A KASP molecular marker associated with early maturity of peanut pods and its application

Abstract

This invention relates to the field of molecular genetic breeding technology, and discloses a KASP molecular marker related to early maturity of peanut pods and its application, comprising: forward primer F1: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTA-3' shown in SEQ ID NO.1; forward primer F2 shown in SEQ ID NO.2; and reverse primer R shown in SEQ ID NO.3. The KASP molecular marker is designed based on the SNP site at 12750868 bp on the peanut A01 chromosome. Through genome-wide association analysis, a functional SNP site in the AhEBF1 promoter region of the peanut A01 chromosome is located. The target SNP is amplified using primer combinations labeled with fluorescent groups, and genotype is determined by fluorescence analysis, establishing a direct association between genotype and phenotype. This significantly improves the linkage tightness between the molecular marker and the target trait, achieving high-throughput genotyping.

Description

Technical Field

[0001] This invention relates to the field of molecular genetic breeding technology, specifically to a KASP molecular marker related to early maturity of peanut pods and its application. Background Technology

[0002] Peanuts (Arachis hypogaea L.) are an important oilseed and economic crop in my country. my country ranks among the world's top countries in both total annual production and planting area. High and stable yields are crucial for ensuring a secure supply of edible oil from plant sources in my country. Early maturity is a key direction for peanut variety improvement, and an important way to alleviate competition for land between grain and oil crops, mitigate climate threats, and meet market demand. Early-maturing peanut varieties have lower light and temperature requirements, making them essential for areas with short frost-free periods (such as high-latitude regions in northern China and less suitable cotton-growing areas in Xinjiang) or regions requiring crop rotation. Furthermore, early-maturing peanut varieties have shorter growth periods and wider planting windows, providing farmers with flexibility to avoid extreme weather (such as spring frosts or autumn droughts). Therefore, breeding early-maturing peanut varieties is an important way to tap the potential for peanut expansion, and it has significant implications and promising applications for improving peanut production capacity and promoting the development of the peanut industry. Identifying early-maturing trait loci in peanuts and developing related molecular markers are effective means to accelerate the breeding of new early-maturing peanut germplasm.

[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 associated with superior traits has become a new direction in peanut breeding, making genotype selection breeding based on molecular markers / microarrays possible. However, there are few reports on loci associated with early maturity in peanuts, and most are designed based on non-functional regions, which cannot directly link to key phenotypic regulatory sites, resulting in low screening efficiency and susceptibility to genetic background interference, thus limiting the progress of molecular marker-assisted early maturity breeding of peanuts. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a KASP molecular marker related to early maturity of peanut pods and its application, solving the problem of low screening efficiency and susceptibility to genetic background interference due to the inability to directly link key phenotypic regulatory sites.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a KASP molecular marker related to early maturity of peanut pods and its application, comprising:

[0006] The forward primer F1 shown in SEQ ID NO.1 is 5'-GGTATTTAGAATTTCACAAATGCTGAGGTA-3';

[0007] The forward primer F2 shown in SEQ ID NO.2 is 5'-GGTATTTAGAATTTCACAAATGCTGAGGTG-3';

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

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

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

[0011] The application of a KASP molecular marker associated with early maturity of peanut pods includes the following steps:

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

[0013] S2. PCR amplification of genomic DNA was performed using primer combination. The PCR reaction system was 5 μl, including 2.43 μl of genomic DNA, 2.5 μl of 2×KASP MasterMix and 0.07 μl of KASPAssay Mix.

[0014] 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.

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

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

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

[0018] If both FAM and HEX signals are detected simultaneously, 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] Step 1: Pre-denaturation at 94℃ for 15 minutes;

[0021] Step 2: Denaturation at 94℃ for 20 seconds, annealing at 61℃ for 1 minute, for a total of 10 cycles, with the annealing temperature decreasing by 0.6℃ in each cycle;

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

[0023] Step 4: Store at 10℃.

[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 the F2 generation population;

[0026] A2. Genotyping of individual plants in the F2 generation population was performed.

