A molecular marker for identifying watermelon seed size, a primer combination and application thereof

Abstract

This invention relates to a molecular marker, primer combination, and their application for identifying watermelon seed size, belonging to the field of molecular marker technology. This invention analyzes resequencing data from watermelon individuals with different phenotypes to identify genes related to watermelon seed size. Cla97 C06 G114610 Two high-quality SNP loci associated with watermelon seed size were identified through screening. Further validation confirmed that the two selected SNP loci were significantly correlated with watermelon seed size (thousand-seed weight). Different genotypes of the SNP loci could effectively distinguish watermelon individuals with large or small thousand-seed weights, indicating that these two SNP loci can serve as SNP molecular markers for identifying watermelon seed size. The SNP molecular markers of this invention help to gain a deeper understanding of watermelon seed variation, providing a new means for detecting watermelon seed size, thereby accelerating the improvement process of watermelon seed size traits and improving the accuracy and selection rate of breeding.

Description

Technical Field

[0001] This invention relates to a molecular marker, primer combination, and its application for identifying the size of watermelon seeds, belonging to the field of molecular marker technology. Background Technology

[0002] watermelon( Citrullus lanatus *Citrus medica* (Thunb.) Matsum. et Nakai is an annual vine belonging to the genus *Citrus* in the family Cucurbitaceae. It is also known as watermelon, water melon, and water rind. Watermelon originated in tropical Africa and is widely cultivated in tropical to temperate regions worldwide, including my country. Rich in nutrients and with a sweet, crisp taste, watermelon contains abundant protein, carbohydrates, dietary fiber, potassium, phosphorus, calcium, iron, sodium, and vitamins A, B, and C. It has gradually become one of the world's most popular fresh fruits.

[0003] Seeds are unique organs in plants and essential for plant life. Seed size is a fundamental agronomic characteristic that influences not only adaptation to environmental stresses and plant fitness but also quality and yield. This is especially important for plants whose primary product organ is the seed. Watermelon seeds serve a dual purpose: watermelons with more flesh typically contain relatively smaller seeds, making small-seeded or seedless watermelons more attractive in the market. In contrast, edible seed watermelons usually contain larger seeds, providing humans with a wealth of nutrients such as protein and oil. Therefore, breeding watermelon varieties with specific seed sizes can meet diverse consumer needs.

[0004] Seed size varies considerably among different watermelon germplasms. In recent years, many genes related to watermelon seed size and their functions have been elucidated, proving that watermelon seed size is a quality trait regulated by complex regulatory mechanisms. Weetman (1937) reported that seed size is controlled by a single gene; Gao et al. (2016) reported that seed size may be regulated by a major QTL; Kim et al. (2015) showed that watermelon seed size is regulated by a single dominant gene; Zhang (2011) found that seed size is determined by a pair of recessive genes and a pair of major genes, but may also involve other modifying factors; Prothro et al. (2012) identified several major QTLs in chr02 and chr06; Meru and McGregor (2013) discovered three major effector QTLs on chr06. Luan et al. (2019) discovered a site called WEG6.1 on chr06 that controls the seed size trait in watermelons. Furthermore, in the study by Wang et al. (2024), QTLS6.2, which is associated with watermelon seed size, was mapped to chr06; Maragal et al. (2022) discovered two qTLs (q_100SW_2.1 and q_100SW_6.1) located on chr06. As can be seen from the above research, with the development of molecular marker technology and the improvement of molecular quantitative genetics, a number of QTL mapping studies have been conducted to reveal the genetic basis of watermelon seed size. However, these studies have mapped large intervals, many candidate genes, and are difficult to screen, resulting in a limited number of molecular markers that can be used for practical production and breeding. This leads to low efficiency in watermelon breeding and seriously restricts the progress of watermelon breeding. Therefore, mining SNP molecular markers associated with watermelon seed size is of great significance for breeding watermelon varieties with different seed sizes and screening high-yield watermelon germplasm resources. Summary of the Invention

[0005] The first objective of this invention is to provide a molecular marker for identifying the size of watermelon seeds.

