Molecular marker and primer combination for identifying size of watermelon seed and application of molecular marker and primer combination

Abstract

The invention relates to a molecular marker for identifying the size of a watermelon seed, a primer combination and application thereof, and belongs to the technical field of molecular markers. According to the invention, re-sequencing data of watermelon individuals with different phenotypes are analyzed, and two high-quality SNP loci related to the size of the watermelon seed are screened out from a gene Cla97C06G114610 related to the size of the watermelon seed. Furthermore, verification proves that the two screened SNP loci are remarkably related to the size (thousand seed weight) of the watermelon seeds, and different genotypes of the SNP loci can effectively distinguish watermelon individuals with large or small thousand seed weight, which indicates that the two SNP loci can be used as SNP molecular markers for identifying the size of the watermelon seeds. The SNP molecular marker disclosed by the invention is helpful to deeply understand variation of the watermelon seeds and provides a new means for detecting the size of the watermelon seeds, so that the improvement process of the size character of the watermelon seeds is accelerated, and the accuracy and the selection rate of breeding are improved.

Description

Technical Field

[0001] The invention relates to a molecular marker, a primer combination and application thereof for identifying the size of watermelon seeds, and belongs to the technical field of molecular markers. Background Art

[0002] watermelon( Citrullus lanatus (Thunb.) Matsum.et Nakai) is an annual vine of the Cucurbitaceae family, also known as cold melon, watermelon, watermelon rind, etc. Watermelon is native to tropical Africa and is widely cultivated in tropical to temperate regions of the world. It is also widely cultivated in various parts of my country. Watermelon is rich in nutrition and tastes sweet and crisp. It contains rich protein, carbohydrates, crude fiber, potassium, phosphorus, calcium, iron, sodium, etc. and vitamins A, B, C, etc. Watermelon has gradually become one of the most popular fresh fruits in the world.

[0003] Seeds are a unique organ in plants and are essential for plant life. Seed size is a fundamental agronomic trait that can affect quality and yield, in addition to influencing adaptation to environmental stresses and the fitness of the plant. This is especially important for plants with seeds as their main product organ. Watermelon seeds have a dual purpose, with fleshier watermelons usually containing relatively small seeds, which makes small-seeded or cultivated seedless watermelons more attractive in the market. In contrast, edible seeded watermelons usually contain larger seeds and provide rich nutrients such as protein and oil for humans. Therefore, breeding watermelon varieties with specific seed sizes can meet the different needs of consumers.

[0004] There is a large difference in seed size between different watermelon germplasms. In recent years, many genes related to watermelon seed size and their functions have been analyzed, and the trait of watermelon seed size has been proved to be a quality trait regulated by a complex regulatory mechanism. 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 other modifying factors may also be involved; Prothro et al. (2012) identified multiple major QTLs in chr02 and chr06; Meru and McGregor (2013) found three major effect QTLs on chr06. Luan et al. (2019) found a locus named WEG6.1 on chr06 that controls the seed size trait of watermelon. In addition, in the study of Wang et al. (2024), QTLSS6.2, which is related to watermelon seed size, was mapped to chr06; Maragal et al. (2022) found two qtls (q_100SW_2.1 and q_100SW_6.1) located on chr06. From the above research content, it can be seen that with the development of molecular marker technology and the improvement of molecular quantitative genetics, a batch of QTL positioning work has been carried out in order to reveal the genetic basis of watermelon seed size. However, the intervals located in these studies are large, and there are many candidate genes that are difficult to screen. There are few molecular markers that can be used for actual production and breeding, resulting in low efficiency of watermelon breeding, which seriously restricts the breeding process of watermelon. Therefore, mining SNP molecular markers related to 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 object of the present invention is to provide a molecular marker for identifying the size of watermelon seeds, and to provide a molecular marker for identifying the size of watermelon seeds.

[0006] The second object of the present invention is to provide a primer combination for detecting the genotype of the molecular marker, and to provide a primer that can quickly and accurately detect the genotype of the molecular marker.

[0007] The third object of the present invention is to provide an application of a molecular marker or primer combination in auxiliary screening and breeding of watermelon seed size, and to provide a method for quickly and accurately breeding watermelon varieties with specific seed sizes.

