Screening method and application of watermelon InDel molecular marker

Abstract

The application belongs to the technical field of plant molecular biology and genetic breeding, and discloses a screening method and application of a watermelon InDel molecular marker. Cla97C01G006190.1 The application constructs a watermelon natural population and determines the root architecture traits of each sample, carries out whole genome resequencing and whole genome association analysis, and screens an InDel molecular marker located at 6,086,594 of the first chromosome of the watermelon reference genome G42 V2 edition, which is in a gene The marker polymorphism is an insertion and deletion variation between a non-insertion allele T and an insertion allele TGAATCA (GAATCA is inserted after T), and is significantly associated with multiple root system traits such as length, projected area, pixel area and branch number. The molecular marker is used for watermelon breeding, can be used to determine the root architecture characteristics of watermelon at the seedling stage, improves the germplasm identification and breeding efficiency, and is suitable for genetic identification and root system improvement breeding of watermelon.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and genetic breeding technology, and relates to a method and application for screening InDel molecular markers in watermelon. Background Technology

[0002] Watermelon is an important horticultural crop worldwide. Its root system architecture directly determines the plant's efficiency in absorbing water and mineral nutrients, thus regulating the plant's growth and development, stress resistance, and ultimately, yield and quality. Due to limitations in the root growth environment, observing and accurately measuring root phenotypes is quite challenging, causing the progress of genetic research on watermelon root traits to lag behind that of aboveground traits for a long time. Traditional root research methods mainly rely on digging and observing after soil cultivation, which is cumbersome, has poor reproducibility, and makes it difficult to achieve high-throughput phenotypic collection, significantly hindering the advancement of watermelon root genetic improvement.

[0003] Current research on the genetic basis of watermelon root traits remains limited. Existing reports mostly focus on the physiological response mechanisms of root morphology under water stress, or on preliminary QTL mapping studies based on a few linkage populations. There are no studies utilizing natural populations combined with whole-genome resequencing technology to systematically analyze the genetic makeup of watermelon root systems. Although whole-genome sequencing of watermelon has been completed, providing a data foundation for discovering root trait-related genes through genome-wide association studies, this approach has not yet been extensively and systematically applied in the field of genetic analysis of watermelon root traits.

[0004] Genome-wide association study (GWAS) has been widely used in the analysis of complex traits in various crops. By detecting the association between high-density molecular markers and target traits across the entire genome, it can efficiently locate candidate gene loci for quantitative traits. Compared with traditional linkage analysis, this technique relies on historical recombination events in natural populations, offering higher localization resolution and directly identifying loci and candidate genes significantly associated with traits. However, root traits are generally regulated by multiple genes, have low heritability, and are easily affected by environmental factors, posing a challenge to the statistical power of GWAS. Currently, there are no reports of successfully identifying key functional genes using this strategy in watermelon root architecture research. Overall, existing technologies lack efficient methods for mining genes related to watermelon root architecture, and have not identified key genes or molecular markers for root traits that can be directly applied to marker-assisted breeding, making it difficult to support the efficient implementation of watermelon root genetic improvement and molecular breeding work. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is that: genetic research on watermelon root traits has long lagged behind that on aboveground traits; traditional root phenotypic observation relies on digging and sampling after soil cultivation, which is cumbersome, has poor reproducibility, and makes it difficult to achieve high-throughput phenotypic collection; existing genetic analyses of watermelon roots mostly focus on physiological responses under stress or preliminary QTL mapping based on a few linked populations, lacking systematic genetic analysis using natural populations combined with whole-genome resequencing; and key molecular markers and functional genes for watermelon root architecture that can be directly applied to molecular marker-assisted breeding have not yet been identified, thus hindering the efficiency of advancing watermelon root genetic improvement.

[0006] To address the aforementioned technical problems, this invention screens and identifies a pleiotropic InDel molecular marker associated with watermelon root architecture, and provides its application in watermelon breeding. It also provides a method for identifying watermelon root architecture using the InDel molecular marker, as well as primer pairs for detecting the InDel molecular marker, aiming to achieve rapid and accurate detection of watermelon root architecture at the seedling stage, and to provide a reliable molecular tool for watermelon germplasm resource identification and root improvement breeding.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, this invention provides the application of products for detecting the InDel molecular marker in watermelon in watermelon breeding. The InDel molecular marker is significantly correlated with the root system architecture of watermelon. The InDel molecular marker is located at position 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2. The polymorphism of the InDel molecular marker is an insertion / deletion variation between T and TGAATCA. The T genotype corresponds to a phenotype of well-developed root system in watermelon plants, while the TGAATCA insertion genotype corresponds to a phenotype of weak root growth in watermelon plants. The root system configuration characteristics include: length, projected area, pixel area, and number of branches.

[0008] Furthermore, in the above applications, the InDel molecular marker is located at the candidate gene. Cla97C01G006190.1 The candidate gene is located at position 3019 in the coding region. Cla97C01G006190.1 Encodes α / β-hydrolase superfamily proteins.

[0009] Furthermore, in the above applications, individuals carrying the T genotype are screened in the watermelon breeding population to breed watermelon varieties with well-developed root systems.

