KASP molecular marker and primer combination for identifying yellow and green leaf veins of watermelons and application of KASP molecular marker and primer combination

Abstract

The invention discloses a KASP molecular marker for identifying yellow and green leaf veins of watermelons, a primer combination and application of the KASP molecular marker and the primer combination, and belongs to the technical field of molecular markers. The KASP molecular marker provided by the invention comprises a K93281 molecular marker and / or a K93136 molecular marker; the polymorphism of the 71st basic group of the K93281 molecular marker is A / G, and the 71st basic group of the K93281 molecular marker is located at the 932281st position of the No.4 chromosome of the watermelon genome; the polymorphism of the 71th basic group of the K93136 molecular marker is G / A, and the 71th basic group of the K93136 molecular marker is located at the 93136th position of the No.4 chromosome of the watermelon genome. Experiments show that the KASP molecular marker is remarkably related to watermelon vein color characters and can be applied to auxiliary identification and breeding of yellow-vein watermelons.

Description

Technical Field

[0001] The present application belongs to the field of molecular marker technology, and in particular relates to a KASP molecular marker, a primer combination and an application thereof for identifying yellow-green veins of watermelon. Background Art

[0002] Watermelon (Citrullus lanatus), a globally cultivated and important commercial crop, is not only beloved by consumers for its sweet taste and rich nutritional value, but also plays a crucial role in agricultural production. With an estimated annual production of approximately 90 million tons, it ranks among the top ten most widely produced fruit crops worldwide.

[0003] Leaf veins are a key characteristic of watermelon plants. Color variation, such as yellow-green veins, has significant ornamental and breeding value, meeting market demand for diverse watermelon varieties. Furthermore, numerous studies have shown that yellowing of leaves may reduce photosynthesis, thereby affecting plant growth and development. Furthermore, given that yellow veins appear in the seedling stage and are easily discernible to the naked eye, current methods for identifying transgenic plants, such as fluorescence detection and gene sequencing, while accurate, lack intuitive visual identification. In contrast, using the key gene for yellow veins as a selectable marker provides a convenient and intuitive method for identifying transgenic plants, significantly improving the efficiency and operability of the identification process. Therefore, in-depth research on the genetic mechanisms underlying these two important traits in watermelon is crucial for improving yield and quality.

[0004] KASP is a high-throughput fluorescence-based SNP genotyping technology developed by LGC in the UK. KASP requires the design of two competitive allele-specific forward primers and a reverse primer based on the sequence preceding and following the target SNP locus. By adding different fluorescent groups to the 5' end of the forward primers, the genotype of the target locus is determined based on the fluorescence signal at the end of the PCR reaction. KASP labeling is a high-throughput genotyping technology that is flexible, inexpensive, and accurate, enabling clear identification of the genotype of samples at the target locus. It has been applied in areas such as gene mapping and population genetics research.

[0005] Leaf veins are a key characteristic of watermelon plants, but the causes of yellowing leaf veins in watermelons remain largely unknown, and candidate genes controlling yellowing have yet to be reported. Watermelon possesses a rich variety of stem and leaf traits, which are closely related to plant vigor, nutrient supply and demand, and yield. Therefore, identifying SNPs associated with leaf vein color in watermelon and developing KASP markers for their identification are of great significance, providing an effective detection method for breeding yellow-veined watermelon varieties and screening yellow-veined watermelon germplasm resources. Summary of the Invention

[0006] In order to overcome the above-mentioned defects in the prior art, the present application provides a KASP molecular marker, primer combination and application thereof for identifying the yellow-green veins of watermelon. The KASP molecular marker is significantly correlated with the yellow-green veins of watermelon and can be used to identify the yellow-green vein trait of watermelon.

[0007] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0008] In one aspect, the present application provides a KASP molecular marker for identifying yellow-green veins of watermelon, wherein the KASP molecular marker includes the K93281 molecular marker and / or the K93136 molecular marker;

[0009] The polymorphism of the 71st base of the K93281 molecular marker is A / G, and the 71st base of the K93281 molecular marker is located at position 93281 of chromosome 4 of the watermelon genome;

[0010] The polymorphism of the 71st base of the K93136 molecular marker is G / A, and the 71st base of the K93136 molecular marker is located at position 93136 of chromosome 4 of the watermelon genome;

[0011] The version of the watermelon genome is Watermelon (97103) v2.5 Genome.