[0008] In order to achieve the above object, a technical solution of a molecular marker for identifying watermelon seed size in the present invention is: A molecular marker for identifying watermelon seed size, wherein the SNP molecular marker consists of a SNP molecular marker 1 and a SNP molecular marker 2; the nucleotide sequence of the SNP molecular marker 1 is shown in SEQ ID NO.1, and the 71st base from the 5' end is A or C; the nucleotide sequence of the SNP molecular marker 2 is shown in SEQ ID NO.2, and the 71st base from the 5' end is A or G.

[0009] The beneficial effect of the above technical scheme is that the molecular marker for identifying the size of watermelon seeds of the present invention is a pioneering invention. The present invention uses a male material with a small seed thousand-grain weight and a female material with a large seed thousand-grain weight to hybridize to obtain the F1 generation. After the F1 generation is self-pollinated to obtain the F2 generation, seeds with a large thousand-grain weight and seeds with a small thousand-grain weight are selected from the F2 generation, and DNA is extracted and resequenced. By analyzing the resequencing data, two high-quality SNP sites related to watermelon seed size were screened out on the watermelon seed size-related gene Cla97C06G114610, and the physical position was anchored at 5540184 bp and 5540261 bp on chromosome 6 of watermelon. It was further verified that the two screened SNP sites were significantly correlated with the size of watermelon seeds (thousand-grain weight), and different genotypes of the SNP sites could effectively distinguish watermelon individuals with large thousand-grain weight and small thousand-grain weight, which shows that the two screened SNP sites can be used as SNP molecular markers for identifying watermelon seed size. The SNP molecular markers of the present invention help to gain a deeper understanding of the variation of watermelon seeds, provide a new means for detecting the size of watermelon seeds, and thus accelerate the improvement process of the watermelon seed size trait and improve the accuracy and selection rate of breeding.

[0010] In order to achieve the above object, a technical solution of a primer combination for detecting the genotype of the molecular marker in the present invention is: A primer combination for detecting the genotype of the molecular marker, the primer combination comprising a primer combination 1 for identifying the genotype of SNP molecular marker 1 and a primer combination 2 for identifying the genotype of SNP molecular marker 1; the nucleotide sequences of the primers in the primer combination 1 are shown in SEQ ID NOs. 3 to 5; the nucleotide sequences of the primers in the primer combination 2 are shown in SEQ ID NOs. 6 to 8.

[0011] The beneficial effect of the above technical solution is that the present invention designs KASP primers for the screened SNP molecular markers, and the primers can effectively detect the genotype of the SNP molecular markers, and have the advantages of rapidity, accuracy and high throughput, providing an effective means for identifying the size of watermelon seeds.

[0012] 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 sequence before and after 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 PCR terminal fluorescence signal.

[0013] More specifically, taking primer combination 1 for identifying the genotype of SNP molecular marker 1 as an example, the nucleotide sequence of the primer shown in SEQ ID NO.3 has a FAM fluorescent label sequence at the 5' end, and the nucleotide sequence of the primer shown in SEQ ID NO.4 has a HEX fluorescent label sequence at the 5' end.

[0014] In order to achieve the above object, the technical solution of the application of a molecular marker or primer combination in auxiliary screening and breeding of watermelon seed size in the present invention is: Application of a molecular marker or primer combination in watermelon seed size-assisted screening and breeding.

[0015] The beneficial effect of the above technical solution is that: the present invention screened and obtained two molecular markers for identifying the size of watermelon seeds, and repeated the test using multiple watermelon materials, confirming that the two SNP molecular markers can be effectively used to identify or assist in identifying the size of watermelon seeds, and can be used in molecular marker-assisted breeding, and have important application value in the exploration and screening of watermelon germplasm resources or varieties with different seed sizes.

[0016] Furthermore, the application of the molecular marker or primer combination of the present invention in auxiliary screening and breeding of watermelon seed size directly uses watermelon genomic DNA as a template, and the SNP site genotype of the KASP molecular marker can be detected in the leaf tissue of the watermelon, which is conducive to convenient and efficient prediction of watermelon seed size and can be applied to watermelon seed size screening and identification before harvest.

[0017] As a further improvement, the watermelon seed size is the thousand-grain weight of the watermelon seeds.