[0027] A3. Statistically analyze the pod maturity phenotypes of 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. Genotyping of individual plants from hybrid offspring;

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

[0031] B3. Self-pollinate AG genotype plants and further screen homozygous AA genotype plants from the offspring.

[0032] Preferably, the hybrid offspring plants in B1 include F2 generation plants produced by crossing early-maturing and late-maturing varieties, as well as individual plants from subsequent self-pollination generations.

[0033] A molecular marker screening kit for early maturity traits in peanuts includes: a KASP molecular marker primer composition, 2×KASP MasterMix, and KASPassay Mix.

[0034] Preferably, the primer concentrations of the KASPAssay Mix are: 10 μM for F1 and F2, and 100 μM for R.

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

[0036] 1. This invention locates functional SNP sites in the AhEBF1 promoter region of peanut chromosome A01 through genome-wide association analysis, amplifies the target SNPs using primer combinations labeled with fluorescent groups, and identifies genotypes by combining fluorescence analysis, establishing a direct association between genotype and phenotype, significantly improving the linkage tightness between molecular markers and target traits, and achieving high-throughput genotyping.

[0037] 2. In this invention, the FAM / HEX fluorescent group is labeled at the 5' end of the forward primers F1 / F2. After KASP-PCR amplification, the genotype is determined by detecting the signal type using a Pherastar fluorescence analyzer. This can shorten the detection cycle and support multi-channel simultaneous analysis, achieving high-throughput automated screening and providing accurate basis for seedling screening.

[0038] 3. This invention optimizes the competitive allele-specific amplification reaction system, uses equal amounts of forward primers to competitively bind to the target SNP site (A / G), and uses an excess of reverse primers to ensure efficient amplification, reducing non-specific products. Furthermore, it simplifies the operation process through premixed kit components, enabling rapid and accurate identification of the genotype of peanut samples to be tested, thereby improving the breeding efficiency of early-maturing peanut varieties. Attached Figure Description

[0039] Figure 1 This is a genome-wide association study (GWAS) diagram of peanut pod maturity according to the present invention.

[0040] Figure 2 This is a graph showing the different haplotypes of peanut AhEBF1 and their correlation with the pod maturity index, as presented in this invention.

[0041] Figure 3 This is a diagram showing the KASP marker genotyping results in the F2 offspring of the hybrid population of this invention. Detailed Implementation

[0042] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Please see the appendix Figure 1 - Appendix Figure 3 This invention provides a KASP molecular marker related to early maturity of peanut pods and its application, including:

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

[0045] The forward primer F2 shown in SEQ ID NO.2 is 5'-GGTATTTAGAATTTCACAAATGCTGAGGTG-3';

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

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

[0048] Specifically, the forward primer F1 (SEQ ID NO.1: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTA-3') can specifically recognize the DNA template of the SNP site at 12750868 bp on the peanut A01 chromosome as the A allele in the KASP-PCR reaction. When the site in the sample is A, the forward primer F1 can accurately pair and bind to the template, initiating DNA chain extension under the action of DNA polymerase, thereby triggering the subsequent amplification reaction. Through specific binding and amplification, the allele can be accurately identified, thus accurately distinguishing samples carrying the A allele. This provides a basis for subsequent determination of whether peanuts have a specific genotype related to early maturity, 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 identify DNA templates with the G allele at the SNP site at 12750868bp on the peanut A01 chromosome. During KASP-PCR, if the site in the sample is G, forward primer F2 will bind complementary to it, thereby guiding DNA polymerase to synthesize and amplify the DNA chain, thus detecting samples carrying the G allele. In conjunction with forward primer F1, it can comprehensively and accurately classify the genotype of the sample, which helps breeders quickly screen 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. It binds complementary to the other strand of the template DNA, providing a starting point for DNA polymerase to synthesize a new strand. This allows the PCR reaction to be successfully amplified from both ends simultaneously. 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. This makes the subsequent detection of specific SNP sites on the peanut A01 chromosome more sensitive and reliable.

