0013] S2. Perform PCR amplification on the genomic DNA using the primer composition. The PCR reaction system is 5 μl, including 2.43 μl of genomic DNA, 2.5 μl of 2×KASP MasterMix, and 0.07 μl of KASP Assay Mix;

[0014] S3. Detect the fluorescence signal of the PCR product by a Pherastar fluorescence analyzer, and judge the genotype of the sample to be tested according to the signal type.

[0015] Preferably, the signal types in S3 include:

[0016] If the FAM fluorescence signal is significant, the genotype is AA, corresponding to the early maturity phenotype;

[0017] If the HEX fluorescence signal is significant, the genotype is GG, corresponding to the late maturity phenotype;

[0018] If both FAM and HEX signals are detected, the genotype is AG, and further screening is required in the self-crossed offspring.

[0019] Preferably, the PCR amplification specifically includes the following steps:

[0020] The first step: pre-denaturation at 94°C for 15 minutes;

[0021] Step 2: Denature at 94°C for 20 seconds, anneal at 61°C for 1 minute, for a total of 10 cycles, with the annealing temperature decreasing by 0.6°C per cycle;

[0022] Step 3: Denature at 94°C for 20 seconds, anneal at 55°C for 1 minute, for a total of 26 cycles;

[0023] Step 4: Store at 10°C.

[0024] Preferably, step S3 specifically includes the following steps:

[0025] A1. Select an early-maturing peanut variety carrying the AA genotype and a late-maturing peanut variety carrying the GG genotype for hybridization to obtain an F2 population;

[0026] A2. Perform genotyping detection on individual plants in the F2 population;

[0027] A3. Statistically analyze the pod maturity phenotypes of the AA, AG, and GG genotypes, and verify the consistency between the early-maturing phenotype and the AA genotype.

[0028] Preferably, the application further includes the following steps:

[0029] B1. Perform genotype detection on individual plants of the hybrid offspring;

[0030] B2. Screen out individual plants with the AA genotype as early-maturing candidate lines;

[0031] B3. Self-cross individual plants with the AG genotype, and further screen out homozygous AA genotype individual plants in the offspring.

[0032] Preferably, the individual plants of the hybrid offspring in B1 include individual plants of the F2 generation and subsequent self-cross generations produced by crossing an early-maturing variety and a late-maturing variety.

[0033] A molecular marker screening kit for peanut early-maturing traits, comprising: a KASP molecular marker primer composition, 2×KASP MasterMix, and KASP Assay Mix.

[0034] Preferably, the primer concentration of the KASP Assay Mix is: 10 μM for each of F1 and F2, and 100 μM for R.

[0035] The present invention provides a KASP molecular marker related to peanut pod early maturity and its application. It has the following beneficial effects:

[0036] 1. Through genome-wide association analysis, the present invention locates the functional SNP locus in the promoter region of AhEBF1 on chromosome A01 of peanut. By using a primer combination labeled with a fluorescent group to amplify the target SNP and combining fluorescence analysis to discriminate genotypes, a direct association between genotypes and phenotypes is established, significantly enhancing the linkage tightness between molecular markers and target traits and achieving high-throughput genotyping.

[0037] 2. In the present invention, the 5' ends of the forward primers F1 / F2 are labeled with FAM / HEX fluorescent groups. After KASP-PCR amplification, the signal type is detected by a Pherastar fluorescence analyzer to discriminate genotypes, which can shorten the detection cycle and support multi-channel synchronous analysis, achieving high-throughput automated screening and providing an accurate basis for seedling stage screening.

[0038] 3. Through the optimization of the competitive allele-specific amplification reaction system, the forward primers competitively bind to the target SNP locus (A / G) equally, and the reverse primers are in excess to ensure efficient amplification, reducing non-specific products. The operation process is simplified through the kit premixed components, enabling rapid and accurate identification of the genotypes of the peanut samples to be tested and improving the breeding efficiency of early-maturing peanut varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the genome-wide association analysis diagram of the peanut pod maturity of the present invention;

[0040] Figure 2 It is the association analysis diagram of different haplotypes of peanut AhEBF1 of the present invention and their relationship with the pod maturity index;

[0041] Figure 3 It is the KASP marker genotyping result diagram in the F2 offspring of the hybrid population of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Please refer to the attached Figure 1 - attached Figure 3 , the embodiment of the present invention provides a KASP molecular marker related to peanut pod early maturity and its application, including:

[0044] The forward primer F1 shown in SEQ ID NO.1: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTA-3';

[0045] Forward primer F2 shown in SEQ ID NO.2: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTG-3';

[0046] Reverse primer R shown in SEQ ID NO.3: 5'-TTCCCCACCCTCCCTCTCTC-3';

[0047] The KASP molecular marker is designed based on the SNP locus at 12750868 bp on chromosome A01 of peanut. The nucleotide polymorphism of the SNP locus is A / G, and the nucleotide polymorphism of the corresponding antisense strand is T / C. The SNP locus is located in the promoter region of the candidate gene AhEBF1.