[0012] Optionally, the nucleotide sequence of the K93281 molecular marker is shown in SEQ ID NO.1;

[0013] The nucleotide sequence of the K93136 molecular marker is shown in SEQ ID NO.2.

[0014] In a second aspect, the present application provides the application of the above-mentioned KASP molecular marker in watermelon molecular marker-assisted breeding.

[0015] In a third aspect, the present application provides a primer combination for detecting the above-mentioned KASP molecular marker;

[0016] The primer combination for detecting the K93281 molecular marker includes two specific forward primers K1-Fam and K1-Hex, and a universal reverse primer K1-R. The nucleotide sequence of K1-Fam is shown in SEQ ID NO.3, the nucleotide sequence of K1-Hex is shown in SEQ ID NO.4, and the nucleotide sequence of K1-R is shown in SEQ ID NO.5.

[0017] The primer combination for detecting the K93136 molecular marker comprises two specific forward primers K2-Fam and K2-Hex, and a universal reverse primer K2-R, the nucleotide sequence of K2-Fam is shown as SEQ ID NO. 6, the nucleotide sequence of K2-Hex is shown as SEQ ID NO. 7, and the nucleotide sequence of K2-R is shown as SEQ ID NO. 8.

[0018] Optionally, the 5' end of K1-Fam and K2-Fam is labeled with a FAM fluorescent tag sequence.

[0019] The 5' end of K1-Hex and K2-Hex is labeled with a HEX fluorescent tag sequence.

[0020] In a fourth aspect, the present application provides an application of the above-mentioned primer combination in preparing a detection reagent or a kit for identifying watermelon yellow-green leaf veins.

[0021] In a fifth aspect, the present application provides a method for identifying watermelon yellow-green leaf veins, comprising the following steps:

[0022] (1) extracting genomic DNA of the watermelon leaves to be tested;

[0023] (2) using the above-mentioned primer combination to perform PCR amplification with the genomic DNA as a template to obtain a PCR product;

[0024] (3) determining the genotype of the PCR product after fluorescence detection, and determining the vein color according to the genotype:

[0025] For K93281: genotype AA or AG is determined as yellow vein, and genotype GG is determined as green vein;

[0026] For K93136: genotype GG or GA is determined as yellow vein, and genotype AA is determined as green vein.

[0027] Optionally, in step (2), the PCR amplification program is as follows: 95℃ pre-denaturation for 10 min; denaturation at 95℃ for 15 s, annealing at 61-55℃ for 60 s, reducing 0.6℃ for each cycle, a total of 10 cycles; denaturation at 95℃ for 15 s, annealing at 55℃ for 60 s, 28-35 cycles.

[0028] In a sixth aspect, the present application provides a watermelon yellow-green leaf vein related gene, comprising Cla97C04G068470 and / or Cla97C04G068530;

[0029] The CDS sequence of Cla97C04G068470 is shown as SEQ ID NO. 9;

[0030] The CDS sequence of Cla97C04G068530 is shown in SEQ ID NO.10.

[0031] In a seventh aspect, the present application provides the use of the above-mentioned watermelon yellow-green vein-related gene in regulating the color of watermelon veins;

[0032] The application includes overexpressing the Cla97C04G068470 gene in watermelon, and / or silencing the Cla97C04G068530 gene, and / or knocking out the Cla97C04G068530 gene to cultivate yellow-veined watermelon varieties.

[0033] Compared with the prior art, this application has the following beneficial effects:

[0034] (1) This application developed a KASP molecular marker related to yellow-green leaf veins, and repeated the test using multiple watermelon materials, confirming that the KASP molecular marker can be effectively used to identify or assist in the identification of yellow-green leaf veins in watermelon.