[0006] A second objective of this invention is to provide a primer combination for detecting the aforementioned molecular marker genotype, and to provide primers capable of rapidly and accurately detecting the aforementioned molecular marker genotype.

[0007] The third objective of this invention is to provide an application of molecular markers or primer combinations in watermelon seed size-assisted screening and breeding, and to provide a method for rapidly and accurately breeding watermelon varieties with specific seed sizes.

[0008] To achieve the above objectives, the present invention provides a molecular marker for identifying the size of watermelon seeds:

[0009] A molecular marker for identifying the size of watermelon seeds, wherein the SNP molecular marker consists of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 71st base from the 5' end being A or C; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 71st base from the 5' end being A or G.

[0010] The beneficial effects of the above technical solution are as follows: This invention, a molecular marker for identifying watermelon seed size, is a pioneering invention. This invention utilizes a paternal parent with small thousand-seed weight and a maternal parent with large thousand-seed weight to obtain the F1 generation. After self-pollination of the F1 generation to obtain the F2 generation, seeds with large and small thousand-seed weights are selected from the F2 generation, and DNA is extracted and resequencing is performed. Analysis of the resequencing data reveals two high-quality SNP loci related to watermelon seed size on the watermelon seed size-related gene Cla97C06G114610, physically anchored at positions 5540184 bp and 5540261 bp on watermelon chromosome 6. Further verification confirms that the two selected SNP loci are significantly correlated with watermelon seed size (thousand-seed weight), and different genotypes of the SNP loci can effectively distinguish between watermelon individuals with large and small thousand-seed weights. This indicates that the two selected SNP loci can serve as SNP molecular markers for identifying watermelon seed size. The SNP molecular markers of this invention help to gain a deeper understanding of watermelon seed variation, provide a new means of detecting watermelon seed size, thereby accelerating the improvement process of watermelon seed size traits and improving the accuracy and selection rate of breeding.

[0011] To achieve the above objectives, a primer combination for detecting the aforementioned molecular marker genotype is provided in this invention:

[0012] A primer combination for detecting the molecular marker genotype, the primer combination comprising primer combination 1 for identifying the SNP molecular marker 1 genotype and primer combination 2 for identifying the SNP molecular marker 1 genotype; the nucleotide sequences of the primers in primer combination 1 are shown in SEQ ID NO. 3-5; the nucleotide sequences of the primers in primer combination 2 are shown in SEQ ID NO. 6-8.

[0013] The beneficial effects of the above technical solution are as follows: This invention designs KASP primers for the screened SNP molecular markers. These primers can effectively detect the genotype of SNP molecular markers and have the advantages of being fast, accurate, and high-throughput, providing an effective means for identifying the size of watermelon seeds.

[0014] Specifically, competitive allele-specific PCR (KSAP) is a high-throughput fluorescence-based SNP genotyping technology developed by LGC in the UK. KASP technology requires the design of two competitive allele-specific forward primers and one reverse primer based on the sequences preceding and following the target SNP site. By adding different fluorescent groups to the 5' end of the forward primer, the genotype of the target site is determined based on the reading of the fluorescence signal at the PCR terminal.

[0015] More specifically, taking primer combination 1 for identifying the SNP molecular marker 1 genotype as an example, the primer with the nucleotide sequence shown in SEQ ID NO.3 has a fluorescent tag sequence of FAM at its 5' end, and the primer with the nucleotide sequence shown in SEQ ID NO.4 has a fluorescent tag sequence of HEX at its 5' end.

[0016] To achieve the above objectives, the technical solution of this invention for the application of molecular markers or primer combinations in watermelon seed size-assisted screening and breeding is as follows:

[0017] Application of a molecular marker or primer combination in watermelon seed size-assisted screening and breeding.