[0051] KASP molecular markers are designed based on significant SNP loci located by genome-wide association analysis. By designing KASP molecular markers targeting these SNP loci, the genotype of peanut plants at that locus can be specifically detected. This allows for rapid and accurate screening of plants with potential early-maturing characteristics in the early stages of peanut growth using simple gene detection techniques, avoiding the long waiting periods required in traditional breeding methods. In peanut breeding practice, detecting the genotype at this locus can accurately predict the early-maturing characteristics of peanut pods, providing a powerful technical means for the targeted breeding of early-maturing peanut varieties and helping to cultivate more early-maturing peanut varieties that meet market demands.

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

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

[0054] The application of a KASP molecular marker associated with early maturity of peanut pods includes the following steps:

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

[0056] S2. PCR amplification of genomic DNA was performed using primer combinations. The PCR reaction system consisted of 5 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, pure DNA is separated from the peanut sample to be tested using S1, 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] The S2 primers selectively amplify specific regions in the extracted genomic DNA associated with early maturity of peanut pods. The primer composition contains forward primers F1 and F2 and a reverse primer R designed for specific SNP sites on the peanut A01 chromosome. Under the suitable reaction conditions provided by 2×KASP Master Mix, the target DNA fragment is replicated in large quantities in vitro using PCR technology, amplifying the originally trace amount of target DNA fragment to a sufficient amount for detection. This facilitates subsequent detection of genotypes by fluorescence signals, improves detection sensitivity, and ensures accurate identification of gene loci associated with early maturity in peanut samples.

[0060] The S3 method is used for the final analysis and judgment of PCR amplification products. Since the 5' ends of the forward primers F1 and F2 are labeled with FAM and HEX fluorescent groups, respectively, during PCR amplification, primers that bind to different alleles will introduce the corresponding fluorescent groups into the amplification products. The Pherastar fluorescence analyzer can detect these fluorescence signals. Different fluorescence signal types represent different allele combinations, thereby determining the genotype of the sample at a specific SNP site. This provides a direct scientific basis for the breeding of early-maturing peanut varieties and accelerates the breeding 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 precocious 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 simultaneously, 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 an early-maturing phenotype. Individual plants with stable early-maturing characteristics can be directly screened, avoiding the problems of long cycles and low efficiency in traditional phenotypic identification. If the HEX fluorescence signal is significant, it indicates that the sample is homozygous G allele (GG genotype), corresponding to a late-maturing phenotype. Individuals that do not meet the breeding goals can be quickly eliminated, 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), suggesting that alleles need to be segregated through self-pollination. Homozygous early-maturing individual plants can then be further screened in the offspring. This preserves potential superior gene resources and ensures the stable inheritance of the early-maturing trait. In other words, through differential detection of fluorescence signals, efficient typing of peanut early-maturing genotypes can be achieved, providing precise and significantly improving the efficiency and accuracy of early-maturing peanut variety selection for molecular marker-assisted breeding, and accelerating the targeted breeding process of high-quality early-maturing germplasm.

[0066] PCR amplification specifically includes the following steps:

[0067] Step 1: Pre-denaturation at 94℃ for 15 minutes;

[0068] Step 2: Denaturation at 94℃ for 20 seconds, annealing at 61℃ for 1 minute, for a total of 10 cycles, with the annealing temperature decreasing by 0.6℃ in each cycle;

[0069] Step 3: Denature at 94℃ for 20 seconds, anneal at 55℃ for 1 minute, for a total of 26 cycles;

[0070] Step 4: Store at 10℃.

[0071] Specifically, the PCR amplification steps achieve highly efficient and specific amplification of the target DNA fragment by precisely controlling temperature and cycling parameters, providing a reliable basis for genotype detection. The first step, pre-denaturation at 94℃ for 15 minutes, fully unwinds the double strand of genomic DNA, activates Taq enzyme activity, and provides a single-stranded template for subsequent reactions. The second step employs gradient annealing (61℃-55℃, decreasing by 0.6℃ per cycle, for 10 cycles). By progressively optimizing the annealing temperature, allele-specific primers (F1, F2) bind precisely to the template DNA, enhancing the recognition of SNP sites (A / G) and reducing non-specific amplification. The third step involves fixed annealing at 55℃ for 26 cycles, amplifying the target fragment in large quantities at the optimal annealing temperature to ensure the product concentration meets the requirements for fluorescence detection. The fourth step involves storage at 10℃ to prevent product degradation and facilitate subsequent fluorescence signal analysis.