[0048] Specifically, forward primer F1 (SEQ ID NO.1: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTA-3') can specifically recognize the DNA template with the A allele at the SNP locus at 12750868 bp on chromosome A01 of peanut in the KASP-PCR reaction. When the locus in the sample is A, forward primer F1 can accurately complement and bind to the template, and under the action of DNA polymerase, initiate the extension of the DNA strand, thus triggering subsequent amplification reactions. Through specific binding and amplification, the accurate identification of this allele can be achieved, so as to accurately distinguish the samples carrying the A allele, providing a basis for subsequent judgment of whether peanuts have specific genotypes related to early maturity, and improving the efficiency and accuracy of screening early-maturing peanut varieties;

[0049] Forward primer F2 (SEQ ID NO.2: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTG-3') is used to recognize the DNA template with the G allele at the SNP locus at 12750868 bp on chromosome A01 of peanut. During the KASP-PCR process, if the locus in the sample is G, forward primer F2 will complementarily bind to it, and then guide DNA polymerase to synthesize and amplify the DNA strand, thus detecting the samples carrying the G allele. Cooperating with forward primer F1, it can comprehensively and accurately genotype the samples of peanuts, helping breeders quickly screen out peanut plants with different genotypes;

[0050] The reverse primer R (SEQ ID NO.3: 5'-TTCCCCACCCTCCCTCTCTC-3') forms a primer pair with the forward primers F1 and F2 in the KASP-PCR reaction, binds complementarily to the other strand of the template DNA, provides a starting point for DNA polymerase to synthesize a new strand, enabling the PCR reaction to amplify simultaneously from both ends smoothly. The presence of the reverse primer R ensures that the target DNA fragment can be amplified efficiently and accurately, improving the specificity and efficiency of PCR amplification, making the subsequent detection of specific SNP sites on peanut chromosome A01 more sensitive and reliable.

[0051] The KASP molecular marker is designed based on the significant SNP sites located by genome-wide association analysis. By designing the KASP molecular marker for this SNP site, the genotype of peanut plants at this site can be specifically detected. Thus, in the early growth stage of peanuts, through simple gene detection techniques, plants that may have the early-maturing trait can be quickly and accurately screened out, avoiding the long waiting time required in traditional breeding methods. In peanut breeding practice, by detecting the genotype of this site, the early-maturing trait of peanut pods can be accurately predicted, providing a powerful technical means for the directional breeding of early-maturing peanut varieties and helping to cultivate more early-maturing peanut varieties that meet market demands.

[0052] The 5' ends of the forward primers F1 and F2 are respectively labeled with FAM and HEX fluorescent groups.

[0053] Specifically, the 5' ends of the forward primers F1 and F2 are respectively labeled with FAM and HEX fluorescent groups, providing fluorescent signal identification for allele-specific amplification. In the subsequent KASP-PCR reaction, when the primers specifically bind to the corresponding target SNP site (A / G) on the template DNA and extend, the primers carrying different fluorescent groups will enter the amplification products along with DNA strand synthesis, making the amplification products of different alleles carry FAM or HEX fluorescent signals respectively. Through scanning with a fluorescence detection device, the genotype (homozygous A, homozygous G, or heterozygous) of the sample can be accurately distinguished according to the presence and intensity of the FAM (corresponding to the A allele) or HEX (corresponding to the G allele) signals.

[0054] An application of a KASP molecular marker related to early maturity of peanut pods includes the following steps:

[0055] S1. Extract the genomic DNA of the peanut sample to be tested;

[0056] S2. Perform PCR amplification on the genomic DNA using a primer composition. The PCR reaction system is 5 μl, including 2.43 μl of genomic DNA, 2.5 μl of 2×KASP MasterMix, and 0.07 μl of KASP Assay Mix;

[0057] S3. Detect the fluorescence signal of the PCR product using a Pherastar fluorescence analyzer, and determine the genotype of the sample to be tested based on the signal type.

