KASP molecular marker of major qtl for peanut seed size and its application

Abstract

This invention relates to KASP molecular markers for major QTLs related to peanut seed size and their applications, belonging to the field of molecular genetics and breeding technology. This invention provides KASP molecular markers for major QTLs related to peanut seed size, named qSSWB06, located on chromosome B06 with a mapping interval of 142.12–146.16 Mb. Closely linked KASP molecular markers include SSW1 and SSW2, with physical locations of 142,219,056 bp and 143,695,716 bp, respectively. This invention provides KASP molecular markers for peanut seed size selection, offering efficient, rapid, convenient, and accurate identification, enabling high-throughput genotyping. It can significantly reduce the complex procedures in peanut underground seed selection, improve breeding efficiency, and has broad application prospects in molecular marker-assisted breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetic breeding technology, specifically involving the KASP molecular marker of the major QTL for peanut seed size and its application. Background Technology

[0002] Cultivated peanut (Arachis hypogaea L.) is one of the world's most important oilseed and economic crops, playing a vital role in meeting global oil and protein demands. The breeding of high-yielding peanut varieties primarily employs the traditional method of sexual hybridization. However, due to peanuts' growth habit of flowering above ground and fruiting underground, conventional breeding methods are inefficient and costly in selecting target individual plants. Molecular marker-assisted breeding can compensate for the shortcomings of conventional methods.

[0003] Marker-assisted selection (MAS) has become an important tool in crop breeding, offering advantages such as speed, accuracy, and independence from environmental factors. It allows for selection through molecular marker genotyping in the early stages of crop growth, shortening generation intervals and accelerating the breeding process. However, aside from resistance to root-knot nematodes, leaf spot, rust, and high oleic acid content, there are few reports on MAS-assisted breeding for other important agronomic traits in peanuts. Commonly used molecular markers, including RFLP, AFLP, SRAP, and SSR, have been applied to the genetic analysis of peanut agronomic traits, but their detection efficiency is low and the procedures are cumbersome. Therefore, there is an urgent need for more efficient, convenient, and rapid molecular marker detection methods.

[0004] KASP markers are designed based on single-base variations of SNPs in the genome. Specific primers are designed using these variation sites to identify plants with traits of interest. Currently, there are few reports on the application of KASP molecular markers developed based on SNP variation sites in marker-assisted breeding for identifying peanut seed size traits. Summary of the Invention

[0005] The purpose of this invention is to provide a KASP molecular marker for the major QTL of peanut seed size and its application. This invention provides a KASP marker for peanut seed size selection, which is efficient, rapid, convenient, and accurate, enabling high-throughput genotyping and significantly reducing the complex breeding process for peanut underground seeds.

[0006] This invention provides a KASP molecular marker for a peanut seed-sized major QTL, named qSSWB06, located on the peanut B06 chromosome with a mapping interval of 142.12–146.16 Mb. KASP molecular markers closely linked to qSSWB06 include SSW1 and SSW2, with physical locations of 142,219,056 bp and 143,695,716 bp, respectively. The mapping interval of qSSWB06 is 4.04 Mb, including SSW1 and SSW2.

[0007] Preferably, the primers for detecting the KASP molecular marker include primers for detecting the SSW1 molecular marker and primers for detecting the SSW2 molecular marker; the primers for detecting the SSW1 molecular marker include a first forward primer, a second forward primer, and a first universal reverse primer; the primers for detecting the SSW2 molecular marker include a third forward primer, a fourth forward primer, and a second universal reverse primer; the nucleotide sequence of the first forward primer is shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.2, the nucleotide sequence of the first reverse primer is shown in SEQ ID NO.3, the nucleotide sequence of the third forward primer is shown in SEQ ID NO.4, the nucleotide sequence of the fourth forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO.6.

[0008] This invention also provides the application of reagents for detecting the KASP molecular markers described in the above technical solutions in the identification or breeding of large- and / or small-seeded peanut varieties.