[0018] The beneficial effects of the above technical solution are as follows: This invention screens and obtains two molecular markers for identifying watermelon seed size, and through repeated testing with multiple watermelon materials, it confirms that these two SNP molecular markers can be effectively applied to identify or assist in identifying watermelon seed size. They can be used in molecular marker-assisted breeding and have important application value in the research of mining and screening watermelon germplasm resources or varieties with different seed sizes.

[0019] Furthermore, the application of molecular markers or primer combinations in watermelon seed size-assisted screening and breeding of the present invention directly uses watermelon genomic DNA as a template, and the SNP locus genotype of KASP molecular markers can be detected in watermelon leaf tissues, which is beneficial for convenient and efficient prediction of watermelon seed size and can be applied to watermelon seed size screening and identification before harvest.

[0020] As a further improvement, the size of the watermelon seeds is the weight of a thousand watermelon seeds.

[0021] Specifically, from a genetic perspective, seed size is a highly heritable trait that is positively correlated with seed weight; that is, the larger the seed, the heavier the seed, and vice versa (Li et al. 2018). Furthermore, multiple studies have used thousand-seed weight as a criterion for evaluating seed size. (Li N, Shang J, Wang J, Zhou D, Li N, Ma S (2018) Finemapping and discovery of candidate genes for seed size in watermelon by genome survey sequencing. Sci Rep 8(1):17843.)

[0022] As a further improvement, competitive allele-specific PCR amplification was performed on the genomic DNA of the watermelon sample to be tested using a primer combination for detecting the SNP molecular marker. The fluorescence signal of the amplification product was read, the genotype of the SNP molecular marker was determined based on the fluorescence signal, and the seed size of the watermelon sample to be tested was determined based on the genotype.

[0023] As a further improvement, when the genotype of SNP molecular marker 1 is CC and / or the genotype of SNP molecular marker 2 is AA, the watermelon sample to be tested exhibits a phenotype with a high thousand-seed weight; when the genotype of SNP molecular marker 1 is AA or CA and / or the genotype of SNP molecular marker 2 is GG or AG, the watermelon sample to be tested exhibits a phenotype with a low thousand-seed weight. Attached Figure Description

[0024] Figure 1 The statistical data of SNPs and InDels among samples in Experiment Example 1 of this invention and their chromosomal distribution are shown below (where A is a visualization of SNPs across the entire genome, B is a Venn diagram of SNP differences among samples, C is the distribution of SNPs on chromosomes, D is a visualization of InDels across the entire genome, E is a Venn diagram of InDel differences among samples, and F is the distribution of InDels on chromosomes; the pie chart from the inside out: the first circle represents the distribution of ED values ​​corresponding to SNPs and InDels, the second circle represents the density distribution of SNPs and InDels, the third circle represents the distribution of genes, and the fourth circle represents chromosome coordinates).

[0025] Figure 2 In Experiment 1 of this invention, the Integrative Genomics Observer (IGV) was used to perform a comparative visualization analysis of the parental genomes.

[0026] Figure 3 This is the SNP molecular marker 1 gene typing result of the watermelon sample to be tested in Experiment Example 3 of this invention;

[0027] Figure 4 This is an association analysis of the SNP molecular marker 1 genotype and thousand-grain weight of the watermelon samples in the F2 population of Experiment Example 3 of this invention (where **** represents p<0.0001, and ns represents no significant difference).

[0028] Figure 5 This is the SNP molecular marker 2 gene typing result of the watermelon sample to be tested in Experiment Example 3 of this invention;

[0029] Figure 6 This is an association analysis of the SNP molecular marker 2 genotype and thousand-grain weight of the watermelon samples in the F2 population of Experiment Example 3 of this invention (where **** represents p<0.0001, and ns represents no significant difference). Detailed Implementation

[0030] From a genetic perspective, seed size is a highly heritable trait, positively correlated with seed weight; that is, larger seeds have higher seed weights, and vice versa. Early studies have shown that seed weight directly affects germination rate, seedling viability, viability, survival rate, and germination period. Therefore, identifying SNP loci associated with watermelon size is crucial for understanding watermelon seed variation and provides detection sites for efficient prediction of watermelon seed size. However, existing molecular markers available for practical production and breeding are limited. Based on this, this invention provides a molecular marker for identifying watermelon seed size.

