KASP marker primer combination for detecting light green / dark green trait of watermelon peel and its application

Abstract

The present invention belongs to the field of molecular biology and provides a KASP marker primer combination for detecting light green / dark green coloration of watermelon peel and its application. The KASP marker primer combination for detecting light green / dark green coloration of watermelon peel comprises the following three primers: upstream primer 1, upstream primer 2, and downstream primer. The present invention also provides applications of the KASP marker primer combination for detecting light green / dark green coloration of watermelon peel. Using the primer combination provided by the present invention to detect light green / dark green coloration of watermelon peel genotypes simplifies the operational process, reduces human error, and achieves high analytical throughput. This method, applied to the selection and breeding of light green / dark green coloration genes for watermelon peel, can significantly save time and labor costs, improve the efficiency of molecular marker-assisted selection, and accelerate the selection and breeding of watermelon peel color traits.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to a KASP (Kompetitive Allele-Specific PCR, i.e. competitive allele-specific PCR) marker primer combination for detecting the light green / dark green trait of watermelon peel and an application thereof. Background Art

[0002] Watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai) is an annual, vine-growing herbaceous plant of the Cucurbitaceae family. With rising consumer spending, research on horticultural crop traits is focusing not only on yield and taste, but also on appearance, which is influencing consumer choices. Consequently, the appearance of horticultural crops has become a growing target for breeders, with peel color receiving significant attention. The composition and content of different pigments are the primary determinants of color diversity in watermelon fruit, and genes controlling pigment types and content play a crucial role in rind color. Therefore, analyzing the genes that control peel color to guide watermelon cultivar selection is crucial. Currently, commonly used molecular marker methods include CAPS, dCAPS, SSR, and RFLP. These techniques require enzyme digestion or electrophoresis verification, which is time-consuming, costly, and cumbersome, limiting their application in molecular breeding. KASP marker technology, however, can detect differences as small as 1 bp, is highly accurate and simple, and can complete large-scale detection in a short period of time, offering significant advantages in its application. Summary of the Invention

[0003] In order to solve the above-mentioned problems existing in the prior art, the present invention provides a KASP marker primer combination for detecting the light green / dark green trait of watermelon peel and its application, and achieves the following invention objectives: by detecting the light green / dark green trait of watermelon through the KASP marker primer combination, the detection time is greatly shortened, and the purpose of precision breeding is achieved.

[0004] In order to achieve the above invention objectives, the technical solutions adopted are as follows:

[0005] A KASP marker primer combination for detecting the light green / dark green trait of watermelon peel, wherein the control gene of the light green / dark green trait of watermelon peel is the WSC gene, and the KASP marker core primer combination includes the following three primers:

[0006] Core sequence of upstream primer 1:

[0007] 5'-AAACGGAGAGCATTAGAGGAGATGG-3';

[0008] Core sequence of upstream primer 2:

[0009] 5'-aaacggagagcattagaggagatgc-3';

[0010] Sequence of downstream primer;

[0011] 5'-ctgaatgaccatgttaacaatcaagaagg-3'.

[0012] The KASP marker primer combination includes the following three primers:

[0013] Sequence of upstream primer 1:

[0014] 5'-gaaggtgaccaagttcatgctaaacggagagcattagaggagatgg-3';

[0015] Sequence of upstream primer 2:

[0016] 5'-gaaggtcggagtcaacggattaaacggagagcattagaggagatgc-3';

[0017] Sequence of downstream primer:

[0018] 5'-ctgaatgaccatgttaacaatcaagaagg-3'.

[0019] Application of a KASP marker primer combination for detecting the light green / dark green trait of watermelon peel, wherein the application uses the KASP marker primer combination to detect the light green / dark green trait of watermelon peel, comprising the following steps:

[0020] (1) Extracting genomic DNA of the watermelon variety to be tested;

[0021] (2) PCR amplification of watermelon genomic DNA was performed using KASP marker primers containing the light green / dark green trait of watermelon peel;

[0022] (3) Based on the differences in PCR fluorescence signals, software was used to analyze and identify the genotype of each watermelon to be tested, that is, watermelon varieties with homozygous GG, CC, and heterozygous GC genotypes were identified.

