KASP marker primer combination for detecting orange / yellow flesh color traits in watermelon and its application

Abstract

The present invention belongs to the field of molecular biology and provides a KASP marker primer combination for detecting the orange / yellow flesh color of watermelon and its application. The KASP marker primer combination for detecting the orange / yellow flesh color of watermelon 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 the orange / yellow flesh color of watermelon. Using the primer combination provided by the present invention to detect the orange / yellow flesh color genotype of watermelon simplifies the operation process, reduces human error, and achieves high analytical throughput. This method, when applied to the breeding of watermelon orange / yellow flesh color genes, can significantly save time and labor costs, improve the breeding efficiency of molecular marker-assisted selection, and accelerate the breeding of watermelon orange / yellow flesh color traits.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically relates to a KASP (Kompetitive Allele-Specific PCR, i.e., competitive allele-specific PCR) marker primer combination for detecting the orange / yellow flesh color of watermelon and its application. A primer combination is provided for rapid screening of the orange / yellow flesh color trait of watermelon plants and molecular marker-assisted breeding, and a novel and simple molecular marker-assisted selection method is provided. Background Art

[0002] Watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai) is an annual, vine-growing herbaceous plant of the Cucurbitaceae family. Fruit color is a key agronomic trait in modern cultivated watermelons, primarily appearing in red, pink, orange, and yellow, creating a striking visual impression on consumers. While the flesh of common cultivated watermelons is mostly red or pink, the flesh of wild-type watermelons is mostly white or pale yellow. The diverse composition and content of carotenoids is the primary driver of the vibrant color of watermelon fruit. Controlling carotenoid synthesis is closely linked to watermelon flesh color, and analysis of related genes can predict watermelon flesh color, thus aiding watermelon breeding. 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. The KASP labeling technology can detect differences as small as 1 bp. It is very accurate and simple, and can complete a large number of tests in a short period of time, which has great advantages in application. Summary of the Invention

[0003] In order to solve the above problems existing in the prior art, the present invention provides a KASP marker primer combination for detecting the orange / yellow flesh color trait of watermelon and its application.

[0004] A KASP marker core primer combination for detecting the orange / yellow flesh color trait of watermelon, wherein the watermelon orange / yellow flesh color gene is the OYC gene, and the KASP marker core primer combination includes the following three primers:

[0005] Core sequence of upstream primer 1: 5′-ggtgaagtttgtgcagagtatgcca-3′;

[0006] Core sequence of upstream primer 2: 5′-ggtgaagtttgtgcagagtatgccg-3′;

[0007] Sequence of the downstream primer: 5′-cttcaattttccatctccaaacacatgtc-3′.

[0008] A KASP marker primer combination for detecting the orange / yellow flesh color of watermelon, wherein the watermelon orange / yellow flesh color gene is the OYC gene, and the KASP marker primer combination includes the following three primers:

[0009] Sequence of upstream primer 1:

[0010] 5'-gaaggtgaccaagttcatgctggtgaagtttgtgcagagtatgcca-3';

[0011] Sequence of upstream primer 2:

[0012] 5'-gaaggtcggagtcaacggattggtgaagtttgtgcagagtatgccg-3';

[0013] Sequence of the downstream primer: 5′-cttcaattttccatctccaaacacatgtc-3′.

[0014] Application of a KASP marker primer combination for detecting the orange / yellow flesh color trait of watermelon, wherein the application is for detecting the orange or yellow flesh color trait of watermelon, comprising the following steps:

[0015] (1) Extracting genomic DNA of the watermelon variety to be tested;

[0016] (2) PCR amplification of watermelon genomic DNA was performed using a KASP marker primer combination containing the detection of watermelon orange / yellow flesh color traits;

[0017] (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 AA, GG, and heterozygous AG genotypes were identified.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The invention relates to an application of a KASP marker primer combination for detecting the orange / yellow flesh color trait of watermelon, wherein the application is to cultivate new plants having the orange / yellow flesh color trait of watermelon.