[0031] The molecular markers for identifying watermelon seed size in this invention consist of SNP molecular marker 1 and SNP molecular marker 2. The nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 71st base from the 5' end being either A or C; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 71st base from the 5' end being either A or G. This invention also designs KASP primers for detecting the genotype of these molecular markers, enabling rapid and convenient detection. Further replication tests using multiple watermelon samples confirmed that these two SNP molecular markers can be effectively used to identify or assist in identifying watermelon seed size, and can be used in marker-assisted breeding. They have significant application value in the research and screening of watermelon germplasm resources or strains with different seed sizes.

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, all reagents, instruments and other items used in the embodiments are commercially available products.

[0033] The test materials used in the following embodiments and experimental examples of this invention are described below:

[0034] The test materials include the maternal parent, paternal parent, and F2 generation population;

[0035] The parent material is (microparticles × 10²B⁴) × 10²B⁴, and the average weight of 1,000 seeds is 16.73g.

[0036] The parent material mentioned is wild in Zhengzhou, with an average thousand-seed weight of 59.06g;

[0037] The aforementioned maternal and paternal parents both came from Luoyang Nongfa Agricultural Biotechnology Co., Ltd., and both the parent plants and offspring were grown on the company's farm.

[0038] The F1 generation obtained by hybridization of the above two materials as parents, and the F2 generation obtained by self-pollination of the F1 generation, were all harvested in 2023 with 275 plants each in the F2 generation.

[0039] The thousand-seed weight of the test materials was determined as follows: Strict self-pollination was performed using standard field management. Each plant was artificially pollinated and labeled. Mature watermelons were harvested 35–43 days after pollination. Seeds were washed, dried, and harvested. Seed weight was measured using an electronic balance, in grams. The average of three replicate measurements was recorded.

[0040] Example 1 of a molecular marker for identifying watermelon seed size

[0041] The molecular markers used to identify the size of watermelon seeds in this embodiment consist of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 71st base from the 5' end being A or C; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 71st base from the 5' end being A or G.

[0042] SNP molecular marker 1 corresponds to the deoxyribonucleotide at position 5540184 on chromosome 6 of the watermelon reference genome version 97103 (v2.5) published by GUGENDBv2 (http: / / cucurbitgenomics.org / v); SNP molecular marker 2 corresponds to the deoxyribonucleotide at position 5540261 on chromosome 6 of the watermelon reference genome version 97103 (v2.5) published by GUGENDBv2 (http: / / cucurbitgenomics.org / v).

[0043] Example 1: Primer combination for detecting the molecular marker genotype

[0044] The primer combinations used in this embodiment for detecting the molecular marker genotype include primer combination 1 for identifying the SNP molecular marker 1 genotype and primer combination 2 for identifying the SNP molecular marker 1 genotype; the nucleotide sequences of the primers in primer combination 1 are shown in SEQ ID NO. 3~5; the nucleotide sequences of the primers in primer combination 2 are shown in SEQ ID NO. 6~8.

[0045] Primers with nucleotide sequences as shown in SEQ ID NO.3 have a fluorescent tag sequence of FAM at the 5' end. Primers shown in SEQ ID NO.3 and SEQ ID NO.5 can amplify the fragment of SNP molecular marker 1 with genotype C, thereby generating a FAM fluorescent signal. Primers with nucleotide sequences as shown in SEQ ID NO.4 have a fluorescent tag sequence of HEX at the 5' end. Primers shown in SEQ ID NO.4 and SEQ ID NO.5 can amplify the fragment of SNP molecular marker 1 with genotype A, thereby generating a HEX fluorescent signal.

