SNP (Single Nucleotide Polymorphism) molecular marker related to muskmelon leaf shape notch character and application of SNP molecular marker

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) molecular marker related to a muskmelon leaf shape notch character and application thereof, and belongs to the technical field of molecular markers. According to analysis, it is found that a non-synonymous mutant gene MELO3C015426 for coding LRR protein is a candidate gene related to regulation and control of muskmelon leaf shape notch characters, by taking the non-synonymous mutant gene MELO3C015426 as a reference, visual analysis is conducted on non-synonymous mutant SNP sites which have obvious differences between parents and are related to the characters in a genome candidate area by utilizing Integrating Genomics Viewer software, and by taking the sequencing depth larger than or equal to 20 as a standard, the non-synonymous mutant SNP sites can be used for identifying the muskmelon leaf shape notch character related to the muskmelon leaf shape notch character related to the muskmelon leaf shape notch character related to the muskmelon leaf shape notch character related to the muskmelon leaf shape notch character. Three SNP sites related to the muskmelon leaf shape notch character are screened out. Furthermore, verification proves that the screened three SNP sites are related to the muskmelon leaf shape notch character. The SNP molecular marker provided by the invention can be used for improving leaf shape screening in a breeding scheme, and is beneficial to understanding the molecular mechanism of the muskmelon leaf shape notch character.

Description

Technical Field

[0001] The invention relates to a SNP molecular marker related to a melon leaf notch trait and application thereof, belonging to the technical field of molecular markers. Background Art

[0002] Muskmelon (Cucumis melo L.), a diploid plant belonging to the Cucurbitaceae family, has attracted much attention due to its unique biological characteristics and economic importance. Leaf shape, as an important horticultural trait, affects the development of melon and thus determines its quality.

[0003] Plant leaves are important nutritional organs of flowering plants. They are the main organs for photosynthesis and have the functions of regulating nutrient distribution, water transport and gas exchange. Each crop plant has a variety of leaf shapes, positions and sizes, which clearly determine the obvious diversity between different species and even similar species.

[0004] It has been found that changes in leaf shape in crop plants can be induced by genetic factors and several extreme environmental changes in temperature conditions. An important part of leaf shape is the leaf margin, which can be non-lobed, serrated or lobed. The functions of leaf shape are reflected in many aspects. For example, notched leaves with deep sinuses can also improve heat transfer, thereby reducing leaf temperature and avoiding sunlight damage to the plant. Therefore, a better understanding of the genes and regulatory networks related to leaf shape will help to further control plant growth and breeding using biotechnology.

[0005] Previous studies have shown that many genes are involved in regulating leaf shape in plants. In Arabidopsis, the LATEMERISTEM IDENTITY 1 (LMI1) gene regulates the formation of serrated leaves. In alfalfa, MtLMI1a and MtLMI1b redundantly trigger serrated expansion of leaf margins. In addition, studies have shown that some genes related to leaf shape are regulated by hormones in plants. The class I KNOTTED1-LIKE HOMEOBOX (KNOX1) gene controls the formation of leaf shape, and the hormone cytokinin (CK) (Aguilar-Martínez et.al. 2014) or gibberellin (GA) levels inhibit the effect of KNOX1 protein on leaf shape. Moreover, in another study, it was reported that the alternating expression of the auxin-responsive and CUP-SHAPED COTYLEDON1 (CUC1) gene regulates the formation of lobes.

[0006] At present, the genetic localization of genes controlling leaf shape has been carried out on a large number of crop plants through multi-omics technology. In cotton, the HD-Zip transcription factor gene (GhLMI1-D1b), as a homologous gene of the LMi1-like gene, is responsible for the main shape variation of cotton leaves. BnA10.LMI1 and BnA10.RCO are located in the 41kb region of chromosome 10 of Brassica napus and are related to the formation of lobed leaves. However, there are few reports on the mechanism of leaf shape formation in melon, which makes it difficult to effectively utilize the advantages of leaf morphology for breeding and quality improvement. Therefore, screening out the genes involved in the regulation of melon leaf shape and clarifying its regulatory network will provide a favorable tool for melon breeding. Summary of the invention

[0007] The first object of the present invention is to provide a SNP molecular marker related to the notch trait of melon leaf shape, and to provide a new molecular marker for regulating the notch trait of melon leaf shape.