[0022] The following steps are involved:

[0023] 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;

[0024] Step 2: Perform genotyping on the BC1F1 generation backcross segregating population using a KASP marker primer combination, select individual plants with a genotype of GG for further backcrossing, and obtain a BC2F1 generation backcross segregating population;

[0025] Step 3: Perform genotyping on the BC2F1 generation backcross segregation population using a KASP marker primer combination, select individual plants with a genotype of GG, and continue backcrossing for n generations to obtain a BCnF1 generation backcross segregation population; wherein n is an integer of 3-8, preferably 4.

[0026] Step 4: For the BCnF1 generation backcross segregating population, the KASP marker primer combination was used to perform genotype detection, and the individual plants with the genotype of GG were selected for self-pollination.

[0027] The beneficial effects of the present invention are as follows:

[0028] 1. Using the KASP marker primer combination for detecting orange / yellow flesh color in watermelon, the present invention provides a simple and efficient procedure, reduces human error, and offers high analytical throughput, making it ideal for simultaneous testing of large numbers of samples. While traditional molecular detection methods require five to six hours, the present method only takes one and a half hours.

[0029] 2. The KASP marker primer combination for detecting the orange / yellow flesh color trait of watermelon provided by the present invention is applied to the breeding of new orange / yellow flesh watermelon varieties, which can greatly save time and labor costs, improve the breeding efficiency of molecular marker-assisted selection, and accelerate the progress of watermelon breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Attachment Figure 1 Allele discrimination diagram in Example 2; in the diagram, blue dots represent that the locus is homozygous genotype AA; red dots represent that the locus is homozygous genotype GG; green dots represent that the locus is heterozygous genotype GA;

[0031] Attachment Figure 2 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by the blue dots;

[0032] Attachment Figure 3 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by green dots;

[0033] 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

[0034] 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.

[0035] Example 1: KASP marker primer combination for detecting orange / yellow flesh color traits in watermelon

[0036] The molecular marker OYC related to the orange / yellow flesh color trait of watermelon was designed and obtained by the following method:

[0037] 1) Comparison of the genome sequences of orange-fleshed and yellow-fleshed watermelon plants revealed a 1-bp difference in the OYC gene between the two at the genomic level. Sequence analysis revealed that this 1-bp mutation occurred in the exon region, indicating that this 1-bp variation would lead to an amino acid change, thus resulting in differences in traits.

[0038] 2) Based on the 1 bp variation in the OYC gene in orange and yellow watermelons, a set of three primers was designed near the region of this variation; the specific sequences of the primer combination are as follows:

[0039] Sequence of FAM-labeled upstream primer 1:

[0040] 5'-gaaggtgaccaagttcatgctggtgaagtttgtgcagagtatgcca-3';

[0041] Sequence of VIC-labeled upstream primer 2:

[0042] 5'-gaaggtcggagtcaacggattggtgaagtttgtgcagagtatgccg-3';

[0043] Sequence of the consensus sequence downstream primer: 5′-cttcaattttccatctccaaacacatgtc-3′;

[0044] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Qingdao).

[0045] That is, the KASP marker primer combination used to detect the orange / yellow flesh color trait of watermelon is a combination of upstream primer 1, upstream primer 2, and downstream primer.

[0046] 3) Genomic DNA of parental seedlings and hybrid offspring seedlings was extracted using the CTAB method. The specific steps are as follows:

[0047] 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.

[0048] ② After crushing, place the tube in a 65°C water bath for 60 minutes, inverting and mixing every 15 minutes.

[0049] 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.

[0050] ④ Take 600 μL of supernatant and add it to 600 μL of pre-cooled isopropanol (in a 96-well deep-well plate), mix gently, and place at -20°C for 30 minutes.

[0051] ⑤ Centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the precipitate once with 70% ethanol, and air-dry at room temperature until there is no ethanol smell.