[0035] (2) The method for identifying the yellow-green vein trait of watermelon established in the present application directly uses watermelon genomic DNA as a template. The SNP site genotype of the KASP molecular marker can be detected in the leaf tissue of watermelon, which is conducive to convenient and efficient prediction of the yellow-green veins of watermelon and can be applied to the screening and identification of the yellow-green veins of watermelon before harvest.

[0036] (3) This application confirmed that the Cla97C04G068470 and Cla97C04G068530 genes play an important regulatory role in the formation of yellow leaf veins by constructing an overexpression vector of the Cla97C04G068470 gene, a transient silencing vector of the Cla97C04G068530 gene, and a CRISPR / Cas9 dual-target knockout vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 This is the phenotypic picture of the parents of this application;

[0039] Figure 2Statistical data of SNPs and InDels among samples of this application and their chromosomal distribution (Note: A represents SNP visualization of the entire genome; B represents InDel visualization of the entire genome; C represents a Venn diagram of SNP differences among samples; D represents a Venn diagram of InDel differences among samples; E represents ED distribution on chromosomes; F represents ED distribution on chromosomes; in addition, the circle diagrams of A and B are from inside to outside: the first circle represents the distribution of ED values ​​corresponding to SNPs and InDels, the second circle represents the density distribution of SNPs and InDels, the third circle represents the distribution of genes, and the fourth circle represents the chromosome coordinates);

[0040] Figure 3 This is the physical map of the yellow-green vein gene in watermelon of this application (Note: A represents the preliminary genetic map obtained from BSA-seq; B represents the mapped region of the yellow-green vein trait locus and the linkage map with genetic distance; C represents the candidate genes and annotations in the mapped interval);

[0041] Figure 4 For this application, the Integrative Genomics Viewer (IGV) was used to perform comparative visual analysis of the parental genomes;

[0042] Figure 5 This is the genotyping result of K93281 in this application;

[0043] Figure 6 This is a statistical analysis chart of the K93281 genotype and leaf vein color phenotype of this application;

[0044] Figure 7 This is the genotyping result of K93136 in this application;

[0045] Figure 8 This is a statistical analysis chart of the K93136 genotype and leaf vein color phenotype of this application;

[0046] Figure 9 This is the overexpression effect of the Cla97C04G068470 gene induced in watermelon leaves of the present application (Note: A represents the PCAMBIA2300-empty phenotype in watermelon leaves; B represents the Cla97C04G068470 gene overexpression phenotype in watermelon leaves; the yellow and green arrows in the figure indicate the injection positions; C represents the expression level of the Cla97C04G068470 gene in PCAMBIA2300-empty and PCAMBIA2300-470 plants measured by real-time RT-PCR from 1 to 5 days. The Cla97C02G026960 gene was used as an internal control);

[0047] Figure 10This is the silencing effect induced by the Cla97C04G068530 gene in watermelon leaves of this application (Note: A represents the TRV-empty phenotype in watermelon leaves; B represents the Cla97C04G068530 gene silencing phenotype in watermelon leaves; the yellow and green arrows in the figure indicate the injection positions; C represents the expression level of the Cla97C04G068530 gene in TRV-empty and TRV-530 plants measured by real-time RT-PCR for 1 to 3 days. The Cla97C02G026960 gene was used as an internal control; D represents the Cla97C04G068530 gene editing knockout mutant plant). DETAILED DESCRIPTION

[0048] The present application will be further described below in conjunction with specific embodiments. The following description is merely a few embodiments of the present application and does not limit the present application in any form. Although the present application discloses the preferred embodiments below, it is not intended to limit the present application. Any person skilled in the art who, without departing from the scope of the technical solution of the present application, makes slight changes or modifications using the above disclosed technical content is equivalent to an equivalent implementation case and falls within the scope of the technical solution.

[0049] Unless otherwise specified, the raw materials in the examples of this application were purchased from commercial channels and used directly without any special treatment.

[0050] Unless otherwise specified, the analytical methods in the examples all adopt conventional settings and conventional analytical methods of instruments or equipment.