[0008] The second object of the present invention is to provide an application of SNP molecular markers related to the melon leaf notch trait in melon breeding, and to provide an effective, accurate and rapid method for screening melon plants with the notch trait.

[0009] In order to achieve the above object, a technical solution of a SNP molecular marker related to the melon leaf notch trait in the present invention is:

[0010] A SNP molecular marker associated with a melon leaf notch trait, the SNP molecular marker consisting of SNP molecular markers 1 to 3; the SNP molecular marker consisting of SNP molecular markers 1 to 3; the nucleotide sequence of the SNP molecular marker 1 is as shown in SEQ ID NO.1, and the sixth base from the 5' end is A or G; the nucleotide sequence of the SNP molecular marker 2 is as shown in SEQ ID NO.1, and the 20th base from the 5' end is C or T; the nucleotide sequence of the SNP molecular marker 3 is as shown in SEQ ID NO.1, and the 102nd base from the 5' end is T or G.

[0011] The beneficial effect of the above technical solution is that: the SNP molecular marker related to the leaf notch trait of melon in the present invention is a pioneering invention. The melon variety '145X1901' has the leaf notch trait, while '1310' has a normal leaf shape. The present invention obtained an F2 population by hybridizing '145X1901' and '1310', and through BSA-Seq analysis, a major candidate region regulating leaf shape changes was found on chromosome 2, and candidate gene prediction was performed based on the RNA-Seq data of '145X1901' melon. Through analysis, it was found that the non-synonymous mutant gene MELO3C015426 encoding Leucine rich repeat (LRR) protein is a candidate gene regulating the leaf notch trait. Taking the candidate gene MELO3C015426 as a benchmark, the Integrative Genomics Viewer software was used to visualize the non-synonymous mutation SNP sites associated with the trait in the candidate regions of the genome that differed significantly between the parents, and three SNP sites associated with the melon leaf shape notch trait were screened out using the sequencing depth ≥ 20 as the standard. It was further verified that the three screened SNP sites were associated with the melon leaf shape notch trait. The SNP molecular markers provided by the present invention can be used to improve leaf shape screening in breeding programs, and help understand the molecular mechanism of the melon leaf shape notch trait, providing a new research target for the development of melon leaf shape.

[0012] In order to achieve the above object, the technical solution of the application of SNP molecular markers related to the melon leaf notch trait in melon breeding in the present invention is:

[0013] The invention discloses an application of a SNP molecular marker related to a leaf notch trait of a melon in melon breeding, wherein the application is to screen melons with leaf notch strains.

[0014] The beneficial effect of the above technical solution is that: the present invention finds a major candidate region regulating the leaf notch trait on melon chromosome 2 through the analysis of BSA-Seq data, and combines RNA-seq and BSA-seq for analysis, and believes that the non-synonymous mutant gene MELO3C015426 of Leucine rich repeat (LRR) protein is a candidate gene regulating the leaf notch trait, and three SNP sites are screened for the gene. KASP primers are designed for the three screened SNP sites, and KASP primers are used to detect the genotypes of the target sites of normal leaf-shaped melons and leaf-shaped notch '145X1901'. The results show that the SNP molecular markers of the present invention successfully distinguish the parents and the F2 population, and leaf notch plants can also be successfully screened out in the F2 offspring of the hybrid population of '145X1901' and '1906', which fully demonstrates that the SNP molecular markers are suitable for screening leaf notch plants to improve the level of melon breeding.

[0015] As a further improvement, the genotype of the SNP molecular marker of the sample to be tested is detected, and individuals whose genotypes of SNP molecular markers 1 to 3 are GG, TT and GG are selected.

[0016] As a further improvement, competitive allele-specific PCR is used to detect the genotype of the SNP molecular marker of the sample to be tested.

[0017] Specifically, competitive allele-specific PCR (KSAP) is an effective means of SNP typing and detecting Indels by using the specific matching of primer terminal bases. This method does not require the synthesis of specific fluorescent primers for each SNP site. It is based on its own unique ARM PCR principle, allowing all site detections to be ultimately amplified using universal fluorescent primers to achieve genotyping detection, which has the advantages of being fast, accurate, and high-throughput.