[0010] Secondly, the present invention provides a method for screening InDel molecular markers related to root architecture traits of watermelon, comprising the following steps: (1) Construct a natural population containing multiple watermelon germplasms and measure the root system architecture of each sample; (2) Whole genome resequencing was performed on natural population samples, the sequencing sequences were aligned to the watermelon reference genome G42V2 version and variant detection was performed, and high-quality variant sites were obtained through quality control; (3) The population structure was corrected by principal component analysis. The principal components were used as covariates, and a linear regression model was used to perform genome-wide association analysis on each root system architecture trait to obtain the set of significant association sites corresponding to each trait. (4) Cross-compare the sets of significant associated sites for different root system architecture traits, count the number of traits associated with each site, and screen candidate sites that are associated with at least five root system architecture traits simultaneously. (5) Gene annotation is performed on the genomic regions 40-60kb upstream and downstream of the candidate sites, and candidate sites containing annotated genes are selected within the regions. (6) The InDel molecular marker located at position 6,086,594 on chromosome 1 of the G42 V2 version of the watermelon reference genome was obtained; the polymorphism of the InDel molecular marker is the insertion / deletion variation between T and TGAATCA.

[0011] Furthermore, in step (1) of the above screening method, complete root systems are collected at the three-leaf-one-heart stage of seedlings, and the root system morphological traits measured include: average diameter, number of branches, number of intersections, fractal dimension, length, number of connections, number of nodes, pixel area, projected area, number of root tips, surface area, and volume.

[0012] Furthermore, in step (2) of the above screening method, whole genome resequencing is performed using the Illumina sequencing platform, variant detection is performed using GATK software, and quality control is performed using PLINK software. The quality control filters sites with a minor allele frequency threshold of 0.1 and a genotype deletion rate threshold of 5%, and removes samples with a genotype deletion rate exceeding 10%.

[0013] Furthermore, in step (3) of the above screening method, the first five principal components are taken as covariates, only the additive genetic effect is retained, and the Bonferroni correction is used to determine the significance threshold of the association.

[0014] Thirdly, the present invention also provides a method for identifying the root system architecture of watermelon, the method comprising the following steps: (1) Extract genomic DNA from the watermelon sample to be tested; (2) Determine the genotype of the watermelon sample to be tested at position 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2 version; (3) Determine the root system architecture of the watermelon sample to be tested based on the genotype determination results. When the locus is T genotype, the watermelon sample to be tested exhibits a phenotype with well-developed roots. When the locus is TGAATCA insertion type genotype, the watermelon sample to be tested exhibits a phenotype with weak root growth.

[0015] Fourthly, the present invention also provides a primer pair for detecting InDel molecular markers related to watermelon root architecture, the primer pair comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO:2; The InDel molecular marker is located at position 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2 version, and its polymorphism is an insertion / deletion variant between T and TGAATCA.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention enables rapid and accurate identification of watermelon root system architecture at the seedling stage. Traditional root phenotypic identification requires manual root digging and observation after planting in soil, which is not only complex but also susceptible to soil environmental interference and can cause irreversible damage to the plant. This invention, by detecting the genotype of the InDel locus, can determine root system architecture characteristics at the three-leaf stage of seedlings, eliminating the need to wait for the plant to mature. This significantly shortens the root phenotypic identification cycle and reduces the difficulty and labor costs of the identification process. This marker was identified based on genome-wide association analysis of over two hundred natural watermelon populations and has been validated by typical materials with significant differences in root phenotypic differences. The correspondence between genotype and root phenotype is stable, and the identification results are accurate and reliable.

[0017] This InDel molecular marker exhibits pleiotropic effects, simultaneously reflecting differences in multiple root architecture traits. This locus is significantly associated with four root architecture traits: length, projected area, pixel area, and number of branches. Statistically significant differences were observed in all of these traits between the two genotype groups. In breeding applications, screening at only this single locus allows for simultaneous targeted selection of multiple root architecture traits, eliminating the need to develop and detect multiple molecular markers for different traits. This effectively reduces the workload of molecular testing and improves the efficiency of multi-trait aggregation breeding.

[0018] This invention identifies candidate genes that regulate watermelon root architecture. Cla97C01G006190.1This discovery supplements the genetic resources available for watermelon root development research. The gene encodes the α / β-hydrolase superfamily protein ClaABH1, and expression level verification showed that its expression level was significantly higher in watermelon materials with well-developed root systems. The identification of this gene fills a research gap in key regulatory genes of watermelon root architecture, providing a clear research target for elucidating the molecular regulatory mechanisms of watermelon root development, and offering important genetic resource support for subsequent gene function verification, gene editing breeding, and molecular design breeding.