[0051] The watermelon reference genome version used in this application is Watermelon (97103) v2.5 Genome.

[0052] Example 1

[0053] Screening of SNPs associated with yellow-green vein traits in watermelon and design of KASP marker primers

[0054] 1. Genomic DNA Extraction Method

[0055] Refer to the Novozymes DNA genomic extraction kit method, the specific steps are as follows:

[0056] 1. Take 2g of fresh watermelon leaves collected, put them into a mortar and add liquid nitrogen to grind them into powder. Transfer the powder to a 1.5ml centrifuge tube, add 400μl buffer A1 and 4μl RNaseA (10mg / ml), vortex and oscillate to mix thoroughly to facilitate lysis.

[0057] 2. Place in a 65°C water bath for 10 minutes. During the water bath, invert the sample every 3 minutes to mix the sample.

[0058] 3. Add 130 μl of buffer A2 to the mixture, mix well and put on ice for 5 min, centrifuge at 1400 rpm for 10 min, and transfer the supernatant to a new 1.5 ml centrifuge tube.

[0059] 4. According to the volume of the supernatant, add 1.5 times the volume of buffer A3, and mix immediately.

[0060] 5. Transfer the mixture obtained in the previous step to FastPure DNA Columns (the adsorption column has been placed in a collection tube), centrifuge at 12000 rpm for 60 s, and discard the filtrate.

[0061] 6. Add 600 μl of buffer AW, centrifuge at 12000 rpm for 30 s, and discard the filtrate.

[0062] 7. Repeat step 6.

[0063] 8. Place the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 min, and remove the rinse solution.

[0064] 9. Place the adsorption column in a new 1.5 ml centrifuge tube, add 50 μl of Elution Buffer preheated to 65°C to the center of the membrane of the adsorption column, and let stand at room temperature for 5 min, and centrifuge at 12000 rpm for 1 min.

[0065] 10. Discard the adsorption column, and store the DNA at -20°C.

[0066] II. Screening of SNP sites related to yellow-green leaf veins of watermelon

[0067] 1. Selection of test materials:

[0068] The test materials include the maternal parent, the paternal parent, and the F2 population;

[0069] The paternal parent material is: yellow skin and green leaf veins;

[0070] The maternal parent material is: small yellow skin and yellow leaf veins;

[0071] The phenotypes of the parents are as shown in Table 1. Figure 1

[0072] The above maternal and paternal parents are from Luoyang Nongfa Agricultural Biotechnology Co., Ltd., and the parent and offspring plants are planted in the farm of the company.

[0073] The F1 generation obtained by crossing the above two materials as the parents, the F2 population obtained by selfing the F1 generation, and the F2 population used in 2024 harvest is 400 plants.

[0074] ​2. Determination of Leaf Vein Color of Test Materials: Strict self-pollination was performed using standard field management. Each plant was hand-pollinated and numbered. Leaf vein color was recorded when the plant was approximately 80 days old.

[0075] 3. BSA Sequencing: Twenty plants with yellow and green veins were selected from the F2 generation. DNA was extracted from each plant, and the concentration and quality of genomic DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). DNA concentrations were adjusted to a uniform concentration and then mixed in equal amounts to create pools. DNA samples were resequenced using the Illumina HiSeq XTen / NovaSeq / BGI platform (Beijing, China) with a 150-bp read length and at least 30× genomic coverage for each sample. Low-quality data were filtered using BcltoFastq 1.8.4 software. Resequencing data from the two extreme pools were realigned to the reference watermelon genome 97103 version 2.5 (http: / / cucurbitgenomics.org / v2) using BWA software. The Mark Dupl icate tool in the Picard online software package (http: / / sourceforge.net / projects / picard / ) was used to remove duplicates, and preprocessing such as local realignment and base quality correction was performed using GATK software to ensure the accuracy of the detected sites. SNP and Indel variants were detected between samples, and association analysis was performed on the differential sites between samples based on alignment information with the watermelon reference genome. The original ED5 was selected as the association value to eliminate background noise. The ED value was fitted using the DISTANCE tool, and the median + 3SD of the fitted values ​​for all sites was used as the association threshold for the analysis. The same method was used to fit the ΔSNP-index and ΔIndel-index, and the values ​​above the threshold were selected as the target regions associated with the yellow-green vein trait of watermelon.