[0018] Specifically, from a genetic point of view, seed size is a trait with high heritability and is positively correlated with seed weight, that is, the larger the seed, the heavier the seed, and vice versa (Li et al. 2018). In addition, many studies have used thousand-grain 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. As a further improvement, the genomic DNA of the watermelon sample to be tested is subjected to competitive allele-specific PCR amplification using a primer combination for detecting the SNP molecular marker, the fluorescence signal of the amplified product is read, the genotype of the SNP molecular marker is determined based on the fluorescence signal, and the size of the seeds of the watermelon sample to be tested is determined based on the genotype.

[0019] 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 has a phenotype of large thousand-grain seed weight; when the genotype of SNP molecular marker 1 is AA, CA and / or the genotype of SNP molecular marker 2 is GG, AG, the watermelon sample to be tested has a phenotype of small thousand-grain seed weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Statistical data of SNPs and InDels between samples and their chromosomal distribution in Experimental Example 1 of the present invention (wherein A is SNP visualization of the entire genome, B is a Venn diagram of SNP differences between samples, C is SNP distribution on chromosomes, D is InDel visualization of the entire genome, E is a Venn diagram of InDel differences between samples, and F is InDel distribution on chromosomes; the circular diagram is from inside to outside: the first circle represents the ED value corresponding to the distribution of 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 the chromosome coordinates); Figure 2 In Experimental Example 1 of the present invention, the Integrative Genomics Viewer (IGV) was used to compare and visualize the parental genomes; Figure 3 The genotyping result of SNP molecular marker 1 of the watermelon sample to be tested in Experimental Example 3 of the present invention; Figure 4This is the association analysis between the genotype of SNP molecular marker 1 and 1000-grain weight of the watermelon samples to be tested in the F2 population in Experimental Example 3 of the present invention (where **** represents p<0.0001, and ns represents no significant difference); Figure 5 The genotyping result of SNP molecular marker 2 of the watermelon sample to be tested in Experimental Example 3 of the present invention; Figure 6 This is the association analysis between the genotype of SNP molecular marker 2 and 1000-grain weight of the tested watermelon samples in the F2 population in Experimental Example 3 of the present invention (wherein, **** represents p<0.0001, and ns represents no significant difference). DETAILED DESCRIPTION

[0021] From a genetic point of view, seed size is a trait with high heritability and is positively correlated with seed weight, that is, the larger the seed, the higher the seed weight, and vice versa. Early studies have shown that seed weight directly affects germination rate, seedling fecundity, activity, survival rate and germination period. Therefore, mining SNP sites related to watermelon size is crucial to understanding the variation of watermelon seeds, and also provides detection sites for efficiently predicting watermelon seed size. However, there are few molecular markers in the prior art that can be used for actual production and breeding. Based on this, the present invention provides a molecular marker for identifying watermelon seed size.

[0022] The molecular marker for identifying the size of watermelon seeds of the present invention is composed of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of the SNP molecular marker 1 is shown in SEQ ID NO.1, and the 71st base from the 5' end is A or C; the nucleotide sequence of the SNP molecular marker 2 is shown in SEQ ID NO.2, and the 71st base from the 5' end is A or G. The present invention also designs KASP primers for detecting the genotype of the molecular marker, which can quickly and easily detect the genotype of the SNP molecular marker. Further repeated testing using multiple watermelon materials confirmed that the two SNP molecular markers can be effectively used to identify or assist in identifying the size of watermelon seeds, can be used in molecular marker-assisted breeding, and have important application value in the research of mining and screening watermelon germplasm resources or lines with different seed sizes.

[0023] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0024] The test materials used in the following embodiments and experimental examples of the present invention are described as follows: The test materials include maternal parent, paternal parent, and F2 generation population; The male parent material is: (microparticles × 102B4) × 102B4, and the average seed thousand-grain weight is 16.73g; The female parent material is: wild in Zhengzhou, with an average seed thousand-grain weight of 59.06g; The above-mentioned female and male parents are both from Luoyang Nongfa Agricultural Biotechnology Co., Ltd., and the parent and offspring plants are planted in the company's farm.

[0025] The F1 generation obtained by hybridizing the above two materials as parents, the F2 generation population obtained by self-pollination of the F1 generation, and the number of F2 generation populations used for harvest in 2023 were 275 plants respectively.

[0026] Among them, the thousand-grain weight of the test materials was determined: strict self-pollination was carried out under conventional field management. Each plant was pollinated manually and numbered. Mature watermelons were collected 35-43 days after pollination. The seeds were washed, dried, and harvested. The weight of the seeds was measured in grams using an electronic balance. The average of 3 repeated measurements was recorded.