[0052] ⑥ Add 200 μL ddH2O and incubate in a 65°C water bath for 20 min. The ddH2O contains 1 μL of 10 mg / mL RNase.

[0053] ⑦Take 1 μL of sample and The sample concentration was detected on N50 and adjusted to be consistent.

[0054] The above method was used to extract genomic DNA from the orange-fleshed plant and the yellow-fleshed plant, respectively; the OYC gene sequences of the orange-fleshed plant and the yellow-fleshed plant were amplified, and the PCR amplification products were submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The PCR amplification method includes using the extracted genomic DNA as a template and performing PCR amplification using primers F and R to obtain PCR amplification products.

[0055] The sequence of primer F was: 5′-atgtcttttgctccttcgttgg-3′;

[0056] The sequence of primer R was: 5′-gcccaaatagccttttgcctctc-3′;

[0057] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0058] The PCR reaction system used was: 100 ng·μL-1 watermelon genomic DNA 2μL, 10μM primer F, 10μM primer R 2μL each, 2×TaqMasterMix 25μL, ddH2O 19μL, and a total volume of 50μL.

[0059] The reaction program of PCR amplification reaction was as follows: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, for a total of 35 cycles; 72°C for 5 min.

[0060] 4) Sequencing revealed that a 1bp variation in the OYC gene was completely consistent with the orange / yellow flesh color trait of watermelon. Therefore, the designed KASP marker primer combination can be used to identify the orange / yellow flesh color trait of watermelon.

[0061] Example 2: Real-time quantitative PCR detection of orange / yellow flesh color traits in watermelon using KASP labeled primer combinations

[0062] 1. Extraction of genomic DNA

[0063] The CTAB method of Example 1 was used to extract genomic DNA from watermelon materials.

[0064] 2. PCR Amplification

[0065] Using the genomic DNA extracted in step 1 as a template, PCR amplification was performed using the KASP marker primer combination for detecting the orange / yellow flesh color trait of watermelon (upstream primer 1, upstream primer 2, downstream primer) described in Example 1 to obtain a PCR amplification product. The PCR reaction system was: 2.5 μL of 50 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.

[0066] The KASP MasterMix is ​​a product of Guangzhou Good Biotechnology Co., Ltd., with catalog number GBS-1016-002.

[0067] 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.

[0068] 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.

[0069] At the same time, a blank control was set up in which no template DNA was added to the reaction system, and 4 blank controls were set up on each PCR plate.

[0070] 3. Fluorescence scanning of PCR amplification products

[0071] 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 AA, GG and heterozygous AG genotypes according to different genotypes to obtain an allele discrimination diagram.

[0072] When the software analyzes dots distributed in the upper left and lower right corners, the corresponding material is homozygous. A blue dot represents the homozygous genotype "AA" at that locus, and a red dot represents the homozygous genotype "GG" at that locus. When a dot appears in the center (green), it indicates the heterozygous genotype "AG" at that locus. A black x mark indicates the 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 AA or GG. 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.

[0073] Experiment 1: 114 watermelon samples from different plants were tested according to the method of Example 2 to obtain allele discrimination diagrams, as shown in the attached figure. Figure 1 The KASP test results of all samples are shown in Table 1.

[0074] Table 1 Statistics of KASP test results for 114 watermelon samples

[0075]

[0076]

[0077]

[0078] In Table 1, the watermelon fruits with the result of AA are all yellow-fleshed varieties from the morphology, and the watermelon fruits with the results of GA and GG are all orange-fleshed varieties from the morphology. Therefore, it can be proved that the molecular markers of the present invention can be used to select watermelon varieties with orange or yellow flesh with high accuracy.

[0079] Experiment 2: Select materials with different fluorescent spots for sequencing

[0080] 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;

[0081] Attachment Figure 2 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by blue dots;

[0082] Attachment Figure 3 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by green dots;

[0083] Attachment Figure 4 The test results in Experiment 1 are the DNA sequencing results of the watermelon samples represented by the red dots.