[0009] This invention also provides the application of reagents for detecting the KASP molecular markers described in the above technical solutions in the breeding of peanuts with long seeds, wide seeds, thick seeds, and heavy kernels.

[0010] The present invention also provides primers for detecting the KASP molecular markers described in the above-mentioned technical solutions. The primers include detection primers for SSW1 molecular markers and detection primers for SSW2 molecular markers. The detection primers for SSW1 molecular markers include a first forward primer, a second forward primer, and a first universal reverse primer. The detection primers for SSW2 molecular markers include a third forward primer, a fourth forward primer, and a second universal reverse primer. The nucleotide sequence of the first forward primer is shown in SEQ ID NO. 1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO. 2, the nucleotide sequence of the first reverse primer is shown in SEQ ID NO. 3, the nucleotide sequence of the third forward primer is shown in SEQ ID NO. 4, the nucleotide sequence of the fourth forward primer is shown in SEQ ID NO. 5, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO. 6.

[0011] Preferably, the kit includes the primer set and reaction solution described in the above technical solution.

[0012] Preferably, the reaction solution comprises HiGeno 2×Probe Mix C.

[0013] This invention also provides a method for identifying or breeding large- and / or small-seeded peanut varieties based on the KASP molecular marker described in the above technical solution, comprising the following steps: using reagents for detecting the KASP molecular marker described in the above technical solution to perform KASP marker PCR amplification on peanut gene haplotypes; if the amplified and identified SSW1 haplotype is CC and the SSW-2 haplotype is AA, then it is a large-seeded peanut variety; if the amplified and identified SSW1 haplotype is AA and the SSW-2 haplotype is CC, then it is a small-seeded peanut variety.

[0014] Preferably, based on a 5 μL reaction system, the KASP-labeled PCR amplification reaction system comprises 1 μL of genomic DNA, 2.5 μL of HiGeno 2×Probe Mix C, 0.07 μL of KASP primer mixing working solution, and 1.43 μL of water; the KASP primer mixing working solution is a mixture of SSW1 molecularly labeled detection primers and water, or a mixture of SSW2 molecularly labeled detection primers and water.

[0015] Preferably, the reaction program for the KASP-labeled PCR amplification is as follows: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 20 s, gradient PCR annealing at 61–55°C for 40 s, decreasing by 0.6°C per cycle, for 10 cycles; denaturation at 95°C for 20 s, annealing at 55°C for 40 s, for 32 cycles.

[0016] This invention provides a KASP molecular marker for the major QTL of peanut seed size. Using this KASP molecular marker, the size of seeds in the underground parts can be detected early in peanut growth through aboveground genomic DNA, improving breeding efficiency. The major QTL of peanut kernel size located by the KASP molecular marker of this invention has high heritability, is technically reliable, economical, and simple, and can achieve high-throughput genotyping. It can simultaneously perform marker-assisted selection on four important yield traits of peanut underground kernel length, width, thickness, and weight per 100 kernels, and can identify large and small seeds accurately and quickly. This effectively avoids the complex procedures of peanut underground seed selection, improves breeding efficiency, and has broad application prospects. It provides a method for early seed trait selection in peanut offspring and further provides a more effective method for marker-assisted breeding of peanut seed size. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results of the combined analysis of seed size genetic loci and candidate genes using QTL-seq and RNA-seq technologies provided by this invention are shown in the figure; where a is the distribution map of SNPs and InDel in the large seed group, b is the distribution map of SNPs and InDel in the small seed group, and c is the distribution map of ΔIndex(SNP+InDel).

[0019] Figure 2 The images show the genotyping results of the KASP marker in the RIL population provided by this invention; the left image shows the genotyping results of the SSW1 marker in the RIL population, and the right image shows the genotyping results of the SSW2 marker in the RIL population.

[0020] Figure 3 The image shows the genotyping results of the KASP marker in the bred varieties; the left image is SSW1 and the right image is SSW2.