[0027] Example 1 of a molecular marker for identifying watermelon seed size The molecular markers for identifying watermelon seed size in this embodiment are composed of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of the SNP molecular marker 1 is shown in SEQ ID NO.1, and the 71st base from the 5' end is A or C; the nucleotide sequence of the SNP molecular marker 2 is shown in SEQ ID NO.2, and the 71st base from the 5' end is A or G.

[0028] SNP molecular marker 1 corresponds to the 5540184th deoxynucleotide on chromosome 6 of the 97103 (v2.5) version of the watermelon reference genome published by GUGENDBv2 (http: / / cucurbitgenomics.org / v); SNP molecular marker 2 corresponds to the 5540261th deoxynucleotide on chromosome 6 of the 97103 (v2.5) version of the watermelon reference genome published by GUGENDBv2 (http: / / cucurbitgenomics.org / v).

[0029] Example 1 of a primer combination for detecting the genotype of the molecular marker The primer combination for detecting the genotype of the molecular marker in this embodiment includes primer combination 1 for identifying the genotype of SNP molecular marker 1 and primer combination 2 for identifying the genotype of SNP molecular marker 1; the nucleotide sequences of the primers in the primer combination 1 are shown in SEQ ID NOs.3~5; the nucleotide sequences of the primers in the primer combination 2 are shown in SEQ ID NOs.6~8.

[0030] The nucleotide sequence of the primer shown in SEQ ID NO.3 has a fluorescent label sequence of FAM at the 5' end, and the primer shown in SEQ ID NO.3 and the primer shown in SEQ ID NO.5 can amplify the fragment of SNP molecular marker 1 with genotype C, thereby generating a FAM fluorescent signal; the nucleotide sequence of the primer shown in SEQ ID NO.4 has a fluorescent label sequence of HEX at the 5' end, and the primer shown in SEQ ID NO.4 and the primer shown in SEQ ID NO.5 can amplify the fragment of SNP molecular marker 1 with genotype A, thereby generating a HEX fluorescent signal.

[0031] The nucleotide sequence of the primer shown in SEQ ID NO.6 has a fluorescent label sequence of FAM at the 5' end, and the primer shown in SEQ ID NO.6 and the primer shown in SEQ ID NO.8 can amplify the fragment of SNP molecular marker 2 with genotype A, thereby generating a FAM fluorescent signal; the nucleotide sequence of the primer shown in SEQ ID NO.7 has a fluorescent label sequence of HEX at the 5' end, and the primer shown in SEQ ID NO.7 and the primer shown in SEQ ID NO.8 can amplify the fragment of SNP molecular marker 1 with genotype G, thereby generating a HEX fluorescent signal.

[0032] Example 1 of the application of a molecular marker or primer combination in auxiliary screening and breeding of watermelon seeds The molecular marker or primer combination of this embodiment is used in auxiliary screening and breeding of watermelon seed size, and the genotype of the SNP molecular marker is detected. 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 large seed 1000-grain 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 small seed 1000-grain weight.

[0033] Experimental Example 1 Screening of SNP loci associated with watermelon seed size In this experimental example, seeds with large thousand-grain weight and small thousand-grain weight were selected from the F2 generation population of the test material, and the genome was resequenced after DNA extraction. Then, the data obtained by resequencing were analyzed to obtain SNP sites related to watermelon seed size. The specific operations are as follows: In the F2 generation, 20 seeds with large 1000-grain weight and 20 seeds with small 1000-grain weight were selected, and DNA of each individual plant was extracted (the genomic DNA extraction method was described in the instructions of the Novozymes DNA Genomic Extraction Kit), and the concentration and quality of genomic DNA were measured using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). After adjusting the DNA concentration to be consistent, equal amounts were mixed to construct a mixed pool.