[0084] By attaching Figure 2-4 The sequencing results show that the DNA samples represented by the blue and red dots are homozygous, while the DNA samples represented by the green dots are heterozygous. Furthermore, the watermelon fruits represented by the blue dots have yellow flesh, the red dots have orange flesh, and the green dots have orange flesh. 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 select watermelon varieties with red or yellow flesh with high accuracy.

[0085] Example 3: A method for cultivating new watermelon plants with orange flesh traits

[0086] Here are the steps:

[0087] 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;

[0088] The parent P1 is a watermelon variety X19-C17 of high quality, with a yellow flesh trait;

[0089] The parent P2 is a watermelon variety DAHORF of average quality, a plant with orange flesh.

[0090] Step 2: Genotype detection was performed on the BC1F1 backcross segregating population using the KASP marker primer combination of Example 1 and the method of Example 2, and individual plants with the genotype of GG were selected for further backcrossing to obtain a BC2F1 backcross segregating population.

[0091] Step 3: Perform genotype detection on the BC2F1 generation backcross segregation population using the KASP marker primer combination of Example 1 and the method of Example 2, select individual plants with a genotype of GG, continue backcrossing for n generations, and obtain a BCnF1 generation backcross segregation population; wherein n is 4.

[0092] Step 4: Genotype detection was performed on the BCnF1 backcross segregating population using the KASP marker primer combination of Example 1 and the method of Example 2. Individual plants with the genotype GG were selected for self-pollination to obtain a homozygous orange-fleshed watermelon variety X19-C17.

[0093] Unless otherwise specified, all percentages and ratios described in the present invention are by mass and by weight.

[0094] 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 orange and yellow flesh color traits in watermelon, characterized by: The gene controlling the orange and yellow flesh color of watermelon is the OYC gene, and the KASP marker primer combination includes the following three primers: Upstream primer F1: 5'-gaaggtgaccaagttcatgctggtgaagtttgtgcagagtatgcca-3'; Upstream primer F2: 5'-gaaggtcggagtcaacggattggtgaagtttgtgcagagtatgccg-3'; Downstream primer: 5'-cttcaattttccatctccaaacacatgtc-3'.

2. Application of a KASP marker primer combination for detecting orange and yellow flesh color traits in watermelon, characterized in that: The application is to detect the orange and yellow flesh color traits of watermelon, comprising 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, the software was used to analyze and identify the genotype of each watermelon to be tested, that is, the watermelon varieties with homozygous AA, GG, and heterozygous GA genotypes were identified; the watermelon with genotype AA was a yellow-fleshed variety, and the watermelon with genotypes GG and GA was an orange-fleshed variety; The watermelon variety is a hybrid offspring of the watermelon variety X19-C17 and the watermelon variety DAHORF.

3. The use of the KASP marker primer combination for detecting orange and yellow flesh traits of watermelon 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 orange and yellow flesh traits of watermelon 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 orange and yellow flesh traits of watermelon 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 orange and yellow flesh color traits in watermelon, characterized in that: The application is to cultivate new plants with orange flesh color traits of watermelon; comprising 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 GG for further backcrossing, and obtain a BC2F1 backcross segregating population; Step 3: Perform genotyping on the BC2F1 generation backcross segregating population using the KASP marker primer combination of claim 1, select individual plants with a genotype of GG, and continue backcrossing for n generations to obtain a BCnF1 generation backcross segregating population, wherein n is an integer of 3-8; Step 4: genotyping the BCnF1 generation backcross segregating population using the KASP marker primer combination described in claim 1, and selecting a single plant with a genotype of GG for self-pollination; The watermelon variety of the parent P1 is X19-C17, and the watermelon variety of the parent P2 is DAHORF.

7. The use of the KASP marker primer combination for detecting orange and yellow flesh traits of watermelon according to claim 6, characterized in that: In step 3, n is 4.

Images