[0021] Figure 4 The haplotype analysis results of the KASP marker provided by this invention on four traits—seed length, seed width, seed thickness, and weight per 100 kernels—in a RIL population are shown in the figure. Detailed Implementation

[0022] This invention provides a KASP molecular marker for a major QTL related to peanut seed size. The major QTL, named qSSWB06, is located on the peanut B06 chromosome, with a mapping interval of 142.12–146.16 Mb. KASP molecular markers closely linked to qSSWB06 include SSW1 and SSW2, with physical locations of 142,219,056 bp and 143,695,716 bp, respectively. The mapping interval of qSSWB06 is 4.04 Mb, including SSW1 and SSW2. This invention utilizes recombinant inbred lines constructed from parents exhibiting significant differences in peanut seed kernel weight. Using QTL-seq and RNA-seq techniques, SNP loci related to peanut seed size were obtained and developed into KASP markers. This provides technical support for peanut seed size selection. The KASP markers of this invention are SNP-based molecular markers located adjacent to peanut chromosomes, demonstrating good effectiveness in identifying peanut seed size. The KASP molecular markers provided by this invention, which are closely linked to peanut seed size traits, are accurate, efficient, and convenient for identification. They enable high-throughput genotyping and can accurately predict the size of underground seeds (large seeds with a 100-kernel weight greater than 85g and small seeds with a 100-kernel weight less than 55g) through the DNA of peanut aboveground tissues. This can significantly reduce the complex procedures for selecting peanut seed size traits, effectively save time in selecting peanut underground seeds, improve breeding efficiency, and provide strong support for molecular marker-assisted breeding.

[0023] In this invention, the primers for detecting KASP molecular markers preferably include primers for detecting SSW1 molecular markers and primers for detecting SSW2 molecular markers; the primers for detecting SSW1 molecular markers preferably include a first forward primer, a second forward primer, and a first universal reverse primer; the primers for detecting SSW2 molecular markers preferably include a third forward primer, a fourth forward primer, and a second universal reverse primer; the nucleotide sequence of the first forward primer is preferably as shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is preferably as shown in SEQ ID NO.2, the nucleotide sequence of the first reverse primer is preferably as shown in SEQ ID NO.3, the nucleotide sequence of the third forward primer is preferably as shown in SEQ ID NO.4, the nucleotide sequence of the fourth forward primer is preferably as shown in SEQ ID NO.5, and the nucleotide sequence of the second reverse primer is preferably as shown in SEQ ID NO.6.

[0024] This invention also provides the application of reagents for detecting the KASP molecular markers described in the above-mentioned technical solutions in the identification or breeding of large- and / or small-seeded peanut varieties. If the genotypes identified in a single peanut plant sample are GG(SSW-1) and AA(SSW-2), it is a large-seeded peanut variety; conversely, if the genotypes identified in a single peanut plant sample are AA(SSW-1) and CC(SSW-2), it is a small-seeded peanut variety. In this invention, the large-seeded peanut is preferably a large-seeded peanut with a 100-kernel weight greater than 85g. In this invention, the small-seeded peanut is preferably a small-seeded peanut with a 100-kernel weight less than 55g.

[0025] This invention also provides the application of reagents for detecting the KASP molecular markers described in the above-mentioned technical solutions in the breeding of peanuts with long seeds, wide seeds, thick seeds, and a high kernel weight. Based on the molecular markers of this invention, peanut varieties containing four excellent yield traits—long seeds, wide seeds, thick seeds, and a high kernel weight—can be bred. When the genotypes GG(SSW-1) and AA(SSW-2) are detected, the peanut seed length, seed width, seed thickness, and kernel weight are high. The large-seed peanut of this invention corresponds to the peanut with long seeds, wide seeds, thick seeds, and a high kernel weight.

[0026] The present invention also provides a set of primers for detecting the KASP molecular markers described in the above-mentioned technical solutions. The primers include detection primers for the SSW1 molecular marker and detection primers for the SSW2 molecular marker. The detection primers for the SSW1 molecular marker include a first forward primer, a second forward primer, and a first universal reverse primer. The detection primers for the SSW2 molecular marker include a third forward primer, a fourth forward primer, and a second universal reverse primer. The nucleotide sequence of the first forward primer is shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.2, the nucleotide sequence of the first reverse primer is shown in SEQ ID NO.3, the nucleotide sequence of the third forward primer is shown in SEQ ID NO.4, the nucleotide sequence of the fourth forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO.6.