[0034] DNA samples were resequenced using the Illumina HiSeq XTen / NovaSeq / BGI platform (Beijing, China) with a sequencing length of 150 bp and a genome sequencing depth of at least 30× coverage for each sample. Low-quality data were filtered using Bcltofastq1.8.4 software, and the resequencing data of the two extreme pools were realigned to the watermelon reference genome 97103V2.5 (http: / / cucurbitgenomics.org / v2) using BWA software. Duplicates were removed using the Mark Duplicate tool of the Picard online software package (http: / / sourceforge.net / projects / picard / ), and local realignment and base quality value correction were performed using GATK software to ensure the accuracy of the detected sites. SNP and InDel variants were detected between samples, and association analysis of differential sites between samples was performed based on the alignment information of the watermelon reference genome 97103V2.5 (http: / / cucurbitgenomics.org / v2). The original ED was selected 5 As the association value to eliminate background noise, the DISTANCE tool was used to fit the ED value, and the median + 3SD of the fitting values ​​of all sites was taken as the association threshold for analysis. The same method was used to fit the ΔSNP-index and ΔIndel-index, and the part with values ​​above the threshold was selected as the target area associated with the seed size trait.

[0035] The intersection of the SNP and Indel association region results obtained by the two association analysis methods was taken to determine the positioning region (such as Figure 1 The parental sequencing results were visualized using the Integrative Genomics Viewer software (as shown in Figure 2 As described above, multiple mutations were observed in a 60.50kb region, and two high-quality SNP sites related to watermelon seed size were screened out with a sequencing depth of ≥20. Both sites were located on the watermelon seed size-related gene Cla97C06G114610, and their physical positions were anchored at 5540184bp and 5540261bp on chromosome 6 of watermelon.

[0036] Note: The Euclidean distance (ED) algorithm is a method that uses sequencing data to find markers with significant differences between mixed pools and evaluate the regions associated with traits. In theory, except for the target trait-related sites, other sites between the two mixed pools constructed by the BSA project tend to be consistent, so the ED value of non-target sites should tend to 0. The larger the ED value, the greater the difference between the markers in the two mixed pools.

[0037] Experimental Example 2 Design of KASP primers for detecting SNP molecular markers In this experimental example, KASP primers were designed for the SNP molecular markers screened in Experimental Example 1. The specific operations are as follows: The identified polymorphic SNPs associated with seed size traits were converted into KASP markers using Poly Marker software. Two allele-specific forward primers and a universal reverse primer based on the flanking sequence around the variant site (SNP) were designed using primer 5 software. The specific primer sequences are shown in Table 1.

[0038] Table 1 KASP primer sequences Taking SNP molecular marker 1 as an example, the 5' end of primer K1-Fam carries a FAM fluorescent label sequence, and K1-Fam and K1-R can amplify a fragment with K5540184 site as C, thereby generating a FAM fluorescent signal; the 5' end of primer K1-Hex carries a HEX fluorescent label sequence, and K1-Hex and K1-R can amplify a fragment with K5540184 site as A, thereby generating a HEX fluorescent signal.

[0039] Experimental Example 3 Application of KASP primers in watermelon seed size-assisted screening and breeding The present invention first uses the KASP primers designed in Experimental Example 2 to perform genotyping on the parents to verify its universality. The results show that the SNP molecular markers screened in Experimental Example 1 have good universality. The designed KASP primers are further used to verify its accuracy in the F2 population. The specific operations for further verification are as follows: Take the F2 generation plant individuals of different test materials selected in Experimental Example 1, extract genomic DNA for KASP typing detection. PCR amplification was performed in a 10 μL reaction system, and the reaction system included 5 μL PCR Mix, 3.9 μL ddH2O, 1 μL DNA template and 0.1 μL primer premix (the concentration of forward and reverse primers was adjusted to 100 μmol / μL, and 50 μL of primer premix was prepared with 10 μL forward primer 1, 10 μL forward primer 2 and 30 μL reverse primer).

[0040] The PCR amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 61-55°C for 60 s, decreasing 0.6°C each cycle, for a total of 10 cycles; denaturation at 95°C for 15 s, annealing at 55°C for 60 s, for 28-35 cycles; and storage at 4°C.

[0041] ABI QuantStudio3 real-time fluorescence quantitative PCR instrument was used for genotyping detection. The plate reading program was 30℃, 1min, and the Allele Discrimination function was selected for analysis. The x-axis and y-axis represent the HEX fluorescence signal and the FAM fluorescence signal, respectively. Clustering was performed based on the fluorescence signal value, and the genotype of the clustered samples was analyzed.