[0046] Primers with nucleotide sequences as shown in SEQ ID NO.6 have a fluorescent tag sequence of FAM at the 5' end. Primers shown in SEQ ID NO.6 and SEQ ID NO.8 can amplify the fragment of SNP molecular marker 2 with genotype A, thereby generating a FAM fluorescent signal. Primers with nucleotide sequences as shown in SEQ ID NO.7 have a fluorescent tag sequence of HEX at the 5' end. Primers shown in SEQ ID NO.7 and SEQ ID NO.8 can amplify the fragment of SNP molecular marker 1 with genotype G, thereby generating a HEX fluorescent signal.

[0047] Example 1: Application of a molecular marker or primer combination in watermelon seed size-assisted screening and breeding

[0048] The application of molecular markers or primer combinations in watermelon seed size-assisted screening and breeding in this embodiment involves detecting the genotype of SNP molecular markers. When the genotype of SNP molecular marker 1 is CC or the genotype of SNP molecular marker 2 is AA, the watermelon sample to be tested has a phenotype of high thousand-seed weight; when the genotype of SNP molecular marker 1 is AA or CA or the genotype of SNP molecular marker 2 is GG or AG, the watermelon sample to be tested has a phenotype of low thousand-seed weight.

[0049] Experiment 1: Screening of SNP sites related to watermelon seed size

[0050] In this experiment, seeds with large and small thousand-seed weights were selected from the F2 generation population of the test material. DNA was extracted and genome resequencing was performed. The resequencing data was then analyzed to obtain SNP loci associated with watermelon seed size. The specific procedures are as follows:

[0051] In the F2 generation, 20 plants with high 1000-seed weight and 20 plants with low 1000-seed weight were selected. DNA was extracted from each plant (the genomic DNA extraction method was described in the instructions for the Novizan DNA Genomic Extraction Kit). The concentration and quality of the genomic DNA were determined using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). After adjusting the DNA concentration to be consistent, equal volumes were mixed to construct a mixed pool.

[0052] DNA samples were resequencing using the Illumina HiSeq XTen / NovaSeq / BGI platform (Beijing, China), with a sequencing length of 150 bp and at least 30× genome-wide sequencing depth per sample. Low-quality data were filtered using Bcltofastq 1.8.4 software, and the resequencing data from two extreme pools were re-aligned to the watermelon reference genome 97103V2.5 (http: / / cucurbitgenomics.org / v2) using BWA software. Duplicates were removed using the Mark Duplicate tool in the Picard online software package (http: / / sourceforge.net / projects / picard / ), and preprocessing was performed using GATK software, including local re-alignment and base quality correction, to ensure the accuracy of the detected loci. SNP and InDel variant detection was performed between samples, and association analysis was conducted on differentially expressed loci based on the alignment information from the watermelon reference genome 97103V2.5 (http: / / cucurbitgenomics.org / v2). The original ED was selected. 5 To eliminate background noise, the DISTANCE tool was used to fit the ED values, and the median + 3SD of the fitted values ​​for all sites was taken as the association threshold for the analysis. The same method was used to fit the ΔSNP-index and ΔIndel-index, and the regions with values ​​above the threshold were selected as the target regions associated with the seed size trait.

[0053] The intersection of the SNP and Indel association region results obtained by the two association analysis methods is used to determine the localization region (e.g., Figure 1 As shown). The parental sequencing results were visualized using the Integrative Genomics Viewer software (e.g., Figure 2As described above), multiple mutations were observed in a 60.50kb region. Based on a sequencing depth of ≥20, two high-quality SNP sites related to watermelon seed size were screened. Both sites are located on the watermelon seed size-related gene Cla97C06G114610, and their physical locations are anchored at 5540184bp and 5540261bp on watermelon chromosome 6.