[0019] This invention provides a well-defined and directly usable primer pair that effectively supports marker-assisted breeding of watermelons. Applying this marker to early screening of breeding populations allows for precise selection of individual plants carrying the target genotype at low generations, eliminating materials that do not meet breeding objectives, significantly reducing the scale of subsequent field planting and phenotypic identification, shortening the breeding selection cycle, and improving the accuracy of root trait selection. The primer pair allows for direct genotype determination via conventional PCR amplification combined with high-resolution gel electrophoresis, eliminating the need for sequencing, simplifying the operation, and demonstrating a verified detection accuracy of 93.36%. The results are stable and reproducible, making it practically valuable for cultivating watermelon varieties with well-developed root systems, high water and fertilizer absorption efficiency, and strong stress resistance. Attached Figure Description

[0020] Figure 1 Manhattan plots were used for genome-wide association analysis of 12 watermelon root traits. Subplots A to L represent mean diameter, number of branches, number of intersections, fractal dimension, length, number of connections, number of nodes, pixel area, projected area, number of root tips, surface area, and volume, respectively. The vertical axis represents the negative logarithm of the p-value for each point; the smaller the p-value, the higher the significance of the association, and the larger the corresponding vertical axis value. The horizontal axis represents the distribution of the loci on the chromosome. The horizontal line in the figure is the threshold line for the significance level of the association.

[0021] Figure 2 UpSet plot showing the overlap of significantly associated sites among different root traits.

[0022] Figure 3 A comparison of seedling phenotypes between watermelon material W48 with well-developed root systems and watermelon material W157 with weaker root systems.

[0023] Figure 4 This is a comparison of the expression profiles of eight genes located within a 50kb interval upstream and downstream of loci 6,086,594 on chromosome 1 in watermelon material W48 (with well-developed root systems) and watermelon material W157 (with weaker root systems). In the figure, ns indicates that the difference in gene expression levels between the two materials is not significant. This indicates a significant difference at the 99% confidence level. This indicates a significant difference at a confidence level of 99.9% or higher.

[0024] Figure 5 The candidate gene is located at position 6,086,594 on chromosome 1. Cla97C01G006190.1 The diagram above shows the location of the T genotype. In the diagram, 0 represents the non-inserted T genotype, 2 represents the presence of an insertion or deletion variant of the TGAATCA sequence at the T genotype, and n represents the number of samples corresponding to the genotype.

[0025] Figure 6 This is a phenotypic comparison of four root system architecture traits (length, projected area, pixel area, and number of branches) between two genotype groups; the p-values ​​in the figure were calculated using a t-test.

[0026] Figure 7 Candidate genes Cla97C01G006190.1 In Arabidopsis thaliana ( Arabidopsis thaliana AT), rice ( Oryza sativa Os) and corn ( Zea mays Figure 1 shows the BLAST alignment results of homologous sequences in the Zm genome.

[0027] Figure 8 This is a predicted protein-protein interaction network diagram for the corresponding homologous protein Q8RX69_ARATH in Arabidopsis thaliana. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified; and the index data are measured using conventional methods unless otherwise specified.

[0030] The test materials used in this embodiment of the invention consisted of 213 natural watermelon germplasm populations, provided by the Polyploid Watermelon Research Group of the Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences. This population covered various watermelon germplasm types, including Amaru watermelon, medicinal watermelon, sticky-seeded watermelon, cultivated varieties, local varieties, and seeded watermelons. The accession numbers, varietal names, and germplasm types of each material are detailed in Table 1.

[0031] Table 1: Experimental material numbers, variety names, and germplasm types of 213 natural watermelon populations

[0032] Example 1: Construction of natural watermelon populations and precise determination of root phenotypic traits.

[0033] This embodiment constructs a natural population by collecting various types of watermelon germplasm and establishes methods for seedling culture and root phenotypic determination to obtain accurate root system architecture data, providing a material basis for subsequent genome-wide association analysis and the discovery of root-related genetic loci.

[0034] 1. Construction of the test natural population The test materials used in this embodiment were 213 watermelon natural population germplasm accessions. The number, variety name and germplasm type of each material are detailed in Table 1.

[0035] 2. Standardized seedling cultivation Twenty-three germplasm materials from the aforementioned population were selected and cultured in a greenhouse environment in Xinxiang, Henan Province, using a modified Hoagland hydroponic system. Fifteen seedlings were planted in each hydroponic pot, and three biological replicates were set up for each germplasm. All tested materials were cultivated simultaneously under uniform experimental conditions to reduce interference from environmental variations and ensure the consistency and reliability of phenotypic data.

[0036] 3. Root phenotypic collection and measurement When the seedlings reached the three-leaf-one-heart stage, the complete root systems of each material were collected, rinsed with deionized water, and then scanned using the LA-S root imaging system from Wanshen Detection Technology. The root images were analyzed using the system's accompanying plant analysis software, and 12 root morphological traits were quantitatively detected: average diameter, number of branches, number of intersections, fractal dimension, length, number of connections, number of nodes, pixel area, projected area, number of root tips, surface area, and volume.

[0037] Example 2: Genome-wide association analysis of whole-genome resequencing and root traits.

[0038] This embodiment is based on the constructed watermelon natural population described above. High-density molecular markers were obtained through whole-genome resequencing, and whole-genome association analysis was carried out in combination with population structure correction to obtain the genomic regions that are significantly associated with root traits.