[0018] As a further improvement, the nucleotide sequences of the detection primers of the competitive allele-specific PCR are shown in SEQ ID NOs. 2-10. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The leaf morphology of the melon plant with normal leaves and the plant with notched leaves on the 35th day of development in Example 1 of the present invention (wherein A is the leaf morphology of the plant with normal leaves, and B is the leaf morphology of the plant with notched leaves);

[0020] Figure 2 Statistics of SNPs and Indels and their distribution on chromosomes in Example 3 of the present invention (Venn diagram of visualization of SNPs in the genome and SNP differences between samples, wherein A is the distribution of SNPs on chromosomes, B is the visualization of Indels in the genome and the Venn diagram of Indel differences between samples, C is the distribution of Indels on chromosomes, D is the first circle in the figure is the chromosome coordinate, the second circle is the gene distribution, the third circle is the density distribution of SNPs and Indels, and the fourth circle is the ED value distribution of SNPs and Indels corresponding to the circle diagram from the outside to the inside);

[0021] Figure 3The three-dimensional principal component analysis of gene expression differences and DEGs enrichment analysis of leaves of normal-leaf type and notched-leaf type melons in Example 4 of the present invention (wherein A is a volcano plot of the two melon genotypes, B is a violin plot of the two melon genotypes, C is a distribution scatter plot of gene expression levels in the two melons, D is a GO analysis of DEGs in leaves of the two melon genotypes ("BP", "CC" and "MF" represent "biological process", "cellular component" and "molecular function", respectively), and E is a heat map of candidate DEGs expression pattern enrichment in the candidate region);

[0022] Figure 4 It is the initial genetic map of ΔSNP / Indel mapping and BSA-seq in Example 5 of the present invention (wherein, A is the preliminary positioning interval of the leaf notch site and the genetic distance of the linkage mapping, B is the fine positioning and recombination events of the leaf notch gene, C is the Venn diagram of BSA-seq and RNA-seq, D is the relative expression of 7 genes in '1310' and '145X1901', and E is the SNP analysis of the coding region of the non-synonymous mutant gene MELO3C015426 (* represents P<0.05, ** represents P<0.01, and ns represents no significant difference));

[0023] Figure 5 The results of the Blast analysis of the parental genome using the Integration Genomics Viewer software in Example 6 of the present invention (parents at SNP 1538515 、SNP 1538529 and SNP 1538611 base variation at the site);

[0024] Figure 6 The genotyping results determined by competitive allele-specific polymerase chain reaction in Example 6 of the present invention (the scatter plot of the selected KASP shows the clustering of varieties on the X-(HEX) and Y-(FAM) axes, the blue dots represent the '145X1901' homozygous strain, the red dots represent the '1310' homozygous strain, and the green dots represent the heterozygous F2 strain). DETAILED DESCRIPTION

[0025] Melon is an important economic crop of the Cucurbitaceae family, which is widely planted around the world and is well-known for its sweet taste, pleasant flavor and high nutritional value. It has been reported that increasing the planting density can increase the yield of melons, and the existing high-density planting system has also increased the planting density of melons. The increase in planting density means that the leaves of different melon plants are more shaded and the utilization rate of sunlight is reduced. At the same time, the heat transfer rate of lobed leaves is greater than that of non-lobed leaves, thereby reducing the temperature of the leaves and preventing the plants from being sunburned; therefore, breeding lobed leaf plants is of great significance to improving the yield and quality of melons. However, there are few reports on the research mechanism of melon leaf shape formation in the prior art, which brings difficulties to the effective use of leaf morphology advantages for breeding and quality improvement. Based on this, the present invention provides a SNP molecular marker associated with the melon leaf notch trait, the SNP molecular marker consisting of SNP molecular markers 1 to 3; the nucleotide sequence of the SNP molecular marker 1 is shown in SEQ ID NO.1, and the 6th base from the 5' end is A or G; the nucleotide sequence of the SNP molecular marker 2 is shown in SEQ ID NO.1, and the 20th base from the 5' end is C or T; the nucleotide sequence of the SNP molecular marker 3 is shown in SEQ ID NO.1, and the 102nd base from the 5' end is T or G.