[0058] Specifically, through S1, pure DNA is isolated from the peanut sample to be tested, removing other impurities and interfering substances, ensuring that the quality and concentration of the DNA meet the requirements of subsequent experiments, and guaranteeing the accuracy of the starting point of the entire detection process.

[0059] Through S2, selective amplification is performed on a specific region related to early maturity of peanut pods in the extracted genomic DNA. The primer composition includes forward primers F1 and F2 and a reverse primer R designed for specific SNP sites on peanut chromosome A01. Under the suitable reaction environment provided by 2×KASP Master Mix, the target DNA fragment is massively replicated in vitro through PCR technology, amplifying the originally trace target DNA fragment to a sufficient amount for detection, facilitating subsequent genotype detection through fluorescence signals, improving the detection sensitivity, and ensuring the accurate identification of gene locus information related to early maturity in peanut samples.

[0060] Through S3, final analysis and judgment are performed on the PCR amplification product. Since the 5' ends of the forward primers F1 and F2 are respectively labeled with FAM and HEX fluorescent groups, during the PCR amplification process, the primers bound to different alleles will introduce the corresponding fluorescent groups into the amplification product. The Pherastar fluorescence analyzer can detect these fluorescence signals. Different fluorescence signal types represent different allele combinations, thereby enabling the determination of the genotype of the sample to be tested at specific SNP sites, providing a direct scientific basis for the breeding of early-maturing peanut varieties and accelerating the cultivation process of early-maturing peanut varieties.

[0061] The signal types in S3 include:

[0062] If the FAM fluorescence signal is significant, the genotype is AA, corresponding to the early-maturing phenotype.

[0063] If the HEX fluorescence signal is significant, the genotype is GG, corresponding to the late-maturing phenotype.

[0064] If both FAM and HEX signals are detected, the genotype is AG, and further screening is required in the self-crossed offspring.

[0065] Specifically, if the FAM fluorescence signal is significant, it indicates that the sample carries the homozygous A allele (AA genotype), corresponding to the early-maturing phenotype. This allows for the direct screening of individual plants with stable early-maturing characteristics, avoiding the problems of long cycle and low efficiency in traditional phenotypic identification. If the HEX fluorescence signal is significant, it means the sample is the homozygous G allele (GG genotype), corresponding to the late-maturing phenotype, enabling the rapid elimination of individuals that do not meet the breeding goals and reducing the retention of ineffective breeding materials. If both FAM and HEX signals are detected simultaneously, it is determined to be a heterozygous genotype (AG), indicating that self-crossing is needed to separate the alleles and further screen for homozygous early-maturing individual plants in the offspring. This not only preserves potential excellent gene resources but also ensures the stable inheritance of the early-maturing trait. That is, through the differential detection of fluorescence signals, efficient genotyping of peanut early-maturing related genotypes is achieved, providing precision for molecular marker-assisted breeding, significantly improving the efficiency and accuracy of early-maturing peanut variety selection, and accelerating the process of directional cultivation of high-quality early-maturing germplasms.

[0066] The PCR amplification specifically includes the following steps:

[0067] The first step: pre-denaturation at 94°C for 15 minutes;

[0068] The second step: denaturation at 94°C for 20 seconds, annealing at 61°C for 1 minute, for a total of 10 cycles, with the annealing temperature decreasing by 0.6°C per cycle;

[0069] The third step: denaturation at 94°C for 20 seconds, annealing at 55°C for 1 minute, for a total of 26 cycles;

[0070] The fourth step: preservation at 10°C.

[0071] Specifically, the PCR amplification steps achieve the efficient and specific amplification of the target DNA fragment by precisely controlling the temperature and cycle parameters, providing a reliable basis for genotype detection. Among them, in the first step, pre-denaturation at 94°C for 15 minutes serves to fully unwind the double-stranded genomic DNA and activate the Taq enzyme activity, providing a single-stranded template for subsequent reactions. In the second step, gradient annealing (61°C - 55°C, decreasing by 0.6°C per cycle for 10 cycles) is adopted. By gradually optimizing the annealing temperature, allele-specific primers (F1, F2) are precisely bound to the template DNA, enhancing the recognition ability for the SNP site (A / G) and reducing non-specific amplification. In the third step, annealing is fixed at 55°C for 26 cycles to amplify the target fragment in large quantities at the optimal annealing temperature to ensure that the product concentration meets the fluorescence detection requirements. In the fourth step, preservation at 10°C prevents product degradation and facilitates subsequent fluorescence signal analysis.