[0023] The PCR amplification used a PCR reaction system of: 2.5 μL of 20-100 ng / μL watermelon genomic DNA, 2.5 μL of KASP Master Mix, and 0.075 μL of Primer mix, for a total of 5.075 μL.

[0024] The Primer mix: upstream primer 1, upstream primer 2, and downstream primer are diluted to 50 μM with ddH2O, and then the upstream primer 1, upstream primer 2, and downstream primer are mixed at a molar concentration ratio of 1:1:3.

[0025] The reaction procedure of the PCR amplification reaction is: pre-denaturation at 95°C for 10 min; 95°C for 15 s, 61°C for 45 s, for a total of 10 cycles, with a temperature drop of 0.6°C during each cycle; 95°C for 15 s, 55°C for 1 min, for a total of 34 cycles.

[0026] Application of a KASP marker primer combination for detecting the light green / dark green trait of watermelon peel, wherein the application includes cultivating new plants having the light green / dark green trait of watermelon; comprising the following steps:

[0027] Step 1: Prepare an F1 hybrid combination with the recipient parent P1 and the donor parent P2; then perform backcrossing with the recipient parent P1 as the recurrent parent to obtain a BC1F1 backcross segregating population;

[0028] Step 2: Genotyping the BC1F1 backcross segregating population using a KASP marker primer combination to detect the light green / dark green trait of the watermelon peel, selecting individual plants with a genotype of CC for further backcrossing to obtain a BC2F1 backcross segregating population;

[0029] Step 3: Genotype detection is performed on the BC2F1 generation backcross segregation population using a KASP marker primer combination to detect the light green / dark green trait of watermelon peel, and individual plants with a genotype of CC are selected and backcrossed for n generations to obtain a BCnF1 generation backcross segregation population, where n is an integer of 3-8, preferably 4.

[0030] Step 4: Genotype detection was performed on the BCnF1 generation backcross segregating population using a KASP marker primer combination to detect the light green / dark green trait of the watermelon peel, and individual plants with a genotype of CC were selected for self-pollination.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. The KASP marker primer combination for detecting the light green / dark green color of watermelon peel, provided by the present invention, is used to detect the light green / dark green color of watermelon peel. The procedure is simple and time-efficient, reducing human error and providing high analytical throughput, making it ideal for simultaneous testing of large numbers of samples. It can detect differences as small as 1 bp, is highly accurate and simple, and can complete large-scale testing in a short period of time, offering significant advantages. Traditional molecular detection methods require five to six hours, while the present method only takes one and a half hours.

[0033] 2. The KASP marker primer combination for detecting the light green / dark green trait of watermelon peel provided by the present invention is applied to the breeding of new varieties of watermelon with light green / dark green peel, which can greatly save time and labor costs, improve the breeding efficiency of molecular marker-assisted selection, and accelerate the process of watermelon breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Attachment Figure 1 Allele discrimination diagram in Example 2; in the diagram, blue dots represent that the site is a homozygous genotype GG; red dots represent that the site is a homozygous genotype CC; and green dots represent that the site is a heterozygous genotype GC.

[0035] Attachment Figure 2 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by blue dots;

[0036] Attachment Figure 3 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by green dots;

[0037] Attachment Figure 4 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by the red dots. DETAILED DESCRIPTION

[0038] To make the purpose, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0039] Example 1: KASP marker primer combination for detecting the light green / dark green trait of watermelon peel

[0040] The molecular marker WSC related to the light green / dark green trait of watermelon rind was designed and obtained using the following method:

[0041] 1) Comparison of the genome sequences of watermelon plants with light green rind and dark green rind revealed a 1-bp variation in the WSC gene in both. Sequence analysis revealed that this 1-bp mutation occurred in the exon region, indicating that this 1-bp variation could lead to amino acid changes, thus resulting in differences in traits.

[0042] 2) Based on the 1bp variation of the WSC gene in light green and dark green watermelons, a set of three primers were designed near the region of this variation;

[0043] The specific sequences of the primer combinations are as follows:

[0044] FAM-labeled upstream primer 1:

[0045] 5'-gaaggtgaccaagttcatgctaaacggagagcattagaggagatgg-3';

[0046] VIC-labeled upstream primer 2:

[0047] 5'-gaaggtcggagtcaacggattaaacggagagcattagaggagatgc-3';

[0048] Common sequence downstream primer:

[0049] 5'-ctgaatgaccatgttaacaatcaagaagg-3'.