[0027] This invention also provides a kit for detecting the KASP molecular marker described in the above-described technical solutions. The kit includes the primer set and reaction solution described in the above-described technical solutions. In this invention, the reaction solution preferably includes HiGeno 2×Probe Mix C.

[0028] This invention also provides a method for identifying or breeding large- and / or small-seeded peanut varieties based on the KASP molecular marker described in the above technical solution, comprising the following steps: using reagents for detecting the KASP molecular marker described in the above technical solution to perform KASP marker PCR amplification on peanut gene haplotypes; if the amplified and identified SSW1 haplotype is CC type and SSW-2 haplotype is AA type, then it is a large-seeded peanut variety; if the amplified and identified SSW1 haplotype is AA type and SSW-2 haplotype is CC type, then it is a small-seeded peanut variety. This invention preferably uses KASP amplification technology to identify the gene haplotype of peanut hybrid progeny plants, and uses the gene haplotype results to screen for large-seeded hybrid progeny peanut plants. In this invention, based on a 5 μL reaction system, the KASP-labeled PCR amplification reaction system preferably includes 1 μL of genomic DNA, 2.5 μL of HiGeno 2×Probe Mix C, 0.07 μL of KASP primer mixing working solution, and 1.43 μL of water; the KASP primer mixing working solution is a mixture of SSW1 molecularly labeled detection primers and water, or a mixture of SSW2 molecularly labeled detection primers and water. In this invention, the water is preferably ultrapure water. In this invention, the preferred reaction program for KASP-labeled PCR amplification is: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 s, gradient PCR annealing at 61–55℃ for 40 s, decreasing by 0.6℃ per cycle, for 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing for 40 s, for 32 cycles. After the reaction is complete, it is preferably stored at 4℃. This invention is preferably performed using AppliedBiosystems. TM QuantStudio TM The PCR results were analyzed using a 6 Pro real-time quantitative PCR system (Thermo Fisher Scientific).

[0029] To further illustrate the present invention, the KASP molecular marker for peanut seed size major QTL and its application are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] This embodiment is based on a hybridization of the large-grained peanut variety Jihua 5 and the small-grained peanut germplasm resource Tifrunner, comprising 265 recombinant inbred lines (RILs). 12 Using a population as experimental material, phenotypic data on four traits—seed length, seed width, seed thickness, and weight of 100 kernels—under seven different environmental conditions were used to evaluate the RIL (Rich Intestine Spectrogen) population. 12The population underwent extreme pooling, including 30 large-grained and 30 small-grained families. Simultaneously, QTL-seq and RNA-seq technologies were used to analyze seed-size genetic loci. The average sequencing depth of resequencing was 24.42×, yielding 350.06 Gb of resequencing data and 1,699,042 SNPs. After quality control filtering, 207,208 SNPs were ultimately selected, identifying a 12.23 Mb candidate region on chromosome B06. Combined RNA-seq analysis annotated 10 candidate genes, which were significantly enriched within a 4.04 Mb (142.14–146.16 Mb) region on chromosome B06 and named qSSWB06. Using the small-particle strain as the mutant strain, the SNP-index and InDel-index of the two progeny pools were calculated. The distribution of the SNP-index and InDel-index on each chromosome was obtained by plotting the data using a 1Mb sliding window and a 1kb step size. Figure 1 The calculated ΔSNP-index and ΔInDel-index were used to perform 1,000 permutation tests, and the SNP and InDel with a 99% confidence level were selected as the screening thresholds.