[0072] Specifically, S3 includes the following steps:

[0073] A1. Select an early-maturing peanut variety carrying the AA genotype and a late-maturing peanut variety carrying the GG genotype for hybridization to obtain an F2 population;

[0074] A2. Genotype the individual plants in the F2 population;

[0075] A3. Statistically analyze whether the ratios of AA, AG, and GG genotypes conform to Mendel's genetic laws, and verify the consistency between the early-maturing phenotype and the AA genotype.

[0076] Specifically, in A1, the genes of early-maturing and late-maturing peanut varieties are recombined through sexual hybridization to create an offspring population with rich genetic diversity. The early-maturing peanut variety carrying the AA genotype contains the genes determining the early-maturing trait, while the late-maturing peanut variety carrying the GG genotype carries the genes related to late maturity. After hybridization, different genes are recombined in the offspring, generating various possible genotype combinations. The F2 generation is obtained because the F2 generation is the generation with the most obvious trait segregation, which can fully display various phenotypes and genotypes after gene recombination, providing rich materials for subsequent screening and research;

[0077] In A2, accurately determine the genotype of each individual plant in the F2 population at the target SNP locus. By using the KASP molecular marker primers mentioned above for PCR amplification and fluorescence signal detection, it is possible to quickly and accurately distinguish whether each individual plant is of the AA, AG, or GG genotype, providing specific gene information for subsequent screening and breeding work, and providing a direct basis for screening out individual plants with early-maturing potential, avoiding the uncertainty and limitations of screening only based on phenotypes;

[0078] In A3, verify the genetic pattern of the studied SNP locus and the correlation between the early-maturing trait and the AA genotype from a genetic perspective. Mendel's genetic laws describe the transmission mode of genes between parents and offspring. If the ratios of AA, AG, and GG genotypes conform to Mendel's genetic laws, it indicates that the inheritance of this SNP locus follows the classical genetic laws, providing theoretical support for its application in breeding. At the same time, verifying the consistency between the early-maturing phenotype and the AA genotype can further confirm that this SNP locus is indeed closely related to the early-maturing trait of peanut pods, providing a scientific basis for the application of this KASP molecular marker in the breeding of early-maturing peanut varieties, and enhancing the reliability and predictability of breeding work.

[0079] The application also includes the following steps:

[0080] B1. Genotype the individual plants of the hybrid offspring;

[0081] B2. Screen out the individual plants with the AA genotype as early-maturing candidate lines;

[0082] B3. Self-cross the individual plants with the AG genotype, and further screen out the homozygous AA genotype individual plants in the offspring.

[0083] Specifically, B1 uses the KASP molecular marker technology to identify the specific genotypes of individual hybrid offspring at the target SNP locus. Due to gene recombination and segregation during hybridization, there are multiple possibilities for the genotypes of individual offspring. Through this detection, the genetic composition of each individual can be clarified;

[0084] B2 selects individual plants carrying the homozygous A allele (AA genotype) from the hybrid offspring according to the detection results of step B1. Since the AA genotype corresponds to the early-maturing phenotype, these individual plants have great early-maturing potential. The plants with potential early-maturing characteristics can be directly locked, narrowing the breeding range to a group with clear genetic advantages and improving the efficiency of screening early-maturing peanut varieties;

[0085] B3 uses self-crossing to cause the alleles of the AG genotype individual plants to segregate and recombine, thereby generating homozygous AA genotype individual plants in the offspring. Since the AG genotype is in a heterozygous state, its early-maturing traits may be unstable. Through self-crossing and further screening, genetically stable early-maturing individual plants can be obtained, increasing the number of homozygous AA genotype individual plants, expanding the population size of the early-maturing candidate lines, and increasing the probability of breeding a stable genetic early-maturing variety.

[0086] The individual hybrid offspring in B1 include the F2 generation produced by crossing an early-maturing variety with a late-maturing variety and the individual plants of subsequent self-crossing generations.

[0087] A molecular marker screening kit for peanut early-maturing traits, comprising: a KASP molecular marker primer composition, 2×KASP MasterMix, and KASP Assay Mix.