[0039] 1. Genomic DNA extraction and whole-genome resequencing Genomic DNA was extracted from young leaf tissues of each watermelon test material in Example 1. Whole-genome resequencing was performed on all samples using the Illumina sequencing platform. After quality control processing of the raw sequencing reads, the effective sequencing sequences were aligned to the watermelon reference genome G42 V2 version. Variation detection was performed using GATK v4.0 software to obtain an initial set of variant sites across the entire genome.

[0040] 2. Quality control of SNP marking PLINK v1.9 software was used to perform quality filtering on the initial variant sites. The filtering parameters were set as follows: sites with a minor allele frequency of less than 0.1 and sites with a genotype deletion rate greater than 5% were removed; samples with a genotype deletion rate of more than 10% were also excluded. After quality control, 677,950 high-quality variant sites were finally obtained for subsequent genome-wide association analysis.

[0041] 3. Population structure correction and genome-wide association analysis Principal component analysis was conducted based on the aforementioned high-quality variant sites, and the first five principal components, PC1 to PC5, were used as correction factors for population structure. A linear regression model in PLINK v1.9 software was used, with PC1 to PC5 as covariates, to perform independent genome-wide association analyses on the 12 root traits obtained in Example 1. Only additive genetic effects were retained in the analysis. The Bonferroni correction method was used to determine the significance threshold of the associations; the corrected significance threshold was 0.05 / 677,950 = 7.37 × 10⁻⁶. -8 Simultaneously, considering the polygenic inheritance characteristics and statistical power of root traits, a relatively lenient threshold is used to assist in identifying candidate sites.

[0042] The results of the correlation analysis in this step are as follows: Figure 1 As shown, Figure 1 Manhattan plots were generated for genome-wide association analysis of the 12 root traits. Subplots A to L correspond to mean diameter, number of branches, number of intersections, fractal dimension, length, number of connections, number of nodes, pixel area, projected area, number of root tips, surface area, and volume, respectively. The vertical axis in each subplot represents the negative logarithmic value of the p-value for each locus; a smaller p-value indicates a higher association significance, and a larger vertical axis value corresponds to a larger association significance. The horizontal axis represents the location of the locus on the chromosome. The horizontal line in the plot represents the association significance threshold. At a significance threshold of 4, a total of 280 significantly associated loci were obtained for the 12 root traits.

[0043] 4. Linkage disequilibrium analysis and candidate genome region partitioning For the significantly associated loci identified through screening, the linkage disequilibrium coefficient between the two loci was calculated using PLINK software, with the analysis window size set to 1000 kb. The connected component method from the R package igraph was then used to calculate the linkage disequilibrium coefficient r. 2 Sites with a linkage disequilibrium of 0.8 or higher are grouped into the same linkage disequilibrium block. The number of sites in each block is counted, and the corresponding candidate genomic intervals are determined based on the genomic range of the block for subsequent screening and identification of candidate genes.

[0044] Example 3: Screening and localization of InDel molecular markers for pleiotropic root configurations.

[0045] Based on the set of significant loci obtained from the aforementioned genome-wide association analysis, this embodiment screens candidate loci with pleiotropic effects through multi-trait association locus overlap analysis, further determines the location of the target InDel molecular marker by combining genome annotation information, and verifies the correspondence between its genotype and root phenotype.

[0046] 1. Overlap analysis of significantly associated loci for multiple traits Based on the genome-wide association analysis results of the 12 root traits obtained in Example 2, a set of significantly associated loci corresponding to each root trait was extracted. Then, the loci sets of different traits were cross-referenced one by one to count how many traits each locus repeated, i.e., to count the number of traits simultaneously associated with each locus. Candidate pleiotropic loci were obtained by using simultaneous association with at least five root traits as the screening criterion. The analysis results of this step are as follows: Figure 2 As shown, Figure 2 UpSet plots show the overlap of significantly associated loci among different root traits, visually illustrating the common distribution characteristics of each locus across various root traits. Statistical analysis identified three loci simultaneously associated with more than five root traits, suggesting that these loci may possess pleiotropic genetic effects regulating multiple root traits.

[0047] 2. Genomic annotation screening of candidate pleiotropic sites For the three pleiotropic candidate loci identified above, genomic regions within a 50kb range upstream and downstream of each locus were extracted. Combined with gene annotation information from the Watermelon Reference Genome G42 V2, statistical analysis was performed on the annotated genes within each region. Analysis revealed that no annotated functional genes were found within the corresponding genomic regions of two candidate loci. The candidate locus located at position 6,086,594 on chromosome 1 contained 10 annotated genes within its 50kb genomic regions upstream and downstream, demonstrating a clearly defined functional gene-carrying region. Therefore, this locus was identified as the target InDel molecular marker for this study.