[0072] S3 specifically 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 the F2 generation population;

[0074] A2. Genotyping of individual plants in the F2 generation population;

[0075] A3. Analyze whether the proportions of AA, AG, and GG genotypes conform to Mendelian inheritance laws, and verify the consistency between the precocious phenotype and the AA genotype.

[0076] Specifically, in A1, genes from early-maturing and late-maturing peanut varieties are recombined through sexual hybridization to create a genetically diverse offspring population. Early-maturing peanut varieties carrying the AA genotype contain genes that determine the early-maturing trait, while late-maturing peanut varieties carrying the GG genotype carry genes related to late maturation. After hybridization, different genes recombine in the offspring, producing various possible genotype combinations. The F2 generation is obtained because it is the generation with the most obvious phenotypic segregation, which can fully demonstrate the various phenotypes and genotypes after gene recombination, providing rich materials for subsequent screening and research.

[0077] In A2, the genotype of each individual plant in the F2 generation at the target SNP site was accurately determined. 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 AA, AG, or GG genotype. This provides specific genetic information for subsequent screening and breeding work, and provides a direct basis for screening individual plants with early maturity potential, avoiding the uncertainty and limitations of screening based solely on phenotype.

[0078] A3 validates the inheritance pattern of the studied SNP locus and the association between the precocious trait and the AA genotype from a genetic perspective. Mendelian inheritance laws describe the way genes are transmitted between parents and offspring. If the ratio of AA, AG, and GG genotypes conforms to Mendelian inheritance laws, it indicates that the inheritance of this SNP locus follows classical genetic laws, providing theoretical support for its application in breeding. At the same time, verifying the consistency between the precocious phenotype and the AA genotype can further confirm that this SNP locus is indeed closely related to the precocious pod trait of peanuts, providing a scientific basis for the application of this KASP molecular marker in the breeding of precocious peanut varieties, and enhancing the reliability and predictability of breeding work.

[0079] The application also includes the following steps:

[0080] B1. Genotyping of individual plants from hybrid offspring;

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

[0082] B3. Self-pollinate AG genotype plants and further screen homozygous AA genotype plants from the offspring.

[0083] Specifically, B1 uses KASP molecular marker technology to identify the specific genotype of the hybrid offspring at the target SNP site. Because genes recombine and segregate during hybridization, there are multiple possibilities for the genotype of the offspring. This test can clarify the genetic composition of each plant.

[0084] Based on the test results of step B1, select individual plants carrying the homozygous A allele (AA genotype) from the hybrid offspring. Since the AA genotype is known to correspond to the early-maturing phenotype, these individual plants have great early-maturing potential. They can directly identify plants that may have early-maturing characteristics, narrowing the breeding scope to a population with clear genetic advantages and improving the efficiency of screening early-maturing peanut varieties.

[0085] B3 utilizes self-pollination to induce allele segregation and recombination in AG genotype plants, thereby producing homozygous AA genotype plants in the offspring. Since the AG genotype is heterozygous, its early-maturing trait may be unstable. Through self-pollination and further selection, genetically stable early-maturing plants can be obtained, increasing the number of homozygous AA genotype plants, expanding the population size of early-maturing candidate lines, and improving the probability of breeding stable genetically early-maturing varieties.

[0086] B1 includes individual plants from the F2 generation (resulting from the cross between an early-maturing variety and a late-maturing variety) and subsequent self-pollination generations.

[0087] A molecular marker screening kit for early maturity traits in peanuts includes: a KASP molecular marker primer composition, 2×KASP MasterMix, and KASPassay Mix.

[0088] Specifically, the kit includes a KASP molecular marker primer composition (containing F1 and F2 specific primers targeting the A / G locus at 12750868 bp on peanut chromosome A01, and a universal reverse primer R, labeled with FAM / HEX fluorescent groups respectively), 2×KASPMaster Mix, and KASPAssay Mix. Its function is to identify and amplify the alleles at the target locus using specific primers. The Master Mix provides the enzymes and buffer system required for the reaction, while the Assay Mix simplifies the operation. It rapidly distinguishes sample genotypes (AA / GG / AG) based on differences in fluorescence signals, supporting high-throughput automated detection without complex processing. It can screen early-maturing AA genotype single plants at the seedling stage, significantly shortening the breeding cycle, reducing screening blindness, providing an efficient and standardized tool for molecular marker-assisted breeding, and promoting the improvement of early-maturing peanut germplasm.