[0026] The present invention obtained 263.89G bp of clean bases and 94.93% Q30 by sequencing a large sample of DNA from normal leaf shape and leaf notch parents and two F2 populations. The results show that the constructed sequencing library is accurate and high-quality, and can be used for the detection of candidate genomic regions and the detection of target phenotype-related genes. SNP and Indel association analysis showed that the 1.59Mb (genomic interval 1.05-2.64Mb) candidate region is associated with the leaf notch trait. The SNPs in the candidate region are further analyzed to select the SNP sites that are most likely to regulate the candidate genes for the leaf notch trait.

[0027] The present invention uses the Integrative Genomics Viewer software to visualize the non-synonymous mutation SNP sites in the candidate region of the parental genome, and finds that multiple SNP sites are mutated in the non-synonymous mutation gene MELO3C015426. Taking the candidate gene MELO3C015426 as a benchmark, the Integrative Genomics Viewer software is used to visualize the non-synonymous mutation SNP sites associated with the trait that are consistent with the trait change and have obvious differences between the parents in the candidate region of the parental genome, and three SNP sites related to the melon leaf shape notch trait are screened out with a sequencing depth of ≥20 as a standard. It is further verified that the three screened SNP sites are related to the melon leaf shape notch trait. The SNP molecular markers provided by the present invention can be used to improve the leaf shape screening in the breeding scheme, and help to understand the molecular mechanism of the melon leaf shape notch trait, providing a new research direction for the development of melon leaf shape.

[0028] 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.

[0029] The plant materials, shape evaluation and phenotypic analysis used in the examples of the present invention are as follows:

[0030] The present invention uses the leaf-normal inbred line 1310 (P1) and the leaf-notched inbred line 145X1901 (P2) and the constructed F2 segregation population containing 284 individual plants as experimental materials. Different from 1310, 145X1901 has a characteristic wrinkled (cup-shaped, curled) leaf phenotype, a shorter stature and many other agronomic traits different from the parent with normal leaves. The seeds of the two parents come from the experimental field of Luoyang Agricultural Development Technology Co., Ltd. in Mengjin District, Luoyang.

[0031] A specific embodiment of a SNP molecular marker associated with the melon leaf notch trait and its application in the present invention:

[0032] Example 1 Field trials and phenotypic analysis

[0033] In order to study the inheritance pattern of leaf notch in muskmelon, an F2 gene mapping population was established by crossing plants with normal leaf shape and plants with notch leaf shape. The male parent '1310' is a round-leaf muskmelon, and the female parent '145X1901' is a notch-leaf muskmelon.

[0034] In April 2023, in the experimental field of Luoyang Agricultural Development Technology Co., Ltd. in Luoyang, Henan, 284F2 individuals and two parent varieties were planted in rows with a spacing of 0.3m between plants and 0.5m between rows. Conventional field management measures were taken for the plants according to their growth conditions. During the vegetative growth stage, the leaf size of individual plants was measured and marked by a vernier caliper and scored according to their different leaf phenotypes. At harvest, traits including leaf shape, hardness, flower quality, and flesh color were evaluated.

[0035] On the 35th day of melon development, we observed the leaf shape of the third axillary bud of melon. The leaves of '1310' were round, and the leaves of '145X1901' were deep notched. All F1 plants had round leaves, and the F2 plants had 266 round plants and 18 notched plants. The separation ratio of normal plants to leaf plants was 15:1 (X 2 =0.004, P = 0.948, Figure 1 , Table 1), indicating that the leaf notch trait is controlled by a recessive gene.

[0036] Table 1 Segregation of leaf notch traits in parents, F1 and F2 populations of '1310' and '145X1901'

[0037]

[0038] Example 2 DNA extraction, quality testing and library construction

[0039] Total genomic DNA was extracted from fresh leaves of young plants using hexadecyl trimethylammonium bromide (CTAB). 18 DNA samples with normal leaf shape were mixed in equal amounts into YN pools, and 18 DNA samples with notched leaf shape were mixed in equal amounts into YD pools. The concentration and quality of total genomic DNA were determined using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). According to the manufacturer's specifications, a DNA library (350 bp) for Illumina / BGI sequencing was constructed for each accession. After the DNA library was constructed, sequencing was performed on the Illumina HiSeq XTen / NovaSeq / BGI platform by a commercial service company (Biomarker Technologies, Beijing, China), and the sequencing length was 150 bp. The reference genome ( / DHL92 / v4.0) can be downloaded from Index of / v2 / ftp / genome / melon / DHL92 / v4.0 (cucurbitgenomics.org). The quality of sequencing data was determined using FASTQC. All downstream analyses were based on high-quality clean data (Liao et al., 2020).