[0048] 3. Confirmation of the association between the variant characteristics of the target InDel molecular marker and the phenotype Sequence variation analysis was performed on the target locus at position 6,086,594 of chromosome 1, confirming that the polymorphism at this locus is an insertion / deletion variation. Specifically, it manifests as a sequence difference between a non-inserted T base and an inserted TGAATCA sequence; one allele is T, and the other is TGAATCA, differing by 6 bases. Association validation using root phenotype data from natural populations revealed a significant correlation between different genotypes at this locus and watermelon root architecture: watermelon plants carrying the non-inserted T genotype generally exhibited a phenotype with well-developed root systems; watermelon plants carrying the TGAATCA inserted genotype generally exhibited a phenotype with weaker root growth. A comparison of seedling phenotypes between W48 (a representative material with well-developed root systems) and W157 (a representative material with weaker root growth) is shown below. Figure 3 As shown, by Figure 3 It is evident that there are significant differences in the overall size and morphology of the root systems between the two, which can intuitively reflect the differences in root system architecture corresponding to different genotypes at this locus.

[0049] Example 4: Candidate Genes ClaABH1 Identification and functional prediction analysis.

[0050] To elucidate the molecular basis of the target InDel site regulating watermelon root architecture, this embodiment uses gene expression verification of differentially expressed materials to identify candidate genes, clarify the specific location of the site on the gene, and conduct functional analysis through homology comparison and interaction network prediction to provide a basis for subsequent mechanism research.

[0051] 1. Validation and preliminary screening of gene expression levels in candidate regions Two watermelon samples with significant differences in root phenotype were selected: W48, with well-developed root systems, and W157, with weaker root growth. Total RNA was extracted from root tissues of both samples, and the expression levels of annotated genes within a 50kb interval upstream and downstream of loci 6,086,594 on chromosome 1 were detected. This genomic interval contains 10 annotated genes, of which 8 were expressed. The expression profiles are shown below. Figure 4 As shown, Figure 4 This is a comparison of the expression profiles of the above 8 genes within the target region in two tested materials, where ns indicates that the difference in gene expression levels between the two materials is not significant. This indicates a significant difference at the 99% confidence level. This indicates a significant difference at a confidence level of 99.9% or higher. Expression profile analysis revealed that... Cla97C01G006150.1 , Cla97C01G006180.1 , Cla97C01G006190.1 , Cla97C01G006220.1 and Cla97C01G006240.1 Five genes were consistently expressed at higher levels in the root-developed material W48, and these genes were preliminarily identified as important candidate genes involved in watermelon root development.

[0052] 2. Localization of sequence variations in target candidate genes Among the candidate genes obtained from the initial screening, for Cla97C01G006190.1 A detailed analysis of the gene's genomic sequence confirmed that the pleiotropic site identified in Example 3 was located at position 3019 in the gene's coding region. The variation at this site was an insertion / deletion variation, specifically a sequence difference between a non-inserted T base and an inserted TGAATCA sequence. The location of this site on the candidate gene is illustrated below. Figure 5 As shown, Figure 5 The gene is located at position 6,086,594 on chromosome 1. Cla97C01G006190.1 The above diagram shows the location of the T genotype at the locus, where 0 represents the non-inserted T genotype with 193 samples, 2 represents the TGAATCA sequence insertion variation at the locus with 17 samples, and n represents the number of samples corresponding to each genotype.

[0053] 3. Validation of the association between candidate gene genotypes and root traits Combining the genotypic grouping results and root phenotypic data of natural watermelon populations, the following was analyzed: Cla97C01G006190.1 The InDel locus on the gene was used to validate phenotypic associations. The test population was divided into two groups according to the genotype at this locus, and a t-test was used to compare the phenotypic differences in root traits between the two groups. The results are as follows: Figure 6 As shown, Figure 6 The figure shows the phenotypic differences in four root system architecture traits—length, projected area, pixel area, and number of branches—between the two genotype groups. The p-values ​​in the figure were calculated using a t-test. The p-values ​​for the differences between the four root system architecture traits were 0.0144, 0.0499, 0.0401, and 0.00585, respectively, all of which reached the statistical significance level, verifying the pleiotropic regulatory effect of this gene locus on watermelon root system architecture.

[0054] 4. Homology alignment of candidate genes and protein family annotation right Cla97C01G006190.1 Functional annotation and cross-species homologous sequence alignment of the gene's coding sequence revealed that it encodes an α / β-hydrolase superfamily protein. Members of this protein family, such as WAV2, KAI2, and D14, have been reported to participate in the regulation of plant root architecture and hormone signaling pathways, suggesting that this candidate gene has the functional potential to regulate watermelon root development. Based on this, it was named... ClaABH1 Homologous sequence BLAST alignments were performed in the genome databases of Arabidopsis thaliana, rice, and maize, respectively. The alignment results are as follows: Figure 7 As shown, Figure 7 Candidate genes Cla97C01G006190.1 In Arabidopsis thaliana ( Arabidopsis thaliana AT), rice ( Oryza sativa Os) and corn ( Zea mays The BLAST alignment results of the Zm genome show that the corresponding homologous genes were found in all three species, suggesting that the gene has conserved sequence characteristics and functional potential in plants.

[0055] 5. Prediction of protein-protein interaction networks of candidate genes Protein interaction network prediction analysis was performed based on the homologous protein Q8RX69_ARATH from Arabidopsis thaliana. The prediction results are as follows: Figure 8 As shown, Figure 8 This is a protein-protein interaction network diagram of the corresponding homologous protein Q8RX69_ARATH in Arabidopsis thaliana. Figure 8 visible, ClaABH1 Homologous proteins of this protein can interact with proteins involved in developmental regulation, such as bZIP transcription factors and TAF12-related proteins, suggesting that... ClaABH1 It may play an integrative role between transcriptional regulation and protein turnover or stability, and participate in the regulation of watermelon root development.