[0050] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Qingdao).

[0051] That is, the KASP marker primer combination used to detect the light green / dark green trait of watermelon peel is a combination of upstream primer 1, upstream primer 2, and downstream primer.

[0052] 3) Genomic DNA of parental seedlings and hybrid offspring seedlings was extracted using the CTAB method. The specific steps are as follows:

[0053] ① Take 0.5 g of young leaves and place them in a 2 mL centrifuge tube. Add 600 μL of CTAB extraction buffer to each tube, add 2 steel balls, and crush them using a Tissuelyser-192 crusher with the frequency set to 60.00 Hz and the time set to 240 s.

[0054] ② After crushing, place in a 65℃ water bath for 60 minutes, inverting and mixing every 15 minutes.

[0055] ③ After water bath, add 200 μL of 3M ammonium acetate, place on ice to cool for 10 min, add 600 mL of chloroform and isoamyl alcohol mixture (the volume ratio of chloroform to isoamyl alcohol is 24:1), mix thoroughly, and centrifuge at 12000 rpm for 10 min.

[0056] ④ Take 600 μL of supernatant and add it to 600 μL of pre-chilled isopropanol (in a 96-well deep-well plate), mix gently, and place at -20°C for 30 minutes.

[0057] ⑤ Centrifuge at 12000rpm for 10min, discard the supernatant, wash the precipitate once with 70% ethanol, and air-dry at room temperature until there is no ethanol smell.

[0058] ⑥ Add 200 μL of ddH2O and incubate in a 65°C water bath for 20 min. The ddH2O contains 1 μL of 10 mg / mL RNase.

[0059] ⑦Take 1 μL of sample and The sample concentration was detected on N50 and adjusted to be consistent.

[0060] The above method was used to extract genomic DNA from plants with light green peel and plants with dark green peel, respectively; the WSC gene sequences of plants with light green peel and plants with dark green peel were amplified, and the PCR amplification products were submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0061] The PCR amplification method comprises the following steps: using the extracted genomic DNA as a template, using primer F and primer R to perform PCR amplification, and obtaining PCR amplification products.

[0062] The sequence of primer F is: 5′-ccaacgttccctctccaact-3′;

[0063] The sequence of primer R was: 5′-gcccgcctgttgaaaattcc-3′;

[0064] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0065] The PCR reaction system used was: 100 ng·μL -1 2 μL of watermelon genomic DNA, 2 μL each of 10 μM primer F and 10 μM primer R, 25 μL of 2×TaqMasterMix, 19 μL of ddH2O, and a total volume of 50 μL.

[0066] The reaction program of PCR amplification reaction was as follows: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 35 cycles; 72°C for 5 min.

[0067] 4) Sequencing revealed that a 1bp variation in the WSC gene was completely consistent with the light green and dark green peel traits of watermelon. Therefore, the designed KASP marker primer combination can be used to identify the light green / dark green peel traits of watermelon.

[0068] Example 2: PCR detection of the light green / dark green trait of watermelon peel using a KASP labeled primer combination

[0069] 1. Extraction of genomic DNA

[0070] The genomic DNA of watermelon material was extracted according to the CTAB method in Example 1.

[0071] 2. PCR Amplification

[0072] The genomic DNA extracted in step 1 was used as a template, and PCR amplification was performed using the KASP marker primer combination (upstream primer 1, upstream primer 2, downstream primer) for detecting the light green / dark green trait of watermelon peel in Example 1 to obtain a PCR amplification product.

[0073] The PCR reaction system was as follows: 2.5 μL of 50 ng / μL watermelon genomic DNA, 2.5 μL of KASP Master Mix, and 0.075 μL of Primermix, for a total of 5.075 μL;

[0074] The KASP MasterMix is ​​a product of Guangzhou Good Biotechnology Co., Ltd., with catalog number GBS-1016-002.

[0075] The Primer mix: upstream primer 1, upstream primer 2, and downstream primer are diluted to 50 μM with ddH2O, and then the upstream primer 1, upstream primer 2, and downstream primer are mixed at a molar concentration ratio of 1:1:3.