[0040] Whole genome resequencing was performed on a library consisting of 18 plants with normal leaf shape (YN) and 18 plants with notched leaf shape (YD) in the parents 1310 and 145X1901 and their F2 population. In order to evaluate the accuracy of the sequencing results, the raw data were screened to obtain high-quality clean data and compared with the reference genome. The results showed that the average efficiency was 95.92%, the average ratios of Q20 and Q30 were 97.35% and 94.93%, respectively, and the GC content of the clean data ranged from 36.23% to 36.48% (as shown in Table 2). More than 95.92% of the data were used for comparison with the reference genome. After comparison with the reference genome, the coverage of 1×, 5× and 10× were 96.87%, 94.09% and 82.09%, respectively, indicating that the comparison results were normal and could be used for mutation detection and correlation analysis.

[0041] Table 2 BSA-Seq sequencing data of parental lines, normal leaf shape and leaf notch pools, and comparison with the melon reference genome

[0042]

[0043] Example 3 BSA-Seq analysis

[0044] The data obtained in Example 2 were filtered to obtain high-quality readings to ensure that subsequent analysis was carried out smoothly. The data were located to the melon reference genome DHL92 V4 for comparison using Burrows-Wheeler Aligner (BWA) software. The calibration file was converted into a BAM file using SAMtools software. Subsequently, we used the GATK tool and SnpEff software for SNP calling and annotation. The variation of all samples was called using the unified genotype function in the GATK software. SNPs were called and Indels were filtered using the Variant Filtration parameter in GATK. In addition, we used SnpEff software for SNP annotation and determined the effects of small indexes (synonymous and non-synonymous mutations) in the genome. All homozygous SNPs / Indels between parents were extracted from the SNPs / indels VCF file. The reads depth information of the above homozygous SNPs / Indels in the offspring pool was obtained, and the SNP / Indel index was calculated. High-quality SNPs / and indels were called and filtered in two mixed pools and parental lines. The simulation uses the 99% quantile of Δ(SNP index) or Δ(Indel index) as the threshold for selecting hits. The difference in gene frequency between mixed pools is explored by calculating Δ(SNP-index). The results of SNP and Indel association regions obtained by the two association analysis methods are intersected, the genotype frequencies of the two allele pools are calculated, and the positioning region of the candidate gene is determined.

[0045] A total of 1,354,198 SNPs were obtained in the mixed pool, of which 23,067 SNPs caused non-synonymous mutations ( Figure 2 The Euclidean distance (ED) algorithm is a method for finding significant difference markers between mixed pools and evaluating regions related to objective traits. According to the relevant threshold, the region above the 99% threshold line (red line) was selected as the region related to the trait, and a total of 20 candidate regions were obtained with a total length of 4.12Mb ( Figure 2 A total of 347,739 small Indels were obtained between the mixed pools ( Figure 2 The same method was used to perform association analysis on Indels. Five significant correlation regions were detected on chromosome 2, with a total length of 3.40 Mb ( Figure 2 (shown in D).

[0046] The results of the association regions corresponding to SNPs and Indels were intersected, and two candidate regions related to the candidate genes of leaf notch traits with a total length of 1.59Mb on chromosome 2 were obtained. A total of 175 genes and 1754 markers (1237 SNPs and 517 Indels) were annotated in the candidate regions. Among the 1237 SNPs, 667 were located upstream, 365 were located downstream, and 205 were located in the intron region. Among the 517 indels, 287 were located upstream, 168 were located downstream, 58 were located in introns, 2 were located at splice sites, one was related to "frameshift mutation", and the other was related to "codon insertion". In addition, a total of 35 non-synonymous mutation genes and 3 frameshift mutation genes that may cause phenotypic variation were annotated between parents.