[0054] Note: The Euclidean Distance (ED) algorithm is a method that uses sequencing data to find significantly different markers between pools and uses this to assess regions associated with traits. Theoretically, in the BSA project, the two pools should be similar except for the loci associated with the target trait; therefore, the ED value for non-target loci should be close to 0. A larger ED value indicates a greater difference in the marker between the two pools.

[0055] Example 2: Design of KASP primers for detecting SNP molecular markers

[0056] In this experimental example, KASP primers were designed for the SNP molecular markers screened in Experiment 1. The specific operation is as follows:

[0057] Polymarker software was used to convert the identified polymorphic SNPs associated with seed size traits into KASP markers. Two allele-specific forward primers and one universal reverse primer based on the flanking sequences around the variant site (SNP) were designed using Primer 5 software. The specific primer sequences are shown in Table 1.

[0058] Table 1 KASP primer sequences

[0059]

[0060] Taking SNP molecular marker 1 as an example, primer K1-Fam carries the fluorescent tag sequence of FAM at its 5' end. K1-Fam and K1-R can amplify the fragment with K5540184 as C, thereby generating the FAM fluorescent signal; primer K1-Hex carries the fluorescent tag sequence of HEX at its 5' end. K1-Hex and K1-R can amplify the fragment with K5540184 as A, thereby generating the HEX fluorescent signal.

[0061] Experiment Example 3: Application of KASP primers in watermelon seed size-assisted screening and breeding

[0062] This invention first uses the KASP primers designed in Experiment 2 to perform genotyping on both parents to verify their universality. The results show that the SNP molecular markers screened in Experiment 1 have good universality. Further verification of the accuracy of the designed KASP primers is then performed in the F2 population. The specific procedures for further verification are as follows:

[0063] Genomic DNA was extracted from F2 generation plants of different test materials selected in Experiment 1 for KASP genotyping. PCR amplification was performed in a 10 μL reaction system, which included 5 μL PCR Mix, 3.9 μL ddH2O, 1 μL DNA template, and 0.1 μL primer premix (the concentrations of the forward and reverse primers were adjusted to 100 μmol / μL, and 50 μL of primer premix was prepared using 10 μL of forward primer 1, 10 μL of forward primer 2, and 30 μL of reverse primer).

[0064] The PCR amplification program is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 15 s, annealing at 61~55℃ for 60 s, decreasing by 0.6℃ per cycle, for a total of 10 cycles; denaturation at 95℃ for 15 s, annealing at 55℃ for 60 s, for 28-35 cycles; store at 4℃.

[0065] Genotyping was performed using an ABI QuantStudio 3 real-time quantitative PCR instrument. The plate reading program was 30℃ for 1 min, and the Allele Discrimination function was selected for analysis. The x-axis and y-axis represent HEX fluorescence signal and FAM fluorescence signal, respectively. Genotyping of clustered samples was analyzed based on fluorescence signal values.

[0066] KASP genotyping results for SNP molecular marker 1: Fluorescence data are presented graphically. Fluorescence signals with the FAM tag located near the Y-axis indicate that the SNP molecular marker 1 genotype of the tested watermelon material is CC; fluorescence signals with the HEX tag located near the X-axis indicate that the SNP molecular marker 1 genotype of the tested watermelon material is AA; fluorescence signals distributed in the middle of the coordinate axes indicate that the SNP molecular marker 1 genotype of the tested watermelon material is CA (e.g., ...). Figure 3 (As shown).

[0067] Statistical analysis was performed on the SNP molecular marker 1 genotyping results and thousand-seed weight results. It was found that the maximum thousand-seed weight for genotype AA was 16.1g, the minimum was 7.7g, and the average was 13.2g; the maximum thousand-seed weight for genotype CA was 36.7g, the minimum was 10.3g, and the average was 21.3g; and the maximum thousand-seed weight for genotype CC was 87.2g, the minimum was 44g, and the average was 63.2g (as shown in Table 2). Significant differences were analyzed (e.g.,...). Figure 4 As shown in the figure, the thousand-seed weight of the CC genotype was significantly higher than that of the AA and CA genotypes (p<0.0001). The results indicate that watermelon materials with the CC genotype have larger thousand-seed weights. Notably, we observed that the CC genotype was only present in the large-seed phenotype, which can serve as a basis for identifying large seeds.