[0076] 4. Polymorphic SNPs associated with the yellow-green vein trait were converted into KASP markers using PolyMarker software. Two allele-specific forward primers and a universal reverse primer based on the flanking sequence surrounding the variant site (SNP) were designed using Primer 5 software. Specific primer sequences are shown in Table 1.

[0077] Table 1 KASP marker primer sequences

[0078]

[0079]

[0080] Primer design for detecting KASP molecular markers K93281 and K93136:

[0081] Taking K93281 as an example, the 5' end of primer K1-Fam carries a fluorescent tag sequence for FAM. K1-Fam and K1-R can amplify the fragment with a G at the K93281 site, thereby generating a FAM fluorescent signal. The 5' end of primer K1-Hex carries a fluorescent tag sequence for HEX. K1-Hex and K1-R can amplify the fragment with an A at the K93281 site, thereby generating a HEX fluorescent signal.

[0082] 5. Method for detecting K93281 and K93136 genotypes using KASP molecular markers

[0083] (1) PCR reaction system

[0084] The universality of the marker was verified by genotyping both parents, and then the KASP marker was validated in the F2 population. PCR amplification was performed in a 10 μL reaction system consisting of 5 μL PCR Mix, 3.9 μL ddH2O, 1 μL DNA template, and 0.1 μL primer premix (adjust the forward and reverse primer concentrations to 100 μmol / μL; prepare 50 μL of primer premix using 10 μL forward primer 1, 10 μL forward primer 2, and 30 μL reverse primer).

[0085] (2) PCR amplification procedure

[0086] Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 61-55°C for 60 s, decreasing the temperature by 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 33 cycles; store at 4°C.

[0087] (3) Fluorescence signal reading

[0088] Genotyping was performed using an ABI QuantStudio 3 real-time fluorescence quantitative PCR instrument with a plate reading schedule of 1 min at 30°C. The Allele Discrimination function was used for analysis. The x-axis represents the HEX fluorescence signal, while the y-axis represents the FAM fluorescence signal. Clustering was performed based on the fluorescence signal values, and the genotypes of the clustered samples were analyzed.

[0089] 6. First, an overexpression vector of the Cla97C04G068470 gene (CDS sequence is shown in SEQ ID NO. 9) was constructed and injected into watermelon leaves. The expression level of the Cla97C04G068470 gene in the transformed watermelon leaves was detected by real-time fluorescence quantitative PCR, and the role of the gene was analyzed. The specific operations are as follows:

[0090] (1) Construction of overexpression vector and transient transformation

[0091] The CDS of Cla97C04G068470 was subcloned into the PCAMBIA2300 vector by BamHI and Sail I digestion, and PCR amplification was performed using primers with homologous arms. The primers used are shown in Table 2, and the constructed vector was named PCAMBIA2300-470. The vector was then transformed into DH5a E. coli, and positive clones were screened by PCR and sequencing. The bacterial solution was stored at -80°C. The recombinant plasmid of the positive strain was then extracted, and PCAMBIA2300-470 and PCAMBIA2300-null were introduced into Agrobacterium tumefaciens GV3101 strain. The bacteria were expanded in LB liquid medium containing kanamycin, and when the OD600 of the bacterial solution was 0.6-0.8, the bacteria were injected into watermelon leaves. The bacteria containing PCAMBIA2300-null were injected into watermelon leaves with four leaves and a heart-shaped center to obtain the control group, and the bacteria containing PCAMBIA2300-470 were injected into watermelon leaves to obtain the test group. The RNA of the control group and the test group was extracted, and qPCR was performed to detect whether Cla97C04G068470 was effectively overexpressed (470RT-F and 470RT-R).