[0047] Example 4 RNA-seq data analysis

[0048] Three biological replicates were set for each of the normal leaf shape and leaf notch parent plants in Table 1, for a total of 6 samples, and samples were taken for transcriptome sequencing analysis. Total RNA was extracted using an RNA isolation kit (QIAGEN, Germany), and its quality was tested using an Agilent 2100 bioanalyzer. Ultra TM RNA Library PrepKit for RNA-seq was performed on the Illumina NovaSeq 6000 (Illumina, USA) platform. One million mapped reads per kilobase of transcript (FPKM) were used to calculate the expression level of the gene. Differentially expressed genes were screened by the criteria of log2 fold change (gene expression ratio between two samples) > 1 and p value (FDR < 0.005). Differentially expressed genes (DEGs) were annotated using the GO database. We selected 7 differentially expressed genes for RT-qPCR to verify the accuracy of the transcriptome analysis. Primer sequences were used Premix Ex Taq TM II kit (Tiangen Biotechnology Co., Ltd., China) was used to perform RT-qPCR reaction mixing on a Roche real-time PCR detection system (Lightcycler 480II, Switzerland). The melon actin gene was used as the internal reference gene and 2 -ΔΔCt Methods The relative gene expression was calculated.

[0049] To elucidate the molecular mechanism of the phenotypic differences between leaf notch type and normal type melon, two cDNA libraries and three biological replicates were sequenced. After filtering out any low-quality sequences and adapters, a clean data set of 39.20Gb with a Q30 ratio ≥ 94.45% was obtained, and the correction rate ranged from 94.45% to 95.17% to evaluate the reliability of the experimental results. PCA analysis found that PC1 and PC2 were 44.9% and 24.4%, respectively, accounting for 69.3% of the principal components ( Figure 3 A total of 3827 differentially expressed genes were found between '1310' and '145X1901', of which 1924 were upregulated and 1903 were downregulated ( Figure 3 As shown in B). The gene expression violin plot shows that the gene expression level distribution of the samples is generally dispersed, and the gene expression abundance of different samples is generally good ( Figure 3 (as shown in C).

[0050] In order to study the expression patterns of differentially expressed genes in normal-leaf melon and notched-leaf melon, the ClusterGVis software package was used to analyze the functional enrichment of differentially expressed genes in notched-leaf melon. GO enrichment analysis showed that the protein binding pathway (GO:0005515) plays an important role in cell growth and development and is the most enriched pathway ( Figure 3 In the candidate region, it was found that the expression level of MELO3C015426 encoding Leucine rich repeat (LRR) protein in this pathway was about twice that of '145X1901', which may play an important role in the leaf notch trait ( Figure 3(shown in E).

[0051] Example 5 Correlation analysis between BSA-seq and RNA-seq

[0052] To identify the key regulatory genes of melon leaf notch, we used BSA-seq technology to identify a 1.59 Mb candidate region ( Figure 4 AB in the figure), and screened 35 non-synonymous mutation genes and 3 frameshift mutation genes. Combining BSA-seq and RNA-seq analysis, 38 candidate genes related to leaf notch traits were identified in the candidate region ( Figure 4 (shown in C). Through further screening of SNP loci, high-quality SNP loci with different genotypes between the two parents and high measurement depth were selected, and 7 candidate genes were obtained. In the leaf notch plant, there were 3 up-regulated genes and 4 down-regulated genes, which were consistent with the RNA-seq sequencing results, proving the authenticity of RNA-seq ( Figure 4 D in the figure, the ordinate is the relative expression level). Combined with the analysis of BSA-seq and RNA-seq sequencing results, we believe that the non-synonymous mutant gene MELO3C015426 encoding Leucine rich repeat (LRR) protein is a candidate gene regulating the leaf notch trait. Based on the candidate gene MELO3C015426, the Integrative Genomics Viewer software was used to visualize the non-synonymous mutation SNP sites associated with the trait that were significantly different between the parents in the candidate genomic region, and three SNP sites related to the melon leaf notch trait were screened out using the sequencing depth ≥20. Figure 4 KASP detection was performed on the SNPs in the 1538511-1538611bp region of chromosome 2 ( Figure 5 ), and tested on the F2 population to determine which markers were most correlated with the leaf notch trait. Based on the candidate gene MELO3C015426, the Integrative Genomics Viewer software was used to visualize the non-synonymous mutation SNP sites in the candidate regions of the parental genome that were consistent with the trait changes and had obvious differences between the parents. The sequencing depth was ≥20 as the standard, and three KASP markers were developed for genotyping based on the base variations at 1538515bp, 1538529bp and 1538611bp on chromosome 2 of the melon leaf notch material '145X1901' and the normal leaf material '1310'. The specific information of the SNP molecular markers is listed in Table 3.