[0032] from Figure 1 As can be seen, SNPs and InDels with a confidence level exceeding 99% are only present on chromosome 16 and are very prominent, with ΔSNP-index and ΔInDel-index values ​​reaching as high as 1.0. Therefore, it is believed that there is a major-effect locus in the candidate region of 4.04 Mb (142.14–146.16 Mb) on chromosome 16, and this region is named qSSWB06. Within this region, combined with resequencing data, 10 significant SNP variant sites were identified and developed as KASP markers. Among them, the two significant SNPs G / A and A / C are significantly associated with four seed size traits of peanut seeds: seed length, seed width, seed thickness, and weight of 100 kernels. Figure 4 When the genotypes were GG(SSW1) and AA(SSW2), the result was large seeds weighing more than 85g per 100 kernels; when the genotypes were AA(SSW1) and CC(SSW2), the result was small seeds weighing less than 55g per 100 kernels.

[0033] KASP markers SSW1 and SSW2 were designed using two significant SNP sites. Primers were designed based on the 250bp sequences upstream and downstream of the SNP sites. The physical locations of the SNPs on chromosome B06 were searched using the "Tifrunner" reference genome, and the 250bp sequences upstream and downstream of each physical location were downloaded. KASP marker primers were developed based on the principles of KASP primer design. See Table 1 for details.

[0034] Table 1 Primer sequences

[0035]

[0036]

[0037] The accuracy and superior haplotypes of the prepared KASP markers have been validated and analyzed in bred varieties. Fifteen markers from the large-grain group and fifteen from the small-grain group of registered varieties were selected. Genomic DNA was extracted from young leaves of the plants, and PCR amplification was performed using the aforementioned KASP markers. The amplification results were analyzed using Applied Biosystems. TM QuantStudio TM Scanning analysis using the 6 Pro real-time quantitative PCR system (Thermo Fisher Scientific).

[0038] The results are shown in Table 2. Figure 2 and Figure 3 This invention was used to develop KASP markers for genotyping two SNP sites in experimental materials. Figure 2 Using SSW1 and SSW2 to test the parents and mixed pools, clear genotyping results were observed. If the genotypes identified in the peanut plant samples were GG(SSW1) and AA(SSW2), the offspring were large-seeded; conversely, if the genotypes identified in the peanut plant samples were AA(SSW1) and CC(SSW2), the offspring were small-seeded. Figure 3 Using SSW1 and SSW2 to test flowering cultivars, if the genotype of the tested sample is GG(SSW1) and AA(SSW2), it is a large-grained cultivar; conversely, if the genotype of the tested sample is AA(SSW1) and CC(SSW2), it is a small-grained cultivar.

[0039] Table 2 KASP tagging results

[0040]

[0041]

[0042] Note: All information on peanut varieties listed in Table 2 is publicly available and can be viewed at the National Peanut Data Center (http: / / peanut.cropdb.cn / variety / ).

[0043] This embodiment performed genotyping identification on the two SNP loci mentioned above. The phenotypic data of the peanut varieties used were all registered with the China Peanut Data Center. Fifteen varieties with a kernel weight >85g and <65g were selected, and genomic DNA was extracted. The extracted DNA was then amplified by PCR using the aforementioned KASP marker primers. The PCR amplification system consisted of a total of 5 μL, including 1 μL of genomic DNA, 2.5 μL of HiGeno2×Probe Mix C, 0.07 μL of KASP primer working solution, and 1.43 μL of ultrapure water. The amplification program was as follows: denaturation at 95℃ for 5 min; denaturation at 95℃ for 20 s, gradient PCR annealing at 61–55℃ for 40 s, decreasing by 0.6℃ per cycle, for 10 cycles; denaturation at 95℃ for 20 s, extension at 55℃ for 40 s, for 32 cycles; storage at 4℃. The results were obtained using Applied Biosystems. TM QuantStudio TM The PCR results were analyzed using a 6Pro real-time quantitative PCR system (Thermo Fisher Scientific). Further haplotype phenotypic difference analysis showed that genotyping of the selected SNP variant sites in the experimental materials was consistent with their phenotypic values, indicating that the KASP markers involved in this embodiment were accurate and reliable, providing a theoretical basis for haplotype identification and screening using molecular marker-assisted selection. In this embodiment, genotyping was performed on the two SNP sites mentioned above, with SSW1 genotype being G / A and SSW2 genotype being C / A, resulting in two haplotypes: G / A and A / C. The phenotypic values ​​of the peanut varieties used were all registered with the China Peanut Data Center. Further haplotype phenotypic difference analysis showed that haplotypes GG (SSW1) and AA (SSW2) were dominant haplotypes, significantly exceeding other haplotypes in four major yield traits: seed length, seed width, seed thickness, and weight per 100 kernels. This provides a theoretical basis for haplotype identification and screening using molecular marker-assisted selection.