[0056] Example 5: A method for identifying watermelon root architecture based on InDel molecular markers.

[0057] Based on the pleiotropic InDel molecular markers obtained from the aforementioned identification, this embodiment establishes a screening method that can be used for rapid identification of watermelon root architecture. By detecting the genotype at the seedling stage, the root architecture characteristics of the plant can be predicted, providing a feasible technical means for watermelon germplasm resource identification and molecular breeding.

[0058] 1. Genomic DNA extraction from the sample to be tested Young leaf tissues from the watermelon plants to be tested were taken, and total genomic DNA was extracted from the samples using plant genomic DNA extraction methods. After passing purity and concentration tests, the samples were used for subsequent genotype testing.

[0059] 2. Genotyping of the target InDel locus Using the extracted genomic DNA as a template, whole-genome resequencing or targeted sequencing was employed to determine the genotype of the sample at locus 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2, clarifying whether the locus is a non-insertion T genotype or a TGAATCA insertion genotype.

[0060] 3. Determination of watermelon root system architecture characteristics The root system architecture characteristics of the tested samples are determined based on the genotype detection results: if the sample carries the non-inserted T genotype, it is determined that the sample has a well-developed root system phenotype, with root system architecture traits such as root length, projected area, pixel area, and number of branches all at a high level; if the sample carries the TGAATCA inserted genotype, it is determined that the root growth of the sample is weak. According to the needs of watermelon breeding, individuals with the T genotype can be screened, i.e., watermelon varieties with well-developed root systems can be bred to improve the plant's water and fertilizer absorption capacity.

[0061] Example 6: Design and PCR identification of specific primer pairs based on InDel molecular markers.

[0062] Based on the InDel molecular marker identified in Example 3, this embodiment provides a detection method for determining the root architecture genotype of watermelon by PCR amplification using specific primer pairs combined with high-resolution gel electrophoresis. This method does not require sequencing, is simple to operate, and is suitable for early screening of large batches of breeding materials.

[0063] 1. Primer pair design and acquisition 1) Design Methods and Parameter Control Based on the flanking sequences upstream and downstream of the InDel site, SnapGene 6.0.2 software was used to visualize the sequences and design candidate primers: a forward primer was designed starting approximately 30 bases before the mutation site, and a reverse primer was designed starting approximately 100 bases after the mutation site, ensuring the forward primer was upstream of the InDel site and the reverse primer was downstream, so that the amplification product completely contained the InDel region. Candidate primers were screened according to a unified standard: Primer lengths were 20–24 nt, melting temperatures (Tm) were close to 60°C, and GC content was controlled between 40% and 60%. The Tm values ​​of the forward and reverse primers differed by no more than 1°C, ensuring efficient amplification at the same annealing temperature. Simultaneously, the amplified product length was controlled to be moderate (approximately 153 bp), guaranteeing PCR amplification efficiency while allowing clear differentiation of 6 bp length differences on high-resolution gels, and excluding candidate primers with hairpin structures or self-dimer risks. The final primers were selected based on a comparison of the Tm value and GC content displayed by the software after selecting candidate sequences.

[0064] 2) Design Results Following the above design and screening, a pair of specific primers was finally obtained, named ClaABH1-F / ClaABH1-R: the nucleotide sequence of the forward primer ClaABH1-F is 5′-AGAAACTTATGACTTGTTGAAAGT-3′ (as shown in SEQ ID NO:1), with an annealing temperature of 60.5℃; the nucleotide sequence of the reverse primer ClaABH1-R is 5′-GAAATATCTTTGAGTGCGTCTT-3′ (as shown in SEQ ID NO:2), with an annealing temperature of 59.5℃. The GC content of both primers is 33.3%~40.9%. When using non-inserted T genotype material as a template, the amplification product length is 153bp, corresponding to a well-developed root phenotype; when using TGAATCA inserted material as a template, the amplification product length is 159bp (i.e., 153bp+6bp), corresponding to a weaker root growth phenotype; the two amplification products differ by 6bp and can be distinguished by high-resolution gel electrophoresis.

[0065] 2. Experimental Materials and Genomic DNA Extraction Experimental materials included: watermelon samples to be tested, primer pairs ClaABH1-F and ClaABH1-R (10 μM working concentration), and 2×Taq Master Mix (containing Taq DNA polymerase, dNTPs, and Mg). 2+ (and buffer), and a 50bp DNA ladder molecular weight marker for confirming product size.

[0066] Take about 100 mg of young leaves from the watermelon sample to be tested, and extract total genomic DNA using the CTAB method or a commercial plant genomic DNA extraction kit. The integrity of the extracted DNA is checked by 1% agarose gel electrophoresis, and the concentration and purity are checked by UV spectrophotometer. The A260 / A280 ratio is considered acceptable when it is between 1.8 and 2.0. Finally, the DNA concentration is diluted to 50-100 ng / μL for later use.