[0088] Specifically, the kit contains a KASP molecular marker primer composition (including F1, F2 specific primers targeting the A / G site at 12750868 bp on chromosome A01 of peanut and a common reverse primer R, labeled with FAM / HEX fluorescent groups respectively), 2×KASP Master Mix, and KASP Assay Mix. Its function is to identify the alleles at the target site through specific primers and amplify. The Master Mix provides the enzymes and buffer system required for the reaction. The Assay Mix simplifies the operation, quickly distinguishes the sample genotypes (AA / GG / AG) using the fluorescence signal difference, supports high-throughput automated detection, requires no complex processing, can screen AA genotype individual plants related to early maturity at the seedling stage, significantly shortens the breeding cycle, reduces the blindness of screening, provides an efficient and standardized tool for molecular marker-assisted breeding, and promotes the improvement of early-maturing peanut germplasm.

[0089] The primer concentrations of the KASP Assay Mix are: 10 μM for each of F1 and F2, and 100 μM for R.

[0090] Specifically, in the KASP Assay Mix, the concentrations of the forward primers F1 and F2 are each 10 μM, and the concentration of the reverse primer R is 100 μM. The competitive amplification reaction is optimized through differential concentration configuration: equal amounts of the forward primers (10 μM) ensure fair competitive binding to the A / G alleles, and the high concentration of the reverse primer (100 μM) ensures its continuous excess to efficiently drive PCR amplification.

[0091] Among them, the early-maturing SNP locus of peanut pods and the KASP molecular marker were obtained through the following methods:

[0092] Previously, 10× whole-genome resequencing was performed on 390 core cultivated peanut germplasm resources, and sequence alignment was carried out with the peanut reference genome "Fuhuasheng". After quality control, 1,048,576 high-quality and high-density SNP loci were obtained.

[0093] At the peanut maturity stage, all the pods on 3 plants were harvested, rinsed with a high-pressure water gun until the mesocarp of the pods was exposed, graded according to the color of the mesocarp rinsed out from the pods, compared with the peanut pod maturity level standard, the color level of each peanut pod was obtained, the number of peanut pods at different levels was counted, and the maturity index was calculated according to the level to which they belonged. The pod maturity index was used as the peanut pod maturity phenotype;

[0094] An association analysis was performed using the peanut pod maturity index and SNP loci, and a mixed linear model (MLM) was used to detect the associated SNP loci. Among them, a cluster of SNP loci significantly associated with the peanut pod maturity index was found on chromosome A01, named qMI_A01. According to LD block analysis, qMI_A01 was located within a 367.5 kb range between 12,525,767 - 12,893,338 bp on chromosome A01, as shown in the appendix Figure 1 shown;

[0095] There are 15 genes in the candidate interval of locus qMI_A01. Based on variant analysis, haplotype analysis, and genomic annotation, it was found that the AhEBF1 gene is a key candidate gene regulating peanut pod maturity. There are 2 variant sites in the AhEBF1 gene. The variant site (A01_12750868) located in the promoter region of the AhEBF1 gene is significantly correlated with peanut pod maturity. The AA genotype is earlier maturing than the GG genotype, as shown in the appendix Figure 2 shown;

[0096] Design primers using the 100bp flanking sequences at both ends of the SNP marker sites to develop KASP markers. Search for KASP marker primers for SNP development through the "Fuhuasheng" reference genome (NCBI database: https: / / www.ncbi.nlm.nih.gov / ; accession number SDMP00000000).

[0097] Example: Verification of the accuracy of KASP markers,

[0098] S101, Select the offspring (F2) of the hybrid population of an early-maturing variety (AA genotype) and a late-maturing variety (GG genotype) as the experimental material. Take young leaves at the seedling stage and extract genomic DNA;

[0099] S102, Perform PCR amplification on the extracted genomic DNA using KASP marker primers. The total PCR reaction system is 5 μl, including 2.43 μl of genomic DNA, 2.5 μl of 2×KASP MasterMix, 0.07 μl of KASP Assay Mix (primer mixed working solution). The KASP primer mixed working solution includes the forward primer (F1) of the specific sequence conjugated with FAM fluorescence, the forward primer (F2) of the specific sequence conjugated with HEX fluorescence, the universal reverse primer (R), and pure water. The PCR reaction program: First step, 94°C, 15 min; Second step, 94°C, 20 s, gradient PCR at 61 - 55°C, 1 min, with a decrease of 0.6°C for each cycle, for 10 cycles; Third step, 94°C, 20 s, 55°C, 1 min, for 26 cycles; Fourth step, store at 10°C;

[0100] S103, Scan and analyze the PCR results using a KASP fluorescence analyzer Pherastar (LGC company).