[0092] Table 2. Fluorescent quantitative primer sequences

[0093] Primer name sequence 470RT-F GGTCATTGGCTGAGGTGT(SEQ ID NO.11) 470RT-R CCAACGCTTTAGAGGTAG(SEQ ID NO.12) 530RT-F530 AGGACCATCCAAGGGCTAT(SEQ ID NO.13) 530RT-R AATTGATTGCCCTGGTGC(SEQ ID NO.14)

[0094] Next, a transient silencing vector and a CRISPR / Cas9 double-target knockout vector of the Cla97C04G068530 gene (CDS sequence shown as SEQ ID NO. 10) were constructed and injected into watermelon leaves. Real-time fluorescent quantitative PCR was used to detect the expression of the Cla97C04G068530 gene in the transformed watermelon leaves, and the function of the gene was analyzed. The specific operation is as follows:

[0095] (2) Construction of silencing vector and transient transformation

[0096] Specific primers were designed to avoid conserved sequences within the Cla97C04G068530 gene. The target fragment was amplified using watermelon leaf cDNA as a template. The recombinant plasmid pTRV2-530 was then digested with BamHI. GV3101 cells were transformed with TRV2-530, TRV2 empty vector, and TRV1 empty vector to obtain positive single clones. The clones were propagated in LB liquid medium containing kanamycin (Kan) and injected into watermelon leaves when the OD600 value reached 0.6-0.8. The vectors used for virus-induced gene silencing (VIGS) testing consisted of TRV1 and TRV2. TRV1 served as a helper plasmid. TRV2 contained multiple cloning sites for inserting foreign genes. Equal volumes of Agrobacterium containing TRV2 and TRV1 were mixed and injected into watermelon leaves at approximately the fourth leaf and first heart stage of the growth cycle to provide a control. Agrobacterium containing the TRV2 vector carrying the target gene was mixed with TRV1 and injected into watermelon leaves to form a test group. RNA was extracted from leaves of the control group and the experimental group, and qPCR assay was performed to detect whether Cla97C04G068530 was effectively silenced (530RT-F and 530RT-R).

[0097] (3) Construction of CRISPR / Cas9 dual-target knockout vector

[0098] Vector construction was performed according to the method of Peng et al. (2023). The pCBC-DT1T2 intermediate vector was used as a template for amplification. The PCR product was recovered and purified, and the pBSE402 gene editing vector was digested and ligated. 10 μL of the product was transformed into competent Escherichia coli DH5α. Positive colonies were screened with kanamycin (Kan) and sent for sequencing. Colonies with the correct sequence were selected and transformed into Agrobacterium tumefaciens EHA105. Correct Agrobacterium colonies were selected for Agrobacterium genetic transformation.

[0099] (4) Real-time fluorescence quantitative PCR detection

[0100] Fluorescent quantitative primers for the Cla97C04G068470 and Cla97C04G068530 genes (Table 2) were designed using Primer Blast from the NCBI website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Detection was performed using the Quantagene Q225 fluorescent quantitative PCR instrument from Beijing Coolbo Technology using the Cham QTM Universal SYBR qPCR Master Mix fluorescent dye from Novozymes. The qPCR procedure was as follows: a 10 μL reaction mixture consisting of 5 μL 2× SYBR Green PCR Master Mix, 0.3 μL primers, and 200 ng cDNA was amplified at 95°C for 5 minutes, followed by 30 cycles of 95°C for 10 seconds, 58°C for 30 seconds, and 95°C for 15 seconds. Three replicates were performed for each sample. In all experiments, Cla97C02G026960 (CDS sequence shown in SEQ ID NO. 15) was used as an internal reference gene, and the expression levels of the tested genes were normalized according to the 2(-ΔΔCT) algorithm.