[0042] KASP typing test results of SNP molecular marker 1: The fluorescence data is presented in a graphical form. The fluorescence signal with the FAM label is located near the Y axis, indicating that the genotype of the SNP molecular marker 1 of the watermelon material to be tested is CC; the fluorescence signal with the HEX label is located near the X axis, indicating that the genotype of the SNP molecular marker 1 of the watermelon material to be tested is AA; if the fluorescence signal is distributed in the middle of the coordinate axis, the genotype of the SNP molecular marker 1 of the watermelon material to be tested is CA (e.g. Figure 3 as shown).

[0043] The results of SNP molecular marker 1 typing and 1000-grain weight were statistically analyzed, and it was found that the maximum 1000-grain weight of seeds with genotype AA was 16.1g, the minimum was 7.7g, and the average 1000-grain weight was 13.2g; the maximum 1000-grain weight of seeds with genotype CA was 36.7g, the minimum was 10.3g, and the average was 21.3g; the maximum 1000-grain weight of seeds with genotype CC was 87.2g, the minimum was 44g, and the average was 63.2g (as shown in Table 2). Figure 4 As shown in Figure 2, the thousand-grain weight of genotype CC was significantly higher than that of genotypes AA and CA (p<0.0001). The results showed that watermelon materials with genotype CC had seeds with larger thousand-grain weight. It is worth noting that we observed that genotype CC only existed in the large seed phenotype, which can be used as a basis for judging large seeds.

[0044] Table 2 Statistics of genotypes and thousand-grain weight of 46 watermelon germplasms KASP typing test results of SNP molecular marker 2: The fluorescence data is presented in a graphical form. The fluorescence signal with the FAM label is located near the Y axis, indicating that the genotype of the SNP molecular marker 2 of the tested watermelon material is AA; the fluorescence signal with the HEX label is located near the X axis, indicating that the genotype of the SNP molecular marker 2 of the tested watermelon material is GG; if the fluorescence signal is distributed in the middle of the coordinate axis, the genotype of the SNP molecular marker 2 of the tested watermelon material is AG (e.g. Figure 5 as shown).

[0045] The results of SNP molecular marker 2 typing and 1000-grain weight were statistically analyzed. The results showed that in the F2 population, the 1000-grain weight of the GG genotype (homozygous genotype (particles × 102B4)) was significantly lower than that of AA (wild homozygous genotype), and there was no significant difference between the heterozygous genotype AG and the homozygous genotype GG (see Table 3 and Figure 6 In SNP 5540261 SNP 5540184 The genotyping results of the same mutation site. It is worth noting that we observed that the genotype AA only existed in the large seed phenotype, which can be used as the basis for judging large seeds.

[0046] Table 3 Statistics of genotypes and thousand-grain weight of 46 watermelon germplasms In summary, the two SNP molecular markers screened by the present invention can be used to identify the size of watermelon seeds. In actual situations, one of the SNP molecular markers can be selected for detection, or the two SNP molecular markers can be combined for watermelon seed size molecular marker-assisted selection breeding.

[0047] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 watermelon seed size, characterized in that: The SNP molecular marker consists of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of the SNP molecular marker 1 is shown in SEQ ID NO.1, and the 71st base from the 5' end is A or C; the nucleotide sequence of the SNP molecular marker 2 is shown in SEQ ID NO.2, and the 71st base from the 5' end is A or G.

2. A primer combination for detecting the molecular marker genotype according to claim 1, characterized in that: The primer combination includes primer combination 1 for identifying the genotype of SNP molecular marker 1 and primer combination 2 for identifying the genotype of SNP molecular marker 1; the nucleotide sequences of the primers in primer combination 1 are shown in SEQ ID NOs.3~5; the nucleotide sequences of the primers in primer combination 2 are shown in SEQ ID NOs.6~8.

3. Use of the molecular marker according to claim 1 or the primer combination according to claim 2 in auxiliary screening and breeding of watermelon seed size.

4. The use of the molecular marker or primer combination according to claim 3 in auxiliary screening and breeding of watermelon seeds, characterized in that: The watermelon seed size refers to the thousand-grain weight of the watermelon seeds.

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

6. Use of the molecular marker or primer combination according to claim 3 or 4 in auxiliary screening and breeding of watermelon seeds, 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 large 1000-grain weight; when the genotype of SNP molecular marker 1 is AA, CA and / or the genotype of SNP molecular marker 2 is GG, AG, the watermelon sample to be tested has a phenotype of small 1000-grain weight.

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