[0067] 3. PCR amplification The total volume of the PCR reaction system is 20 μL, specifically: 10 μL of 2×Taq Master Mix (final concentration 1×), 1 μL of 10 μM forward primer ClaABH1-F (final concentration 0.5 μM), 1 μL of 10 μM reverse primer ClaABH1-R (final concentration 0.5 μM), 1 μL of 50~100 ng / μL template DNA (approximately 50~100 ng), and ddH2O to bring the total volume to 20 μL.

[0068] The PCR reaction program is as follows: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 30 s, for a total of 35 cycles; then a final extension at 72℃ for 5 min; and finally storage at 4℃. To obtain the optimal annealing temperature, temperature gradient PCR optimization can be performed within the range of 55~62℃. Based on the primer Tm value (approximately 60℃), it is recommended to start screening at 58℃.

[0069] 4. Gel electrophoresis detection of PCR products The identification principle of PCR amplification combined with high-resolution gel electrophoresis is as follows: the InDel site differs in length by 6 bp between the two genotypes. After PCR amplification, the product lengths of different genotypes differ, and high-resolution gel electrophoresis can directly distinguish the genotypes based on the band migration position. A 3.5%–4% agarose gel is used, with 1×TBE as the electrophoresis buffer (its resolution is higher than TAE buffer). 8–10 μL of PCR product is loaded into each well, and electrophoresis is performed slowly at a low voltage of 80–100 V for 60–90 min. The gel is stained with GelRed or ethidium bromide (EB), and a 50 bp DNA ladder is used as a molecular weight control. On the 3.5%–4% agarose gel, the non-inserted band migrates slightly faster (approximately 153 bp), while the inserted band migrates slightly slower (approximately 159 bp), allowing them to be distinguished.

[0070] 5. Genotype determination Genotypes were determined based on the number and location of bands: A short band of approximately 153 bp indicated a non-insertion homozygous genotype (T / T), characterized by well-developed roots; a longer band of approximately 159 bp indicated an insertion homozygous genotype (TGAATCA / TGAATCA), characterized by weaker root growth; and the presence of both 153 bp and 159 bp bands indicated a heterozygous genotype (T / TGAATCA), with a root phenotype intermediate between the two. The correspondence between this genotype and the watermelon root architecture phenotype is consistent with Examples 3 and 5, and will not be repeated here.

[0071] 6. Result Validation and Quality Control To ensure accurate and reproducible identification results, a positive control was set up using known genotype materials W48 (non-inserted) and W157 (inserted) as controls; a negative control was set up using ddH2O instead of template DNA to detect system contamination; each sample was subjected to at least 2-3 independent PCR replicates; for samples with questionable electrophoresis results, the PCR products were directly subjected to Sanger sequencing to confirm the true genotype at position 6,086,594 of chromosome 1.

[0072] Example 7: Validation of InDel molecular marker and specific primer detection method.

[0073] This embodiment uses 213 natural watermelon populations constructed in Example 1 as the verification object to verify the accuracy and reliability of the specific primer pair ClaABH1-F / ClaABH1-R described in Example 6 for determining the genotype of the target InDel site by high-resolution gel electrophoresis, and to determine the root system architecture of watermelon accordingly.

[0074] 1. Verification materials The 213 watermelon natural population materials constructed in Example 1 and whose root system configuration traits have been measured were used as verification materials. Each material has root system configuration trait data such as total length, projected area, pixel area and number of branches obtained by Wanshen LA-S type root imaging system.

[0075] 2. Primer PCR-High-Resolution Gel Electrophoresis Genotyping Genomic DNA was extracted from 213 validation samples. Using these samples as templates, amplification was performed using the specific primer pairs ClaABH1-F / ClaABH1-R described in Example 6, along with the PCR reaction system and procedure. The amplified products were separated by high-resolution agarose gel electrophoresis at a mass fraction of 3.5%–4%. The genotype of each material at position 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2 was determined according to the genotyping criteria in Example 6: Genotypes with an amplified product length of approximately 153 bp and rapid band migration were identified as non-insertion T genotypes, and their root systems were predicted to be well-developed; genotypes with an amplified product length of approximately 159 bp and slow band migration were identified as TGAATCA insertion genotypes, and their root systems were predicted to be weak. Each material underwent three independent PCR replicates to ensure reproducibility of the genotyping results.

[0076] 3. Comparison of genotype determination results with actual root system architecture The genotypes determined by molecular markers and specific primers according to this invention, and the root architectures predicted by them, were compared one by one with the actual root architecture phenotypes obtained by root imaging systems for each material. The proportion of genotype detection results matching the actual root architectures was statistically analyzed. Root architecture phenotype determination criteria: length <180cm, projected area <6cm². 2 Pixel area < 5cm 2 A root system with fewer than 500 branches and meeting all the above conditions is considered to have an underdeveloped root system phenotype; conversely, a root system with a length ≥180cm and a projected area ≥6cm² is considered to have an underdeveloped root system phenotype. 2 Pixel area ≥ 5cm 2 Roots with ≥500 branches and meeting the above conditions are considered to have a well-developed root system phenotype. The results are shown in Table 2.