[0076] The reaction program of PCR amplification reaction was as follows: pre-denaturation at 95°C for 10 min; 10 cycles at 95°C for 15 s, 61°C for 45 s, with the temperature decreasing by 0.6°C during each cycle; 34 cycles at 95°C for 15 s, 55°C for 1 min.

[0077] At the same time, a blank control in which no template DNA is added to the reaction system is set up, and 4 blank controls are set up on each PCR plate.

[0078] 3. Fluorescence scanning of PCR amplification products

[0079] PCR reactions were performed on a QuantStudio 6Flex instrument using Qantstudio TM Real-Time PCR Software is used to check the typing status and directly obtain the analysis results. The software automatically divides the test samples into homozygous GG, CC genotypes and heterozygous GC genotypes according to different genotypes to obtain an allele discrimination diagram.

[0080] When the software analyzes dots distributed in the upper left and lower right corners, the corresponding material is homozygous. A blue dot indicates the homozygous genotype "GG" at that locus, and a red dot indicates the homozygous genotype "CC." When a dot appears in the center (green), it indicates the heterozygous genotype "GC." A black x mark indicates NTC, which is the water control. When no heterozygote is present, no dot appears in the center, and the blue dot in the upper left corner turns green, indicating that all the material is homozygous. Because the two upstream primers (upstream primer 1 and upstream primer 2) are preceded by different fluorescent linkers, FAM and VIC, respectively, if the material being tested is homozygous, one of the corresponding primers will amplify during amplification, and the difference in fluorescence will distinguish whether the material being tested is GG or CC. If the material being tested is heterozygous, both primers will amplify during amplification, producing fluorescence different from that of the homozygous material, thus achieving the purpose of distinguishing heterozygous genotypes.

[0081] Experiment 1: 80 watermelon plants of different varieties were tested according to the method described in Example 2 to obtain allele discrimination diagrams, as shown in the attached figure. Figure 1 The test results of all samples are shown in Table 1.

[0082] Table 1 Statistics of test results of 80 watermelon samples

[0083]

[0084]

[0085]

[0086] In Table 1, the watermelon fruits with a result of GG are all light green varieties, and the watermelon fruits with a result of GC or CC are all dark green varieties. Therefore, it can be demonstrated that the accuracy of the present invention for selecting watermelon varieties with light green or dark green peel is very high.

[0087] Experiment 2: Select materials with different fluorescent spots for sequencing

[0088] Select the samples with red, blue and green test results in test 1, amplify the DNA fragments and submit them to Sangon Biotech (Shanghai) Co., Ltd. (Qingdao) for sequencing. The results of DNA map analysis of the sequenced samples are shown in the attached figure. Figure 2-4 As shown;

[0089] Attachment Figure 2 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by blue dots;

[0090] Attachment Figure 3 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by green dots;

[0091] Attachment Figure 4 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by the red dots.

[0092] The sequencing results show that the DNA samples represented by blue and red dots are all homozygous, while the DNA samples represented by green dots are all heterozygous. Furthermore, the watermelon fruit represented by blue dots has a light green peel color, while the watermelon fruit represented by red dots has a dark green peel color. The sequencing results are consistent with the phenotypic results. Therefore, it can be demonstrated that the molecular markers of the present invention can be used to assist in the selection of watermelon varieties with light green or dark green peels with high accuracy.

[0093] Example 3 A method for cultivating new plants with dark green rind characteristics of watermelon

[0094] Here are the steps:

[0095] Step 1: Prepare an F1 hybrid combination with the recipient parent P1 and the donor parent P2; then perform backcrossing with the recipient parent P1 as the recurrent parent to obtain a BC1F1 backcross segregating population;

[0096] The parent P1 is a watermelon variety X19-09 of high quality, with a light green rind.

[0097] The parent P2 is a watermelon variety X19-02 of average quality, a plant with dark green rind.

[0098] Step 2: For the BC1F1 backcross segregation population, the KASP marker primer combination for detecting the light green / dark green trait of watermelon peel described in Example 1 was used. By the method of Example 2, individual plants with the CC genotype were selected for further backcrossing to obtain a BC2F1 backcross segregation population.

[0099] Step 3: Using the KASP marker primer combination for detecting the light green / dark green trait of watermelon peel described in Example 1, select individual plants with the CC genotype using the method of Example 2, and continue backcrossing for n generations to obtain a BCnF1 backcross segregating population, where n is 4.