[0044] The KASP markers developed in this embodiment have suitable genetic distances, are technically reliable, economical, and simple, and can achieve high-throughput genotyping. They can be used for marker-assisted selection of peanut underground seed size. When conducting yield breeding, seedling genomic DNA can be extracted after indoor germination before sowing. Using the two KASP markers in this embodiment for joint genotyping, large-seed and small-seed peanut offspring can be screened simultaneously. This establishes an early marker-assisted selection technology for hybrid offspring of large and small seed traits, which can effectively avoid the complex procedures of peanut underground seed breeding and improve breeding efficiency.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of primers for detecting KASP molecular markers in the identification or selection of large- and / or small-seeded peanuts, characterized in that, The primers for detecting KASP molecular markers include primers for detecting SSW1 molecular markers and primers for detecting SSW2 molecular markers; the primers for detecting SSW1 molecular markers include a first forward primer, a second forward primer, and a first universal reverse primer; the primers for detecting SSW2 molecular markers include a third forward primer, a fourth forward primer, and a second universal reverse primer; the nucleotide sequence of the first forward primer is shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.2, the nucleotide sequence of the first universal reverse primer is shown in SEQ ID NO.3, the nucleotide sequence of the third forward primer is shown in SEQ ID NO.4, the nucleotide sequence of the fourth forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the second universal reverse primer is shown in SEQ ID NO.6; If the genotype of the SSW1 molecular marker is GG and the genotype of the SSW2 molecular marker is AA, then it is a large-seeded peanut. If the genotype of the SSW1 molecular marker is AA and the genotype of the SSW2 molecular marker is CC, then it is a small-seeded peanut.

2. The application of primers for detecting KASP molecular markers in the breeding of peanuts with long seeds, wide seeds, thick seeds, and heavy kernels, characterized in that... The primers for detecting KASP molecular markers include primers for detecting SSW1 molecular markers and primers for detecting SSW2 molecular markers; the primers for detecting SSW1 molecular markers include a first forward primer, a second forward primer, and a first universal reverse primer; the primers for detecting SSW2 molecular markers include a third forward primer, a fourth forward primer, and a second universal reverse primer; the nucleotide sequence of the first forward primer is shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.2, the nucleotide sequence of the first universal reverse primer is shown in SEQ ID NO.3, the nucleotide sequence of the third forward primer is shown in SEQ ID NO.4, the nucleotide sequence of the fourth forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the second universal reverse primer is shown in SEQ ID NO.6; If the genotype of the SSW1 molecular marker is GG and the genotype of the SSW2 molecular marker is AA, then it is a large-seeded peanut. If the genotype of the SSW1 molecular marker is AA and the genotype of the SSW2 molecular marker is CC, then it is a small-seeded peanut.

3. A method for identifying or selecting large- and / or small-seeded peanuts, characterized in that, The method includes the following steps: using the primers for detecting KASP molecular markers as described in claim 1 to perform KASP marker PCR amplification on peanut gene haplotypes; if the genotype of the amplified and identified SSW1 molecular marker is GG and the genotype of the amplified and identified SSW2 molecular marker is AA, then it is a large-seed peanut; if the genotype of the amplified and identified SSW1 molecular marker is AA and the genotype of the amplified and identified SSW2 molecular marker is CC, then it is a small-seed peanut.

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