[0077] Table 2: Genotype and phenotype of 213 natural population materials

[0078] Table 2 shows that among the 213 natural population materials, 2 materials did not have a detectable genotype and were therefore not included in the valid samples. Statistical analysis of the remaining 211 samples revealed that the genotypes of 14 samples did not match the actual root phenotypes, while the genotypes of the remaining samples matched the phenotypes. The detection accuracy rate was (211-14) / 211=93.36%.

[0079] The InDel molecular marker combined with specific primers for PCR-high resolution gel electrophoresis detection of watermelon root architecture achieved an accuracy of 93.36%, meaning that the root architecture determined based on this molecular marker for the vast majority of materials was consistent with their actual root architecture.

[0080] The above results indicate that the InDel molecular markers of the present invention can stably and accurately indicate the root architecture of watermelon. The specific primer pairs and detection methods are reliable and reproducible, and can be used for early seedling screening of large quantities of materials in watermelon germplasm resource identification and molecular marker-assisted breeding.

[0081] The above embodiments can well illustrate the technical solution of the present invention, but they are only describing preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the present invention.

Claims

1. The application of products containing the InDel molecular marker from watermelon in watermelon breeding, characterized in that... The watermelon InDel molecular marker is significantly associated with root architecture traits in watermelons. The watermelon InDel molecular marker is located at position 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2. The polymorphism of the watermelon InDel molecular marker is an insertion / deletion variation between T and TGAATCA. Among them, the T genotype corresponds to the phenotype of well-developed root system in watermelon plants, while the TGAATCA insertion genotype corresponds to the phenotype of weak root growth in watermelon plants. The root system configuration characteristics include: length, projected area, pixel area, and number of branches.

2. The application according to claim 1, characterized in that, The InDel molecular marker is located in the candidate gene. Cla97C01G006190.1 The candidate gene is located at position 3019 in the coding region. Cla97C01G006190.1 Encodes α / β-hydrolase superfamily proteins.

3. The application according to claim 1, characterized in that, Individuals carrying the T genotype are selected from watermelon breeding populations to breed watermelon varieties with well-developed root systems.

4. A method for screening InDel molecular markers related to watermelon root architecture traits, characterized in that, Includes the following steps: (1) Construct a natural population containing multiple watermelon germplasms and measure the root system architecture of each sample; (2) Whole-genome resequencing was performed on natural population samples, the sequencing sequences were aligned to the watermelon reference genome G42 V2 version and variant detection was performed, and high-quality variant sites were obtained through quality control; (3) The population structure was corrected by principal component analysis. The principal components were used as covariates, and a linear regression model was used to perform genome-wide association analysis on each root system architecture trait to obtain the set of significant association sites corresponding to each trait. (4) Cross-compare the sets of significant associated sites for different root system architecture traits, count the number of traits associated with each site, and screen candidate sites that are associated with at least five root system architecture traits simultaneously. (5) Gene annotation is performed on the genomic regions 40-60kb upstream and downstream of the candidate sites, and candidate sites containing annotated genes are selected within the regions. (6) The InDel molecular marker located at position 6,086,594 on chromosome 1 of the G42 V2 version of the watermelon reference genome was obtained; the polymorphism of the InDel molecular marker is the insertion / deletion variation between T and TGAATCA.

5. The screening method according to claim 4, characterized in that, In step (1), complete root systems are collected at the three-leaf-one-heart stage of seedlings. The root system morphological characteristics measured include: average diameter, number of branches, number of intersections, fractal dimension, length, number of connections, number of nodes, pixel area, projected area, number of root tips, surface area, and volume.

6. The screening method according to claim 4, characterized in that, In step (2), whole genome resequencing was performed using the Illumina sequencing platform, variant detection was performed using GATK software, and quality control was performed using PLINK software. The quality control used a minor allele frequency threshold of 0.1 and a genotype deletion rate threshold of 5% to filter sites and remove samples with a genotype deletion rate of more than 10%.

7. The screening method according to claim 4, characterized in that, In step (3), the first five principal components are taken as covariates, only the additive genetic effect is retained, and the Bonferroni correction is used to determine the significance threshold of the association.

8. A method for identifying the root system architecture of watermelon, characterized in that, The method for identifying watermelon root system architecture includes the following steps: (1) Extract genomic DNA from the watermelon sample to be tested; (2) Determine the genotype of the watermelon sample to be tested at position 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2 version; (3) Determine the root system architecture of the watermelon sample to be tested based on the genotype determination results. When the locus is T genotype, the watermelon sample to be tested exhibits a phenotype with well-developed roots. When the locus is TGAATCA insertion type genotype, the watermelon sample to be tested exhibits a phenotype with weak root growth.

9. A primer pair for detecting InDel molecular markers related to watermelon root architecture, characterized in that, The primer pair includes a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:2; The InDel molecular marker is located at position 6,086,594 on chromosome 1 of the watermelon reference genome G42 V2 version, and its polymorphism is an insertion / deletion variant between T and TGAATCA.

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