[0101] 7. The SNP and InDel association region results obtained by the two association analysis methods were intersected to determine the preliminary positioning region of 1.6Mb (0-1090000bp, 2710000-2790000bp and 2970000-3400000bp) ( Figure 2 ). The parental sequencing results were visualized using Integrative Genomics Viewer software ( Figure 3 ), six KASP markers were designed in this region, and the candidate region was further located to K93281-K298046. Through gene annotation, it was noted that Cla97C04G068530 was annotated as magnesium chelatase ( Figure 3 ), one of the key enzymes in chlorophyll synthesis, is speculated to be a candidate gene that may be involved in regulating the yellow-green veins of watermelon leaves. In addition, using a sequencing depth of ≥20 as the standard, two high-quality SNP sites associated with the yellow-green veins of watermelon leaves were screened. Both sites are located on the watermelon yellow-green vein-related gene Cla97C04G068470, and are physically anchored at 93281bp and 93136bp on chromosome 4 of watermelon ( Figure 4 ), Cla97C04G068470 was hypothesized to be a possible candidate gene. The identified trait-associated polymorphic SNPs were converted into KASP molecular markers, designated K93281 (nucleotide sequence shown in SEQ ID NO. 1) and K93136 (nucleotide sequence shown in SEQ ID NO. 2), and applied to molecular-assisted selection breeding.

[0102] 8. Application of K93281 molecular marker in watermelon yellow-green leaf vein assisted breeding

[0103] The fluorescence data is displayed in a graphical result, the fluorescence signal with FAM label is near the Y axis, indicating that the K93281 genotype of the watermelon material to be tested is GG. The fluorescence signal with HEX label is near the X axis, indicating that the K93281 genotype of the watermelon material to be tested is AA. The fluorescence signal is distributed in the middle position of the coordinate axis, then the K93281 genotype of the watermelon material to be tested is AG. Figure 5

[0104] Statistical analysis of K93281 genotyping results and leaf vein color phenotype results found that genotype AA showed yellow leaf veins, genotype AG showed yellow leaf veins, and genotype GG showed green leaf veins (Table 3, Figure 6

[0105] Table 3 Genotype and leaf vein color statistics of 346 watermelon germplasm

[0106]

[0107] 9. Application of K93136 molecular marker in watermelon yellow-green leaf vein assisted breeding

[0108] In order to further verify the reliability of the K93281 molecular marker, another K93136 molecular marker is used to verify in 46 F2 populations. Figure 7 ) The KASP molecular marker is used to detect the genotype of K93136 site by the same method as above, and the results show that in the F2 population, the AA genotype leaf vein shows green, and the heterozygous genotype GA and the homozygous genotype GG show yellow leaf veins (Table 4, Figure 8 ) The same genotyping results as the K93281 molecular marker are observed on the SNP93136 mutation site. Therefore, the two KASP markers can be combined for practical work of watermelon yellow-green leaf vein molecular marker assisted selection breeding.

[0109] Table 4 Genotype and leaf vein color statistics of 46 watermelon germplasm

[0110]

[0111] 10. Regulatory role of Cla97C04G068470 and Cla97C04G068530 genes in the formation of watermelon yellow leaf veins

[0112] ​​To further explore the role of the two genes in regulating watermelon vein color, this application constructed an overexpression vector (PCAMBIA2300-470) and a silencing vector (TRV2-530) to overexpress the Cla97C04G068470 gene and silence the Cla97C04G068530 gene in watermelon. PCAMBIA2300-empty and TRV-empty were used as controls. A sterile medical syringe was used to inject the recombinant plasmid and empty vector bacterial solution into the leaves of 30 watermelon seedlings, and the growth status of the leaves was observed and samples were taken every day.

[0113] By observing the color of watermelon leaves infected with virus vectors for one week, it was found that the leaves were different from those infected with TRV-empty vector ( Figure 9 Compared with infected leaves in A), after overexpression of Cla97C04G068470 gene, the injected area of ​​the leaves turned yellow ( Figure 9 B), after silencing the Cla97C04G068530 gene, compared with the empty vector ( Figure 10 A), the injection area of ​​the leaf turns yellow ( Figure 10 B in the figure). This indicates that the Cla97C04G068470 and Cla97C04G068530 genes play a regulatory role in the formation of yellow veins. Total RNA was extracted from watermelon leaves 5 days after treatment, and cDNA was obtained by reverse transcription and identified by fluorescence quantitative PCR. Data analysis was performed using GraphPad software. Fluorescence quantitative results showed that the transient expression system was successfully established in watermelon leaves ( Figure 9 C and Figure 10 C in the figure). CRISPR / Cas9 dual-target knockout mutant plants showed yellowing of the entire leaf or the veins ( Figure 10 D) in.