[0053] Table 3 Genotypes of three specific SNP molecular markers of melon leaf notch gene and their location information on chromosome 2

[0054]

[0055] Example 6 Molecular marker development and genotyping

[0056] The polymorphic SNPs identified as being associated with the leaf notch trait were converted into KASP markers using Poly Marker software. For the three SNPs obtained in Example 5, two allele-specific forward primers and a universal reverse primer based on the flanking sequence around the variable site (SNP) were designed using primer 5 software. The polymorphic SNP primers were converted into KASP markers, and their universality was verified by genotyping the parents, and then the KASP markers were verified in the entire F2 population. The designed KASP primer sequences are shown in the sequences in Table 4.

[0057] Table 4 KASP primer sequences

[0058]

[0059] PCR amplification was performed in a 10 μL reaction system, including 5 μL PCR Mix, 3.9 μL ddH2O, 1 μL DNA template and 0.1 μL primer premix (the concentrations of forward and reverse primers were 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).

[0060] PCR amplification program: 95℃ pre-denaturation for 10min; denaturation at 95℃ for 15s, annealing at 61-55℃ for 60s, reducing 0.6℃ for each cycle, 10 cycles in total; denaturation at 95℃ for 15s, annealing at 55℃ for 60s, 28-35 cycles; stored at 4℃. ABIQuantStudio3 real-time fluorescence quantitative PCR instrument was used for genotyping detection, 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 according to the fluorescence signal value, and the genotype of the clustered samples was analyzed.

[0061] The above three markers were used to detect the genotypes of the target sites of the melon '1310' with normal leaf shape and the '145X1901' with leaf notch. The results showed that the signal points of the melon '145X1901' with leaf notch were blue, aggregated near the y-axis, and the 5' end primer connected to the FAM fluorescent marker sequence was competitively amplified. The signal points of the melon '1310' with normal leaf shape were red, aggregated near the x-axis, and the primer connected to the HEX fluorescent marker sequence at the 5' end was competitively amplified. These three markers successfully distinguished parents and populations, indicating that these three KASP markers are suitable for screening plants with leaf notches to improve the level of melon breeding ( Figure 6 ). The three KASP markers were verified in 60 plants from another F2 population formed by the cross between '145X1901' and '1906'. The notch genotype was GTG, and its trait segregation was consistent with the actual survey trait segregation, further confirming its accuracy ( Figure 4 In Figure E, F2-15, F2-35, F2-104, F2-12, F2-60, and F2-74 show some of the test results).

[0062] 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 SNP molecular marker associated with the leaf notch trait of melon, characterized in that: The SNP molecular marker consists of SNP molecular markers 1 to 3; the nucleotide sequence of the SNP molecular marker 1 is shown in SEQ ID NO.1, and the 6th base from the 5' end is A or G; the nucleotide sequence of the SNP molecular marker 2 is shown in SEQ ID NO.1, and the 20th base from the 5' end is C or T; the nucleotide sequence of the SNP molecular marker 3 is shown in SEQ ID NO.1, and the 102nd base from the 5' end is T or G.

2. An application of the SNP molecular marker associated with the melon leaf notch trait as claimed in claim 1 in melon breeding, characterized in that: The application is to screen for melons with leaf notch strains.

3. The use of the SNP molecular marker associated with the melon leaf notch trait in melon breeding according to claim 2, characterized in that: The genotype of the SNP molecular markers of the sample to be tested is detected, and individuals whose genotypes of SNP molecular markers 1 to 3 are GG, TT and GG are selected.

4. The use of the SNP molecular marker associated with the melon leaf notch trait in melon breeding according to claim 3, characterized in that: Competitive allele-specific PCR was used to detect the genotype of the SNP molecular markers in the sample to be tested.

5. The use of the SNP molecular marker associated with the melon leaf notch trait in melon breeding according to claim 4, characterized in that: The nucleotide sequences of the competitive allele-specific PCR detection primers are shown in SEQ ID NOs. 2-10.

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