[0089] The primer concentrations for KASPAssay Mix were: 10 μM each for F1 and F2, and 100 μM for R.

[0090] Specifically, the concentrations of the F1 and F2 forward primers in the KASPAssay Mix 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: the equal amount of forward primer (10 μM) ensures fair competitive binding to the A / G alleles, while the high concentration of reverse primer (100 μM) ensures its continuous excess to efficiently drive PCR amplification.

[0091] The early-maturing SNP sites and KASP molecular markers of peanut pods were obtained through the following methods:

[0092] In the early stage, 10× whole genome resequencing was performed on 390 core peanut cultivar resources and sequenced with the peanut reference genome “Fuhuasheng”. After quality control, 1,048,576 high-quality, high-density SNP loci were obtained.

[0093] Harvest all pods from 3 peanut plants at the peanut maturity stage, wash them with a high-pressure water gun until the mesocarp of the pod is exposed, grade them according to the color of the washed mesocarp, compare them with the peanut pod maturity grade standard to obtain the color grade of each peanut pod, count the number of peanut pods of different grades, calculate the maturity index according to the grade, and use the pod maturity index as the peanut pod maturity phenotype.

[0094] Association analysis was performed between the peanut pod maturity index and SNP loci. A mixed linear model (MLM) was used to detect associated SNP loci. A cluster of SNP loci significantly associated with the peanut pod maturity index was found on chromosome A01 and named qMI_A01. Based on LD block analysis, qMI_A01 was located within a 367.5 kb range on chromosome A01, between 12525767 and 12893338 bp, as shown in the attached diagram. Figure 1 As shown;

[0095] The candidate region of locus qMI_A01 contains 15 genes. Based on variation analysis, haplotype analysis, and genome annotation, the AhEBF1 gene was identified as a key candidate gene regulating peanut pod maturity. Two variation sites exist within the AhEBF1 gene. The variation site (A01_12750868) located in the AhEBF1 gene promoter region is significantly associated with peanut pod maturity. The AA genotype matures earlier than the GG genotype, as shown in the attached figure. Figure 2 As shown;

[0096] Primers were designed using the 100bp flanking sequences at both ends of the SNP marker site to develop KASP markers. KASP marker primers for the SNP were developed by searching the *Peanuta fusiforme* reference genome (NCBI database: https: / / www.ncbi.nlm.nih.gov / ; accession number SDMP00000000).

[0097] Example: Accuracy verification of KASP tags,

[0098] S101, the offspring (F2) of the hybrid population of an early-maturing variety (AA genotype) and a late-maturing variety (GG genotype) were selected as the experimental material. Young leaves were taken during the seedling stage and genomic DNA was extracted.

[0099] S102, PCR amplification of extracted genomic DNA was performed using KASP-labeled primers. The total PCR reaction volume was 5 μl, including 2.43 μl of genomic DNA, 2.5 μl of 2×KASP MasterMix, and 0.07 μl of KASPassay Mix (primer mixing working solution). The KASP primer mixing working solution included a FAM fluorescently bound specific sequence forward primer (F1), a HEX fluorescently bound specific sequence forward primer (F2), a universal reverse primer (R), and pure water. The PCR reaction program was as follows: Step 1, 94℃, 15 min; Step 2, 94℃, 20 s, 61–55℃ gradient PCR, 1 min, decreasing the temperature by 0.6℃ per cycle, for 10 cycles; Step 3, 94℃, 20 s, 55℃, 1 min, for 26 cycles; Step 4, stored at 10℃.

[0100] S103 was analyzed using a Pherastar fluorescence analyzer (LGC). PCR results were scanned and analyzed.