[0114] The above descriptions are merely a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application discloses the preferred embodiments as above, they are not intended to limit the present application. Any technical personnel familiar with the present profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A KASP molecular marker for identifying yellow-green veins of watermelon, characterized in that: The KASP molecular marker includes the K93281 molecular marker and / or the K93136 molecular marker; The polymorphism of the 71st base of the K93281 molecular marker is A / G, and the 71st base of the K93281 molecular marker is located at position 93281 of chromosome 4 of the watermelon genome; The polymorphism of the 71st base of the K93136 molecular marker is G / A, and the 71st base of the K93136 molecular marker is located at position 93136 of chromosome 4 of the watermelon genome; The version of the watermelon genome is Watermelon (97103) v2.5 Genome.

2. The KASP molecular marker for identifying yellow-green veins of watermelon according to claim 1, characterized in that: The nucleotide sequence of the K93281 molecular marker is shown in SEQ ID NO.1; The nucleotide sequence of the K93136 molecular marker is shown in SEQ ID NO.

2.

3. Use of the KASP molecular marker according to claim 1 or 2 in watermelon molecular marker-assisted breeding.

4. A primer combination, characterized in that The primer combination is used to detect the KASP molecular marker according to claim 1 or 2; The primer combination for detecting the K93281 molecular marker includes two specific forward primers K1-Fam and K1-Hex, and a universal reverse primer K1-R. The nucleotide sequence of K1-Fam is shown in SEQ ID NO.3, the nucleotide sequence of K1-Hex is shown in SEQ ID NO.4, and the nucleotide sequence of K1-R is shown in SEQ ID NO.

5. The primer combination for detecting the K93136 molecular marker includes two specific forward primers K2-Fam and K2-Hex, and a universal reverse primer K2-R. The nucleotide sequence of K2-Fam is shown in SEQ ID NO.6, the nucleotide sequence of K2-Hex is shown in SEQ ID NO.7, and the nucleotide sequence of K2-R is shown in SEQ ID NO.

8.

5. A primer combination according to claim 4, characterized in that, The 5' ends of K1-Fam and K2-Fam both carry a FAM fluorescent tag sequence; The 5' ends of the K1-Hex and K2-Hex both carry a HEX fluorescent tag sequence.

6. Use of the primer combination according to claim 4 or 5 in preparing a detection reagent or a detection kit for identifying the yellow-green veins of watermelon.

7. A method for identifying yellow-green veins of watermelon, characterized in that: The steps include: (1) Extracting genomic DNA from the watermelon leaves to be tested; (2) using the genomic DNA as a template and performing PCR amplification using the primer combination of claim 4 or 5 to obtain a PCR product; (3) The genotype of the PCR product is determined after fluorescence detection, and the vein color is determined based on the genotype: For K93281: When the genotype is AA or AG, the leaf veins are yellow; when the genotype is GG, the leaf veins are green; For K93136: When the genotype is GG or GA, the leaf veins are judged to be yellow; when the genotype is AA, the leaf veins are judged to be green.

8. The method for identifying yellow-green veins of watermelon according to claim 7, characterized in that: In step (2), the PCR amplification procedure is 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.

9. A gene related to yellow-green veins of watermelon, characterized in that: including Cla97C04G068470 and / or Cla97C04G068530; The CDS sequence of Cla97C04G068470 is shown in SEQ ID NO.9; The CDS sequence of Cla97C04G068530 is shown in SEQ ID NO.

10.

10. Use of the watermelon yellow-green vein-related gene according to claim 9 in regulating the color of watermelon veins; The application includes overexpressing the Cla97C04G068470 gene in watermelon, and / or silencing the Cla97C04G068530 gene, and / or knocking out the Cla97C04G068530 gene to cultivate yellow-veined watermelon varieties.

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