[0101] The test results show that using the KASP markers designed by the present invention to genotype the SNP sites of the experimental material, it is found that these markers can well distinguish between AA and GG genotypes, as shown in the appendix Figure 3 shown, indicating that the KASP markers are accurate and reliable and can be used for molecular breeding assistant selection.

[0102] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A KASP molecular marker primer composition related to early maturity of peanut pods, characterized in that: include: Forward primer F1 shown in SEQ ID NO.1: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTA-3'; Forward primer F2 shown in SEQ ID NO.2: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTG-3'; Reverse primer R shown in SEQ ID NO.3: 5'-TTCCCCACCCTCCCTCTCTC-3'; The KASP molecular marker is designed based on the SNP site at 12750868bp on the peanut A01 chromosome. The nucleotide polymorphism of the SNP site is A / G, and the nucleotide polymorphism of the corresponding antisense strand is T / C. The SNP site is located in the promoter region of the candidate gene AhEBF1.

2. The KASP molecular marker primer composition related to early maturity of peanut pods according to claim 1, characterized in that: The 5' ends of the forward primers F1 and F2 are labeled with FAM and HEX fluorescent groups, respectively.

3. An application of KASP molecular markers related to early maturity of peanut pods, characterized in that: The KASP molecular marker primer composition related to early maturity of peanut pods as claimed in claim 1 or 2 comprises the following steps: S1. Extracting genomic DNA from peanut samples to be tested; S2. PCR amplification of genomic DNA was performed using the primer combination. The PCR reaction system was 5 μl, including 2.43 μl genomic DNA, 2.5 μl 2×KASP Master Mix and 0.07 μl KASP Assay Mix; S3. Detect the fluorescence signal of the PCR product using a Pherastar fluorescence analyzer, and determine the genotype of the sample to be tested based on the signal type.

4. The use of a KASP molecular marker related to early maturity of peanut pods according to claim 3, characterized in that: The signal types in S3 include: If the FAM fluorescence signal is significant, the genotype is AA, corresponding to the precocious phenotype; If the HEX fluorescence signal is significant, the genotype is GG, corresponding to a late-maturing phenotype; If both FAM and HEX signals are detected, the genotype is AG and further screening is required in the self-pollinated progeny.

5. The use of a KASP molecular marker related to early maturity of peanut pods according to claim 3, characterized in that: The PCR amplification specifically The following steps are involved: Step 1: pre-denaturation at 94°C for 15 minutes; Step 2: denaturation at 94°C for 20 seconds, annealing at 61°C for 1 minute, for a total of 10 cycles, with the annealing temperature decreasing by 0.6°C per cycle; Step 3: denaturation at 94°C for 20 seconds and annealing at 55°C for 1 minute, for a total of 26 cycles; Step 4: Store at 10°C.

6. The use of a KASP molecular marker related to early maturity of peanut pods according to claim 3, characterized in that: The S3 specifically includes the following steps: A1. Select an early-maturing peanut variety carrying the AA genotype and a late-maturing peanut variety carrying the GG genotype for hybridization to obtain an F2 generation population; A2, performing genotyping detection on individual plants in the F2 generation population; A3. Statistically determine whether the proportions of AA, AG, and GG genotypes conform to Mendel's laws of inheritance, and verify the consistency of the precocious phenotype with the AA genotype.

7. The use of a KASP molecular marker related to early maturity of peanut pods according to claim 3, characterized in that: The application further comprises the following steps: B1. Conduct genotype detection on individual hybrid offspring; B2. Select the individual plants with AA genotype as early-maturing candidate lines; B3. Self-pollinate the AG genotype plants and further select homozygous AA genotype plants in the progeny.

8. The use of a KASP molecular marker related to early maturity of peanut pods according to claim 7, characterized in that: The hybrid offspring plants in B1 include the F2 generation produced by hybridization of early-maturing varieties and late-maturing varieties and the subsequent self-pollination generations.

9. A molecular marker screening kit for early maturity traits of peanut, characterized in that: The KASP molecular marker primer composition related to early maturity of peanut pods as described in claim 1 or 2 comprises: a KASP molecular marker primer composition, 2×KASP MasterMix and KASP Assay Mix.

10. The molecular marker screening kit for early maturity traits of peanut according to claim 9, characterized in that: The primer concentrations of the KASPAssay Mix are: 10 μM for each of F1 and F2, and 100 μM for R.

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