[0101] Experimental results show that using the KASP marker designed in this invention to genotype SNP sites in experimental materials, the marker can effectively distinguish between AA and GG genotypes, as shown in the attached figure. Figure 3 As shown, this demonstrates that the KASP marker is accurate and reliable, and can be used for molecular breeding-assisted selection.

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

Claims

1. A KASP molecular marker primer composition associated with early pod maturity in peanut, characterized in that, Comprise: 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: 5'-GGTATTTAGAATTTCACAAATGCTGAGGTG-3'; a reverse primer R as shown in SEQ ID NO. 3: 5'-TTCCCCACCCTCCCTCTCTC-3'; the 5' end of the forward primers F1 and F2 are labeled with FAM and HEX fluorescent groups respectively; the KASP molecular marker is designed based on a SNP site at 12750868 bp on peanut A01 chromosome, the nucleotide polymorphism of the SNP site is A / G, the nucleotide polymorphism of the antisense strand is T / C, and the SNP site is located in the promoter region of the candidate gene AhEBF1.

2. Use of a peanut pod early maturity associated KASP molecular marker characterized in that, A KASP molecular marker primer composition for the early maturity related to peanut pod of claim 1, comprising the following steps: S1, extracting the genomic DNA of the peanut sample to be tested; S2, PCR amplifying 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 Master Mix and 0.07 μl of KASP Assay Mix; S3, detecting the fluorescence signal of the PCR product by Pherastar fluorescence analyzer, and judging the genotype of the sample to be tested according to the signal type; the signal type in S3 comprises: if the FAM fluorescence signal is significant, the genotype is AA, corresponding to the early maturity phenotype; if the HEX fluorescence signal is significant, the genotype is GG, corresponding to the late maturity phenotype; if FAM and HEX signals are detected at the same time, the genotype is AG, which needs to be further screened in the selfed progeny.

3. The use of a peanut pod early maturity associated KASP molecular marker according to claim 2, characterized in that, The PCR amplification specifically comprises the following steps: first step: 94℃ pre-denaturation for 15 minutes; second step: 94℃ denaturation for 20 seconds, 61℃ annealing for 1 minute, a total of 10 cycles, and the annealing temperature decreases by 0.6℃ for each cycle; third step: 94℃ denaturation for 20 seconds, 55℃ annealing for 1 minute, a total of 26 cycles; fourth step: 10℃ storage.

4. The use of a peanut pod early maturity associated KASP molecular marker according to claim 2, characterized in that, S3 specifically comprises the following steps: A1, crossing the early maturity peanut variety carrying AA genotype with the late maturity peanut variety carrying GG genotype to obtain F2 population; A2, genotyping detection on single plants in the F2 population; A3, whether the AA, AG and GG genotype proportions conform to Mendelian inheritance law is counted, and the consistency of early maturity phenotype and AA genotype is verified.

5. The use of a peanut pod early maturity associated KASP molecular marker according to claim 2, wherein the marker is selected from the group consisting of: KASP marker ARMS-1 (SEQ ID NO: 1) and KASP marker ARMS-2 (SEQ ID NO: 2). The application further comprises the following steps: B1, genotyping detection on single plants of hybrid progeny; B2, screening single plants with AA genotype as early maturity candidate lines; B3, selfing single plants with AG genotype, and further screening single plants with homozygous AA genotype in the progeny.

6. The use of a peanut pod early maturity associated KASP molecular marker according to claim 5, wherein the marker is selected from the group consisting of: KASP marker ARMS-1 (SEQ ID NO: 1) and KASP marker ARMS-2 (SEQ ID NO: 2). The single plants of hybrid progeny in B1 comprise single plants of F2 generation and subsequent selfing generations produced by crossing early maturity varieties with late maturity varieties.

7. A molecular marker screening kit for early maturity trait of peanut, characterized by, The KASP molecular marker primer composition for the early maturity related to the peanut pod of claim 1 comprises: a KASP molecular marker primer composition, a 2x KASP Master Mix and a KASP Assay Mix.

8. The molecular marker screening kit for early maturity trait of peanut according to claim 7, characterized in that, The primer concentration of the KASP Assay Mix is: 10 μM of F1 and F2 respectively, and 100 μM of R.

Images