[0100] Step 4: For the BCnF1 generation backcross segregating population, continue to use the KASP marker primer combination for detecting the light green / dark green trait of watermelon peel described in Example 1. Using the method of Example 2, select a single plant with the CC genotype for self-pollination to obtain a homozygous watermelon variety X19-09 dark green peel strain.

[0101] Unless otherwise specified, all percentages and ratios described in the present invention are by mass and by weight.

[0102] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and many variations are possible. Any variations that can be directly derived or imagined by a person skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A KASP marker primer combination for detecting the light green and dark green traits of watermelon peel, characterized by: The control gene for the light green and dark green traits of the watermelon peel is the WSC gene, and the KASP marker primer combination includes the following three primers: Upstream primer 1: 5'-gaaggtgaccaagttcatgctaaacggagagcattagaggagatgg-3'; Upstream primer 2: 5'-gaaggtcggagtcaacggattaaacggagagcattagaggagatgc-3'; Downstream primer: 5'-ctgaatgaccatgttaacaatcaagaagg-3'.

2. Application of a KASP marker primer combination for detecting the light green and dark green traits of watermelon peel, characterized in that: The application is used to detect the light green and dark green traits of watermelon peel, and includes the following steps: (1) Extracting genomic DNA of the watermelon variety to be tested; (2) performing PCR amplification on watermelon genomic DNA using the KASP marker primer combination described in claim 1; (3) Based on the difference in PCR fluorescence signals, software was used to analyze and identify the genotype of each watermelon to be tested, that is, watermelon varieties with homozygous GG, CC, and heterozygous GC genotypes were identified; watermelons with genotype GG were light green varieties, and watermelons with genotypes GC and CC were dark green varieties; The watermelon variety is the offspring of the hybrid of watermelon variety X19-09 and watermelon variety X19-02.

3. The use of the KASP marker primer combination for detecting the light green and dark green traits of watermelon peel according to claim 2, characterized in that: The PCR reaction system used in the PCR amplification is: 20-100 ng / μL watermelon genomic DNA 2.5 μL, KASP MasterMix 2.5 μL, Primer mix 0.075 μL, a total of 5.075 μL.

4. The use of the KASP marker primer combination for detecting the light green and dark green traits of watermelon peel according to claim 2, characterized in that: The Primer mix: upstream primer 1, upstream primer 2, and downstream primer are diluted to 50 μM with ddH2O, and then the upstream primer 1, upstream primer 2, and downstream primer are mixed at a molar concentration ratio of 1:1:

3.

5. The use of the KASP marker primer combination for detecting the light green and dark green traits of watermelon peel according to claim 2, characterized in that: The reaction procedure of the PCR amplification reaction is: pre-denaturation at 95°C for 10 min; 95°C for 15 s, 61°C for 45 s, for a total of 10 cycles, with a temperature drop of 0.6°C during each cycle; 95°C for 15 s, 55°C for 1 min, for a total of 34 cycles.

6. Application of a KASP marker primer combination for detecting light green and dark green traits of watermelon peel, characterized in that: The application is to cultivate new plants with dark green rind of watermelon, which comprises the following steps: Step 1: Prepare an F1 hybrid combination with the recipient parent P1 and the donor parent P2; then perform backcrossing with the recipient parent P1 as the recurrent parent to obtain a BC1F1 backcross segregating population; Step 2: Perform genotyping on the BC1F1 backcross segregating population using the KASP marker primer combination described in claim 1, select individual plants with a genotype of CC and continue backcrossing to obtain a BC2F1 backcross segregating population; Step 3: Perform genotyping on the BC2F1 generation backcross segregating population using the KASP marker primer combination described in claim 1, select individual plants with a genotype of CC, and continue backcrossing for n generations to obtain a BCnF1 generation backcross segregating population, where n is 3-8; Step 4: genotyping the BCnF1 generation backcross segregating population using the KASP marker primer combination of claim 1, and selecting a single plant with a CC genotype for self-pollination; The watermelon variety of the parent P1 is X19-09, and the watermelon variety of the parent P2 is X19-02.

7. The use of the KASP marker primer combination for detecting the light green and dark green traits of watermelon peel according to claim 6, characterized in that: The n is 4.

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