[0068] Table 2. Statistical table of genotypes and thousand-grain weight of 46 watermelon germplasm samples.

[0069]

[0070] KASP genotyping results for SNP molecular marker 2: Fluorescence data are presented graphically. Fluorescence signals with the FAM tag located near the Y-axis indicate that the SNP molecular marker 2 genotype of the tested watermelon material is AA; fluorescence signals with the HEX tag located near the X-axis indicate that the SNP molecular marker 2 genotype of the tested watermelon material is GG; fluorescence signals distributed in the middle of the coordinate axes indicate that the SNP molecular marker 2 genotype of the tested watermelon material is AG (e.g., ...). Figure 5 (As shown).

[0071] Statistical analysis was performed on the SNP molecular marker genotyping results and thousand-grain weight results. The results showed that in the F2 population, the thousand-grain weight of the GG genotype (homozygous genotype (particle × 10² B4)) was significantly lower than that of the AA genotype (wild-type homozygous genotype), while there was no significant difference between the heterozygous genotype AG and the homozygous genotype GG (as shown in Table 3 and ). Figure 6 (As shown). In SNP 5540261 Mutation sites were observed to be related to SNPs 5540184 Genotyping results with the same mutation site. Notably, we observed that the AA genotype was only present in the large-seed phenotype, which can serve as a basis for identifying large seeds.

[0072] Table 3. Statistical table of genotypes and thousand-grain weight of 46 watermelon germplasm samples.

[0073]

[0074] In summary, the two SNP molecular markers obtained by screening in this invention can be used to identify the size of watermelon seeds. In practice, one of the SNP molecular markers can be selected for detection, or the two SNP molecular markers can be combined for molecular marker-assisted selection breeding of watermelon seeds.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A molecular marker for identifying the size of watermelon seeds, characterized in that: The molecular markers consist of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 71st base from the 5' end being A or C; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 71st base from the 5' end being A or G.

2. A primer combination for detecting the molecular marker of claim 1, characterized in that: The primer sets include primer set 1 for identifying the SNP molecular marker 1 genotype and primer set 2 for identifying the SNP molecular marker 2 genotype; the nucleotide sequences of the primers in primer set 1 are shown in SEQ ID NO. 3~5; the nucleotide sequences of the primers in primer set 2 are shown in SEQ ID NO. 6~8.

3. The application of a primer combination for detecting the molecular marker as described in claim 1 or the primer combination as described in claim 2 in watermelon seed size-assisted screening and breeding.

4. The application of the primer combination according to claim 3 in watermelon seed size-assisted screening and breeding, characterized in that: The size of the watermelon seeds is the weight of 1000 watermelon seeds.

5. The application of the primer combination according to claim 3 or 4 in watermelon seed size-assisted screening and breeding, characterized in that: Competitive allele-specific PCR amplification was performed on the genomic DNA of the watermelon sample to be tested using the primer combination for detecting the molecular marker. The fluorescence signal of the amplification product was read, and the genotype of the SNP molecular marker was determined based on the fluorescence signal. The seed size of the watermelon sample to be tested was determined based on the genotype.

6. The application of the primer combination according to claim 3 or 4 in watermelon seed size-assisted screening and breeding, characterized in that: When the genotype of SNP molecular marker 1 is CC and / or the genotype of SNP molecular marker 2 is AA, the watermelon sample to be tested has a phenotype of high thousand-seed weight; when the genotype of SNP molecular marker 1 is AA or CA and / or the genotype of SNP molecular marker 2 is GG or AG, the watermelon sample to be tested has a phenotype of low thousand-seed weight.

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